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The Molecular Basis of Substrate Recognition, Catalysis, and Regulation in Sortase Enzymes

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Jacobitz, Alex William

Publication Date
2015

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 UNIVERSITY OF CALIFORNIA

Los Angeles

The Molecular Basis of Substrate Recognition, Catalysis, and Regulation in

Sortase Enzymes

A dissertation submitted in partial satisfaction of the requirements for the degree

Doctor of Philosophy in Biochemistry and Molecular Biology

by

Alex William Jacobitz

2015
 ABSTRACT OF THE DISSERTATION

The Molecular Basis of Substrate Recognition, Catalysis, and Regulation in

Sortase Enzymes

By

Alex William Jacobitz

Doctor of Philosophy in Biochemistry and Molecular Biology

University of California, Los Angeles, 2015

Professor Robert T. Clubb, Chair

Gram-positive bacteria utilize sortase enzymes to catalyze a transpeptidation reaction that

covalently links protein substrates to the cell wall or polymerizes proteinaceous pili. Sortase

enzymes are membrane anchored cysteine transpeptidases that recognize a cell wall sorting

signal motif at the C-terminus of their primary protein substrate and covalently attach it to an

amino-nucleophile located in their secondary substrate. For cell wall anchoring sortases, this

secondary substrate is the cell wall precursor lipid II, while for pilin polymerizing sortases it is a

lysine sidechain within their pilin protein substrate. A number of major questions have remained

unanswered concerning the binding interactions that govern the substrate specificity of these

enzymes, their catalytic mechanism, and the mechanism through which appendages that contact

the active site regulate their activity. This dissertation advances the understanding of the sortase

mechanism through the elucidation of new sortase structures, and the characterization of their

dynamic behavior.

ii
 Chapter three of this thesis describes the solution structure of the class D sortase from

Bacillus anthracis by NMR. Class D enzymes anchor proteins involved in bacterial sporulation

to the cell wall, and this is the first structure of a class D sortase to ever be determined. NMR

analysis of the enzyme uncovered a rigid substrate binding pocket and a novel dimerization

interface. Chapter four describes the crystal structure of the class B sortase from Staphylococcus

aureus bound to a substrate analog. This work provided new insight into the biophysical basis of

substrate recognition. The structure combined with computational modeling and molecular

dynamics simulations led to the discovery of a substrate-stabilized oxyanion hole that is used to

stabilize tetrahedral reaction intermediates. Molecular dynamics simulations and the high degree

of sequence conservation inherent in these enzymes suggest that all members of the sortase

superfamily will stabilize high energy reaction intermediates in a similar manner. Chapter five

investigates the additional structural features some sortases use to regulate access to their active

site, focusing on sortase C-1 from Streptococcus pneumoniae as a model. Using NMR,

mutagenesis, and biochemical experiments this work demonstrated that the enzyme utilizes a

rigid N-terminal appendage, termed the “lid,” to maintain the enzyme in an inactive state. The

results of in vitro biochemical assays indicate that the lid prevents cleavage of the primary

substrate by preventing access to the catalytic cysteine residue. Both in vitro activity and access

to the active site cysteine could be increased by the incorporation of mutations which were

shown by NMR to destabilize the lid and increase its flexibility. Based on these results we

propose that on the cell surface, lid containing class C enzymes exist in a dormant state and are

only activated during pilus biogenesis by interactions with either their substrate or other factors

on the cell surface. The results of these experiments have provided new insight into substrate

binding, catalysis, and regulation in sortase enzymes.

iii
The dissertation of Alex Jacobitz is approved.

James U. Bowie

Beth A. Lazazzera

Robert T. Clubb, Committee Chair

University of California, Los Angeles

2015

iv
 DEDICATION

To my father, who taught me the value of hard work and perseverance, and always

faced adversity with humor and grace. This would not have been possible without

his strength and guidance.

- Thought is the wind, knowledge the sail, and mankind the vessel -

v
 TABLE OF CONTENTS

Chapter 1. Introduction to Sortase Enzymes ..............................................................................1

1.1 Overview ..............................................................................................................................2

1.2 Introduction to Sortase Biology ...........................................................................................3

1.3 References ............................................................................................................................7

Chapter 2. Structures and mechanism of the sortase superfamily of transpeptidases .........13

2.1 Overview ............................................................................................................................14

2.2 Introduction ........................................................................................................................15

2.3 The Staphylococcus aureus SrtA Paradigm .......................................................................17

2.4 Classification of Sortases ...................................................................................................19

2.5 Structural Biology of Sortase Enzymes .............................................................................20

2.6 Molecular Basis of Substrate Recognition .........................................................................29

2.7 The Molecular Mechanism of Sortase Enzymes ...............................................................38

2.8 Sortases as Powerful In Vitro Biochemical Reagents ........................................................40

2.9 Development of Structure-Based Therapeutics .................................................................42

2.10 Conclusion .........................................................................................................................46

2.11 Figures................................................................................................................................49

2.12 References ..........................................................................................................................60

vi
Chapter 3. Solution structure of the sortase required for efficient production of infectious

Bacillus anthracis spores..................................................................................................76

3.1 Overview ............................................................................................................................78

3.2 Introduction ........................................................................................................................79

3.3 Results and Discussion ......................................................................................................82

3.3.1 Design and Validation of Soluble BaSrtC .............................................................82

3.3.2 NMR Structure of BaSrtCΔ55 .................................................................................83

3.3.3 Disordered Loops Positioned near the Active Site Might Mediate in vitro ...............

Dimerization ..........................................................................................................89

3.3.4 Class A and D Sortases Are Structurally Related ..................................................89

3.3.5 Model of the BaSrtCΔ55–Sorting Signal Complex .................................................90

3.4 Experimental Procedures ...................................................................................................93

3.4.1 Cloning, Protein Expression, and Purification.......................................................93

3.4.2 NMR Spectroscopy and Structure Calculations ....................................................95

15
3.4.3 N Relaxation Measurements ...............................................................................96

3.4.4 Analytical Ultracentrifugation and Enzyme Assays ..............................................97

3.5 Figures................................................................................................................................98

3.6 References ........................................................................................................................108

3.7 Supplementary materials ..................................................................................................117

Chapter 4. Structural and Computational Studies of the Staphylococcus aureus Sortase B-

Substrate Complex Reveal a Substrate-stabilized Oxyanion Hole............................119

vii
4.1 Overview ..........................................................................................................................121

4.2 Introduction ......................................................................................................................122

4.3 Results ..............................................................................................................................124

4.3.1 Crystal Structure of the SrtB-NPQT* Complex and Computational Modeling

of the Thioacyl Intermediate ................................................................................124

4.3.2 Identification of Enzyme-Substrate Interactions Required for Catalysis ............127

4.3.3 Molecular Dynamics Simulations of SrtA and SrtB Reveal a Conserved

Mechanism through which the Substrate Stabilizes the Positioning of the

Active Site Arginine Residue...............................................................................129

4.4 Discussion ........................................................................................................................132

4.5 Experimental Procedures .................................................................................................139

4.5.1 Production, Crystallization, and Structure Determination of SrtB-NPQT*

Complex ...............................................................................................................139

4.5.2 Transpeptidation Assay........................................................................................140

4.5.3 Computational Modeling and Molecular Dynamics ............................................141

4.6 Figures..............................................................................................................................143

4.7 References ........................................................................................................................154

Chapter 5. The “lid” of Streptococcus pneumoniae SrtC1 is not Dynamic in Solution and

regulates substrate access to the active site .................................................................164

5.1 Overview ..........................................................................................................................166

5.2 Introduction ......................................................................................................................167

5.3 Results ..............................................................................................................................171

viii
 5.3.1 Initial NMR analysis indicates SpnSrtC1 forms dimers in solution ....................171

5.3.2 NMR dynamics data indicate that the SpnSrtC1 lid is not dynamic in vitro .......172

5.3.3 Mutations to the “lid” of SpnSrtC1 make the region highly dynamic in

solution.................................................................................................................175

5.3.4 Mutation of the lid is required to induce in vitro hydrolysis ...............................176

5.3.5 A closed lid is important for protecting the active site cysteine from

oxidation ..............................................................................................................178

5.4 Discussion ........................................................................................................................178

5.5 Experimental Procedures .................................................................................................184

5.5.1 Cloning, protein expression and purification .......................................................184

5.5.2 NMR spectroscopy...............................................................................................185

5.5.3 In vitro cleavage assay .........................................................................................186

5.5.4 In vitro oxidation assay ........................................................................................187

5.6 Figures..............................................................................................................................188

5.7 References ........................................................................................................................196

ix
 LIST OF FIGURES

Figure 2.1 Biological mechanism of sortase ...........................................................................49

Figure 2.2 Structure of sortase ................................................................................................50

Figure 2.3 Structural variation by class of sortase ..................................................................53

Figure 2.4 Sorting signal recognition ......................................................................................54

Figure 2.5 The substrate stabilized oxyanion hole ..................................................................55

Figure 2.6 Molecular mechanism of sortase enzymes ............................................................56

Figure 3.1 NMR spectra and amino acid sequence of BaSrtC ................................................98

Figure 3.2 MALDI-TOF demonstrating BaSrtCΔ55 can cleave the LPNTA-

containing peptide ................................................................................................100

Figure 3.3 Structures of B. anthracis class D sortase ............................................................101

Figure 3.4 Dynamics and ultracentrifugation analysis ..........................................................104

Figure 3.5 Comparison of representative structures from class A–D sortases ......................106

Figure 3.6 Substrate binding and active site architecture ......................................................107

Figure S3.1 Residue by residue T2, T1 and 15N{1H} Heteronuclear NOE data for BaSrtCΔ55 117

Figure 4.1 Structure of the SrtB-NPQT* complex ................................................................143

Figure 4.2 Structure and interactions of the NPQT* modifier in complex with SrtB...........145

Figure 4.3 Expanded view of the active site in the energy-minimized model of the SrtB-

NPQT thioacyl intermediate ................................................................................147

Figure 4.4 Transpeptidation activity of wild-type and mutant SrtB......................................148

Figure 4.5 Alignment of SrtB-NPQT* and SrtA-LPAT* .....................................................150

Figure 4.6 Structures and free energy profiles of the SrtA and SrtB thioacyl complexes ....151

x
Figure 4.7 The SrtB transpeptidation mechanism showing how the sorting signal may

stabilize the oxyanion hole...................................................................................153

Figure 5.1 S. pneumoniae SrtC1 structure and lid motif .......................................................188

Figure 5.2 NMR analysis shows SpnSrtC1 is dimeric in vitro..............................................189

Figure 5.3 NMR dynamics of SpnSrtC1 ...............................................................................190

Figure 5.4 NMR shows SpnSrtC1 mutants have more dynamic lids ....................................192

Figure 5.5 SpnSrtC1 lid mutants are more active in vitro .....................................................194

Figure 5.6 The lid of SpnSrtC1 protects the active site cysteine from modification by a

sulfhydryl modifier ..............................................................................................195

xi
 LIST OF TABLES

Table 2.1 Structurally characterized sortase enzymes ...........................................................51

Table 2.2 Activity of sortase enzymes in vitro ......................................................................58

Table 3.1 Structural Statistics for the Solution Structure of BaSrtCΔ55 ...............................102

Table 4.1 Crystallographic data collection and refinement statistics...................................144

xii
 ACKNOWLEDGEMENTS

First and foremost I would like to thank my advisor, Dr. Robert Clubb for his guidance,

support, and for the unlimited supply of coffee he provided: without which my PhD would not

have been possible. My collaborators, Dr. Jeff Wereszczynski, Dr. J. Andrew McCammon, Dr.

Sung Wook Yi, and Dr. Michael Jung for helping in the design and execution of numerous

experiments, and I would like to additionally thank Jeff specifically for teaching me to run

molecular dynamics simulations. Dr. Michael Sawaya for patiently teaching me X-ray

crystallography. Dr. Robert Peterson for helping me to better understand NMR and for writing

new pulse programs when necessary. My rotation mentors, Dr. Todd Yeates and Dr. William

Gelbart for their advice on topics in and out of the lab. My committee members: Dr. James

Bowie, Dr. Pascal Egea, Dr. Beth Lazazzera, and Dr. Joseph Loo for their thoughts and

guidance. The undergraduates, John Cho, Allen Tsiyer, and Scott McConnell, and rotation

students, Brendan Amer, Grace Huang, Angelyn Nguyen, Reid O’Brien Johnson, Kevin Eden,

Mimi Ho, and Jessica Davis, whose efforts in the lab proved to be invaluable. Members of the

Clubb lab, Dr. Scott Robson, Dr. Valerie Villareal, Dr. Thomas Spirig, Dr. Ethan Weiner, Dr.

Reza Malmirchegini, Dr. Albert Chan, Megan Sjodt, Brendan Amer, Grace Huang, Michele

Kattke, and Ramsay Macdonald, whose friendship, scientific discourse, and moral support

helped to get me through graduate school. My friends and family for putting up with me for the

last 6 years. The Cellular and Molecular Biology Training Grant, Audree Fowler Fellowship in

Protein Science, John M. Jordan Memorial Award for Research in Biochemistry, and the UCLA

Graduate Division Dissertation Year Fellowship for supporting me throughout my graduate

xiii
career, and for the additional opportunities for enriching my scientific development that they

have provided.

Chapter two of this dissertation is a version of a manuscript in preparation titled:

Structures and Mechanism of the Sortase Superfamily of Transpeptidases. Jacobitz, A. W.,

Kattke, M. D., Wereszczynski, J., and Clubb, R. T. 2015.

Chapter three of this dissertation is a version of the manuscript: Robson S. A., Jacobitz A.

W., Clubb R.T., Phillips M.L., Clubb R.T. Solution Structure of the Sortase Required for

Efficient Production of Infectious Bacillus anthracis Spores. Biochemistry. 2012; 51(40):7953-

7963. doi:10.1021/bi300867t.http://pubs.acs.org/doi/abs/10.1021/bi300867t. Reproduced with

permission. Copyright 2011, American Chemical Society. We express great appreciation to Dr.

Joseph Loo and members of the Clubb laboratory for their assistance.

Chapter four of this dissertation is a version of the manuscript: Jacobitz, A. W.,

Wereszczynski, J., Yi, S. W., Amer, B. R., Huang, G. L., Nguyen, A. V., Sawaya, M. R., Jung,

M. E., McCammon, J. A., Clubb, R. T. Structural and computational studies of the

Staphylococcus aureus Sortase B-substrate complex reveal a substrate-stabilized oxyanion hole.

Journal of Biological Chemistry. 2014; jbc.M113.509273.

http://doi.org/10.1074/jbc.M113.509273 Reproduced with permission. Copyright 2014, Journal

of Biological Chemistry. This work was supported, in whole or in part, by National Institutes of

Health Grants AI52217 (to R. T. C. and M. E. J.), GM31749 (to J. A. M.), and T32

GM007185 (to A. W. J. and G. L. H.). This work was also supported by the National Science

Foundation, the Howard Hughes Medical Institute, the Center for Theoretical Biological Physics,

the National Biomedical Computation Resource, and the National Science Foundation

Supercomputer Centers.

xiv
 Chapter Five of this dissertation is a version of a manuscript in preparation titled: The

“lid” of S. pneumoniae SrtC1 is not dynamic in solution and regulates substrate access to the

active site. Jacobitz A. W., Yi, S. W., McConnell, S., Peterson, R., Jung, M. E., and Clubb, R. T.

2015.

xv
 Vita

2007 – 2009 Bachelors of Arts, Chemistry

California State University, Channel Islands

2010 – 2011 Teaching Assistant, Department of Chemistry and Biochemistry

University of California, Los Angeles

2011 Candidate in Philosophy, Biochemistry and Molecular Biology

University of California, Los Angeles

2011 – 2014 Cellular and Molecular Biology Training Grant

2013 – 2014 Audree Fowler Fellowship in Protein Science

2014 John M. Jordan Memorial Award for Research in Biochemistry

2014 – 2015 UCLA Graduate Division Dissertation Year Fellowship

xvi
 PUBLICATIONS

Jacobitz, A. W., Wereszczynski, J., Yi, S. W., Amer, B. R., Huang, G. L., Nguyen, A. V., Sawaya, M.

R., Jung, M. E., McCammon, J. A. & Clubb, R. T. Structural and computational studies of the

Staphylococcus aureus Sortase B-substrate complex reveal a substrate-stabilized oxyanion hole. J. Biol.

Chem. (2014). doi:10.1074/jbc.M113.509273

Robson, S. A., Jacobitz, A. W., Phillips, M. L. & Clubb, R. T. Solution Structure of the Sortase Required

for Efficient Production of Infectious Bacillus anthracis Spores. Biochemistry. 51, 7953–7963 (2012).

doi:10.1021/bi300867t

King, N. P., Jacobitz, A. W., Sawaya, M. R., Goldschmidt, L. & Yeates, T. O. Structure and folding of a

designed knotted protein. Proc. Natl. Acad. Sci. (2010). doi:10.1073/pnas.1007602107

xvii
 Chapter 1

Introduction to the Biology of Sortase Transpeptidase Enzymes

1
1.1 Overview

Sortase enzymes are ubiquitous in Gram-positive bacteria. These enzymes perform a

transpeptidation reaction that covalently attaches proteins to the cell wall, or polymerizes protein

substrates into long filamentous pili. These surface anchored proteins and pili serve to augment

the pathogenic potential of their bacterial proprietor by allowing the organism to more

effectively interact with host cells and other bacteria. Sortase anchored surface proteins perform

a variety of functions including adhesion, immune evasion, nutrient acquisition, and spore

formation. Numerous studies have shown that the removal of sortase enzymes is generally not

lethal to the bacterium, but often renders the bacterium avirulent. This observation has led to a

wealth of research into the biology, structures, mechanism, regulation, and inhibition of these

enzymes.

2
1.2 Introduction to Sortase Biology

Gram-positive organisms are recognized by the presence of a thick cell wall. While its

function in protecting the cell against mechanical and osmotic stresses is well known, the cell

wall also provides an additional function as a scaffold for the display of a large number of

surface proteins by sortase enzymes1–3. All sortase enzymes catalyze the formation of a peptide

bond that covalently joins two substrates together. For most sortases, this transpeptidation

reaction links a substrate protein bearing an appropriate cell wall sorting signal (CWSS)

containing a C-terminal 5 residue recognition motif and transmembrane helix to the cell wall, but

a select subgroup of the enzymes instead catalyze the formation of an isopeptide bond between a

CWSS containing protein and a lysine sidechain of a pilin motif containing protein. Based on

sequence similarity and structural homology, sortases can be categorized into 6 distinct families.

These families each typically operate to anchor a specific type of substrate, with the class A

enzymes being constitutively expressed and anchoring a number of diverse proteins to the cell

surface, and each of the other types being relegated to a separate operon and only expressed

along with their substrates under specific conditions. Most Gram-positives have at least one

sortase gene, with certain bacteria harboring genes for multiple sortases which typically function

non-redundantly to attach a specific subset of surface proteins to the cell wall. For bacteria with a

single sortase, that sortase is typically class A and will attach multiple proteins to the cell wall,

with the most prolific of these enzymes being sortase A from Listeria monocytogenes, which is

predicted to display 43 distinct proteins4,5. Other enzymes, like Staphylococcus aureus sortase B,

are instead dedicated to mounting a single substrate6.

The majority of sortase enzymes studied to date catalyze the covalent attachment of their

substrates to the cell wall, and many of these enzymes have been shown to be particularly

3
important in virulence. Genetic knock-outs of sortase A and B have both been shown to reduce

virulence in a mouse model of infection for the human pathogens S. aureus7,8, and Bacillus

anthracis9, without affecting the ability of these organisms to grow in rich media. Sortase A KOs

have also been shown to reduce virulence of several other human pathogens including Listeria

monocytogenes10,11, and Streptococcus pneumoniae12–14. These studies have highlighted the

importance of this class of enzymes and led to the development of a number of small-molecule

sortase inhibitors which may eventually lead to the production of a novel anti-virulence

therapeutic15.

In addition to their importance as virulence factors, additional work has gone into

understanding the need for multiple classes of sortase and these studies have shown that the

different classes function non-redundantly to attach specific substrates to specific regions of the

cell wall. While all sortase enzymes are known to recognize a CWSS, the individual classes

generally recognize different 5 residue sorting signal motifs, and the structural basis of this

recognition is only beginning to be understood2,16,17. Beyond their ability to recognize different

sorting signals, these different classes of enzymes have also been shown to anchor their

substrates to distinct locations on the cell wall. While most sortases mount their proteins to the

outermost surface of the cell wall, certain class B enzymes which mount proteins involved in

delivering heme-iron to the cell have been shown to anchor proteins to a more buried location

within the cell wall, which is likely necessary to develop a heme transport conduit to deliver

heme from the surface of the cell wall to the cell membrane18,19. Sortase enzymes and their

substrates have also been shown to colocalize to distinct foci on the bacterial surface. Class A

sortases in cocci have been shown to specifically anchor substrates at division septa, where

subsequent growth and expansion will lead to a ring-like distribution of surface proteins across

4
the cell wall20–22. For rod shaped bacteria, cell surface anchoring has been localized to helical

patterns along the cell surface1,23. Certain class C sortases which are typically involved in pilus

polymerization have also been implicated in surface protein localization, and class A and C

sortases have been shown to anchor pili to different locations on the cell surface24. While it is

now well established that certain sortases are capable of localizing their substrates to discrete

locations on the cell wall, further study is needed to understand the mechanisms that cause this

localization to occur.

Instead of attaching proteins to the cell wall, a subset of sortase enzymes catalyze the

polymerization of pili. These sortases are typically found clustered in genomic islands along with

their substrates, and some bacteria can harbor genomic islands coding for the production of

multiple distinct of pili25. These pilus islands typically harbor at least 2 sortases and at least 2

substrates, and the molecular mechanism by which they coordinate to assemble pili is only

beginning to be understood4. Typically these operons code for one primary pilus protein that is

polymerized to make up the shaft of the pilus, and then one or two accessory proteins that

function as either the base of the pilus for attachment to the cell wall, or as an additional adhesin

to be attached as the tip, or elaborate the length of the shaft26. Many of the class C sortases have

high sequence identity, and some studies have suggested that some of these proteins are

multifaceted, polymerizing the pilus, attaching accessory pilus proteins, and attaching the pilus

to the wall, and that other sortases in the operon may function semi-redundantly24,27–29.

Interestingly, several more recent studies have indicated that individual sortases may each be

dedicated to specific steps in the pilin polymerization process, recognizing different pilin motifs

at from separate domains within a single pilin protein substrate to build more complex branched,

or “knobbed” structures30,31.

5
 While much is known about the basic biology of sortase enzymes, structural studies in

this field have answered, and will continue to answer questions about their substrate recognition,

molecular mechanism, regulation, and the interactions they have with other proteins on the cell

surface that are all necessary to build a complete understanding of the sorting reaction that these

enzymes perform on the cell surface.

6
1.3 References

1. Liew PX, Wang CLC, Wong S-L. Functional characterization and localization of a

Bacillus subtilis sortase and its substrate and use of this sortase system to covalently

anchor a heterologous protein to the B. subtilis cell wall for surface display. J Bacteriol.

2012;194(1):161-175. doi:10.1128/JB.05711-11.

2. Paterson GK, Mitchell TJ. The biology of Gram-positive sortase enzymes. Trends

Microbiol. 2004;12(2):89-95. doi:10.1016/j.tim.2003.12.007.

3. Desvaux M, Dumas E, Chafsey I, Hébraud M. Protein cell surface display in Gram-

positive bacteria: from single protein to macromolecular protein structure. FEMS

Microbiol Lett. 2006;256(1):1-15. doi:10.1111/j.1574-6968.2006.00122.x.

4. Spirig T, Weiner EM, Clubb RT. Sortase enzymes in Gram-positive bacteria. Mol

Microbiol. 2011;82(5):1044-1059. doi:10.1111/j.1365-2958.2011.07887.x.

5. Boekhorst J, de Been MWHJ, Kleerebezem M, Siezen RJ. Genome-wide detection and

analysis of cell wall-bound proteins with LPxTG-like sorting motifs. J Bacteriol.

2005;187(14):4928-4934. doi:10.1128/JB.187.14.4928-4934.2005.

6. Mazmanian SK, Ton-That H, Su K, Schneewind O. An iron-regulated sortase anchors a

class of surface protein during Staphylococcus aureus pathogenesis. Proc Natl Acad Sci U

S A. 2002;99(4):2293-2298. doi:10.2307/3057958.

7. Mazmanian SK, Liu G, Jensen ER, Lenoy E, Schneewind O. Staphylococcus aureus

sortase mutants defective in the display of surface proteins and in the pathogenesis of

7
 animal infections. Proc Natl Acad Sci U S A. 2000;97(10):5510-5515.

doi:10.1073/pnas.080520697.

8. Weiss WJ, Lenoy E, Murphy T, et al. Effect of srtA and srtB gene expression on the

virulence of Staphylococcus aureus in animal models of infection. J Antimicrob

Chemother. 2004;53(3):480-486. doi:10.1093/jac/dkh078.

9. Zink SD, Burns DL. Importance of srtA and srtB for growth of Bacillus anthracis in

macrophages. Infect Immun. 2005;73(8):5222-5228. doi:10.1128/IAI.73.8.5222.

10. Bierne H, Mazmanian SK, Trost M, et al. Inactivation of the srtA gene in Listeria

monocytogenes inhibits anchoring of surface proteins and affects virulence. Mol

Microbiol. 2002;43(4):869-881. http://www.ncbi.nlm.nih.gov/pubmed/11929538.

Accessed August 20, 2015.

11. Garandeau C, Réglier-Poupet H, Dubail I, Beretti J-L, Berche P, Charbit A. The sortase

SrtA of Listeria monocytogenes is involved in processing of internalin and in virulence.

Infect Immun. 2002;70(3):1382-1390.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=127754&tool=pmcentrez&ren

dertype=abstract. Accessed August 21, 2015.

12. Chen S, Paterson GK, Tong HH, Mitchell TJ, DeMaria TF. Sortase A contributes to

pneumococcal nasopharyngeal colonization in the chinchilla model. FEMS Microbiol Lett.

2005;253(1):151-154. doi:10.1016/j.femsle.2005.09.052.

8
13. Paterson GK, Mitchell TJ. The role of Streptococcus pneumoniae sortase A in

colonisation and pathogenesis. Microbes Infect. 2006;8(1):145-153.

doi:10.1016/j.micinf.2005.06.009.

14. Kharat AS, Tomasz A. Inactivation of the srtA Gene Affects Localization of Surface

Proteins and Decreases Adhesion of Streptococcus pneumoniae to Human Pharyngeal

Cells In vitro. Infect Immun. 2003;71(5):2758-2765. doi:10.1128/IAI.71.5.2758-

2765.2003.

15. Suree N, Jung ME, Clubb RT. Recent advances towards new anti-infective agents that

inhibit cell surface protein anchoring in Staphylococcus aureus and other gram-positive

pathogens. Mini Rev Med Chem. 2007;7(10):991-1000.

doi:10.2174/138955707782110097.

16. Jacobitz AW, Wereszczynski J, Yi SW, et al. Structural and computational studies of the

Staphylococcus aureus Sortase B-substrate complex reveal a substrate-stabilized oxyanion

hole. J Biol Chem. February 2014:jbc.M113.509273. doi:10.1074/jbc.M113.509273.

17. Suree N, Liew CK, Villareal VA, et al. The Structure of the Staphylococcus aureus

Sortase-Substrate Complex Reveals How the Universally Conserved LPXTG Sorting

Signal Is Recognized. J Biol Chem. 2009;284(36):24465-24477.

doi:10.1074/jbc.M109.022624.

18. Marraffini LA, Schneewind O. Anchor Structure of Staphylococcal Surface Proteins V.

ANCHOR STRUCTURE OF THE SORTASE B SUBSTRATE IsdC. J Biol Chem.

2005;280(16):16263-16271. doi:10.1074/jbc.M500071200.

9
19. Mazmanian SK, Skaar EP, Gaspar AH, et al. Passage of Heme-Iron Across the Envelope

of Staphylococcus aureus. Science (80- ). 2003;299(5608):906-909.

doi:10.1126/science.1081147.

20. Raz A, Fischetti VA. Sortase A localizes to distinct foci on the Streptococcus pyogenes

membrane. Proc Natl Acad Sci U S A. 2008;105(47):18549-18554.

doi:10.1073/pnas.0808301105.

21. DeDent A, Bae T, Missiakas DM, Schneewind O. Signal peptides direct surface proteins

to two distinct envelope locations of Staphylococcus aureus. EMBO J. 2008;27(20):2656-

2668. doi:10.1038/emboj.2008.185.

22. DeDent AC, McAdow M, Schneewind O. Distribution of protein A on the surface of

Staphylococcus aureus. J Bacteriol. 2007;189(12):4473-4484. doi:10.1128/JB.00227-07.

23. Bruck S, Personnic N, Prevost M-C, Cossart P, Bierne H. Regulated Shift from Helical to

Polar Localization of Listeria monocytogenesCell Wall-Anchored Proteins. J Bacteriol.

2011;193(17):4425-4437. doi:10.1128/JB.01154-10.

24. Fälker S, Nelson AL, Morfeldt E, et al. Sortase-mediated assembly and surface topology

of adhesive pneumococcal pili. Mol Microbiol. 2008;70(3):595-607. doi:10.1111/j.1365-

2958.2008.06396.x.

25. Mandlik A, Swierczynski A, Das A, Ton-That H. Pili in Gram-positive bacteria:

assembly, involvement in colonization and biofilm development. Trends Microbiol.

2008;16(1):33-40. doi:10.1016/j.tim.2007.10.010.

10
26. Hendrickx APA, Budzik JM, Oh S-Y, Schneewind O. Architects at the bacterial surface -

sortases and the assembly of pili with isopeptide bonds. Nat Rev Microbiol.

2011;9(3):166-176. doi:10.1038/nrmicro2520.

27. Mora M, Bensi G, Capo S, et al. Group A Streptococcus produce pilus-like structures

containing protective antigens and Lancefield T antigens. Proc Natl Acad Sci U S A.

2005;102(43):15641-15646. doi:10.1073/pnas.0507808102.

28. Ton-That H, Marraffini LA, Schneewind O. Sortases and pilin elements involved in pilus

assembly of Corynebacterium diphtheriae. Mol Microbiol. 2004;53(1):251-261.

doi:10.1111/j.1365-2958.2004.04117.x.

29. Neiers F, Madhurantakam C, Fälker S, et al. Two crystal structures of pneumococcal pilus

sortase C provide novel insights into catalysis and substrate specificity. J Mol Biol.

2009;393(3):704-716. doi:10.1016/j.jmb.2009.08.058.

30. El Mortaji L, Terrasse R, Dessen A, Vernet T, Di Guilmi AM. Stability and Assembly of

Pilus Subunits of Streptococcus pneumoniae. J Biol Chem. 2010;285(16):12405-12415.

doi:10.1074/jbc.M109.082776.

31. El Mortaji L, Fenel D, Vernet T, Di Guilmi AM. Association of RrgA and RrgC into the

Streptococcus pneumoniae pilus by sortases C-2 and C-3. Biochemistry. 2012;51(1):342-

352. doi:10.1021/bi201591n.

11
 Chapter 2

Structures and Mechanism of the Sortase Superfamily of

Transpeptidases

12
The work described in this chapter is a version of a manuscript to be submitted for publication.

Jacobitz, A. W., Kattke, M. D., Wereszczynski, J., and Clubb, R. T., 2015.

13
2.1 Overview

Sortase enzymes are ubiquitous in Gram-positives and catalyze a transpeptidation

reaction that can be used to anchor surface proteins to the peptidoglycan or polymerize them into

long filamentous pili. Gram-positive organisms require these surface proteins to effectively

interact with their environment and to mount infections. Because many of these surface proteins

are necessary for pathogenesis, sortase enzymes are considered virulence factors, and this

designation has led to extensive characterization of their structures and mechanism. To date, over

40 structures of sortase enzymes have been deposited in the PDB and this wealth of structural

information combined with numerous in vitro and in vivo analyses of their catalytic activity has

led to a thorough understanding of the molecular mechanism of substrate binding and catalysis.

These structural and mechanistic observations have prompted a number of computational studies

to understand the dynamic behavior of these enzymes and to develop inhibitors. Here we review

what is known about the structures and mechanism of these enzymes.

14
2.2 Introduction

Gram-positive organisms utilize a thick cell wall composed of cross-linked peptidoglycan

to protect themselves against environmental stressors and as a scaffold for the display of a large

number of surface proteins by sortase enzymes. Sortase enzymes are cysteine transpeptidases

that link their two substrates together via the formation of a new covalent bond1–3. Many of these

enzymes catalyze the covalent attachment of surface proteins to peptidoglycan, while a subset of

the family instead catalyzes the polymerization of filamentous pili on the cell surface. Since their

discovery over 15 years ago4, genome sequencing efforts have revealed that Gram-positive

bacteria harboring a conventional cell wall typically encode multiple sortase enzymes5–7. The

surface proteins which these enzymes attach to the cell wall are important for adhesion, nutrient

acquisition, spore formation, and immune evasion2,6,8, and it has been shown that sortase knock-

outs are defective in establishing persistent infections9,10. Since many surface proteins are

necessary for efficiently interacting with a host organism, and their deletion results in a defect in

pathogenesis, sortase enzymes have been classified as virulence factors9,10. This designation has

led to the development of several small-molecule inhibitors of sortase, and the search for

molecules with increased potency and specificity is a subject of ongoing research8,11–15. Beyond

their importance as a potential drug target, the ability of these enzymes to covalently join two

distinct substrates through the formation of a new peptide bond has led to their development as a

biochemical reagent to covalently attach proteins to each other, attach fluorophores or drugs to

proteins or antibodies, to cyclize proteins, and to immobilize peptides on solid surfaces16–18.

The sortase superfamily is large. At present over 800 sortase enzymes in 300 species of

bacteria have been identified6. Based on their primary sequences these enzymes have been

classified into six distinct families, whose members share related atomic structures and

15
frequently have similar functions. On the cell surface all sortases characterized to date function

as cysteine transpeptidase enzymes that attach surface proteins bearing a C-terminal cell wall

sorting signal (CWSS) consisting of a C-terminal hydrophobic domain19 with a positively

charged cytoplasmic anchor and a 5 residue recognition motif to an amino nucleophile located on

the cell surface. While the basic chemistry of this reaction appears to be highly conserved,

sortases can function to either attach proteins to the cell wall, or assemble pili (Fig. 1). All

sortases characterized to date are believed to be associated with the extracellular membrane via

an N-terminal trans-membrane helix and a short positively charged cytoplasmic anchor7,20,21.

Additionally, sequence conservation and a number of structural and biochemical studies have

determined that the active site of all sortase enzymes studied to date consists of a His-Cys-Arg

catalytic triad22,23. In atomic structures of sortases, these residues are invariantly clustered in the

active site such that the cysteine residue is bracketed by the histidine and arginine residues.

Sortase enzymes are also known to harbor a highly conserved TLXTC motif leading to, and

encompassing the catalytic cysteine24 which could be important for stabilizing the position of the

active site cysteine residue and maintaining it in a catalytically competent orientation. While the

basic architecture is conserved, unique class specific variations enable distinct CWWS and

nucleophiles to be recognized, and in some instances the variations appear to play critical

regulatory roles.

A number of excellent reviews have been written regarding the overall function of

sortases in biology, their specific role in pilin assembly, their development as biochemical

reagents and efforts to discover inhibitors of this important family2,7,8,25–29. Here we review what

is known about the structural basis of catalysis and sortase function on the cell surface,

highlighting recent structural and computational studies that have revealed unique family

16
specific structural variations, as well as the mechanism of sorting signal recognition and enzyme

regulation.

2.3 The Staphylococcus aureus SrtA Paradigm

The majority of early work to understand the structure and mechanism of sortase

enzymes was conducted on the Staphylococcus aureus SrtA (SaSrtA) enzyme, and this enzyme

is typically considered to be the archetypal member of the family. Here we briefly summarize

what has been learned about the molecular basis of its function from biochemical and structural

studies. The first structure of the enzyme was determined by the Clubb group in 200130 (Fig. 2).

Initial analysis of the protein sequence revealed the presence of an N-terminal signal sequence,

and transmembrane helix preceding a 181 residue soluble domain containing the absolutely

conserved catalytic cysteine residue4. The structure of this active soluble domain, SaSrtAΔ59 was

determined by NMR and showcases the now canonical closed 8 stranded beta barrel architecture.

This represented the first example of a new protein fold, now defined by the Structural

Classification of Proteins (SCOP) project31 as a “sortase fold.” This core domain architecture is

highly conserved across all families of sortase with small alterations present in the various other

classes (expanded upon below). The structure is well resolved with the exception of a single loop

between strands β6 and β7. Subsequently, a crystal structure of the same construct was solved at

2.0 Å resolution32. The two structures are very similar with an RMSD of 1.97 over all Cα, but

there are several slight differences: first, the crystal structure showcases elongated β7 and β8

strands, and a short helix in the β6/β7 loop in two of the 3 proteins in the asymmetric unit. This

loop was unstructured in the solution NMR structure, and the authors cite the lack of a single

structure for these residues in the asymmetric unit as additional evidence for its flexibility. The

17
active site architecture is also similar in both structures with the catalytic histidine, cysteine, and

arginine residues positioned together within a deep groove. The conserved TLXTC motif which

ends with the catalytic cysteine residue in all sortases largely forms stabilizing interactions with

other residues in the protein’s core, with the exception of the “X” residue (an Ile for SaSrtA)

which extends below the active site cysteine to form a portion of the hydrophobic pocket that

surrounds the active site. SaSrtA has additionally been shown to require Ca2+ for efficient

catalysis, and removal of the ion results in 5 fold lower activity30. NMR chemical shift mapping

revealed that the Ca2+ ion binds in a pocket between the β3/β4 loop and the β6/β7 loop, where it

is coordinated by the sidechains of E105, E108, D112, E171 and a backbone carbonyl from

N11433. NMR dynamics experiments also indicated that binding of calcium altered the mobility

of the β6/β7 loop which contacts the active site, replacing fast, ps-ns timescale motions with

slower, µs-ms timescale motions which are typically associated with ligand binding and

catalysis.

Substrates for the enzyme are recognized by the presence of a (CWSS) harboring the

canonical LPXTG sorting signal. Catalysis begins with a nucleophilic attack from the active site

cysteine residue on the carbonyl carbon in the peptide bond between the Thr and Gly residues in

the sorting signal. This forms a tetrahedral intermediate that quickly collapses to form a semi-

stable thioacyl intermediate covalently attaching the substrate to the sortase’s active site

cysteine7,24,34. The secondary substrate, a pentaglycine cross-bridge peptide on the cell wall

precursor lipid II, then performs a nucleophilic attack on the carbonyl carbon in the newly

formed thioacyl bond between the sortase and substrate, forming a second tetrahedral

intermediate that collapses to form a new peptide bond between the primary substrate and lipid

II34–36. This covalent complex is then incorporated into the cell wall via the standard cell wall

18
synthesis machinery. If the secondary substrate is not available, the reaction can proceed via a

secondary, terminal pathway wherein water performs the second nucleophilic attack and cleaves

the thioacyl bond between sortase and substrate, hydrolyzing the peptide and releasing it to

solvent (Fig. 2D). In this latter reaction, the enzyme acts as a protease. On the cell surface this

would have the presumably negative effect of cleaving the protein from its membrane anchor

where it would be then free to be released from the microbe.

2.4 Classification of Sortases

Members of the sortase-superfamily have been classified into distinct sub-families based

on their primary sequences7. Many organisms utilize multiple sortases simultaneously to

elaborate their surfaces with a diverse array of protein substrates. These enzymes frequently

operate non-redundantly with each sortase displaying proteins that contain specific sorting signal

motifs. This 5 residue sorting signal is generally similar to the canonical LPXTG motif seen for

SaSrtA with variations to the Leu and Gly positions of the recognition motif occurring most

frequently among signals for the different classes, while the Pro and Thr residues are largely

invariant6,37. Class A and B enzymes are prevalent in bacteria in the Firmicutes phylum. Many

class A enzymes are thought to perform a “housekeeping” role in that they are constitutively

expressed and generally anchor multiple protein substrates to the cell wall with a variety of

functions dependent on the organism. Typically in organisms with only a single sortase, the

sortase will be of the class A variety. Most class B enzymes also anchor proteins to the cell wall.

In many instances these enzymes anchor proteins involved in iron acquisition and typically exist

on a separate, iron regulated operon with their substrate(s). Not all class B enzymes are involved

in heme acquisition, and some members of this group are instead responsible for assembling

19
pili38,39. Class C enzymes are the most unique members of the sortase superfamily. Most

members of this class function to construct pili, requiring them to function as pilus polymerases

by joining the CWSS of one pilin subunit to a lysine residue in a second pilin subunit. The

polymerization of these subunits results in the construction of long (up to 1 µm) filamentous

fibers which are used for adhesion to host cells and have also been implicated in biofilm

formation7,40,41. The remaining types of sortases are less well studied but appear to attach

proteins to the cell wall like their class A and B counterparts. Many species of bacilli encode

class D enzymes that attach proteins involved in spore formation to the cell wall. Members of the

class E enzymes are predominantly found in Actinobacteria species. They are thought perform a

housekeeping role similar to class A enzymes in Firmicutes and have also been shown to anchor

proteins involved in the formation of aerial hyphae6,42. Class F enzymes are also present in

Actinobacteria, but their function is currently unknown. Here we focus on the structural

variations inherent in each class of sortase, as the biological implications of these enzymes and

their substrates has been thoroughly reviewed elsewhere7,24,25,27,28.

2.5 Structural Biology of Sortase Enzymes

At present, over 40 structures of sortase enzyme have been reported (Table 1). All

sortases studied to date are constructed from the same conserved 8 β-stranded sortase fold shown

in Fig. 2. However, members from each class exhibit unique variations that may be reflective of

their distinct functionalities in the cell. Generally the most significant discrepancies can be

localized to 4 distinct structural foci: (i) the N-terminal segment of the enzyme between the

transmembrane helix and the first β-strand of the barrel, (ii) the loop between strands β6 and β7

(the β6/β7 loop), (iii) the loop between strands β7 and β8 (the β7/β8 loop), and (iv) the C-

20
terminus of the enzyme which for some class C sortases harbors an additional transmembrane

helix, although this has not been visualized in any reported structure thus far20,21 (Fig. 3). In

many instances these unique structural differences have been shown to influence the enzyme’s

ability to recognize its specific sorting signal motif, and in others they are postulated to perform

a regulatory role by controlling substrate access to the active site. In particular, N-terminal

appendages in some enzymes have been shown to modulate sorting signal access to the active

site, while large structural variation in the β6/β7 and β7/β8 loops has been implicated in substrate

binding. Below we compare the known structures of sortases, highlighting class specific

differences and the functional implications of these differences where known.

Class A enzymes. Beyond the archetypal SaSrtA enzyme from S. aureus described in

detail above, class A enzyme structures have been reported for Streptococcus pyogenes43,

Bacillus anthracis44, Streptococcus agalactiae45, and Streptococcus mutans46. All of these

enzymes are believed to perform a housekeeping role and recognize the LPXTG sorting signal.

The β6/β7 loop of these enzymes contains a short helix that in structures of SaSrtA and BaSrtA

bound to their cognate sorting signal plays a critical role in signal recognition47,48. Interestingly,

members of the class A family differ in the mobility of this key active site loop. In SaSrtA the

loop is disordered in the absence of substrate, whereas all other class A enzymes display an

ordered β6/β7 loop and thus maintain a preformed binding site for the sorting signal. Many of the

structures also differ substantially from one another in the surface near the active site histidine

residue. This difference was first highlighted in the crystal structure of S. pyogenes SrtA

(SpySrtA)43. The catalytic core of this enzyme assumes the same canonical 8 stranded β-barrel

sortase fold, but unlike SaSrtA, the active site Cys-S is oriented towards the active site His-δN, at

a distance of 5.4 Å (in SaSrtA these groups are pointed away from another and separated by 6.5

21
Å). Most notably, a unique groove runs between these two residues whose walls are defined by

residues in helix H1 and the β7/β8 loop, and whose base is defined by residues in the β4/β5 loop.

This groove provides a potential exit tunnel for the substrate that could accommodate binding of

a full length protein bearing amino acids C-terminal of the LPXTG motif: a feature which was

not obvious in SaSrtA structures wherein the active site histidine residue is actually occluded

from solvent and this channel closed by interactions between residues in the β7/β8 loop and helix

H1. The structure of the S. agalactiae SrtA (SagSrtA) has also been determined and is most

similar to the SpySrtA structure, maintaining the same exit channel between His118 and Cys184

active site residues with its most distinguishing feature being an altered conformation for its

β6/β7 loop which is likely a byproduct of its crystallization conditions as this loop is involved in

coordinating a Zn2+ ion in conjunction with a Glu158 from an adjacent protein in the asymmetric

unit45. The active site His118 residue is also involved in coordinating a Zn2+ ion along with

His136 from a neighboring SagSrtA in the asymmetric unit, but this interaction does not seem to

move the residue into a non-physiological position, as it aligns well with the histidine residues

from active sites of other SrtA enzymes. The B. anthracis SrtA (BaSrtA) structure is noteworthy

in that it possesses an N-terminal appendage which wraps behind helix H1 and contacts the

active site, potentially acting as a cap to prevent access by the secondary substrate, lipid II (Fig.

3A)44. The most recently determined sortase structure, that of S. mutans SrtA (SmSrtA), was

crystallized with a significant portion of its N-terminus intact46. Interestingly, this enzyme

crystallized in an apparent dimer in which an extended N-terminal helix interacts with residues in

the active site of a symmetry related molecule. The authors suggest that this interaction is likely

an artifact of crystallization, but it is interesting to consider that in many recent sortase structures

solved with extended N-termini (especially when considering other classes of sortase, discussed

22
further below), these regions frequently maintain some form of interaction with the active site. It

should also be noted that an additional structure of a class A enzyme, SrtA from Streptococcus

pneumoniae (SpnSrtA), exists in the PDB as a β-strand-swapped dimer, in which the β6/β7 loop

extends from one monomer into an adjacent monomer in the asymmetric unit placing the β7 and

β8 strands in the β-barrel core of an adjacent molecule in the asymmetric unit. No publications

associated with this PDB entry could be found in PubMed, and it is unclear whether this

uncharacteristic result is biologically relevant.

Class B enzymes. The class B sortases which are typically involved in iron acquisition are

largely similar in structure to the class A sortases discussed previously with the exception of a

longer, structured β6/β7 loop. The first structure of a class B enzyme was of the S. aureus SrtB

(SaSrtB) enzyme, solved by X-ray crystallography in 200449 in the presence of various

sulfhydryl modifiers. When compared to the canonical SaSrtA structure, the most obvious

differences are in the presence of two additional helices N-terminal of the first β-strand and a

much longer loop inserted between strands β6 and β7 which contains an additional α-helix (Fig.

3B). These structural differences could potentially be important for dictating the class B

enzyme’s preference for anchoring substrates to buried, uncrosslinked portions of the cell

wall50,51. The unmodified structures of SaSrtB and B. anthracis SrtB (BaSrtB) were solved later

the same year and exhibit remarkably similar structures (Cα RMSD = 3.23Å). The main

differences between these two enzymes are localized to a slight conformational change in the

β6/β7 loop, a lack of density for a section of BaSrtB’s β7/β8 loop, and an altered conformation of

the short loop between helices H1 and H2. An active site cysteine mutant of the SrtB enzyme

from Clostridium difficile (CdSrtB) was also solved earlier this year by X-ray crystallography52

and displays an almost identical structure to SaSrtB (RMSD = 1.93 Å for all Cα). Interestingly,

23
this class B enzyme may bear a more generalized function as a genetic analysis has predicted 7

possible substrates, none of which are iron associated as is typical of other class B enzymes52.

Beyond these fairly similar class B sortases, the structure of two additional pilin polymerizing

class B sortases from S. pyogenes (SpySrtB) and S. pneumoniae (SpnSrtB) (also referred to as

SrtG-1) have also been solved38,53. These proteins are extremely unique in that their genetic

localization and biochemical analysis would have grouped them with class C pilin polymerizing

sortases, but their structures indicate that they are in fact most similar to class B types (Fig. 3).

The SpySrtB and SpnSrtB structures have a similar arrangement of secondary structure elements,

including the helix containing β6/β7 loop, but also bear an additional short β-strand after this

conserved helix. This alteration adds an uncommon 9th β-strand to these proteins, but this strand

is not inserted into the conserved β-barrel core and instead pairs with a portion of strand β6 that

extends above the core of the beta barrel. Understanding how these class B sortases fulfill the job

of the structurally distinct class C sortases to polymerize pili instead of attaching proteins to the

cell wall will likely be an extremely important step towards understanding the structural

characteristics that are necessary for these disparate functions.

Class C enzymes. Class C sortase structures are most notable for the presence of a

conserved, elongated N-terminal region that occludes the active site, termed “the lid” (Fig.

3C)7,28,54,55. This class has also been the recipient of a significant number of structural studies,

with a total of 15 structures currently deposited in the PDB. Interestingly, every class C, pilin-

polymerizing sortase studied to date contains this lid region, which invariably contains a

DP(F/W/Y) motif. In all but 1 of the class C sortases studied to date, the conserved DP(F/W/Y)

motif contacts the active site through a hydrogen bond between the Asp residue in the lid and the

arginine residue in the active site as well as through a sulfur-aromatic interaction between the

24
active site Cys-S and the aromatic sidechain in the conserved lid motif. Because of these key

interactions, these two residues have been referred to as “anchors” of the lid54,55. The first class C

enzymes to be structurally characterized were SrtC-1 and SrtC-3 from S. pneumoniae (SpnSrtC1

and SpnSrtC3) by Manzano and coworkers in 200854. Upon analyzing the structures the authors

noted that the B-factors for the lid region covering the active site were the highest in the structure

and suggested that the lid was likely mobile in solution and that this mobility would unmask the

active site allowing for recognition of the substrate. Subsequent structures of class C sortases

from S. agalactiae21,56,57, A. oris58, and S. pneumoniae59 furthered this idea with additional

structures showing similar patterns of elevated B-factors or stretches of missing electron density

in regions flanking the anchor residues in the lid. Although many organisms utilize multiple class

C sortases to polymerize pili, systems for which multiple SrtC enzymes have been solved for an

individual organism have shown minimal structural variation. These systems typically show only

small shifts in the position or amount of density found for the N-terminal extension harboring the

lid, with perhaps the most significant deviation from the norm being the addition of a short C-

terminal α-helix opposite the active site in the structure of SpnSrtC3 which has not been

functionally characterized54. Although the structures of most class C sortases show the lid

blocking access to the active site, a structure of S. agalactiae SrtC-1 (SagSrtC1) bound to

MTSET, a nonspecific sulfhydryl modifier, also maintains the lid in a closed state indicating that

the active site cysteine for this enzyme is at least partially accessible even while the lid is

closed56. Mutations to the lid region have shown no effect on transpeptidation in vivo20,21, while

similar mutations have instead been shown to decrease enzyme stability55 and increase the rate of

hydrolysis in vitro21 leading to the theory that the lid performs a regulatory function.

Interestingly SagSrtC1 has been solved in multiple crystal forms, and one of these structures,

25
solved in space group C2, showcases the lid in an “open” conformation providing a model for

what is presumed to be a necessary lid-opening event expected to precede catalysis (Fig. 3C,

compare left to right)56. The enzyme maintains the typical sortase fold and, excluding the N-

terminal extension preceding the β-barrel core, is extremely similar to SagSrtC1 structures

previously solved in space groups P212121 and P312 with an average backbone RMSD of 0.72 Å.

However, in space group C2, residues A38-E71 which typically form the flexible lid structure,

instead form an extended helical structure with the aromatic lid anchor residue of the conserved

DP(F/W/Y) motif, Y51, displaced from the active site by over 30 Å to a position where it stacks

against the backbone of helix H2. An additional crystal structure of S. suis SrtC-1 (SsSrtC1) also

maintains a similar open form of the lid, with the same extended helix replacing what was

expected to be a closed lid60.

Class D enzymes. Class D sortases are predicted to perform a specialized function in

spore formation and have received less attention than classes A-C, with only two structures

currently available in the PDB. The first class D structure was determined by NMR for the B.

anthracis SrtD enzyme (BaSrtD, and also previously referred to as SrtC) in 201261. The BaSrtD

solution structure is most structurally similar to class A enzymes, as it lacks the lid seen in class

C enzymes and the elongated β6/β7 loop that is characteristic of class B enzymes; however,

unlike SaSrtA, BaSrtD reveals an ordered 310 helix within the β6/β7 loop, indicating a rigid

binding pocket for recognition of substrates containing an LPNTA motif (Fig. 3D). This solution

structure reveals a catalytic core formed by residues 56-198, which adopts the conserved eight,

β-stranded sortase fold with two short helices positioned on opposite faces of the protein. The N-

terminus of BaSrtD is predicted to contain a single transmembrane helix formed by residues 1-

19, followed by an experimentally-determined, unstructured linker region formed by

26
approximately 30 amino acids preceding the catalytic core. Interestingly, NMR studies of

BaSrtD also indicate that the β2/β3 and β4/H1 surface loops adjacent to the active site histidine

are structurally disordered, as residues for these regions are completely unassigned, and the

β7/β8 loop following and preceding the active site cysteine (Cys173) and arginine (Arg185),

respectively, is also poorly ordered. BaSrtD was also shown to form dimers in vitro, and these

disordered loops could possibly contribute to a dimerization interface, or this surface

surrounding the active site may be responsible for associating BaSrtD with its lipid II substrate.

Recently, a 1.99Å resolution crystal structure was solved for Clostridium perfringens SrtD

(CpSrtD), representing only the second characterized enzyme from this class62. The CpSrtD

structure contains eight β-strands, adopting the characteristic β-barrel sortase fold with the

catalytic triad residues (Cys171, His109, and Arg178) positioned within the enzyme’s active site.

Interestingly, based on sequence conservation, this enzyme was originally assigned to the class E

family of sortases, and CpSrtD contains two alpha helices at its N-terminus, a marked difference

from the BaSrtD structure. CpSrtD also exhibits catalytic activity in vitro, which is enhanced in a

magnesium-dependent manner, making it one of only two sortases (along with SaSrtA) known to

be modulated by the presence of metal ions. In further contrast to the BaSrtD enzyme, both the

β2/β3 and β4/H1 loops are well ordered in the CpSrtD crystal structure, with the β2/β3 loop

adopting a two-turn alpha helix. Furthermore, CpSrtD is thought to exist as a monomer,

supported through crystallographic and dynamic light scattering studies. Together, these

observations suggest that BaSrtD and CpSrtD could represent distinct subclasses of the sortase D

family.

Class E enzymes. In Actinobacteria, class E sortases have been shown to act as

housekeeping enzymes, similar to class A enzymes in Firmicutes, and to be involved in the

27
production of aerial hyphae. These enzymes have been minimally characterized, with the first

and only structure for a class E enzyme determined recently by the Clubb group63. Two sortase

enzymes from Streptomyces coelicolor, ScSrtE1 and ScSrtE2, have been confirmed as bonafide

class E enzymes in that they selectively process the unique LAXTG substrate motif in vitro64.

These enzymes are each predicted to contain a single transmembrane domain, formed by

residues 139-161 in ScSrtE1 and residues 31-53 in ScSrtE2, followed by an extracellular domain

containing the characteristic sortase fold with the conserved catalytic triad (Cys320, His251,

Arg329) positioned within the active site. Although most seemingly related to class C enzymes,

the ScSrtE1 structure reveals a largely disordered N-terminus. ScSrtE1 also maintains an

elongated β6/β7 loop with a short, preformed helix, which suggests a rigid substrate-binding

pocket.

Class F enzymes. Lastly, class F enzymes are very poorly understood, with unknown

function, unknown substrate motif specificity, and no structural characterization to date. Here,

we attempt to gain insight to class F enzymes by analyzing five remaining sortase genes from S.

coelicolor that belong to the class F sortase family7. Two of these five enzymes are predicted to

contain an N-terminal transmembrane helix based on analysis with TMHMM65. Extracellular

domains of the remaining three enzymes were identified using sequence alignment with

ClustalW2, and homology models of the extracellular domains of these five class F enzymes

generated with the Phyre 2 server have low sequence coverage, ranging from 69-83%, indicating

their divergence from other sortase classes. These SrtF enzymes are apparently most similar to

class D sortases, as the BaSrtD structure was utilized as the top template in all five models.

Notable features of these SrtF models include a preformed helix within the β6/β7 loop for four

out of five enzymes, as well as a disordered N-terminus that is predicted with high confidence.

28
 In addition to structural variations within enzyme classes, some sortases have also been

shown to localize to distinct foci on the membrane66–68, leading to the hypothesis that sortases

may associate with one another on the cell surface69. Several sortases have already been shown

to form dimeric assemblies in vitro61,70, and recently, the functional role of SaSrtA dimerization

was investigated in vivo71. Zhu et al. generated a triple alanine mutant of three residues of

SaSrtA, which was shown to disrupt dimerization in vivo. S. aureus strains expressing this

monomeric SaSrtA showed improved surface display of adhesive proteins, as well as more

efficient invasion of host mammalian cells, suggesting that the monomeric form has increased

activity in vivo. This is in stark contrast to an earlier study which claimed that SaSrtA was more

active when chemically cross-linked to maintain its dimeric form in vitro70. Interestingly, MD

simulations of WT SrtAΔ59 and the monomeric mutant form of the protein showed that the triple

alanine mutation induces a “fixed” β6/β7 loop, and the authors proposed that monomer-dimer

equilibrium regulates the enzymatic activity of SaSrtA most likely by influencing the

conformation of the β6/β7 loop71. Determining the prevalence of in vivo complex formation for

other sortase homodimers and exploring the potential of heterodimeric and higher order

assemblies between different classes of sortases and/or other cell surface translocation or cell

wall synthesis machinery present on the cell surface is an active area of research.

2.6 Molecular Basis of Substrate Recognition

Sorting signal recognition has become one of the most intriguing areas of recent study for

sortase enzymes. Many sortase enzymes have been shown to recognize their substrates with high

specificity and have the ability to differentiate between the appropriate sorting motif or their

substrates and that of other sortase enzymes in vitro and in vivo47,59,72–76. This strict recognition

29
allows the sortases to function non-redundantly to catalyze reactions with specific substrates

even when coexpressed in the same cell. In addition to this strict specificity for primary

substrates, different species of bacteria utilize distinct cross-bridge peptides within their lipid II

cell wall precursor molecule which are typically only recognized as a secondary substrate by the

cell-wall associated sortases for that species. The pilin associated sortases on the other hand are

required to recognize a 4 residue lysine-containing pilin motif located within their protein

substrate. This motif is typically different for different sortases, and is thought to regulate

specificity in systems with multiple class C pilin associated sortases75,76. Understanding the

principles that guide substrate recognition for sortase enzymes continues to be an extremely

difficult area of study. Since sortase enzymes will perform an off target hydrolysis reaction in the

absence of an appropriate nucleophile, slowly cleaving their substrate, structural studies of these

complexes have required the implementation of modified sortases or substrates to form

complexes stable enough for structural studies. To date, 4 structures have been solved of

complexes between sortases and their respective substrates or substrate analogs32,37,47,48. To

facilitate discussion of the binding interactions that govern recognition of the sorting signal

substrate, we henceforth refer to residues in each sorting signal in relation to their distance from

the scissile bond, where residues in an L-P-X-T-G sorting signal are referred to as P4-P3-P2-P1-

P1’, and the binding sites for those residues on the enzyme as S4-S3-S2-S1-S1’, respectively.

The first example of a sortase-substrate complex was a crystal structure of an SaSrtA-

C193A mutant with an LPETG peptide soaked into the crystal32. While the complex appeared to

show the LPETG peptide lying across the surface of the enzyme near the active site, the validity

of this complex was quickly called into question as the highly conserved Leu residue at P4 of the

substrate and the Thr residue at P1 were both seen pointing into solvent. Given that these

30
residues are highly conserved across all substrates for this enzyme6, and that an in vitro analysis

with peptide libraries had shown that both of these residues are extremely important for substrate

recognition73, it was expected that these residues would be recognized by numerous contacts to

the enzyme. Additionally, there was only density for the bound peptide in 1 of the 3 proteins in

the asymmetric unit, indicating extremely weak binding. A breakthrough came when the Jung

group at UCLA synthesized an LPAT* substrate mimic, where T* is a modified threonine

containing a sulfhydryl moiety in place of its carboxyl oxygens. This molecule mimics the native

LPXTG substrate, but can form a disulfide bond with the active site cysteine leading to the

production of a stable enzyme-substrate complex suitable for structural studies77. Subsequent

NMR analysis of the SaSrtA-LPAT* complex led to the localization of the substrate binding

site78, and eventually the structure of a covalent SaSrtA-LPAT* complex47 (Fig. 4A). This

structure revealed a reorganization of both the β6/β7 loop to include the appearance of a short 310

helix, and an opening of the β7/β8 loop upon binding the substrate, potentially indicating a

regulatory mechanism which would open the loop to expose the active site histidine residue to

solvent and allow for binding of the secondary substrate only after the primary substrate had
15
bound. Additional N relaxation analysis revealed that the β6/β7 loop, which was highly

dynamic in the unbound state, becomes more rigid upon binding of the substrate47. In addition to

significant changes in the active site of the enzyme, the structure also revealed the major residues

that make up the binding pocket for the substrate and showed that the previous peptide-soaked

crystal structure had most likely displayed the peptide non-specifically bound. This non-specific

binding could have either been due to the low affinity of the interaction (Km = 7.33 mM)79, or

due to crystal packing interactions preventing the reorganization of the β6/β7 loop which appears

to be necessary to appropriately construct the binding pocket for recognition of the peptide. One

31
of the most notable features of SaSrtA-LPAT* complex is the large hydrophobic S4 pocket made

up of residues V161, V166, V168, and L169 in the reordered β6/β7 loop, and I199 on strand β8

which binds the P4 Leu residue. The complex also explains the requirement for a Pro residue at

position P3, as this is seen forming a kink in the peptide backbone which directs the rest of the

peptide towards the active site cysteine. Interestingly, the residue in the P2 position which, based

on sequences of known substrates6 and biochemical data73 can be any amino acid, is seen

pointing into the active site where it makes contact with A118 and I182. This space does not

appear to be large enough to accommodate the presence of larger amino acid side chains which

was curious given that these are known to be recognized by the enzyme. Additionally, the P1

threonine residue which is conserved in over 95% of all sortase substrates is seen here pointing

out of the active site towards solvent, only making a single hydrogen bond to the active site

histidine side chain. The positions of the active site residues in this structure are compatible with

biochemical data and the placement of the active site arginine residue hydrogen bonding

exclusively with the backbone carbonyl of the P2 Ala residue seemed to indicate that this

absolutely conserved residue is likely involved in orienting the substrate for catalysis as opposed

to activating the active site Cys or acting as a general base in catalysis as had been proposed

previously.

A subsequent crystal structure of SaSrtB covalently bound to an NPQT* substrate mimic

of its native NPQTN substrate served to confirm several observations from the SaSrtA-LPAT*

structure and clarify some of the more ambiguous ones (Fig. 4B)37. The P4 residue, an

asparagine in this sorting signal, is primarily recognized by contacts to the β6/β7 loop, as in the

SaSrtA-LPAT* structure, although in a more solvent exposed position, concordant with the more

hydrophilic nature of the asparagine sidechain. This is also compatible with biochemical data

32
from the McCafferty group which has shown that replacement of the β6/β7 loop of SaSrtA with

that of SaSrtB is sufficient for altering the specificity of the resultant chimeric protein to the

SaSrtB sorting signal’s NPQTN motif80. The P3 proline residue also appears to provide the same

function as seen in the SaSrtA-LPAT* structure, kinking the peptide to point it towards the

catalytic cysteine. There are also some notable differences between this and the SaSrtA-LPAT*

structure, one of which being that the β6/β7 loop for this enzyme appears to be maintained in a

binding-competent “lock and key” conformation since the structure of the complex was almost

identical to that of the unbound crystal structure (RMSD = 0.44 Å for all Cα). The P2 and P1

residues also appear to be bound in a unique conformation in the SaSrtB-NPQT* complex. The

P2 Gln residue rests along the wall of the pocket, pointing out towards solvent, as opposed to

pointing into the base of the active site as the P2 Ala residue does in the SaSrtA-LPAT*

structure. Interestingly, the P1 Thr residue in the SaSrtB-NPQT* structure is buried in the active

site (Thr-in position) where it forms two hydrogen bonds with the active site arginine residue,

instead of pointing towards solvent and hydrogen bonding with the active site histidine residue as

seen in the SaSrtA-LPAT* structure (Thr-out position).

The third and most recent structure utilizing the same type of disulfide bond forming

substrate mimic, that of BaSrtA-LPAT*, bears similarities to both of the previously described

structures (Fig. 4C)48. The substrate mimic peptide in this structure maintains the same general

orientation as seen in both previous structures providing further evidence that the β6/β7 loop is

instrumental in building the S4 site and dictating specificity for the P4 residue, while the P3 Pro

residue is necessary for providing a kink in the peptide backbone to orient the P1 and P2 residues

toward the active site cysteine. Interestingly, this enzyme substrate complex maintains a Thr-in

conformation, reminiscent of that seen in the SaSrtB-NPQT* substrate complex, where the P1

33
Thr is buried within the binding pocket leading to the active site, but here it does not interact

with the active site arginine. The active site arginine instead forms a hydrogen bond with a

backbone carbonyl in the peptide P3 Pro residue, similar to what was seen in the SaSrtA-LPAT*

structure, albeit in this structure the H-bond was to the adjacent P2 Ala carbonyl. Unlike the

flexible β6/β7 loop found in the unmodified SaSrtA, the β6/β7 loop of BaSrtA was already

shown to be structured in the absence of substrate44. The structure of the modified complex

confirmed the presence of a preformed binding pocket as the backbone atoms for these residues

exhibit an RMSD of 0.76 Å. Conversely, the β7/β8 loop, which was disordered in the

unmodified BaSrtA structure, undergoes a disordered to ordered transition when bound to the

substrate mimic which is correlated with a ~7 Å displacement of the active site cysteine residue

upon binding the peptide. Taken together, these three substrate-mimic bound structures show

conclusively that the S4 site is built by residues in the β6/β7 loop which is essential in

determining the specificity of these enzymes for their P4 residue, and that the highly conserved

Pro residue and P3 is necessary to properly orient the residues at positions P2 and P1 for

interaction with the active site (Fig. 4D). While both the SaSrtB-NPQT* and SaSrtA-LPAT*

structures share an overall similar conformation for the P1 and P2 residues, the interactions seen

between the P1 Thr and the catalytic triad are not consistent across the three structures and likely

indicate internal flexibility inherent to this portion of the active site for some members of this

enzyme family. These inconsistencies could also be caused, or compounded, by potential slight

inaccuracies in complexes with these substrate mimics caused by the use of an unnatural

disulfide bond in place of the native thioester.

An energy minimized model of the thioacyl intermediate that exists in the SaSrtB-NPQT

complex provided additional information about the nature of the catalytic mechanism. This

34
model (Fig. 5) shows that the highly conserved P1-Thr residue acts through a hydrogen bonding

network to stabilize the position of the active site arginine residue so that it is maintained in close

proximity to the thioacyl bond. In this position it can act as part of an oxyanion hole, in concert

with the backbone amide of E224, immediately C-terminal to the active site cysteine, to stabilize

the two high-energy tetrahedral intermediates which occur during catalysis37. This novel

“substrate-stabilized oxyanion hole” could serve as a regulatory mechanism to help increase

specificity for the substrate and thereby prevent unwanted catalysis, as biochemical analysis

shows that the enzyme is intolerant to even conservative mutations (Ser or Val) to the Thr at the

P1 position of its substrate37. A similar energy minimized model was produced for the BaSrtA-

LPAT complex which displayed an analogous hydrogen bonding interaction between the P1 Thr

residue and the active site arginine, along with a similar orientation for the P1 Thr carbonyl in

the thioacyl bond that places it in proximity to the arginine guanidino group for stabilization of

the negatively charged tetrahedral intermediate48. It should be noted however, that the geometry

of these two models is not identical, and that the active site arginine in the BaSrtA structure is in

a more extended structure that allows it to make additional hydrogen bonding interactions with

the backbone carbonyl of the P3 Pro residue, in place of a direct hydrogen bonding interaction

with the thioacyl carbonyl oxygen as seen in the SaSrtB-NPQT model.

To rectify the discrepancies between the SaSrtA-LPAT* Thr-out and SaSrtB-NPQT*

Thr-in conformations, Jacobitz et. al. performed molecular dynamics (MD) simulations for three

thioacyl intermediate systems: SaSrtA-LPAT, SaSrtB-NPQT, and SaSrtB-NPAT37. Through the

use of umbrella sampling calculations, the free energy landscape of transitions between the Thr-

out and Thr-in states was mapped for each complex, and it was found that SaSrtA-LPAT could

adopt both the Thr-in and Thr-out states with equal probability, while for both NPQT and NPAT

35
substrates, SaSrtB only samples the Thr-in state. Based on these results, it appears as if the Thr-

in conformation is likely the more evolutionarily conserved state, and that the inherent flexibility

of SaSrtA allows for the Thr-out conformation with an LPAT substrate that was captured by

Suree et al47.

Additional MD studies with SaSrtA have provided additional insight into the recognition

process of sortase enzymes. By performing conventional and accelerated MD simulations of both

the sorting signal free and bound states, Kappel et al. proposed that sorting signal binding is a

mixture of conformational selection and induced fit mechanisms81. For example, the β6/β7 loop

appears to follow the conformational selection paradigm: it sampled a range of stable

conformations in the apo state, some of which were relatively close to the bound configurations.

In contrast, the β7/β8 “open” state from the NMR structure was only stable in the presence of a

bound sorting signal, suggesting an induced fit mechanism. In addition, analysis of the sorting

signal-bound conformations showed that an allosteric network runs throughout the protein,

linking the calcium, sorting signal, and proposed lipid-II binding regions to one another. In a

complementary study, Moritsugu et al. used the multiscale enhanced sampling method to probe

the allosteric effects of the calcium ion and sorting signal82. Simulations of each combination of

bound states showed that binding of both molecules is required to stabilize the β6/β7 and β7/β8

loops in conformations observed in the NMR-LPAT* structure. Overall, these simulations point

towards a mechanism in which calcium, sorting signal, and potentially lipid II binding are

modulated by a dynamic network that includes the β6/β7 loop region in SaSrtA.

Other aspects of the SaSrtA recognition and catalytic processes have also been explored

by computational studies. Biswas et al. used a hybrid MD and biochemical experimental

approach to probe the roles of the conserved sorting signal Leu and Pro residues in substrate

36
binding83. Comparative simulations with LPAT, APAT, and LAAT substrates demonstrated that

contacts between the leucine sidechain and SaSrtA help to stabilize the β6/β7 loop, whereas the

kink that is induced by the proline appears to be essential for recognition. In another study, Tian

and Eriksson performed simulations in which His120 and Cys184 were in their zwitterionic and

neutral forms84. Their results showed that Arg197 adopts distinct conformations based upon the

charged state of the protein, which helps to stabilize the catalytically active form. It should be

noted that each of these studies was performed with the sorting signal in the Thr-out state.

Although the global effects of the Thr-in and Thr-out states on the induced fit/conformational

selection process, allosteric networks, and recognition processes are likely similar, subtle

differences may exist that influence some of the fine details that resulted from these simulations.

When compared to the recent accumulation of information regarding the binding of the

primary sorting signal substrate, there is relatively little known about the location of the binding

pocket or interactions involved in recognizing the secondary substrate of sortase enzymes.

Progress on this front has been slowed most significantly by the poor solubility and difficulty

inherent in obtaining large quantities of the intact lipid II substrate. To date, the most convincing

line of evidence stems from NMR chemical shift mapping of the SaSrtA-LPAT* complex with a

triglycine peptide titrated into the sample to mimic the pentaglycine cross-bridge present in the S.

aureus lipid II molecule by Suree et al47. Analysis of the chemical shift perturbations resulting

from this addition revealed a continuous surface spanning portions of the β7/β8 loop, β4/H2

loop, and an N-terminal segment of helix H1. While this surface lies near the active site and

bound peptide, it is too large to definitively assign a single binding pocket to the secondary

substrate. It is also interesting to note that these chemical shift changes were not observed when
15
titrating the triglycine peptide against an unbound N labeled SaSrtA but only occur after the

37
SaSrtA-LPAT* complex has formed, presumably due to a rearrangement of the β7/β8 loop

which creates a more open conformation necessary for substrate binding in this enzyme. An X-

ray structure of SaSrtB bound to MTSET, a sulfhydryl modifier, with a triglycine peptide soaked

into the crystal has also been solved and shows the triglycine peptide bound exclusively to the

β7/β8 loop85. While this is compatible with NMR chemical shift data from the SaSrtA-LPAT*

complex, it does not fit the accepted view of the mechanism which would have the incoming

nucleophile deprotonated by the active site histidine residue. In this complex the N-terminal

amine of the triglycine peptide is 6.4 Å from the active site histidine which is occluded from

solvent by a closed β7/β8 loop. This is also incompatible with the clear groove extending past the

active site histidine and cysteine residues visible in SpySrtA, SagSrtA, and SmSrtA structures

that has been hypothesized to be the binding groove for the secondary substrate in these

enzymes43,45,46. Confidently discerning the true location of the secondary substrate binding site

will be an important addition to the current understanding of the sortase mechanism.

2.7 The Molecular Mechanism of Sortase Enzymes

The culmination of the past 16 years of research since the discovery of the sortase

enzymes has been a relatively complete understanding of the molecular mechanism, compiled

from a large number of studies encompassing in vitro biochemical data, mutagenesis, X-ray and

NMR structures, NMR dynamics, bioinformatics, computational modeling, and MD simulations.

While there will still undoubtedly be variations, improvements, and exceptions added to the

current understanding of the mechanism, the existing model is backed by a large and ever

growing body of evidence. The current model of the molecular mechanism is presented in Figure

6. Kinetic studies suggest that catalysis occurs through a ping-pong mechanism that begins when

38
the sortase recognizes the CWSS of a membrane anchored protein as it binds in a groove made

by residues in the β6/β7 loop, strands β4, β7, β8, and closed by the β2/β3 and β3/β4 loops (Fig.

4D). Here the sorting signal’s L-shaped structure dictated by the highly conserved proline

residue at P3 (>90% conserved) will orient residue P4 for recognition in subsite S4 at the β6/β7

loop, as the remainder of the peptide is directed deeper into the active site. In order for catalysis

to proceed, the enzyme must contain a properly charged active site with cysteine and histidine

residues in their thiolate and imidazolium ion forms, respectively. Based on pKa measurements

of the SaSrtA79,86 and BaSrtA44 enzymes, and the similarly slow in vitro catalytic rates of all

known sortases studied to date, this is expected to comprise <1% of the available population of

enzymes at physiological pH. This is also confirmed by structural analysis of SaSrtA wherein

none of the residues in the active site are close enough to support the presence of any ion pairing,

with a Cys-S-His-δN distance of 6.5 and 7.6 Å for NMR and crystal structures, respectively. If

the cysteine residue is appropriately charged it can perform a nucleophilic attack on the P1 Thr’s

carbonyl carbon, leading to the formation of the first tetrahedral intermediate, which is likely

coordinated by the substrate-stabilized oxyanion hole postulated to exist between the active site

arginine residue and a backbone amide in the β7/β8 loop37. This intermediate quickly collapses

to form a semi-stable thioacyl intermediate between the substrate’s P1-Thr and the active site

cysteine, while the leaving peptide is protonated by the active site histidine residue. The active

site arginine residue may also participate in the reaction at this stage and others by hydrogen

bonding with the backbone carbonyls of the bound substrate to maintain its orientation in the

active site47,48,84. This interaction is potentially dependent on the position (Thr-in or Thr-out) of

the bound P1 Thr residue and also on the particular sortase in question as existing structures of

sortase-substrate complexes have depicted multiple binding positions for this residue and

39
molecular dynamics (MD) studies have indicated that it can sample multiple conformations in

some enzymes which could potentially influence the position of the active site arginine residue37.

In the next step of the reaction, the secondary substrate enters the active site where its amine

group is thought to be deprotonated by the active site histidine residue before performing a

nucleophilic attack on the thioacyl bond. This results in a second tetrahedral intermediate which

is again thought to be stabilized by the substrate-stabilized oxyanion hole. This second

tetrahedral intermediate quickly collapses to form a new peptide bond between the incoming

nucleophile and the P1-Thr residue which then exits the active site to be incorporated into the

cell wall via the standard cell wall synthesis machinery, or added to additional protein subunits in

the case of pilin polymerizing sortases.

2.8 Sortases as Powerful In Vitro Biochemical Reagents

Many of the sortase enzymes have been studied using a variety of different methods in

vitro. The first demonstration of in vitro activity was performed by Ton-That and coworkers

using the prototypical SaSrtA enzyme87. Their assay was the first of many to utilize FRET based

reporters of activity by incorporating donor and quencher fluorophores onto both ends of an

LPXTG sorting signal peptide. Cleavage of this peptide, either through hydrolysis,

hydroxylaminolysis, or the native transpeptidation reaction, liberates the donor from the

quencher and this activity can be detected by an increase in fluorescence. While the initial study

demonstrated only cleavage and hydroxylaminolysis, subsequent studies also demonstrated the

ability of SaSrtA to catalyze the native transpeptidation reaction in vitro using only short peptide

substrates where one contains a sorting signal motif, and the other a series of 1 to 5 glycines88,89.

While this assay was useful for demonstrating a preference for secondary substrates containing at

40
least 2 glycines at the N-terminus89, and for initial characterization of the active site histidine

cysteine and arginine residues22,23, early kinetics analysis was found to be plagued by the inner

filter effect in which very high concentrations of substrate will lead to a donor quenching effect

even after cleavage of the FRET pair, leading to inaccuracies in kinetic measurements90.

Development of an HPLC assay and subsequent revision of the kinetic parameters based on

fitting the parameters to a ping-pong hydrolytic shunt model to more accurately account for loss

of activity due to hydrolysis of the peptide revealed relatively weak binding affinity for the

LPETG sorting signal (Km = 7.33 ± 1.01 mM) and secondary Gly5 substrate (Km = 196 ± 64 µM)

and low kcat of 0.28 ± 0.02 s-1 79. Although this is a fairly low turnover number, the measured

pKas of the active site residues indicate that the majority of the enzyme is actually reverse-

protonated, with only a small percentage (~0.06%) of the enzyme actually in the appropriately

charged, active form in vitro. Accounting for this low population of appropriately charged

enzyme indicates that this subpopulation of the enzyme must actually be extremely active, with a

kcat of over 105 M-1 79. It has also been noted that the reaction occurs extremely rapidly (<3 min

from expression to surface attachment) in vivo19 indicating that there are likely other factors

involved in the reaction that are not adequately replicated in the in vitro systems described to

date. This effect could simply be due to the reduction of the search problem from 3 dimensions

to 2 by the incorporation of membrane anchors to both sortase and substrate, or the existence of

as yet uncharacterized interactions between other cell surface factors that either activate the

sortase or modulate the positions of protein and substrate for more efficient catalysis. Kinetic

analysis has also been conducted on several other sortase enzymes, but these have all reported

even slower rates of in vitro catalysis indicating a similar mechanism which is likely plagued by

similar shortcomings in all simplified in vitro systems43,44,80. A number of additional studies have

41
measured the endpoints of reactions at a defined incubation time and lack the comprehensive

analysis necessary to extract kinetic parameters. A summary of the results of in vitro reactions

with sortases performed to date is available in Table 2. The fact that many of these analyses are

highly simplified, along with numerous differences in substrate and buffer requirements for the

various members of the sortase family have made a comparison of the various kinetic rates of

these enzymes difficult.

2.9 Development of Structure-Based Therapeutics

As a validated virulence factor, the sortase superfamily represents an attractive antibiotic

target for which inhibitor development is being actively pursued91. Many classes of sortase

inhibitors have been identified with high-throughput screening (HTS) via monitoring FRET-

based substrate cleavage in vitro or virtual docking of small molecule libraries against sortase

structures in silico91. Several sortase inhibitor classes function through covalent modification of

the active site cysteine, resulting in irreversible inactivation of the enzyme, and several Cys-

linked, inhibitor:sortase complexes have been structurally characterized. Many of these

structures display sortase enzymes covalently linked with general thiol modifiers; however,

several structures exhibit complexes with novel, sortase-specific compounds that provide insight

into active site interactions and potential derivatization strategies. Furthermore, structure-activity

relationship (SAR) and molecular docking studies for several lead compounds have facilitated

rational design of inhibitors with improved biochemical and pharmacological properties,

bringing a novel class of therapeutics for Gram-positive infections closer within reach.

Maresso et al. identified AAEK inhibitors using an in vitro FRET-based HTS, and

subsequently, solved the crystal structures of BaSrtB linked to AAEK1 and AAEK2 inhibitors13.

42
Sortases specifically activate AAEK inhibitors via a β-elimination reaction that generates an

olefin intermediate which covalently modifies the active site cysteine. AAEK compounds

currently inhibit different class sortases from staphylococci and bacilli species with IC50s in the

low micromolar range (~5-50 µM). A comparison of the structures of native and inhibitor-bound

BaSrtB reveals several key differences, including rotation of the active site cysteine by ~180° to

accommodate the ligand, displacement of the conserved arginine away from active site, and

higher mobility and different conformations of residues from the H5/β6 loop. Interestingly, the

aryl group of the thienylpropanone adduct engages in stacking interactions with a critical

tyrosine (Tyr138) within the BaSrtB active site. Furthermore, the inhibitor-bound form reveals

two available binding sites, a cationic site above and anionic site below the aryl group that could

be exploited to improve potency and selectivity of the AAEK inhibitor class.

To identify more potent SrtA inhibitors, Suree et al. performed an in vitro HTS of a

30,000 compound library that resulted in three promising leads, the rhodanine, pyridazinone, and

pyrazolethione classes11. All three lead classes inhibited SaSrtA and BaSrtA reversibly with

IC50s in the low to sub-micromolar range. SAR studies of the most promising class, the

pyridazinone lead, identified critical regions of the chemical scaffold that are required for sortase

inhibition. The binding modes of these inhibitors were modeled using induced-fit docking

protocol and the SaSrtA-LPAT* structure. In this model, the pyridazinone phenyl ring is buried

in a hydrophobic pocket consisting of side chains from Ile199 on the β8 strand and Val166 and

Val168 from the β6/β7 loop of SaSrtA, while the carbonyl oxygen on the pyridazinone ring is

positioned towards the active site Arg197 side chain. The inhibitor’s thiol group points towards

the conserved His120, and mutation with a chloro group significantly reduces activity.

43
Interestingly, mutating the R1 substituent to an ethoxy moiety is well-tolerated, presumably due

to additional hydrophobic contacts with side chains of H1 residues, Pro94 and Ala92.

To improve the success rate of virtual screening against the substrate-bound form of

SaSrtA, Chan et al. virtually screened over 55,000 ligands, the top-ranked 500 of which were

docked onto the NMR structure of SaSrtA with a relaxed complex scheme to account for protein

receptor flexibility12. This approach identified 24 unique leads, and in vitro validation of eight

out of 19 compounds yielded a 42.2% success rate with IC50s ranging from 47-368 µM.

Interestingly, the 2-phenyl-2,3-dihydro-1H-perimidine scaffold, which was the most active

compound experimentally, did not rank highly during the first round of virtual screening, but

ranked substantially higher when docked to the ensemble of molecular dynamics (MD) simulated

sortase conformers. This compound appears to exploit regions of the enzyme that have evolved

for substrate binding, as computational docking studies suggest that the DHP group within the 2-

phenyl-2,3-dihydro-1H-perimidine lead scaffold, will bind in the S4 site adjacent to the β6/β7

loop. Interestingly, the naphthalene ring moves within the hydrophobic pocket during MD

simulations, suggesting that adding non-polar substituents here could improve binding. Finally,

the phenyl group of the lead scaffold is positioned towards the conserved catalytic triad residues

and the attached carboxyl moiety is wedged between and the active site histidine and arginine

residues where it forms hydrogen bonds with both.

Zhulenkovs et al. identified benzisothiazolinone-based inhibitors via in vitro screening,

followed by secondary screening by HSQC and subsequent structural determination of the

inhibitor bound to SaSrtA using NMR92. Secondary screening of inhibitor binding by HSQC

provided unique benefits, including identifying interacting sortase residues, estimating potency

based on adduct accumulation, and determining reversibility of binding, and also highlighted the

44
susceptibility of in vitro screening to yield false positives (11 of 41 compounds showed no

binding by HSQC). The experimentally determined NMR structure of the enzyme-inhibitor

complex was then used to design analog molecules, which irreversibly inhibited SaSrtA with

IC50s in the low micromolar range (~3-7 µM). The NMR structure indicates various inhibitor

conformations, although interacting groups are nearly identical, which is possibly due to the

dynamic nature of the β6/β7 and β7/β8 loops. The high affinity interaction is facilitated by

covalent binding of a benzo[d]isothiazol-3(2H)-one heterocycle moiety to the catalytic cysteine

side chain; however, positions 4, 5, and 6 of this moiety did not show contacts with SaSrtA,

suggesting that derivatization to larger substituents could facilitate additional contacts. Within

the structure ensemble, the phenyl group of the inhibitor adduct can interact with L97 and A118

or participate in pi-pi stacking interactions with W194. The linker region preceding the

adamantyl moiety was varied to contain one or two carbonyl groups, which may hydrogen bond

with the active site arginine. In addition, the length of the linker region could be varied to

optimally bury the adamantyl moiety within the enzyme’s hydrophobic pocket, where it was

experimentally determined to interact with V166, V168, L169 in the β6/β7 loop and T180, I182,

and I199 from the β7 and β8 strands.

Recently, the structure of SmSrtA was solved with the natural product flavonoid

precursor, trans-chalcone46. The structure depicts chalcone bound outside of the SmSrtA active

site; however, this compound was verified biochemically to covalently modify the active site

cysteine residue through Michael addition. Wallock-Richards et al. generated a model of the

Cys-linked SmSrtA-trans-chalcone complex utilizing the crystal structures of SaSrtA-LPETG

and BaSrtB-AAEK. The fortuitous interaction of the N-terminus from the symmetry-related

molecule within the SmSrtA active site guided overlay of the aromatic rings of chalcone at the

45
phenyl rings of Phe67 and Phe69, which presumably represents the binding mode of this

compound. This binding mode seems to be shared among other sortase inhibitor classes,

including methyl(2E)-2,3-bus(4-methoxyphenyl)prop-2-enoate in which correct positioning of

two phenyl groups is essential for SrtA inhibition93. In fact, the criteria for two aromatic regions

seems to be a common feature of SrtA inhibitors and has been implemented to filter compounds

using a pharmacophore model generated from all SrtA inhibitors available in the literature before

completing virtual screening and MD simulations94.

Structure-based HTS is an attractive approach to identify and optimize sortase inhibitors.

The wealth of structural information available for sortase enzymes is evolving virtual screening

strategies to account for the dynamic nature of SrtA by implementing docking against multiple

structures and models generated through MD simulations12,95. These structure-based screening

strategies, in combination with SAR of lead compounds, have produced many promising

inhibitor classes with IC50s reaching the low- and sub-micromolar range11,15, as well as informed

our understanding of important active site contacts that mediate binding affinity and exploited

the knowledge of these contacts for further optimization.

2.10 Conclusion

Sortase enzymes are ubiquitous in Gram-positive organisms and are commonly

associated with increased pathogenicity. Beyond their medical relevance, these enzymes have

also been shown to be extremely useful tools for site specifically linking proteins and small

molecules in vitro. Because of this, a considerable effort has been put forth to understand the

structural features that dictate substrate and small molecule inhibitor binding, as well as the

molecular mechanism of catalysis. Recent studies have uncovered a conserved mechanism of

46
substrate binding, shed light on the importance of variable regions of sortase structure, elucidated

the molecular mechanism, and produced a number of small molecule sortase inhibitors. In spite

of the many discoveries that have been made in the last decade, many questions remain to be

answered. No conclusive evidence has emerged to pinpoint the secondary substrate binding site

and very little is known about this latter half of the mechanism. While there are a number of

hypotheses, each with varying degrees of experimental backing, there has been no consensus in

the field. It is conceivable that this lack of consensus is the result of individual enzymes or

specific classes recognizing their secondary substrates in very different ways. This could

potentially stem from the needs of certain classes to attach their primary protein substrates to

different locations on the cell surface or recognize a secondary protein substrate instead of lipid

II. Exploration of this interaction in a number of different enzymes will be necessary to draw

useful conclusions about the nature of secondary substrate recognition. A number of enzymes

have also been shown to harbor N-terminal extensions which in some cases contact or

completely occlude the active site. For class C enzymes it appears that lid removal must be a

necessary step in uncovering the active site for catalysis, and while structures of class C enzymes

with their lids displaced have emerged recently, it remains to be seen whether these structures are

physiologically relevant, what would be responsible for removing the lid in vivo, and how this lid

removal would take place. Understanding the various ways sortase enzymes utilize these

additional features in substrate recognition and catalysis is an area of active research. Finally,

although many small molecule sortase inhibitors have been identified, to date none has been

successful enough to move forward towards becoming a therapeutic for treating Gram-positive

infections in humans. Given the rising prevalence of antibiotic resistant bacteria in recent years,

47
pressure to develop viable sortase inhibitors as therapeutics is rising and will undoubtedly lead to

the discovery and characterization of more potent and specific compounds in the future.

48
2.11 Figures

Figure 2.1 Biological mechanism of sortase.

Sortase and substrate are both membrane bound. 1 Sortase recognizes a sorting signal motif (here

the LPXTG sorting signal for SrtA types is shown) within CWSS and performs a nucleophilic

attack. 2 LPXTG sorting signal is cleaved to produce sortase-substrate thioacyl intermediate.

Mechanism for cell wall anchoring proceeds through “a” steps (top) and mechanism for pilus

assembly proceeds through “b” steps (bottom). 3a Cross-bridge peptide from lipid II molecule

performs a nucleophilic attack on the thioacyl intermediate. 4a new peptide bond is formed

between the lipid II molecule and surface protein which is then incorporated into mature cell

wall. 3b a lysine residue within the pilin motif performs nucleophilic attack on thioacyl

intermediate. 4b covalently linked pilin proteins can either be attached to cell wall as in 3a and

4a or polymerized further by additional rounds of 3b.

49
Figure 2.2 Structure of sortase.

(A) SaSrtA NMR structure shown as cartoon, showcasing 8 Stranded β-barrel, with active site

residues shown as sticks. (B) SaSrtA NMR structure shown as a surface representation in green

with active residues Arg in blue and Cys in yellow. The active site His is occluded by a closed

β7/β8 loop, and there is no obvious groove for a full length peptide to exit the active site. (C)

SpySrtA structure shown as a surface representation, colored as in (B), with active site His

residue also shown in cyan. An open β7/β8 loop creates a clear channel that can be seen running

between active Cys and His residues indicating the likely path of binding for a full length peptide

substrate. (D) Sortase can catalyze both a reversible transpeptidation reaction, and an irreversible

hydrolysis reaction (indicated by dashed arrow).

50
Table 2.1 Structurally characterized sortase enzymes.
Organism and Sortase PDB Bound Ligands or Substrates Method

Class A

S. aureus SrtA 1IJA N/A NMR

S. aureus SrtA-C184A 1T2O N/A Xray

S. aureus SrtA 1T2P N/A Xray

S. aureus SrtA 1T2W LPETG Xray

S. aureus SrtA 2KID Cbz-LPAT*, Ca2+ NMR

S. aureus SrtA 2MLM benzo[d]isothiazol-3-one based inhibitor NMR

B. anthracis SrtA 2KW8 N/A NMR

B. anthracis SrtA unpub Cbz-LPAT* NMR

S. pyogenes SrtA 3FN5 N/A Xray

S. pyogenes SrtA 3FN6 Cys in suflphenic acid form Xray

S. pyogenes SrtA 3FN7 N/A Xray

S. agalactiae SrtA 3RCC Zn2+ Xray

S. pneumoniae SrtA 4O8L N/A Xray

S. pneumoniae SrtA-C207A 4O8T N/A Xray

S. mutans SrtA 4TQX chalcone Xray

Class B

S. aureus SrtB 1NG5 N/A Xray

S. aureus SrtB 1QWZ MTSET Xray

S. aureus SrtB 1QX6 E-64 Xray

S. aureus SrtB 1QXA Gly3 Xray

S. aureus SrtB 4FLD Cbz-NPQT* Xray

B. anthracis SrtB 1RZ2 N/A Xray

B. anthracis SrtB 2OQW AAEK1 Xray

B. anthracis SrtB 2OQZ AAEK2 Xray

S. pyogenes SrtB 3PSQ Zn2+, Cl- Xray

C. difficile SrtB 4UX7 N/A Xray

51
 Class C

A. oris SrtC-1 2XWG Ca2+ Xray

S. pneumoniae SrtC-1 2W1J glycerol X-ray

S. pneumoniae SrtC-3 2W1K N/A Xray

S. pneumoniae SrtC-1-H131D 2WTS alanine Xray

S. pneumoniae SrtC-2 3G66 N/A X-ray

S. pneumoniae SrtC-2 3G69 SO42- Xray

S. agalactiae SrtC-1Pilus Island-2a 3O0P N/A Xray

S. agalactiae SrtC-1 Pilus Island-1 3RBI N/A Xray

“Type III”

S. agalactiae SrtC1- Pilus Island-1 3RBJ N/A Xray

C184A; KDPYS to IPNTG

S. agalactiae SrtC-1 Pilus Island-1 3RBK N/A Xray

“Type II”

S. agalactiae SrtC1 Pilus Island-1 3TB7 N/A Xray

“Type I” - open lid

S. agalactiae SrtC-1 Pilus Island-1 3TBE MTSET Xray

S. agalactiae SrtC-2 Pilus Island-1 4G1H Ca2+ Xray

S. agalactiae SrtC-1 Pilus Island-1 4G1J N/A Xray

S. suis SrtC-1 3RE9 N/A Xray

Class D

B. anthracis SrtD 2LN7 N/A NMR

C. perfringens SrtD 4D70 none Xray

Class E

S. coelicolor SrtE-1 unpub N/A Xray

52
Figure 2.3 Structural variation by class of sortase.

Sortases representative of the major themes seen for each class are displayed as a cartoon

representation, with active site residues shown as sticks. The hallmark sortase beta barrel is

highlighted in blue. Major sources of structural variability are highlighted: N-terminus, red;

β6/β7 loop, orange; β7/β8 loop, green.

53
Figure 2.4 Sorting signal recognition.

(A) The SaSrtA-LPAT* complex. (B) The SaSrtB-NPQT* complex. (C) The BaSrtA-LPAT*

complex, shown with N-terminal appendage removed from view for clarity. Enzymes are shown

as surface representations with SrtA types in light green and SaSrtB in light blue, Substrate

mimics are shown as grey sticks. Active site Cys and Arg residues are shown as gold and blue

surfaces, respectively. (D) Conserved recognition sites for sortase enzymes. Left, SaSrtA shown

as a transparent surface representation with recognition subsites determined from the

combination of sortase structures color coded as follows: S4 is shown in red, S3 in orange, S2 in

green, and S1 in magenta, and active site Arg in blue, Cys in gold, and His in cyan. Right,

Cartoon diagram of SaSrtA with secondary structure elements that contribute to substrate binding

labeled for clarity.

54
Figure 2.5 The substrate stabilized oxyanion hole.

The energy minimized model of the SaSrtB-NPQT thioacyl intermediate displayed with SaSrtB

as light blue cartoon and residues in the active site and oxyanion hole shown as sticks. NPQT

substrate shown as grey sticks. The substrate’s P1 Thr residue’s sidechain hydroxyl, and

backbone carbonyl participate in a hydrogen bonding network with the active site Arg, and the

backbone amide of Glu224 that together build an oxyanion hole to stabilize the high energy

tetrahedral reaction intermediates. Reproduced from Jacobitz et al 2012.

55
Figure 2.6 Molecular mechanism of sortase enzymes.

The active site of sortase consists of a His-Cys-Arg triad, and in its active form, the His and Cys

residues will form a thiolate-imidazolium ion pair (a). The first step in the reaction is the

recognition of an appropriate sorting signal (here the LPXTG sorting signal for SrtA types is

shown), and the active site Cys residue will perform a nucleophilic attack on the carbonyl carbon

at the substrate’s P1 position (b). This leads to a tetrahedral intermediate whose oxyanion is

stabilized by the nearby Arg residue which is likely held in proximity by interactions with the

side chain of the substrate’s P1 residue which is a threonine in over 95% of all substrates (c). The

active His residue concomitantly donates a proton to the leaving group and the tetrahedral

transition state then collapses to form a semi-stable thioacyl intermediate between the substrate’s

P1 residue and the active site Cys (d). Next, the secondary substrate, (Here shown as the lipid II

molecule used by cell wall anchoring sortases) enters the active site where its terminal amine is
56
deprotonated by the active His residue before it performs a nucleophilic attack on the carbonyl

carbon in the thioacyl bond (e) forming a second tetrahedral intermediate (f) before collapsing to

form a new peptide bond between the two substrates, which is then released to leave the

regenerated active site (a).

57
Table 2.2 Activity of sortase enzymes in vitro.

Sortase Primary Substrate Secondary Cleavage Transpeptidation

(Km mM) Substrate kcat (s-1) kcat (s-1)
(Km µM)

Class A

SaSrtAΔ2479 Abz-LPETG-Dap(Dnp) Gly5 0.086 ± 0.015 0.28 ± 0.02

(7.33 ± 1.01) (196 ± 64)

SaSrtAΔ2490 Abz-LPETG-Dap(Dnp) Gly5 NR 0.27

(5.5 mM) (140)
SaSrtAΔ2496 Abz-LPETGG-Dap(Dnp) Gly5 NR 1.10 ± 0.06 a
(8.76 ± 0.78) a (NR)

SaSrtAΔ5997 Abz-LPETGK-(Dnp) Gly3 NR 1.5 ± 0.2

(7.6 ± 0.5) (140 ± 30)
SaSrtAΔ59 Abz-LPETGK-(Dnp) Gly3 NR 4.8 ± 0.8
Evolved (0.17 ± 0.03) (4800 ± 700)
tetramutant97
SpySrtAΔ8143 Abz-LPETGG-Dap(Dnp) Ala2 NR 0.0136 ± 0.0011
(0.83 ± 0.11) (NR)
BaSrtAΔ5644 Abz-LPETG-Dap(Dnp) m-DAP* 0.0004 ± 0.0001† *
(0.038 ± 4)†

BaSrtAΔ5648 Abz-LPETG-Dap(Dnp) m-DAP* 3.6 ± 0.2 x10-5 *

(0.306 ± 0.023)
BaSrtAΔ6448 Abz-LPETG-Dap(Dnp) m-DAP* 5.7 ± 0.2 x 10-5 *
(0.173 ± 0.011)
SmutSrtAΔ4046 Dabcyl-QALPETGEE-Edans NR Yes NR
(0.0904 ± 0.0047)†

Class B

SaSrtBΔ2180 Abz-KVENPQTNAGT- Gly5 NR 5.4 ± 0.5 x 10-4

Dap(DNP) (NR)
(7.8 ± 2)
SaSrtBΔ3137 SNKDKVENPQTNAGT Gly5 NR 1.010-4
(1.8) (NR)
BaSrtBΔ3798 Abz -KTDNPKTGDEA- NR Yes NR
Dap(DNP)

CdSrtBΔ2699 KIVKSPKTGDETQLMK Gly4/5 or NR NR

KPPVPPKTGDSTTIGK Ala-D-Glu-
DAP

58
 Class C

SpnSrtC117-228 54 IPQTG in RrgB30-633 YPKN in No Yes

RrgB30-633
SagSrtC143-254 Dabcyl-KKVTIPQTGGIGT- NR Yes NR
from PI-2a21 Edans
(0.0138)

SagSrtC142-305 Dabcyl-RPPGVFPKTGGIG- NR 1.16 ± 0.044 x10-3 NR

from PI-157 Edans
(0.01358 ± 0.00063)
Dabcyl-RPSIPNTGGIG-Edans NR 1.77 ± 0.101 x10-3 NR
(0.03100 ± 0.00462)

Dabcyl-RGGLIPKTGEQQ- NR 0.77 ± 0.038 x10-3 NR

Edans
(0.01639 ± 0.00250)
SagSrtC242-283 Dabcyl-RPPGVFPKTGGIG- NR 1.04 ± 0.058 x10-3 NR
from PI-157 Edans
(0.006385 ± 0.00142)
Dabcyl-RGGLIPKTGEQQ- NR 4.36 ± 0.256 x10-4 NR
Edans
(0.02733 ± 0.00435)
Dabcyl-RPSIPNTGGIG-Edans NR 5.56 ± 0.174 x10-3 NR
(0.05715 ± 0.00354)

Class D

BaSrtDΔ5561 VQGEKLPNTASNN m-DAP (NR) Yes NR

BaSrtDΔ55100 Abz-GEKLPNTASNN-Dnp m-DAP (NR) Yes NR
CpSrtDΔ23-18762 Aβ1-16-LPQTGS NR Yes NR

Sorting signals for all substrates are highlighted in bold

Errors are reported where published

“Yes” indicates the reaction was performed in vitro but kinetics parameters were not reported

NR – Not Reported

m-DAP – meso-diaminopimelic acid

a
These values calculated assuming a hydrolytic shunt mechanism

† Values reported from fluorescence assay and subject to inner filter effect and are likely underestimates

of true parameters

* The enzyme reportedly does not perform this reaction in vitro

59
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75
 Chapter 3

Solution Structure of the Sortase Required for Efficient Production

of Infectious Bacillus anthracis Spores

76
 The work described in this chapter has been reproduced with permission from:

Robson, S. A., Jacobitz, A. W., Clubb, R. T., Phillips, M. L., Clubb, R. T. Solution Structure of

the Sortase Required for Efficient Production of Infectious Bacillus anthracis Spores.

Biochemistry. 2012 51(40):7953-7963.

doi:10.1021/bi300867t.http://pubs.acs.org/doi/abs/10.1021/bi300867t

Copyright 2011 American Chemical

Society

I contributed in vitro mass spectrometry experiments and analysis, as well as comparative

structural and genetic analysis between the sortase structure presented here and existing sortase

enzymes.

77
3.1 Overview

Bacillus anthracis forms metabolically dormant endospores that upon germination can

cause lethal anthrax disease in humans. Efficient sporulation requires the activity of the SrtC

sortase (BaSrtC), a cysteine transpeptidase that covalently attaches the BasH and BasI proteins to

the peptidoglycan of the forespore and predivisional cell, respectively. To gain insight into the

molecular basis of protein display, we used nuclear magnetic resonance to determine the

structure and backbone dynamics of the catalytic domain of BaSrtC (residues Ser56–Lys198).

The backbone and heavy atom coordinates of structurally ordered amino acids have coordinate

precision of 0.42 ± 0.07 and 0.82 ± 0.05 Å, respectively. BaSrtCΔ55 adopts an eight-stranded β-

barrel fold that contains two short helices positioned on opposite sides of the protein.

Surprisingly, the protein dimerizes and contains an extensive, structurally disordered surface that

is positioned adjacent to the active site. The surface is formed by two loops (β2−β3 and β4–H1

loops) that surround the active site histidine, suggesting that they may play a key role in

associating BaSrtC with its lipid II substrate. BaSrtC anchors proteins bearing a noncanonical

LPNTA sorting signal. Modeling studies suggest that the enzyme recognizes this substrate using

a rigid binding pocket and reveals the presence of a conserved subsite for the signal. This first

structure of a class D member of the sortase superfamily unveils class-specific features that may

facilitate ongoing efforts to discover sortase inhibitors for the treatment of bacterial infections.

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3.2 Introduction

Bacterial surface proteins play key roles in microbial physiology and pathogenesis.

Gram-positive bacteria display surface proteins using sortase enzymes, a large superfamily of

cysteine transpeptidases, which covalently join proteins bearing an appropriate C-terminal cell

wall sorting signal (CWSS) to strategically positioned amino groups located on the cell

surface.(1-7) Typically, bacteria encode multiple sortases that either attach proteins to the cross-

bridge peptide of the cell wall or assemble pili, long proteinaceous structures that extend from

the cell surface. Both processes occur through a related transpeptidation reaction that is best

characterized for the sortase A enzyme from Staphylococcus aureus (SaSrtA). SaSrtA anchors

proteins to the cell wall containing a CWSS that consists of an LPXTG motif (where X denotes

any amino acid), followed by a segment of hydrophobic amino acids, and a tail that is comprised

primarily of positively charged residues. The C-terminal charged tail presumably retards export,

positioning the protein for processing by the extracellular membrane-associated SaSrtA enzyme.

Catalysis occurs through a ping-pong mechanism that is initiated when the active site cysteine

residue nucleophilically attacks the backbone carbonyl carbon of the threonine residue within the

LPXTG motif, breaking the threonine–glycine peptide bond to create a sortase–protein complex

in which the components are linked via a thioacyl bond.(8, 9) SaSrtA then transfers the protein to

the cell wall precursor lipid II, when the amino group in this molecule nucleophilically attacks

the thioacyl linkage to create a peptide-linked protein–lipid II product. The transglycosylation

and transpeptidation reactions that synthesize the cell wall then incorporate this product into the

peptidoglycan, resulting in a surface-displayed protein that is covalently attached to the cross-

bridge peptide. Instead of anchoring proteins to the cell wall, some sortases assemble pili.(2, 3)

These sortases operate through a similar mechanism; however, they link pilin subunits together

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by joining a lysine amino nucleophile located in one protein to the CWSS of another. A

molecular-level understanding of sortase function could lead to new therapeutics for the

treatment of bacterial infections, as many clinically significant pathogens are attenuated in their

virulence when their sortase enzymes are genetically eliminated.(4, 10)

On the basis of primary sequence homology, most sortases in pathogenic Gram-positive

bacteria can be grouped into four distinct families, called class A–D enzymes.(7, 11, 12) Class A

enzymes are most closely related to SaSrtA. They appear to perform a housekeeping role in

different species of bacteria as members of this group have been shown to anchor a large number

of functionally distinct proteins to the cell wall that generally contain an LPXTG motif within

their CWSS. In contrast, class B–D enzymes appear to have more specialized functions as

representative members of these groups display a limited number of proteins that frequently

contain noncanonical sorting signals. Class B enzymes are present in Firmicutes and can have

distinct functions. Some members of this group attach heme receptors to the peptidoglycan,(13)

while others function as polymerases that assemble pili. Class C enzymes are also broadly

distributed in Gram-positive bacteria and function as pilin polymerases.(14, 15) Finally, class D

enzymes are present in many bacilli (Bacillus cereus, Bacillus anthracis, and Bacillus

thuringiensis). A single member of this family has been characterized, the class D enzyme from

B. anthracis, which has been shown to attach proteins to the cell wall that facilitate

sporulation.(16, 17) Atomic structures of representative class A–C enzymes have been

determined, revealing a conserved eight-stranded β-barrel fold that houses three essential active

site residues: His120, Cys184, and Arg197 (SaSrtA numbering).(1, 2, 7) The roles of these

residues in catalysis have been defined for SaSrtA and are likely conserved in other sortases:

Cys184 acts as a nucleophile that attacks the carbonyl atom in the scissile peptide bond of the

80
CWSS, Arg197 stabilizes the binding of the sorting signal substrate, and His120 may act as a

general acid or base.

B. anthracis is a Gram-positive facultative anaerobe that causes lethal anthrax

disease.(18) Like other bacterial species within the genus Bacillus, it forms dormant endospores

(spores) that are capable of surviving for long periods of time under harsh conditions. In humans,

anthrax caused by the inhalation of aerosolized B. anthracis spores has a high rate of mortality

and has led to their use as a bioterrorism agent.(19) Efficient sporulation of B. anthracis is

dependent upon the activity of SrtC (BaSrtC), a class D sortase that attaches the BasH and BasI

proteins to distinct cellular structures.(16, 17) BasI is attached to the peptidoglycan of

predivisional cells, while BasH is exclusively attached to the forespore presumably by BaSrtC

inherited from the mother cell before the polar division takes place. B. anthracis also encodes a

housekeeping class A enzyme (BaSrtA) that anchors a different set of proteins to the cell

wall.(20, 21) Interestingly, BaSrtC and BaSrtA specifically recognize very closely related sorting

signals.(16, 17) BaSrtA recognizes a canonical LPXTG-type sorting signal present in seven B.

anthracis proteins (three of these proteins are involved in collagen adhesion, while the functions

of the other proteins are not known). In contrast, BaSrtC anchors BasH and BasI that contain

LPNTA sorting signals. B. anthracis also encodes a third sortase, BaSrtB.(22) This class B

enzyme anchors the IsdC heme binding protein to the cell wall by recognizing its unique

NPQTN sorting signal. While the sortase enzymes in B. anthracis anchor different proteins to the

cell wall by recognizing distinct sorting signals, they are believed to attach these proteins to the

same chemical group, the m-diaminopimelic acid (m-DAP) side chain of the cross-bridge

peptide.(17, 23) In this paper, we describe the structure and dynamics of the B. anthracis BaSrtC

enzyme, which is the first reported structure of a class D enzyme. The structure provides insight

81
into the mechanism of protein anchoring in pathogenic B. anthracis, as well as the evolutionary

relationship between different types of enzymes within the sortase enzyme superfamily.

3.3 Results and Discussion

3.3.1 Design and Validation of Soluble BaSrtC

We studied the B. anthracis BaSrtC enzyme to gain insight into the molecular basis

through which it selectively anchors proteins to the cell wall during bacterial sporulation. BaSrtC

is 198 amino acids in length and contains a nonpolar N-terminal region (residues Met1–Tyr19)

that likely functions to embed the protein in the membrane. This is followed by a C-terminal

region (residues Lys20–Lys198) that has been shown to mediate the in vitro cleavage of an

LPNTA peptide that contains the amino acid sequence of the CWSS found in its BasI protein

substrate.(16) Inspection of the amino acid sequence of BaSrtC reveals that the C-terminal region

contains residues that are homologous to the catalytic domain of SaSrtA as well as a less

conserved polypeptide segment that connects the presumed catalytic domain to the N-terminal

nonpolar region (Figure 1A). Structural studies have revealed that many sortases can contain

ordered appendages that either precede or follow the amino acid sequence of the catalytic

domain. We therefore used NMR to study two polypeptides, BaSrtCΔ19 (residues Lys20–

Lys198), which contains the entire C-terminal region, and BaSrtCΔ55 (residues Ser56–Lys198),

which contains only amino acids within the presumed catalytic domain. The 1H–15N HSQC

spectra of these proteins reveal a similar set of well-dispersed backbone amide chemical shifts

indicating that these fragments adopt similar three-dimensional structures (Figure 1B). However,

the spectrum of BaSrtCΔ19 also contains an additional ∼30 cross-peaks that exhibit narrow line

widths and random coil chemical shifts. Surprisingly, this indicates that unlike sortases from

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other classes, the linker preceding the catalytic domain in BaSrtC is unstructured in the isolated

protein.

Previous studies have shown that the intact C-terminal region of BaSrtC selectively

cleaves an LPNTA peptide derived from the CWSS of its BasI protein substrate.(16) To

ascertain whether only the conserved catalytic domain is required for this enzymatic activity, the

ability of BaSrtCΔ55 to cleave a peptide containing the LPNTA sequence was determined.

MALDI-TOF analysis of a reaction mixture containing BaSrtCΔ55 and the peptide reveals that

the isolated catalytic domain cleaves the peptide between the threonine and alanine residues

(Figure 2). Importantly, BaSrtCΔ55 exhibits specificity for the BasI sorting signal, as it is unable

to cleave LPATG and LPETG peptides derived from proteins that are anchored to the cell wall

by the BaSrtA sortase (data not shown and ref 16). Combined, these data indicate that in vitro the

catalytic domain within BaSrtC is folded and sufficient for hydrolytic activity.

3.3.2 NMR Structure of BaSrtCΔ55

The structure of BaSrtCΔ55 was determined using multidimensional heteronuclear NMR

and simulated annealing methods. A total of 2584 experimental restraints were employed,

including 2330 interproton distance, 196 dihedral angle, and 58 hydrogen bond restraints. Figure

3A displays the ensemble of the 20 lowest-energy conformers of BaSrtCΔ55. Each structure

exhibits good covalent geometry and has no NOE or dihedral angle violations greater than 0.5 Å

or 5°, respectively (structural and restraint statistics are listed in Table 1). The structure of

BaSrtCΔ55 is generally well defined by the NMR data; the backbone and heavy atom coordinates

of residues Lys62–Ala85, Val94–Gly115, Leu124–Thr172, and Arg185–Lys198 have root-

mean-square deviations (rmsd's) from the average structure of 0.42 ± 0.07 and 0.82 ± 0.05 Å,

83
respectively. The structure of BaSrtCΔ55 also contains three disordered surface loops that

surround the active site (colored red and orange in Figure 3A), two of which (red) likely mediate

protein dimerization (vide infra).

BaSrtCΔ55 adopts an eight-stranded β-barrel fold that contains two short helices

positioned on opposite sides of the protein (Figure 3B). The structure is initiated by strand β1

(Val67–Ile72), which after a short reverse turn interacts with strand β2 (Lys77–Tyr82) in an

antiparallel manner. A poorly ordered 11-amino acid loop then leads into strand β3 (Val94–

Met96), which is positioned parallel and antiparallel to strands β2 and β4 (His110–Gly115),

respectively. The active site His116 residue is located immediately after strand β4 and is

followed by a large polypeptide segment containing helix H1 (Leu124–Gln126) that stretches to

the opposite face of the protein. Strand β5 (Thr132–Glu136) then joins with β1 in an antiparallel

manner before the peptide reverses direction to form strand β6 (Thr141–Thr152). Strand β6

wraps around the enzyme toward the active site and contains a sharp point of curvature created

by a β-bulge at residues Gln147 and Lys148. A long loop containing helix H2 then leads into

strand β7 (Ile167–Thr172), which lies parallel with respect to strand β4. A large loop then

reverses the directionality of the chain before forming strand β8 (Arg185–Tyr197) that lies

antiparallel with respect to strands β6 and β7. Strand β8 is extensive as it contains a β-bulge at

residues Thr193 and Gly194 that allows it to form a continuous set of hydrogen bonding

interactions with strand β6 that together contribute residues to both faces of the protein structure.

84
3.3.3 Disordered Loops Positioned near the Active Site Might Mediate in vitro

Dimerization

On the basis of primary sequence homology, the active site in BaSrtCΔ55 is formed by

three spatially adjacent amino acids: His116, Cys173, and Arg185. These residues are conserved

in all sortases, and in BaSrtCΔ55, they are located at the end of a large cleft formed by strands β4,

β7, and β8. Cys173 is located at the end of strand β7, and in the prototypical SaSrtA enzyme, the

analogous residue mediates the nucleophilic attack on the threonine carbonyl carbon within the

sorting signal.(38, 39) Surrounding the thiol are the side chains of Arg185 (strand β8) and

His116 (strand β4), which in SaSrtA may stabilize binding of the sorting signal substrate and

facilitate acid–base chemistry, respectively.(1) Surprisingly, unlike previously characterized

sortases, BaSrtCΔ55 contains three disordered surface loops that are positioned immediately

adjacent to the active site (loops colored red and orange in Figure 3A). The loops are formed by

residues that immediately follow the active site cysteine so as to connect strand β7 to β8 (the

β7−β8 loop, residues Tyr173–Asp184), residues immediately following the active site histidine

that connect strand β4 to H1 (the β4–H1 loop, residues His116–Asp123), and residues

immediately adjacent to His116 that connect strands β2 and β3 (the β2−β3 loop, residues Trp83–

Gly93). The loops are structurally disordered because an insufficient number of interproton

distance restraints were identified in the NOESY spectra to define their position. For the β2−β3

and β4–H1 loops, few NOEs were observed because many of their residues exhibit resonance

line broadening. The broadening is most severe for residues Leu90–Lys92 (β2−β3 loop) and

His116–Phe121 (β4–H1 loop), which are completely absent from the NMR spectra. This

suggests that residues within the β2−β3 and β4–H1 loops experience fluctuations in their

85
magnetic environments that occur on a micro- to millisecond time scale, presumably because

they are flexible or because they reside within a surface that mediates protein aggregation.

To gain insight into the origin of structural disorder in the three active site loops, we
15 15
quantitatively probed N–H bond motions in BaSrtCΔ55 by measuring N spin–spin (T2), N

spin–lattice (T1), and {1H}–15N heteronuclear NOE relaxation parameters (Figure S1 of the

Supporting Information). In general, the relaxation data are compatible with the structure of

BaSrtCΔ55 as residues within regular secondary structural elements whose coordinates are well

defined in the ensemble of conformers have an average {1H}–15N NOE value of 0.79, indicating

that they undergo only small amplitude motions on the picosecond time scale. Interestingly, an

analysis of the relaxation data suggests that the structurally disordered β2−β3, β4–H1, and β7−β8

loops may participate in transient protein oligomerization. As the chemical shifts of many

residues within the disordered β2−β3 and β4–H1 loops are unassigned, the relaxation data do not

directly report on their dynamic status. However, several assigned surrounding residues within

the β2−β3 and β4–H1 loops, as well as in the adjacently positioned β7−β8 loop, exhibit

substantially shortened T2 values compared to those of other residues in the protein (Figure S1A

of the Supporting Information). Intriguingly, this indicates that their backbone amide atoms

experience fluctuating magnetic environments on the micro- to millisecond time scale that could

result from motions within the loops themselves and/or because the loops reside within or near a

molecular surface involved in transient oligomerization.

A more detailed analysis of the NMR relaxation data indicates that active site loops likely

form a surface that mediates protein oligomerization. Initially, we attempted to model the

dynamics of BaSrtC using the Lipari–Szabo model-free formalism, which yields for each

backbone N–H bond vector a generalized order parameter (S2).(35) S2 ranges from 0 to 1 and

86
describes the rigidity of the amide bond on the picosecond time scale, with a value of 1

indicating that it is completely immobilized. Surprisingly, fitting of our data consistently yielded

many S2 values of precisely 1.0, which is suggestive of protein aggregation. This is illustrated in

Figure 4A, which shows a scatter plot of experimentally derived T1 and T2 values for each

residue overlaid with model-free predicted values of T1 and T2 for a range of S2 values [colored

lines indicate values of T1 and T2 expected for backbone amide nitrogens containing S2 values

ranging from 0.5 to 1 (see the key)]. The plot reveals that data for most residues are inconsistent

with all reasonable values of S2 because their T2 and T1 times are conjointly too short; this results

in the software attempting a “best fit” by assigning values of 1 to S2. We hypothesized that these

anomalous results are caused by protein aggregation. As protein aggregation is concentration-

dependent, we remeasured the T1 and T2 times using a sample of BaSrtCΔ55 that was diluted to

0.125 mM. As shown in Figure 4B, the data for the dilute sample are in better agreement with

values predicted by the model-free formalism primarily because the residues now have longer T2

values. This is most likely because in the dilute sample a smaller fraction of the protein forms

higher-molecular weight oligomers that influence the measured relaxation parameters.

To measure the stoichiometry and affinity of protein aggregation, we performed

sedimentation equilibrium ultracentrifugation experiments (Figure 4C). The resultant data are

best fit by a monomer–dimer equilibrium model that yields a dimer dissociation constant (KD) of

89 μM. Thus, at the protein concentrations used in our NMR studies, ∼90% of BaSrtCΔ55 likely

exists in its dimeric form. This explains our difficulty in fitting the relaxation data with the

model-free formalism and why analysis of the relaxation data yielded an overall molecular

correlation time of ∼16 ns, a value that is significantly longer than expected for a protein with

BaSrtCΔ55’s molecular mass. Taken together, the NMR and centrifugation data indicate that

87
residues within the β2−β3 and β4–H1 loops likely form a dimerization interface and that these

residues are broadened beyond detection in the NMR data because of monomer–dimer exchange

that occurs at a rate that is intermediate on the chemical shift time scale. This notion is

compatible with the positioning of many residues that exhibit anomalously short T2 times at high

protein concentrations as they are positioned proximal to the β2−β3 and β4–H1 loops (orange

points in Figure 4B and orange spheres in Figure 4D). Apparently, these residues also experience

monomer–dimer exchange broadening, however to a lesser extent, allowing us to still detect

them. In addition to forming a dimer interface, residues within the three disordered loops may

also exhibit elevated mobility as several residues within the β2−β3 and β7−β8 loops exhibit

depressed {1H}–15N heteronuclear NOEs indicating that they undergo faster picosecond motions.

The biological significance of the weak in vitro dimerization surface detected by NMR

and centrifugation is unclear. As BaSrtC is embedded in the membrane, it may indeed dimerize

on the cell surface because it undergoes limited two-dimensional diffusion. If this is the case,

then class D enzymes might be unique, as previously reported structures of class A–C sortases

have shown that they are monomeric(40-55) (it should be noted that biochemical studies have

shown that the S. aureus SaSrtA enzyme dimerizes with weak affinity, but this finding is

controversial(56)). Alternatively, it is also possible that the in vitro dimerization we have

observed occurs only because BaSrtC is being studied in isolation. In this scenario, dimerization

via the large structurally disordered surface occurs because the appropriate binding partner that

normally interacts with this surface is missing. On the cell surface, the missing binding partner

could be, among others, protein factors involved in secretion (e.g., the SEC translocon), a

component of the cell wall, or BaSrtC’s intact lipid II and protein substrates. This is supported by

microscopy studies that have shown that the SrtA and SecA proteins in Streptococcus pyogenes

88
colocalize at the cross wall compartment where the cell wall is synthesized, and at polar sites

where surface protein anchoring also occurs.(57) It would also explain the in vitro catalytic

properties of BaSrtC as we and others have been able to demonstrate only that it can hydrolyze

its sorting signal (Figure 2 and ref 16). The full transpeptidation reaction using m-DAP as a

nucleophile has never been demonstrated biochemically. This is presumably because to be fully

active, BaSrtC needs to either associate with other factors on the cell surface or bind to its intact

lipid II and protein substrates. The notion that the disordered surface mediates substrate

recognition on the cell surface is supported by NMR and X-ray studies of SaSrtA and SaSrtB

that have implicated the β7−β8 loop in binding the cross-bridge peptide.(49, 53) Furthermore, it

is interesting to note that of all the sortase enzymes whose structures have been determined only

the sortases from B. anthracis (BaSrtA, BaSrtB, and BaSrtC) contain a disordered β7−β8 loop

and only these enzymes attach proteins to lipid II molecules with a m-DAP group.(50, 51) Thus,

on the cell surface, the large disordered loop regions identified by NMR may play a key role in

associating BaSrtC with protein factors and substrates that facilitate cell surface protein

anchoring.

3.3.4 Class A and D Sortases Are Structurally Related

Structures of representative class A–C sortases have been determined. Despite a limited

degree of sequence homology, BaSrtC is most structurally similar to class A enzymes based on a

DALI analysis. Three structures of class A enzymes have been reported: SaSrtA, BaSrtA, and

the SrtA enzyme from S. pyogenes (SpSrtA).(41, 48-50, 52) The sequence of BaSrtC is only 23–

24% identical with the sequences of these enzymes; however, the coordinates of its carbon atoms

that participate in regular secondary structures can be superimposed with rmsd's of 2.4, 1.9, and

89
2.0 Å, respectively. Notably, both class A and D sortases contain a short 310-helix located within

the β6−β7 loop (Figure 5, yellow). In SaSrtA, NMR studies have shown that this loop interacts

with the leucine-proline portion of the LPXTG sorting signal, suggesting that BaSrtC will also

recognize its distinct LPNTA sorting signal through a generally similar mechanism (vide

infra).(49) Interestingly, class A enzymes also exhibit structural heterogeneity in their N-termini,

because BaSrtA possesses a long N-terminal appendage that contacts the active site histidine.

This N-terminal appendage is disordered in the structures of BaSrtC, SaSrtA, and SpSrtA (Figure

5, green). Although the BaSrtC enzyme adopts a canonical sortase fold, its structure differs

markedly from class B and C enzymes. For example, in contrast to class B enzymes that possess

a large structured β6−β7 loop that contains an α-helix, the analogous loop in BaSrtC is

substantially shorter and lacks a similarly positioned helix (Figure 5, yellow).(42, 51, 53) Unlike

class B enzymes, BaSrtCΔ55 is also missing the N-terminal α-helices that precede the catalytic

domain (Figure 5, green). The structure of BaSrtCΔ55 also differs substantially from that of class

C enzymes as it is missing an N-terminal “lid” appendage that has been proposed to regulate

access to the active site in several members of this group.(44, 46, 54, 58)

3.3.5 Model of the BaSrtCΔ55–Sorting Signal Complex

To gain insight into how BaSrtCΔ55 recognizes its sorting signal, we modeled the

structure of its substrate complex by superimposing the backbone coordinates of BaSrtCΔ55 onto

the coordinates of our previously determined solution structure of the SaSrtA enzyme covalently

bound to an LPAT peptide analogue.(49, 59) Inspection of the model reveals that BaSrtC and

SaSrtA can bind to their respective sorting signals in a similar manner as there is minimal atomic

overlap between the peptide and BaSrtC atoms in the model of the BaSrtC–peptide complex

90
(Figure 6A). In the model, the sorting signal peptide adopts a kinked structure and rests in a

groove whose base is formed by residues in strands β4 and β7 and whose walls are formed by the

β6−β7, β7−β8, β3−β4, and β2–H2 surface loops. Because the experimentally determined

structure of the apo form of BaSrtC can readily accommodate the peptide, this suggests that the

enzyme may recognize its sorting signal substrate through a lock-and-key mechanism. This is in

marked contrast to SaSrtA, which undergoes major changes in the structure and dynamics of the

β6−β7 loop upon signal binding.(49, 60) This can be seen in Figure 6B. The left panel shows

apo-SaSrtA (red) aligned with our BaSrtC structure (green). While the active site residues of

apo-SaSrtA (pink) align well with the active site residues of BaSrtC (gray), the agreement is

significantly better than that of the holo-SaSrtA (SaSrtA–LPAT*) structure in Figure 6B [middle

panel (blue)]. Furthermore, the BaSrtC active site residues more closely resemble the position of

holo-SaSrtA than that of the closely related apo form of the class A sortase from B. anthracis,

apo-BaSrtA (Figure 6B, right panel, orange). This result further suggests that BaSrtC exists in an

“active-ready” form prior to binding its cognate sorting signal.

To facilitate further discussion of the recognition process, hereafter we refer to amino

acids within the signal as P and P′ if they precede and follow the scissile peptide bond,

respectively [e.g., the LPNTA signal-recognized BaSrtC is Leu (P4)-Pro (P3)-Asn (P2)-Thr (P1)-

Ala (P′1)]. Corresponding binding sites on the enzyme for these residues are termed S and S′,

respectively. The model clearly defines enzyme subsites that can accommodate the leucine (P4)

and proline (P3) residues in the LPNTA signal. The leucine residue is recognized by a small

pocket on the enzyme that is formed by the β6−β7 loop and residues in the underlying β-sheet.

Within the S4 subsite, the leucine is contacted by the side chains of Val166, Pro168, Val173, and

Val174 on the β6−β7 loop and Val198 on strand β8. The S3 subsite, which recognizes the

91
LPNTA signal’s proline residue, is formed by residues in the underlying β-sheet [Ala124 (β4)

and Ile185 (β7)] and Val110 within the adjacent β3−β4 loop. The structures of the class A

BaSrtA and SpSrtA enzymes also contain similarly shaped nonpolar subsites,(48, 50) which is

compatible with bioinformatics studies that have predicted that the majority of sorting signals

processed by class A and D enzymes contain proline and leucine amino acids at positions P3 and

P4, respectively.(11) In B. anthracis, the class A BaSrtA and class D BaSrtC enzymes anchor

proteins to the cell wall that contain closely related LPXTG and LPNTA sorting signals,

respectively. Our modeling studies suggest both enzymes recognize the conserved leucine-

proline element of these signals through structurally conserved subsites that do not require

substrate binding to form. How each distinguishes differences that occur at the P1′ position

cannot be predicted as the coordinate precision of amino acids that contact this residue is poorly

defined in the NMR ensembles of both enzymes. However, BaSrtC may in part distinguish the

unique signal present in its BasH and BasI substrates by interacting with the P2 amino acid that

is invariably an asparagine, as the bottom of BaSrtC’s S2 subsite contains Ser114, which can

presumably favorably interact with the asparagine amide group through hydrogen bonding

(Figure 6A, gray), whereas BaSrtA contains a hydrophobic residue (Ala124).

At present, more than 800 sortase genes have been identified in nearly 300 species of

bacteria. The functions of the vast majority of these enzymes are not known because most

bacteria contain multiple sortases with unknown substrate specificities. Work described here lays

the foundation for more detailed structure–function studies that will elucidate the molecular basis

of substrate specificity and could facilitate future efforts to predict sortase function and to

discover therapeutically useful sortase inhibitors. Structural data could aid inhibitor discovery

efforts in several ways.(61) First, atomic structures can be used to discover new chemical

92
scaffolds that are likely to bind and inhibit sortases. In this approach, an in silico screen of

compound libraries could be performed to identify small molecules that have the appropriate

physicochemical properties to efficiently interact with the enzyme active site. These molecules

serve as new leads for further development after their inhibitory properties have been confirmed

experimentally. Second, the structures can be used to optimize the binding affinity and

selectivity of established sortase inhibitors. In this approach, computational methods are used to

dock the lead inhibitor molecule to the structure of the enzyme. Analogues that are likely to have

increased potency are then identified in silico by modifying the bound inhibitor and calculating

an algorithm-dependent binding energy. The most promising of these analogues are then tested

biochemically to ascertain whether they have improved activity. Finally, if the structure of the

enzyme–substrate complex is known, computational methods can be used to identify small

molecules with physicochemical features similar to those of the substrate. These molecules

presumably bind and inhibit the enzyme that can be tested experimentally. As several sortases

with known structures recognize sorting signals with distinct amino acid sequences, their unique

active site features in combination with the aforementioned computational methods may allow

class- and/or bacterial species-specific inhibitors to be developed. Such molecules are urgently

needed as infections caused by MRSA and other multidrug-resistant bacteria are a major health

concern.

3.4 Experimental Procedures

3.4.1 Cloning, Protein Expression, and Purification

BaSrtCΔ19 and BaSrtCΔ55 were amplified via polymerase chain reaction (PCR) from

genomic B. anthracis DNA (Sterne strain) with primers that placed an NdeI restriction site and a

93
BamHI restriction site on the 5′ and 3′ ends of the PCR product, respectively. Each PCR product

was digested with NdeI and BamHI restriction enzymes, as was empty vector pET15B (Qiagen).

Digested PCR products and pET15B plasmid were ligated together and transformed into

Escherichia coli XL-1 cells (Stratagene). Successful transformants were confirmed by DNA

sequencing. Plasmids where then transformed into E. coli BL21(DE3) (Stratagene) for

expression.

Protein for enzymatic assays was expressed in BL21(DE3) cells in standard Luria-Bertani

broth (LB), at 37 °C. Isotopically labeled protein for nuclear magnetic resonance (NMR) studies
15
was expressed in BL21(DE3) cells grown in M9 medium supplemented with NH4Cl and/or

[13C6]glucose. At an A600 of ∼0.7, expression was induced by the addition of 1 mM isopropyl β-

d-thiogalactopyranoside (IPTG). In the case of LB cultures, cells were harvested after 4 h by

centrifugation at 6000g and stored at −80 °C. Cells grown in M9 media where shifted to 22 °C

after induction, with induction allowed to progress overnight. Pellets were resuspended in

BugBuster (Novagen) with the addition of phenylmethanesulfonyl fluoride (PMSF) and

benzamidine (the final concentration of each was 2 mM) and allowed to lyse at room

temperature for 30 min. The lysate was cleared by centrifugation at 13000g for 30 min at 4 °C.

The soluble fraction was incubated with TALON His-affinity resin (Clontech). The resin was

washed with 20 mL of lysis buffer followed by 10 mL of 10 mM imidazole followed by a final

washing with thrombin cleavage buffer [20 mM Tris (pH 8.0), 150 mM NaCl, and 2.5 mM

CaCl2]. Resin was then treated with thrombin at 37 °C for 2 h, releasing untagged BaSrtC

proteins. Protein was further purified by Sephacryl-100 gel filtration in either NMR buffer [20

mM HEPES (pH 6.0)] or peptide cleavage assay buffer (see below).

94
3.4.2 NMR Spectroscopy and Structure Calculations

BaSrtCΔ55 was concentrated to 1 mM in NMR buffer [20 mM HEPES (pH 6.0) in 7%

D2O]. In addition, PMSF and ethylenediaminetetraacetic acid (EDTA) were added to final

concentrations of 1 mM each. A second sample was produced by lyophilizing BaSrtCΔ55 in NMR

buffer and resolubilizing it with an equal volume of 99.999% D2O. NMR spectra were recorded

at 298 K on Bruker Avance 500, 600, and 800 MHz spectrometers equipped with triple-

resonance cryogenic probes. All NMR spectra were processed using NMRPipe(24) and analyzed

using CARA (version 1.8.4).(25) Chemical shift assignments (1H, 13C, and 15N) were obtained by

analyzing the following experiments: HNCA, HNCACB, CBCA(CO)NH, HNCO, HN(CA)CO,

HNHA, HNHB, HBHA(CO)NH, CC(CO)NH, HCCH-TOCSY, HCCH-COSY,

(HB)CB(CGCDCE)HE, and (HB)CB(CGCD)HD.(26, 27) TALOS+(28) was used to obtain φ

and ψ dihedral angle restraints. Additional φ and ψ dihedral angle restraints were determined by

measuring relative HNHAi and HNHAi–1 NOE intensities. Distance restraints were obtained
15 13
from three-dimensional N- and C-edited NOESY spectra. The collection of residual dipolar

couplings (RDCs) was attempted in a PEG/hexanol mixture; however, protein alignment was not

achieved. Initial NOE assignments were determined using ATNOS/CANDID(29, 30) and

simulated annealing in NIH-XPLOR.(31) These NOE assignments were subsequently transferred

back to CARA for validation and additional manual picking of NOE assignments. Hydrogen

bonds were identified initially on the basis of calculated structures and NOE patterns, verified

using deuterium exchange of backbone amides, and included in NIH-XPLOR calculations using

the HBDB algorithm.(32) A total of 200 final structures were calculated. Structures with no NOE

or dihedral angle violations greater than 0.5 Å or 5°, respectively, were selected with the 20

lowest-energy conformers presented. The BaSrtC–LPAT* complex was modeled by aligning the

95
SaSrtA–LPAT* structure [Protein Data Bank (PDB) entry 2KID] with the BaSrtC structure in

PyMOL.(33) The enzyme portion of the SaSrtA–LPAT* complex was removed, leaving behind

the substrate only. Manual adjustment of the remaining LPAT* substrate was done to remove

atom clashes. The NMR structure of the apoenzyme has been deposited as PDB entry 2LN7.

3.4.3 15N Relaxation Measurements

15
N relaxation data (T1 and T2) and heteronuclear {1H}–15N NOE data were collected on a

Bruker Avance 600 MHz spectrometer equipped with a triple-resonance cryogenic probe at an

initial protein concentration of 1 mM. Relaxation data and parameters were analyzed in

SPARKY,(34) which generated raw relaxation and NOE parameters. Relaxation parameters were

analyzed using programs kindly provided by Arthur G. Palmer III (Columbia University, New

York, NY) in an attempt to perform “ModelFree” analysis.(35) Using the data from a protein

concentration of 1 mM, the ModelFree formalism failed to generate meaningful results.

However, we noticed that the R2R1_tm software that utilizes the R2/R1 ratio to calculate an

approximate correlation time (τc) resulted in an unusually high number for a protein of ∼16.5

kDa (τc at 1 mM ∼ 16 ns). We conjectured that monomer–dimer exchange phenomena might

result in aberrant tumbling and relaxation behavior, so we measured T2 and T1 values at an

additional protein concentration 0.125 mM. To compare our relaxation data to the ModelFree

formalism of Lipari and Szabo,(36, 37) we used eqs 7a and 7b from ref 36 to plot T1 versus T2

for order parameters from S2 = 1 to S2 = 0.6.

96
3.4.4 Analytical Ultracentrifugation and Enzyme Assays

Runs of sedimentation equilibrium were performed at 25 °C on a Beckman Optima XL-A

analytical ultracentrifuge in 12 mm path length double-sector cells. All samples were in 20 mM

HEPES (pH 6.0) and 100 mM NaCl with the addition of 5 mM TCEP. Absorption was

monitored at 280 nm at sample concentrations of 0.03, 0.015, and 0.0075 mM. Sedimentation

equilibrium profiles were measured at 40000 and 50000 rpm. The data were initially fit with a

nonlinear least-squares exponential fit for a single ideal species using the Beckman Origin-based

software (version 3.01). Analysis of the association behavior used the global analysis software

(the multifit option of the software mentioned above) to analyze four scans simultaneously,

corresponding to protein at a concentration of 0.03 mM at 40000 and 50000 rpm and a protein at

a concentration of 0.015 mM at 40000 and 50000 rpm. Partial specific volumes were calculated

from the amino acid composition.

Peptides were synthesized by NEO Biosciences and used without further purification.

BaSrtC (10 μM) was incubated with 200 μM peptide substrate in 5 mM CaCl2 and 20 mM

HEPES (pH 7.5) at room temperature. Samples (1 μL) were removed and plated with 0.5 μL of

2,5-dihydroxybenzoic acid (DHB) matrix (Acros Organics) dissolved in 50% ethanol and 0.1%

trifluoroacetic acid (TFA) and analyzed by MALDI-TOF immediately upon addition of enzyme,

and again after 24 h. Each reaction was performed in triplicate, and all samples were spotted and

analyzed in triplicate.

97
3.5 Figures

Figure 3.1 NMR spectra and amino acid sequence of BaSrtC.

(A) Sequence alignment of B. anthracis (Ames strain) class A sortase (BaSrtA), class B sortase

(BaSrtB), and class D sortase (BaSrtC). The sequence alignment was performed by ClustalW.63

Conserved residues are colored orange, while identical residues are colored red. The predicted

98
transmembrane region (TM) is indicated by a cylinder labeled TM. Secondary structure features

of BaSrtC are indicated by cylinders or arrows above the sequence. (B) 1H–15N HSQC spectra of

BaSrtCΔ19 (left) and BaSrtCΔ55 (right). BaSrtCΔ19 contains approximately 30 more peaks, the

majority of which reside toward the center of the spectrum. The BaSrtCΔ55 HSQC spectrum has

its residue assignments indicated.

99
Figure 3.2 MALDI-TOF demonstrating BaSrtCΔ55 can cleave the LPNTA-containing

peptide.

A peptide substrate, VQGEKLPNTASNN, was incubated with BaSrtCΔ55 for 24 h at room

temperature. MALDI spectra of the peptide were taken immediately upon addition of the enzyme

(A) and after incubation with BaSrtCΔ55 for 24 h (B).

100
Figure 3.3 Structures of B. anthracis class D sortase.

(A) Stereoview of the overlay of the ensemble of NMR structures of BaSrtCΔ55 (PDB entry

2LN7). The unassigned β2–β3 and β4–H1 loops are colored red. The assigned but poorly ordered

β7–β8 loop is colored orange. (B) Ribbon diagram with secondary structure elements labeled.

Loop structures are labeled with text. Active site residue side chains for Arg185, Cys173, and

His86 are colored green.

101
Table 3.1 Structural Statistics for the Solution Structure of BaSrtCΔ55a

⟨SA⟩ ( SA )r

rmsd from NOE restraints (Å)b 0.029 ± 0.001 0.046

all (2330) 0.032 ± 0.001 0.058
sequential (|i − j| = 1) (617) 0.039 ± 0.002 0.054
medium-range (|i − j| ≤ 4) (292) 0.027 ± 0.001 0.040
long-range (|i − j| ≥ 5) (977) 0.021 ± 0.001 0.034
intraresidue (444) 0.021 ± 0.001 0.034
rmsd from dihedral angles restraints (deg)c (196) 0.182 ± 0.087 0.095
deviation from idealized covalent geometry
bonds (Å) 0.007 ± 0.003 0.004
angles (deg) 0.414 ± 0.172 0.523
impropers (deg) 0.191 ± 0.081 0.493
PROCHECK results (%)d
most favorable region 85.6 ± 2.6 89.0
additionally allowed region 14.4 ± 2.6 11.0
generously allowed region 0.0 ± 0.0 0.0
disallowed region 0.0 ± 0.0 0.0
coordinate precision (Å)e,f
protein backbone 0.42 ± 0.07
protein heavy atoms 0.82 ± 0.05

a
The notation of the NMR structures is as follows. ⟨SA⟩ represents the final 20 simulated

annealing structures. ( SA )r represents the average energy-minimized structure. The number of

terms for each restraint is given in parentheses.

b
None of the structures exhibited distance violations of >0.5 Å or dihedral angle violations of

>5°.

102
c
The experimental dihedral angle restraints were as follows: 95 ϕ, 94 ψ, and39 χ1 angular

restraints.
d
Determined using PROCHECK. (62)
e
The coordinate precision is defined as the average atomic rmsd of the 20 individual SA

structures and their mean coordinates. These values are for residues 63−85, 94−113, 124−172,

and 182−198 of BaSrtCΔ55. Backbone atoms are N, Cα, and C′. Assignments were made for

residues 58−89, 93−115, 122−183, and 184−198 of BaSrtCΔ55.

f
Structure calculations also included 58 hydrogen bonds. These bonds were included in xplor

structure calculations as HBDB terms as described in ref 32.

103
Figure 3.4 Dynamics and ultracentrifugation analysis.

(A) Plot of experimentally determined T2 vs T1 data (black dots) for 1 mM BaSrtCΔ55 (assignable

backbone 15N resonances). Also plotted are lines that correspond to calculated T2 and T1 values

for various S2 order parameter values and correlation times (correlation times increase along each

line in a clockwise fashion). (B) Same as panel A, but T2 and T1 data were collected at a protein

concentration of 0.125 mM. Residues with T2 times of <0.07 s are colored orange. (C)

Analytical ultracentrifugation equilibrium data demonstrating that BaSrtC is a dimer with a KD

104
of 89 µM. (D) Structure of BaSrtC with unassigned residue backbone nitrogens colored red.

Residue backbone nitrogens with short T2 times identified in panel B are colored orange.

105
Figure 3.5 Comparison of representative structures from class A–D sortases.

B. anthracis class D enzymes are structurally similar to class A enzymes. BaSrtC lacks the long,

structured, β6–β7 loop (yellow) present in class B enzymes, but the appearance of a short 310-

helix in this region likely indicates the existence of a rigid substrate-binding pocket, similar to

BaSrtA. BaSrtC also lacks the structured N-terminal lid present in class C enzymes (green).

106
Figure 3.6 Substrate binding and active site architecture.

(A) Model of the CWSS peptide structure from the SaSrtA–LPAT* complex on the substrate

binding surface of BaSrtC. The peptide (green) is taken from the SaSrtA–LPAT* structure

without modification. Active site residues are indicated along with the potential Ser114 binding

site. The hydroxyl oxygen of Ser114 (Ser114 is colored gray, while its OH group is colored

white) comes into close contact with the side chain of position X. (B) Close-up overlay of the

BaSrtC structure (green) with apo-SaSrtA (red, left), holo-SaSrtA (blue, center), and apo-BaSrtA

(orange, right). Active site arginine, cysteine, and histidine residues are denoted. The active site

residues of apo-BaSrtC overlay best with those of holo-SaSrtA (SaSrtA–LPAT*).

107
3.6 References

1. Clancy, K. W., Melvin, J. A., and McCafferty, D. G. (2010) Sortase transpeptidases:

Insights into mechanism, substrate specificity, and inhibition Biopolymers 94, 385– 396.

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44. Manzano, C., Contreras-Martel, C., El Mortaji, L., Izore, T., Fenel, D., Vernet, T.,

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3.7 Supplementary Materials

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Figure S3.1 Residue by residue T2, T1 and 15N{1H} Heteronuclear NOE data for

BaSrtCΔ55.

Panels A and B are T2 relaxation data at 1 mM and 0.125 mM respectively.

Panels C and D are T1 relaxation at 1 mM and 0.125 mM respectively. Panel contains the
15
N{1H} heteronuclear NOE data. Labeled bars indicate flexible loops. Absence of data

for a particular residue indicates either a proline residue or unassigned residues.

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 Chapter 4

Structural and Computational Studies of the Staphylococcus aureus

Sortase B-Substrate Complex Reveal a Substrate-Stabilized

Oxyanion Hole

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 The work described in this chapter has been reproduced with permission from:

Jacobitz, A. W., Wereszczynski, J., Yi, S. W., Amer, B. R., Huang, G. L., Nguyen, A. V.,

Sawaya, M. R., Jung, M. E., McCammon, J. A., Clubb, R. T. (2014). Structural and

computational studies of the Staphylococcus aureus Sortase B-substrate complex reveal a

substrate-stabilized oxyanion hole. Journal of Biological Chemistry, jbc.M113.509273.

http://doi.org/10.1074/jbc.M113.509273

Copyright 2014

Journal of Biological Chemistry

I was the primary author of this work. I designed experiments, performed biochemical assays,

crystallography, computational model building, and analyzed data.

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4.1 Overview

Sortase cysteine transpeptidases covalently attach proteins to the bacterial cell wall or

assemble fiber-like pili that promote bacterial adhesion. Members of this enzyme superfamily are

widely distributed in Gram-positive bacteria that frequently utilize multiple sortases to elaborate

their peptidoglycan. Sortases catalyze transpeptidation using a conserved active site His-Cys-Arg

triad that joins a sorting signal located at the C terminus of their protein substrate to an amino

nucleophile located on the cell surface. However, despite extensive study, the catalytic

mechanism and molecular basis of substrate recognition remains poorly understood. Here we

report the crystal structure of the Staphylococcus aureus sortase B enzyme in a covalent complex

with an analog of its NPQTN sorting signal substrate, revealing the structural basis through

which it displays the IsdC protein involved in heme-iron scavenging from human hemoglobin.

The results of computational modeling, molecular dynamics simulations, and targeted amino

acid mutagenesis indicate that the backbone amide of Glu224 and the side chain of Arg233 form

an oxyanion hole in sortase B that stabilizes high energy tetrahedral catalytic intermediates.

Surprisingly, a highly conserved threonine residue within the bound sorting signal substrate

facilitates construction of the oxyanion hole by stabilizing the position of the active site arginine

residue via hydrogen bonding. Molecular dynamics simulations and primary sequence

conservation suggest that the sorting signal-stabilized oxyanion hole is a universal feature of

enzymes within the sortase superfamily.

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4.2 Introduction

Surface proteins in bacteria play key roles in the infection process by promoting

microbial adhesion to host tissues, nutrient acquisition, host cell entry, and the suppression of the

immune response. In Gram-positive bacteria, virulence factors are displayed by sortase enzymes,

a superfamily of cysteine transpeptidases that join proteins bearing a cell wall sorting signal to

the cell wall or to other proteins to construct pili (1–6). Sortases have proven to be useful

molecular biology tools to site-specifically attach proteins to a variety of biomolecules and are

considered a potential drug target because they display virulence factors. The clinically

important pathogen, Staphylococcus aureus, displays surface proteins using two sortase

enzymes, sortase A (SrtA)(2) and sortase B (SrtB). srtA− strains of S. aureus are significantly

attenuated in virulence, whereas srtB− strains establish less persistent infections (7–9). SrtA

plays a “housekeeping” role in the cell, covalently mounting a variety of proteins to the cell wall,

whereas SrtB anchors the heme transporter IsdC, a key component of the iron-regulated surface

determinant system that captures heme-iron from hemoglobin (10–12). The mechanism of

catalysis is best understood for SrtA, the archetypal member of the sortase superfamily. Proteins

anchored by SrtA possess a C-terminal cell wall sorting signal that consists of an LPXTG motif

that is processed by the enzyme, followed by a hydrophobic transmembrane segment and a

positively charged C-terminal tail (13). SrtA operates through a ping-pong mechanism that

begins when its active site cysteine residue nucleophilically attacks the carbonyl carbon of the

threonine in the LPXTG motif. This results in a tetrahedral intermediate that, after cleavage of

the threonine-glycine peptide bond, generates a semistable enzyme-substrate thioacyl

intermediate (14, 15). The protein is then transferred by SrtA to the cell wall precursor, lipid II,

when the amino group in this molecule nucleophilically attacks the thioacyl linkage creating a

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second tetrahedral intermediate that collapses to form the covalently linked protein-lipid II

product (16–18). Transglycosylation and transpeptidation reactions that synthesize the cell wall

then incorporate this product into the peptidoglycan. SrtB anchors the IsdC protein to the cell

wall through a similar mechanism. However, unlike SrtA, SrtB recognizes a unique NPQTN

sorting signal, and it attaches IsdC to un-cross-linked peptidoglycan instead of heavily cross-

linked peptidoglycan (19, 20).

Sortase enzymes adopt an α/β sortase fold that contains three proximally positioned

active site residues: His130, Cys223, and Arg233 (SrtB numbering). Although the nucleophilic

role of the cysteine residue is well established, various catalytic functions have been proposed

for the histidine and arginine residues. His130 was originally thought to activate the cysteine by

forming a histidine-cysteine ion pair (21), but more recent data suggest that it instead functions

as a general acid/base (14, 22). Arg233 has been proposed to either stabilize substrate binding

(23–26), function as a general base (27), or directly stabilize the tetrahedral catalytic

intermediates (14, 22, 23). Other residues within the active sites of sortases have also been

proposed to participate in catalysis, including Asp225 in SrtB, which was postulated to

participate in a His130-Cys223-Asp225 catalytic triad (28). The catalytic mechanism has

remained poorly understood because the tetrahedral and acyl intermediates of catalysis are too

short lived to be characterized by either NMR or x-ray crystallography. Several sortase structures

have been determined in the absence of their substrates (28–32) or covalently bound to generic

sulfhydryl modifiers (33). However, only a single structure of a sortase enzyme covalently bound

to its sorting signal substrate has been reported (the NMR structure of SrtA bound to an LPAT

substrate analog) (1, 24). This structure revealed that the active site in this enzyme undergoes

substantial changes in its structure and dynamics that facilitate specific recognition of the sorting

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signal, but it did not provide an atomic level view of the positioning of atoms within the active

site because their coordinates were not well defined in the NMR structure because of resonance

line broadening. Thus, the structural features utilized by sortases to stabilize key tetrahedral and

thioacyl reaction intermediates remain poorly understood.

Here we report the 2.5 Å crystal structure of SrtB covalently bound to an analog of its

NPQTN sorting signal. The structure of the complex closely resembles the thioacyl intermediate

formed during catalysis, laying the groundwork for MD simulations to investigate the catalytic

mechanism. The results of these simulations and in vitro transpeptidation measurements suggest

that Arg233 and the backbone amide of Glu224 form an oxyanion hole that stabilizes high

energy tetrahedral catalytic intermediates. Interestingly, a highly conserved threonine residue

within the sorting signal actively participates in constructing the oxyanion hole by hydrogen

bonding to the active site arginine residue. MD simulations of SrtA, as well as primary sequence

conservation, suggest that all sortases will use a similar substrate-stabilized mechanism to anchor

proteins to the cell wall or to assemble pili.

4.3 Results

4.3.1 Crystal Structure of the SrtB-NPQT* Complex and Computational Modeling of the

Thioacyl Intermediate

Sortase catalyzed transpeptidation reactions occur via covalent enzyme-substrate acyl and

tetrahedral intermediates that are too short-lived to be resolved by NMR or x-ray crystallography

(2, 14, 43). To overcome this problem, we synthesized a Cbz-NPQT* sorting signal analog,

where Cbz is a carbobenzyloxy protecting group, and T* is a threonine derivative that replaces

the carboxyl group with -CH2-SH ((2R,3S)-3-amino-4-mercapto-2-butanol). The peptide contains

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the sorting signal sequence recognized by the S. aureus SrtB sortase and forms a disulfide bond

via its T* moiety to the thiol of Cys223, generating a SrtB-NPQT* complex that structurally

mimics the thioacyl catalytic intermediate (Fig. 1a). A soluble version of SrtB lacking its 30-

amino acid N-terminal membrane anchor (SrtB, residues 31–244) was disulfide-bonded to the

NPQT* substrate analog, and a 2.5 Å crystal structure of the SrtB-NPQT* complex was

determined (Table 1). The data were refined to 2.5 Å resolution in accordance compatible with

CC1/2 statistics (36, 37), although the data extended only to 2.9 Å using more conventional

statistics such as Rmerge and I/σ. In the complex, SrtB adopts an α/β sortase fold containing eight

β-strands that are flanked by five α-helices (Fig. 1b). The positioning of the sorting signal

substrate is well defined, as evidenced by an Fo − Fc omit map of the complex (Fig. 2a). The

solvent-exposed Cbz group at the N terminus of the peptide is only partially modeled in the

structure of the complex because its electron density was not strong enough to fully define its

position. The signal adopts an L-shaped structure and is connected via the sulfhydryl of the T*

moiety to the active site cysteine, Cys223. It is nestled within a narrow groove whose base is

formed by residues within strands β4 and β5 and whose walls are formed by residues projecting

from loops connecting strands β6 to β7 (the β6/β7 loop), β7 to β8 (the β7/β8 loop), and β2 to β3

(the β2/β3 loop) (Fig. 1, b and c). To facilitate a discussion of the molecular basis of substrate

recognition, we henceforth utilize the nomenclature developed by Schechter and Berger (56),

where P and P′ refer to amino acids on the N-terminal and C-terminal sides of the scissile peptide

bond of the sorting signal, respectively. For the NPQTN sorting signal, the N-terminal N is P4, P

is P3, Q is P2, T is P1, and the C-terminal N, occurring after the scissile bond, is P1′. The base of

the NPQT binding pocket is defined by residues Asn92, Tyr128, Tyr181, Ile182, and Ser221,

with the walls defined by residues Leu96, Thr177, Cys223, Glu224, and Arg233 (Fig. 2, b and

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c). The side chain of the disulfide linked T* residue is buried inside of a deep groove where its

methyl group contacts the side chains of Tyr128 and Ile182. This positions the hydroxyl oxygen

on the threonine residue to accept two hydrogen bonds from the side chain of Arg233 in the

active site: a 3.0 Å hydrogen bond to the ϵ-nitrogen atom and a 3.3 Å hydrogen bond to the η-

nitrogen atom in the guanidino group (Fig. 2, b and c). The side chain of the glutamine residue at

position P2 points out of the binding pocket where it is packed against the side chain of Leu96

and donates a hydrogen bond to the backbone carbonyl group of Glu224. Additional enzyme

interactions to the backbone of P2 Gln residue stabilize the positioning of the sorting signal. The

backbone carbonyl group is held in place by a hydrogen bond from the side chain amide nitrogen

of Asn92, and its backbone nitrogen donates a hydrogen bond to a sulfate ion, which in turn is

coordinated by the side chain of His93 and the backbone amide of Asn92. The P3 Pro residue

rests on top of Ile182 and forms a kink that causes the bound peptide to adopt an L-shaped

structure that positions the side chain of the P4 Asn to donate a hydrogen bond to the backbone

carbonyl of Thr177 within the β6/β7 loop. Substrate binding induces only small changes in the

structure of SrtB as the Cα coordinates of the SrtB-NPQT* complex can be superimposed with

the previously determined structure of the unmodified enzyme (28) with a root mean square

deviation of 0.44 Å.

To gain insight into how sortase stabilizes reaction intermediates, an energy-minimized

model of the thioacyl complex was generated by replacing the disulfide link in the structure of

the SrtB-NPQT* complex with a thioacyl bond (Fig. 3). This required only small changes

including the removal of the methylene group in between the Cys223 thiol and the P1 Thr

residue of the sorting signal that decreased their separation by ∼1 Å. The coordinates of the

thioacyl intermediate were then energy-minimized while the restraints on the initial atom

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positions were gradually removed. To verify that the final model contained the proper orientation

of the thioacyl bond, two initial models of the intermediate were energy-minimized, one in which

the carbonyl oxygen pointed toward Arg233 and a second model in which the thioacyl bond was

rotated by 180° (carbonyl oxygen pointing away from Arg233). After minimization, both starting

models converged to nearly identical structures (root mean square deviation = 0.71 Å for all Cα)

that closely resembled the SrtB-NPQT* complex. Importantly, in both refined models, the

thioacyl linkage adopts a similar conformation in which the P1 threonine residue in the signal is

poised to accept hydrogen bonds from the active site Arg233 residue and the backbone amide

from Glu224 (Fig. 3). This key sorting signal-enzyme interaction may hold the active site

Arg233 side chain in a catalytically competent conformation that also stabilizes higher energy

tetrahedral reaction intermediates that form during catalysis (see “Discussion”).

4.3.2 Identification of Enzyme-Substrate Interactions Required for Catalysis

The catalytic importance of active site and sorting signal amino acids was investigated in

vitro using SNKDKVENPQTNAGT (sorting signal in bold) and GGGGG peptides that mimic

the sorting signal and secondary lipid II substrates, respectively. HPLC separation of the reaction

products indicates that SrtB used for crystallographic studies is fully functional in vitro (kcat and

Km values for SrtB of 1.0 × 10−4 s−1 and 1.8 mM, respectively) (Fig. 4a) (57). Based on sequence

conservation, residues His130, Cys223, and Arg233 in SrtB have been postulated to form a triad

that mediates catalysis. In addition, it has been proposed that the side chain of Asp225,

positioned near the active site, may also play a critical role in catalysis by stabilizing and

activating His130 (28). To investigate the relative importance of these residues, we purified four

single amino acid mutants of SrtB and assayed them for their ability to catalyze transpeptidation.

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H130A, C223A, and R233A mutant enzymes had no detectable activity after 24 h, whereas

D225A exhibited nonspecific proteolytic activity (Fig. 4b). This suggests that unlike the

conserved active site residues (His130, Cys223, and Arg233), Asp225 is not required for early

steps in transpeptidation that involve the formation of the first thioacyl intermediate. The D225A

mutation may disrupt the active site architecture of SrtB, allowing recognition of various

sequences as primary substrates, but it does not appear to play a direct role in the catalytic

mechanism. Interestingly, similar promiscuous activity has been observed in several SrtA

mutants (57, 58), as well as the wild-type SrtB enzyme from Listeria monocytogenes (59).

The importance of enzyme-substrate interactions visualized in the structure of the

complex was also tested using the HPLC assay. Sorting signal peptides containing alanine

substitutions at sites P1 (NPQAN), P3 (NAQTN), and P4 (APQTN) were unreactive (Fig. 4c),

compatible with extensive enzyme contacts to the side chains of these residues in the structure of

the complex. In contrast, peptides containing an alanine substitution at either site P2 (NPATN) or

P1′ (NPQTA) could be processed by the enzyme to yield transpeptidation products, although

they were less reactive than the native signal. The ability of SrtB to process the P2 mutant

peptide (NPATN) is compatible with the structure of the complex as the side chain of residue P2

projects out of the binding pocket toward the β7/β8 loop. The SrtA enzyme also processes

signals containing a range of amino acids at site P2 in its LPXTG sorting signal (60), suggesting

that this promiscuity is evolutionarily conserved.

The structure of the SrtB-NPQT* complex and computational model of the thioacyl

intermediate reveal that the threonine side chain within the sorting signal likely plays a key role

in catalysis by stabilizing the positioning of Arg233 through hydrogen bonding (Figs. 2b and 3).

To determine the importance of this interaction in catalysis, we tested how efficiently NPQSN

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and NPQVN peptides were used as transpeptidation substrates. These peptides are identical to

the native sorting signal peptide but contain threonine to serine and threonine to valine mutations

at the P1 position of the signal, respectively. The threonine to serine substitution preserves the

hydroxyl group that hydrogen bonds to Arg233 but removes the methyl group of threonine that

interacts with the side chains of Tyr128 and Ile182. In contrast, introduction of a valine

substitution eliminates the hydroxyl group but does not significantly alter the size or shape of the

P1 residue. Both peptides were unreactive, suggesting that each type of substrate-enzyme

interaction is important for catalysis (Fig. 4c).

4.3.3 Molecular Dynamics Simulations of SrtA and SrtB Reveal a Conserved Mechanism

through which the Substrate Stabilizes the Positioning of the Active Site Arginine

Residue

The atomic structures of only two sortases covalently bound to their substrates have been

determined: the NMR structure of SrtA bound to an LPAT* peptide (24) and the structure of the

SrtB complex reported here (Fig. 5). A comparison reveals a generally similar mode of binding

in which the signals adopt an L-shaped structure enabling extensive enzyme contacts to the side

chains of residues located at positions P4 and P3 (Leu-Pro and Asn-Pro in the SrtA and SrtB

substrates, respectively). Interestingly, although both bound sorting signals contain a conserved

threonine residue at the P1 position, the side chain of this amino acid is oriented differently in the

structures of the SrtA-LPAT* and SrtB-NPQT* complexes. As described above, in SrtB the side

chain of the P1 Thr residue faces “in” and interacts with the active site arginine, and the side

chain of the P2 Gln residue projects “out” toward the β7/β8 loop. In contrast, in the SrtA-LPAT*

structure the P1 Thr points out toward the solvent and the P2 Ala residue points in toward the

129
bottom of the binding pocket. This conformational difference occurs because the P1 and P2

residues in each signal have distinct backbone torsional angles; the P2 ϕ and ψ angles are 51 and

98° for SrtA-LPAT*, and −75 and 127° for SrtB-NPQT*, respectively, and the P1 T* pseudo ϕ

and ψ angles are −71 and −17° for SrtA-LPAT* and −119 and 25° for SrtB-NPQT*,

respectively. This key structural difference is not caused by a lack of coordinate precision in the

NMR structure of the SrtA-LPAT* complex because several NOEs define the positioning of the

side chains of the P1 and P2 residues (see Fig. 1c in Ref. 24).

It is possible that the sorting signals bound to SrtA and SrtB are flexible and thus capable

of undergoing motions in which the P1 Thr side chain moves into, and out of, the active site. To

investigate this issue, we performed MD simulations of both complexes using the method of

umbrella sampling with Hamiltonian replica exchange (54). The free energy profile for

transitions between the Thr-in (SrtB-like) and Thr-out (SrtA-like) states for the bound signals in

each complex was then calculated. MD calculations using the coordinates of the SrtA-peptide

complex indicate that the peptide can transition from the Thr-out conformation observed in the

NMR structure of the SrtA-LPAT* complex (Fig. 6a, middle panel) to a Thr-in orientation

observed in the structure of the SrtB-NPQT complex (Fig. 6a, left panel). To investigate the

energetics associated with this transition, we calculated the free energies of intermediate

structures on this pathway, which are represented by a two-dimensional coordinate system (Fig.

6b). The first coordinate (x axis) reports on the positioning of the P1 and P2 residues, and the

second reaction coordinate (y axis) reports on the positioning of the remainder of the sorting

signal relative to the enzyme. The former is defined as a collective coordinate that describes the

structure of residues P1 and P2 relative to the catalytic cysteine, and the latter is defined as the

radius of gyration of residues P3 and P4 with select atoms in the β sheet of the sortase molecule

130
(described further under “Experimental Procedures”). In the free energy profile for the covalent

SrtA-LPAT complex, there are three dominant energy wells all with minima within 0.4 kcal/mol

of one another. The region we refer to as well 1 corresponds to a sorting signal structure similar

to that observed in the SrtB-NPQT* complex in which the sorting signal contacts the arginine

(Thr-in) (Fig. 6a, left panel), whereas well 2 conformers resemble the NMR structure of the

SrtA-LPAT* complex in which the P1 threonine side chain points away from the active site

(Thr-out) (Fig. 6a, middle panel). Interestingly, this analysis reveals that a low energy pathway

exists between these two states (Fig. 6b), suggesting that the P1 Thr and P2 Ala residues can

alter their conformation within the active site of SrtA. This structural transition is documented in

supplemental Video S1, which shows select snapshots from the MD trajectory in which the P2

Thr transitions from the Thr-out to Thr-in state where it engages the active site arginine residue.

Interestingly, the Thr-out to Thr-in transition can also occur through a third low energy

intermediate (well 3) in which the hydrophobic residues P3 and P4 do not contact SrtA but are

instead exposed to solution. This entropically stabilized state is presumably not significantly

populated in vivo when the enzyme contacts larger protein substrates that contain a full cell wall

sorting signal.

In contrast to SrtA, MD simulations of the SrtB-NPQT complex reveal only a single,

narrow free energy minimum in which the threonine remains projected into the active site (Thr-

in) where it contacts Arg233 (Fig. 6, a, right panel, and c). This indicates that the Thr-out

conformer observed in SrtA is disfavored in SrtB. The larger size of the P2 residue in the SrtB

sorting signal could, in principle, cause this difference. In the SrtA-LPAT complex, the P2 Ala

residue adopts a positive ϕ angle, and the side chain projects toward the base of the binding

pocket, whereas the larger Gln P2 residue in the SrtB bound peptide adopts a negative ϕ angle

131
and rests on the surface of the enzyme. Because reduced steric stress enables amino acids with

smaller side chains to more readily adopt positive ϕ angles, it is possible that the smaller size of

the alanine side chain in the LPAT signal facilitates formation of the Thr-out state. To test this

hypothesis, a third free energy profile was computed for SrtB bound to NPAT, which changes its

P2 residue to alanine (Fig. 6d). This change expanded the range of conformers accessible to the

peptide bound to SrtB (Fig. 6, compare c and d), but it did not encourage sampling of the Thr-out

state observed in SrtA. This suggests that features of the SrtB-peptide complex other than the

identity of its P2 residue are important for dictating how the P1 Thr residue is positioned

(described below). In sum, our MD simulations indicate that sorting signals bound to both SrtA

and SrtB can adopt conformations in which the P1 Thr side chain is located within the active site

for stabilizing interactions with the active site arginine. In SrtB this is the predominant state of

the signal, whereas in SrtA, it is one of two possible low energy binding conformations.

4.4 Discussion

Members of the sortase superfamily catalyze transpeptidation reactions that covalently

attach proteins to the bacterial cell wall or assemble pili (1–3). At present, over a thousand

sortase enzymes have been identified that, based on their primary sequences and functions, can

be partitioned into at least six distinct families (called class A to F enzymes) (1). The structure of

the SrtB-NPQT* complex reveals how class B sortases recognize their substrates. In bacterial

pathogens, class B enzymes typically anchor heme-binding proteins to the cell wall that enable

the bacterium to utilize host derived heme-iron as a nutrient (1, 10–12). Our results indicate that

SrtB recognizes its NPQTN sorting signal via a large groove adjacent to the active site formed by

residues in the β6/β7 loop and residues within strands β4 and β7. Structurally, class B enzymes

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are distinguished from other members of the sortase superfamily by the presence of a large β6/β7

loop that contains a long α-helix. In the complex, this loop plays a major role in signal

recognition because it contacts the Asn (P4), Pro (P3), and Thr (P1) residues of the peptide,

which are highly conserved in sorting signals recognized by class B enzymes. These substrate-

enzyme contacts are important for catalysis because alanine substitutions at these sites in the

sorting signal disrupt transpeptidation (Fig. 4c). As in the prototypical SrtA enzyme, site P2 in

the SrtB sorting signal is tolerant to alanine substitution, which is consistent with the positioning

of the glutamine residue at this site, which rests on the surface of the enzyme. Although not

visualized in our structure, the P1′ residue following the scissile peptide bond is also tolerant to

alanine mutation (Fig. 4c), distinguishing SrtB from the prototypical class A SrtA enzyme, which

only recognizes glycine at the P1′ position (61). Given that the signal sequences and known

structures of class B enzymes are highly similar, it is likely that they all recognize their

substrates in a fashion similar to what is seen in the SrtB-NPQT* complex. Interestingly, our

results indicate that SrtA and SrtB enzymes recognize their cognate sorting signals in a generally

similar manner, despite the fact that the enzymes share only 26% sequence identity and that they

recognize distinct LPXTG and NPQTN sorting signals, respectively. A comparison of the SrtB-

NPQT* and previously reported NMR structure of the SrtA-LPAT* complex reveals that the

bound sorting signals both adopt an L-shaped conformation in which the proline residue at

position P3 redirects the polypeptide to position the side chain of the P4 residue so that it

contacts the β6/β7 loop. This general mode of binding is likely a conserved feature of substrate

recognition by sortase enzymes as an inspection of sorting signals predicted to be processed by

these enzymes shows that over 90% of them contain a proline at P3 (60).

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 Computational modeling of the thioacyl enzyme-substrate reaction intermediate suggests

that SrtB facilitates catalysis by forming a substrate-stabilized oxyanion hole. In both the

experimental and energy-minimized model of this reaction intermediate, the conserved active

site arginine (Arg233) is held in place near the scissile bond by donating a hydrogen bond from

its ϵ-nitrogen to the hydroxyl group of the threonine located at the P1 position in the sorting

signal (Figs. 2b and 3). In the model of the thioacyl complex, this interaction positions the

arginine so that its guanidino group and the backbone amide of Glu224 donate hydrogen bonds

to the oxygen atom in the thioacyl bond. During catalysis, two oxyanionic transition states form;

the first precedes the generation of the thioacyl intermediate emulated by the SrtB-NPQT*

structure, and the second occurs after nucleophilic attack of the thioacyl bond by the amino

group present in lipid II. It seems likely that Arg233 and Glu224 form an oxyanion hole that

stabilizes these high energy reaction intermediates because only small changes in the positioning

of the oxygen atom within the thioacyl bond are expected to occur when the carbonyl carbon

transitions from its planar sp2 configuration to its tetrahedral sp3 state. This is distinct from the

oxyanion hole discovered in penicillin binding proteins that perform a similar transpeptidation

reaction but use two backbone amides to stabilize the tetrahedral intermediate (62). The

threonine residue in the sorting signal appears to play a significant role in stabilizing this

oxyanion hole because even conservative mutations to either serine or valine disrupt

transpeptidation (Fig. 4c). Its catalytic role is also substantiated by the high degree of sequence

conservation at site P1 in all known SrtB substrates. The putative oxyanion hole reported here is

compatible with results obtained by McCafferty and co-workers (22, 23), who demonstrated that

the analogous arginine in the SrtA enzyme is intolerant to mutation, except when substituted

with citrulline, an arginine isostere that lacks a formal positive charge.

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 MD simulations indicate that the SrtA enzyme can also form a substrate stabilized

oxyanion hole that could facilitate catalysis. In the structures of the SrtB-NPQT* and SrtA-

LPAT* complexes, the bound sorting signals adopt similar L-shaped conformations. However,

the positioning of the P1 Thr residue in each sorting signal differs substantially; in SrtB-NPQT*,

the threonine side chain interacts with the active site arginine residue (Thr-in conformation)

(described above), whereas in the SrtA complex, the analogous threonine residue projects away

from the active site (Thr-out conformation). Because the threonine in the SrtA complex is not

positioned to form stabilizing interactions with the active site arginine residue, we wondered

whether the Thr-in conformer observed in the SrtB complex could also be sampled by the bound

SrtA sorting signal to stabilize its tetrahedral reaction intermediates. To investigate this issue,

MD simulations of the SrtA thioacyl complex were performed, revealing that the P1 threonine

residue can transition from the Thr-out to the Thr-in state presumably needed to construct the

oxyanion hole (supplemental Video S1 and Fig. 6). Conformations of the SrtA-LPAT complex in

which the threonine side chain of the sorting signal forms interactions with the active site

arginine were obtained without major rearrangement of the structure of the enzyme.

Interestingly, a previous MD study based on the SrtA-LPAT* NMR structure reports that the

active site arginine residue functions only to position the sorting signal substrate by hydrogen

bonding to its backbone carbonyl atoms at positions P2 and P4 (25). Our work is compatible

with this conclusion but suggests that this stabilizing interaction will only occur when the P1 Thr

samples the out position that is presumably not catalytically active. Thus, based on primary

sequence conservation and the demonstrated importance of the P1 Thr in catalysis in both SrtA

and SrtB, we conclude that the Thr-in conformation observed in the SrtB structure, and

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accessible to SrtA, represents a catalytically competent form of the peptide that is essential for

stabilizing the tetrahedral transition state.

The active site of SrtB appears to be more conformationally restrictive than SrtA because

MD simulations of the SrtB-NPQT thioacyl complex reveal that only the Thr-in conformer is

accessible to the bound peptide. This is compatible with structural and NMR relaxation data,

which have shown that the active site of SrtA contains a flexible β6/β7 loop that becomes

ordered upon substrate binding (24, 63), whereas SrtB contains a preformed, rigid binding pocket

for its sorting signal substrate. Interestingly, inspection of the MD data suggests that SrtA can

form unique contacts to the sorting signal that may stabilize the Thr-out conformer. In the NMR

structure of the SrtA-LPAT* structure, the conserved active site His101 residue is positioned to

form a 3.1 Å hydrogen bond from its ϵ-nitrogen to the P1 Thr hydroxyl. In contrast, although the

SrtB active site histidine is about the same distance from the active site cysteine as the analogous

residues in SrtA (∼5.2 Å from His δ-N to Cys S), this potential stabilizing interaction for the

Thr-out conformer is obstructed by the side chain of Leu96, which is inserted between His130

and Cys223 in SrtB. Thus, the more restrictive active site of SrtB and the lack of stabilizing

interactions may prevent the signal bound to SrtB from adopting the catalytically nonproductive

Thr-out conformer. It is unlikely that the ability of the SrtA peptide to sample the less reactive

Thr-out state impacts the kinetics of transpeptidation because the half-life of the long-lived

thioacyl intermediate presumably far exceeds the time needed for the threonine residue to

transition between its Thr-in and Thr-out conformers.

It seems likely that nearly all members of the sortase superfamily will employ a substrate

stabilized oxyanion hole to anchor proteins to the cell wall or to assemble pili. Based on

structural and mutagenesis data, the transpeptidation reaction will be initiated when the sorting

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signal of the partially secreted protein substrate binds to the groove on sortase formed by

residues in the β6/β7 loop and residues within strands β4 and β7. Most sorting signals can be

expected to adopt an L-shaped structure when bound to the enzyme because they contain a

proline residue at position P3 (∼90% of predicted sorting signals) (60). For catalysis to proceed,

the sortase must contain a properly charged active site in which the cysteine and histidine

residues are in their thiolate and imidazolium ionization states, respectively. In isolation, only a

small fraction of the enzyme may be properly ionized based on pKa measurements of the SrtA

enzymes from S. aureus and Bacillus anthracis (14, 29, 64). If properly ionized, the cysteine

thiolate attacks the carbonyl carbon of the P1 residue, forming the first tetrahedral intermediate.

Our results suggest that the oxyanion in this intermediate is stabilized by hydrogen bonding from

the active site arginine residue and a backbone amide group located in the β7/β8 loop (in SrtB

Arg233 and the backbone amide of Glu224) (Fig. 7). The threonine residue within the sorting

signal, at position P1, plays a key role in constructing the oxyanion hole by stabilizing the

positioning of the arginine side chain via hydrogen bonding. This oxyanion hole is presumably

used by most sortase enzymes to facilitate catalysis because they all contain conserved active site

arginine residues, and ∼95% of their predicted sorting signal substrates contain a threonine at the

P1 position (60). Breakage of the scissile bond is then facilitated by protonation of the amide

group by His130, resulting in a semistable thioacyl intermediate. It remains unclear where the

amino nucleophile on lipid II enters the active site in SrtB. A crystal structure of SrtB

noncovalently bound to a triglycine peptide, meant to mimic the lipid II substrate, localized the

binding site for the peptide to the β7/β8 loop (33). However, the specificity of this interaction is

suspect because the peptide is expected to bind weakly (Km for GGGGG binding is 140 μM (43))

and because the complex was not co-crystallized (the peptide was soaked into the crystal). It is

137
also important to note that in this and all crystal structures of SrtB solved to date, side chains

from residues Asp225–Tyr227 in the β7/β8 loop are involved in crystal lattice contacts to

symmetry related molecules in the crystal. Although it is possible that these contacts simply

reinforce the existing, predominant conformation of this loop, it is also possible that they have

captured one of many possible conformations that could be involved in mediating substrate

access to the active site. Alternatively, as originally proposed by Joachimiak and co-workers

(28), the pentaglycine peptide in lipid II may enter the active site via a groove located between

the β7/β8 and β2/β3 loops. This is compatible with NMR chemical shift perturbation studies of

SrtA (24), high resolution crystal structures of other sortase enzymes, which also contain a

similarly positioned groove (30, 32), and the presence of the highly conserved histidine residue

in this groove that has been proposed to function as a general base that deprotonates lipid II. Our

model of the thioacyl intermediate does not rule out either of these entry points. However, it is

most compatible with lipid II entering via the groove between the β7/β8 and β2/β3 loops because

from this direction, attack of the carbonyl carbon by the amino nucleophile will generate a

tetrahedral intermediate whose negative charge is positioned to be stabilized by the oxyanion

hole formed by Arg233. The transpeptidation reaction would then be completed by the collapse

of the second tetrahedral intermediate into the final, covalently linked, protein-lipid II product.

Additional hybrid quantum mechanics/molecular mechanics simulations are currently underway

to quantitatively investigate the role of the oxyanion hole in catalysis. Beyond providing

fundamental insight into the process of protein display and pilin assembly in bacteria, the new

mechanistic insights reported in this paper could guide the rational design of therapeutically

useful transition state analog inhibitors of sortases and facilitate protein engineering efforts to

expand the utility of sortases as biochemical reagents.

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4.5 Experimental Procedures

4.5.1 Production, Crystallization, and Structure Determination of SrtB-NPQT* Complex

DNA encoding SrtB (residues 31–244) was amplified by PCR from S. aureus genomic

DNA, cloned into a pE-SUMO vector (LifeSensors) and transformed into Escherichia coli

Rosetta (DE3) pLysS cells (Novagen). Protein expression was induced by addition of 1 mM

isopropyl β-D-1-thiogalactopyranoside and allowed to continue for 16 h at 16 °C. Protein was

purified by affinity purification using HisPur cobalt resin (Thermo) per the manufacturer's

instructions. The His6-SUMO tag was then cleaved by incubating the protein overnight at 4 °C

with recombinant ULP1 protease and removed by reapplying the protein mixture to the HisPur

cobalt resin. Cbz-NPQT* (where T* is (2R,3S)-3-amino-4-mercapto-2-butanol, and Cbz is a

carbobenzyloxy protecting group) was synthesized as in Ref. 34 and added to purified SrtB in

modification buffer (10 mM Tris-HCl, pH 7.0, 20 mM NaCl, 1 M L-proline) at a ratio of 10:1 for

a final concentration of 1 mM Cbz-NPQT* to 100 μM SrtB. The reaction was first reduced with

1 mM DTT for 4 h, then oxidized by addition of 10 mM CuCl2, and allowed to rock gently at

room temperature for 7 days. Production of stable complex was confirmed by MALDI mass

spectrometry.

Crystals of the SrtB-NPQT* complex were produced from a stock of 150 μM SrtB-

NPQT* in 10 mM Tris-HCl, pH 7.0, 20 mM NaCl. Crystals were grown using the hanging drop,

vapor diffusion method in 2.8 M ammonium sulfate, 70 mM sodium citrate, pH 5.0. Data were

collected on Beamline 24-ID-C at 100 K at the Advanced Photon Source (λ = 0.964 Å). Three

data sets were scaled, integrated, and merged using XDS and XSCALE (35). Using conventional

criteria, the resolution boundary for the data set might have been drawn at 2.9 Å given that I/σ in

139
this shell (2.98–2.90 Å) is 2.0, and Rmerge is 72%, with a completeness of 95% and a multiplicity

of 5.2. However, recent studies from Karplus and Diederichs (36) have indicated that the CC1/2

statistic has superior properties as an indicator of data precision compared with Rmerge. Moreover,

in their study, and in the following work (37), the authors show that high resolution data

typically discarded because of high Rmerge values (i.e., over the conventionally acceptable

threshold value of ∼60–80%), and low I/σ values (i.e., under the conventionally acceptable

threshold value of 2) actually contain information that can improve the quality of the model if

used in refinement. Given these results, we thought our model would improve if we used the

more generous resolution cutoff (2.5 Å) indicated by the CC1/2 statistic (50.4% in the 2.5 Å

shell), rather than a conventionally accepted limit (2.9 Å) indicated by the Rmerge and I/σ

statistics. Thus, data extending to 2.5 Å resolution were used for the refinement process,

although by conventional standards, the structure should be considered to be resolved at 2.9 Å

resolution.

Phases were determined by molecular replacement using the unmodified SrtB structure

(28) (Protein Data Bank code 1NG5) as a search model in the program Phaser (38). The NPQT*

modifier was modeled into positive density using COOT (39, 40), and the model was prepared

through successive iterations of manual adjustment in COOT and refinement in BUSTER (41,

42).

4.5.2 Transpeptidation Assay

Active site mutants were produced using the QuikChange site-directed mutagenesis kit

(Stratagene) as per the manufacturer's instructions, confirmed by DNA sequencing, and

expressed and purified as described for the wild-type protein. In vitro transpeptidation reactions

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were performed based on the method developed by Kruger et al. (43). 100 μM SrtB (wild-type or

mutant) was incubated with 2 mM GGGGG and 200 μM peptide substrate in 100 μl of assay

buffer (300 mM Tris-HCl and 150 mM NaCl) at 37 °C for 24 h. The reactions were quenched by

adding 50 μl of 1 M HCl and injected onto a Waters XSelect HSS C18 reversed phase HPLC

column. Peptides were eluted by applying a gradient from 3 to 23% acetonitrile (in 0.1%

trifluoroacetic acid) over 25 min at a flow rate of 1 ml/min. Elution of the peptides was

monitored by absorbance at 215 nm. Peak fractions were collected, and their identities were

confirmed by MALDI-TOF mass spectrometry.

4.5.3 Computational Modeling and Molecular Dynamics

Molecular dynamics simulations were performed with NAMD (44), using the

AMBER99SB-ILDN force field (45), a 2-fs time step, and the SHAKE algorithm to constrain all

hydrogen containing bonds (46). Nonbonded interactions were truncated at 10 Å, with the use of

a smoothing function beginning at 9 Å, and long range electrostatics were handled with the

particle mesh Ewald method using a maximum grid spacing of 1 Å and a cubic B spline (47).

Parameters for the Cys-Thr linkage were generated with GAFF (48, 49), with the charges derived

from a RESP fit (48). Constant temperature was maintained through the use of Langevin

dynamics with a damping coefficient of 2 ps−1, whereas the barostat was controlled through a

Nosè-Hoover method with a target pressure of 1 atm, a piston period of 100 fs, and a damping

time of 50 fs (50, 51).

Models of the thioacyl intermediate were originally constructed from the SrtB-NPQT*

structure by replacing the disulfide bond with a thioester in PyMOL (52). The models were

solvated in a periodic water box with a solvent distance of 10 Å and parameterized in tLeap (53).

141
Models were then energy-minimized and equilibrated in NAMD (44) by slowly removing

restraints from the initial atom positions over 1 ns with 2-fs steps. For simulations of SrtA, the

NMR structure 2KID was utilized (24).

Potential of mean force calculations were performed using two-dimensional replica-

exchange umbrella sampling calculations (54). For the first dimension (the x coordinate in Fig. 6,

b–d), a vector was defined based on the difference in positions between residues in the SrtA

structure 2KID and the SrtB structure presented here for the heavy atoms in the backbones of

residues P1, P2, the catalytic cysteine, and the three residues upstream of it in the sortase

molecule, along with the heavy atoms in the backbone of residues P1 and the catalytic cysteine.

The second coordinate (the y axis in Fig. 6, b–d) was defined as the radius of gyration for the C

atoms in residues 98–100 and 142–144 in SrtA (or residues 171–173 and 235–237 in SrtB) along

with the heavy atoms of residues P4 and P3. Restraints for umbrella sampling were evenly

spaced every 10 Å from −100 to 100 Å in the first coordinate and every 0.5 Å from 5 to 14 Å in

the second coordinate. This created a total of 399 simulation “windows,” each of which were

simulated for 10 ns. Positions were exchanged between adjacent windows every 1 ps based upon

a Metropolis criteria with a temperature of 300 K. The weighted histogram analysis method was

used for computing the potential of mean force based upon the umbrella sampling calculations

(55). Analysis of subsamples from these simulations indicate that the overall free energy profiles

require on the order of 5 ns to equilibrate; thus the first 5 ns of each window is discarded in the

weighted histogram analysis method analysis presented here.

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4.6 Figures

Figure 4.1 Structure of the SrtB-NPQT* complex.

(a) a comparison of the chemical bonds that join the peptide substrate to the SrtB enzyme in the

SrtB-NPQT* complex (left panel) and the SrtB-NPQT thioacyl catalytic intermediate (right

panel). Atoms from SrtB are colored red. (b) ribbon diagram of the SrtB-NPQT* complex.

Green, β2/ β3 loop; orange, β6/β7 loop; red, β7/β8 loop. Active site residues and substrate

analog are shown as sticks. (c) surface representation of SrtB in the complex utilizing the same

color scheme as in (b). Active site residue Arg233 is highlighted in blue, and Cys223 is in

yellow.

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Table 4.1 Crystallographic data collection and refinement statistics

Parameters SrtB-NPQT*
Data Collection
Space Group P21
Cell Dimensions
a, b, c (Å) 102.6, 59.12, 72.49
Resolution (Å) 72.49-2.49 (2.58-
2.49)
Rmerge 26.6 (212.4)
I/σ(I) 3.8 (0.7)
Completeness (%) 92.2 (94.2)
Multiplicity 5.1 (5.0)
CC1/2 98.6 (50.4)a
CC* 99.6 (81.8) a

Refinement
Resolution (Å) 72.49-2.49
No. Reflections 28147
Rwork/Rfree 21.5/27.1
CCwork 94.4 (76.4) a
CCfree 87.7 (75.5) a
No. atoms
Protein 7248
Ligand/ion 176
Water 27
B factors
Mean 72.5
Wilson 56.2
Protein 72.3
Ligand/ion 87.6
Water 29.9
RMSD
Bond lengths (Å) 0.010
Bond angles (˚) 1.26
Values in parentheses are for highest-resolution shell.
a
Values reported as a percentage (%)

144
Figure 4.2 Structure and interactions of the NPQT* modifier in complex with SrtB.

(a) Fo − Fc map contoured at 3 σ. Electron density (gray mesh) was generated by removing the

NPQT* peptide from the final model and repeating refinement. The map shown is an average of

145
the density from all four chains in the asymmetric unit. The peptide extends from the active site

cysteine. (b) stereo view of the NPQT* peptide (gray sticks) and interacting residues (blue

sticks). Hydrogen bonds are indicated by dashed green lines. (c) diagram of the interactions

between SrtB (blue) and the NPQT* peptide (gray). Hydrogen bonds are indicated by dashed

green lines. SrtB residues that make only hydrophobic contacts are depicted as blue circles

positioned near their most significant point of contact to the peptide.

146
Figure 4.3 Expanded view of the active site in the energy-minimized model of the SrtB-

NPQT thioacyl intermediate.

Interactions between SrtB and the threonine residue in the sorting signal are shown. The thioacyl

carbonyl oxygen atom is positioned to accept hydrogen bonds from the η-nitrogen atom of

Arg233 and the backbone nitrogen atom of Glu224. The side chain of Arg233 is held in position

by a hydrogen bond between its ϵ-nitrogen atom and the hydroxyl group on the Thr residue

located at the P1 position of the sorting signal.

147
Figure 4.4 Transpeptidation activity of wild-type and mutant SrtB.

(a) representative HPLC chromatograms showing the reaction products that are produced when

SrtB was incubated with SNKDKVENPQTNAGT (sorting signal in bold type) and GGGGG

peptides that mimic its sorting signal and secondary lipid II substrates, respectively. Reactions

performed in the presence (left panel) and absence (right panel) of SrtB are shown. Only when

SrtB is present (left panel) is the appropriate transpeptidation peptide product produced

SNKDKVENPQTGGGGG). (b) transpeptidation activity of SrtB mutants. The indicated SrtB

mutant was incubated with 200 μM peptide containing an NPQTN sorting signal and

pentaglycine and monitored by HPLC as described above. The dark shaded bars indicate the

amount of full-length peptide remaining after reaction with the enzyme. The lightly shaded bar

indicates the amount of transpeptidation product that was formed. An asterisk indicates that no

transpeptidation product could be detected after 24 h. The error bars represent the standard

148
deviation of three reactions. (c) transpeptidation activity of sorting signal amino acid mutants.

Sorting signal peptides containing select alanine substitutions were assayed for their ability to be

utilized by SrtB as a substrate and monitored by HPLC as described above. The amount of

transpeptidation product formed for each mutant peptide is expressed as a percentage of the

amount formed from reaction of SrtB with the native NPQTN sorting signal.

149
Figure 4.5 Alignment of SrtB-NPQT* and SrtA-LPAT* (24).
SrtB is shown as blue ribbons, and SrtA is shown as green ribbons.

150
Figure 4.6 Structures and free energy profiles of the SrtA and SrtB thioacyl complexes.

(a) selected structures obtained from MD simulations of the SrtA and SrtB thioacyl complexes.

The left and middle panels show structures of the SrtA thioacyl complex in which the threonine

side chain in the sorting signal either interacts with the active site arginine (left panel, SrtA-

LPAT Thr-“In”) or projects away from the active site (middle panel, SrtA-LPAT Thr-“Out”).

The right panel shows the structure of the SrtB-NPQT thioacyl complex in which the threonine

side chain interacts with the arginine residue (Thr-in). The structures are displayed with the SrtA

and SrtB surfaces colored green and blue, respectively. Surface representations of labeled active

site residues were calculated independently of the remaining protein surface, which allows for

the visualization of SrtB His130 (cyan) behind Leu96 (dark gray) in the right panel, even though

this residue would not normally be considered solvent-accessible in this conformation. (b–d) free

151
energies were calculated from positions sampled during a Hamiltonian replica exchange

simulation for the SrtA-LPAT (b), SrtB-NPQT (c), and SrtB-NPAT (d) thioacyl complexes. The

x axis records the position of P1 and P2 residues in the bound sorting signal relative to the active

site cysteine residue. The y axis describes the positioning of residues P3 and P4 in the sorting

signal relative to the body of the protein.

152
Figure 4.7 The SrtB transpeptidation mechanism showing how the sorting signal may

stabilize the oxyanion hole.

(a) the SrtB active site with His130 in its imidazolium form and Cys223 in its thiolate form. (b)

the NPQTN substrate binds with its P1 Thr residue in the in position within the active site. The

Cys223 thiolate performs a nucleophilic attack on the P1 Thr carbonyl carbon. (c) the first

tetrahedral intermediate is formed. The P1 Thr residue interacts with Arg233 to construct a

substrate-stabilized oxyanion hole in which the side chain of Arg233 and the backbone amide of

Glu224 hydrogen bond to the oxyanion. The His130 imidazolium group donates a proton to the

leaving group to complete breakage of scissile bond. (d) the P1 Thr residue maintains hydrogen

bonds with Arg233 to stabilize its interaction with the thioacyl intermediate. (e) the incoming

GGGGG peptide from lipid II acts as a nucleophile that attacks the carbonyl carbon of the

thioacyl bond. (f) the second tetrahedral intermediate is formed and is again stabilized by the

substrate-stabilized oxyanion hole. This intermediate then collapses, releasing the

NPQTGGGGG transpeptidation product and returning the enzyme to its active form (a).

153
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surface proteins to the cell wall of Staphylococcus aureus. III. Lipid II is an in vivo

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17. Ruzin, A., Severin, A., Ritacco, F., Tabei, K., Singh, G., Bradford, P. A., Siegel, M. M.,

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18. Schneewind, O., Fowler, A., and Faull, K. F. (1995) Structure of the cell wall anchor of

surface proteins in Staphylococcus aureus. Science 268, 103–106

19. Marraffini, L. A., and Schneewind, O. (2005) Anchor Structure of Staphylococcal

Surface Proteins V. ANCHOR STRUCTURE OF THE SORTASE B SUBSTRATE

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21. Ton-That, H., Mazmanian, S. K., Alksne, L., and Schneewind, O. (2002) Anchoring of

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23. Bentley, M. L., Lamb, E. C., and McCafferty, D. G. (2008) Mutagenesis Studies of

Substrate Recognition and Catalysis in the Sortase A Transpeptidase from

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24. Suree, N., Liew, C. K., Villareal, V. A., Thieu, W., Fadeev, E. A., Clemens, J. J., Jung,

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25. Tian, B.-X., and Eriksson, L. A. (2011) Catalytic Mechanism and Roles of Arg197 and

Thr183 in the Staphylococcus aureus Sortase A Enzyme. J. Phys. Chem. B 115, 13003–

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E., and Clubb, R. T. (2004) Localization and mutagenesis of the sorting signal binding

site on sortase A from Staphylococcus aureus. FEBS Lett. 571, 221–226

27. Marraffini, L. A., Ton-That, H., Zong, Y., Narayana, S. V. L., and Schneewind, O.

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Schneewind, O., and Joachimiak, A. (2004) Structures of Sortase B from Staphylococcus

aureus and Bacillus anthracis Reveal Catalytic Amino Acid Triad in the Active Site.

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29. Weiner, E. M., Robson, S., Marohn, M., and Clubb, R. T. (2010) The Sortase A enzyme

that attaches proteins to the cell wall of Bacillus anthracis contains an unusual active site

architecture. J. Biol. Chem. 285, 23433–23443

30. Race, P. R., Bentley, M. L., Melvin, J. A., Crow, A., Hughes, R. K., Smith, W. D.,

Sessions, R. B., Kehoe, M. A., McCafferty, D. G., and Banfield, M. J. (2009) Crystal

Structure of Streptococcus pyogenes Sortase A. J. Biol. Chem. 284, 6924–6933

31. Ilangovan, U., Ton-That, H., Iwahara, J., Schneewind, O., and Clubb, R. T. (2001)

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Narayana, S. V. (2011) Structural Differences between the Streptococcus agalactiae

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 Chapter 5

The “lid” of S. pneumoniae SrtC1 is not dynamic in solution and

regulates substrate access to the active site

164
 The work described in this chapter is a version of a manuscript to be submitted for publication.

Jacobitz A. W., Yi, S. W., McConnell, S., Peterson, R., Jung, M. E., and Clubb, R. T. 2015.

I was the primary author of this work. I designed experiments, performed biochemical assays and

NMR experiments, and analyzed data.

165
5.1 Overview

Sortase enzymes perform a transpeptidation reaction that covalently links their two

substrates via a new peptide bond. While most sortase enzymes are known for catalyzing this

transpeptidation between their primary protein substrate and the cell wall peptidoglycan, a subset

of these enzymes instead covalently links proteins to each other via isopeptide bonds to produce

elongated protein polymers known as pili. These protein-polymerizing class C sortases are

structurally distinguished by the presence of an N-terminal extension that occludes the active

site. Numerous publications have hypothesized that the lid is mobile in solution as this would be

necessary for the binding and catalysis of the enzyme’s substrate. Here we show using NMR

dynamics measurements and in vitro assays that the lid of wild-type Streptococcus pneumoniae

SrtC1 is rigid in solution and does not experience conformational dynamics on a mechanistically

relevant timescale. Additionally, we show that point mutations to the lid induce dynamic

behavior that correlates with an increase in both the hydrolysis of the primary substrate and

general access to the active site cysteine residue as evidenced by increased oxidation. These

results suggest that the lid of the S. pneumoniae SrtC1 enzyme performs a regulatory function,

and they imply that interaction with other regions of the full length protein substrate, or other as

yet unnamed surface factors, are likely required to activate the enzyme in vivo.

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5.2 Introduction

Gram-positive bacteria commonly utilize their thick cell walls as a scaffold for anchoring

proteins necessary for interacting with their environment. Many of these proteins are covalently

attached to the cell wall through a reaction catalyzed by sortase enzymes. A particular subtype of

sortases, the class C sortases, are not only implicated in the attachment of surface proteins to the

cell wall of Gram-positive organisms but have also been shown to polymerize pili: long

filamentous protein fibers that are used by some bacteria to make initial, long-range contacts to

host cells1–3. These pili are thought to provide a preliminary point of attachment to the host cell

which acts like a grappling hook, allowing the bacterium to swing closer to the cell, thereby

facilitating the formation of additional close-range adhesive interactions via auxiliary surface

anchored adhesive proteins3. Pili have subsequently been shown to increase the virulence of

certain bacterial species3–5, participate in immune system evasion6, are implicated in biofilm

formation7,8 and have also been heavily researched for their potential as vaccine components due

to their ability to elicit an immune response9–11.

Sortase enzymes are ubiquitous in Gram-positive bacteria and can be classified into 5

distinct classes based on their sequence and structural similarities12,13. Classes A, B, D, E, and F

are generally considered to be cell wall anchoring sortases, while the class C sortases stand alone

in their ability to polymerize their protein substrates (it should be noted that there have recently

been two sortases which have been structurally defined as class B that are in fact pilus associated

sortases)14,15. The cell wall anchoring sortases all recognize lipid II, a precursor to cell wall

synthesis, as their secondary substrate. Class A enzymes are often referred to as “housekeeping”

enzymes; they are constitutively expressed, and generally anchor a variety of proteins to the cell

wall. Class B, D, E, and F enzymes on the other hand are generally utilized for attaching a

167
particular subclass of proteins to the cell wall and are typically expressed along with their

substrate(s) only under specific conditions. The class C enzymes are similar to the class B, D, E

and F enzymes in that they are also sequestered in specific genomic islands along with their pilus

protein substrates, but they are unique in that most class C enzymes are known to recognize two

distinct positions on a single protein to polymerize it into a pilus instead of utilizing lipid II as

their secondary substrate. When fully polymerized, these pili can contain hundreds of individual

pilin protein monomers and be over 1 micron in length.

A large number of sortase structures have been solved and they all share a common 8-β-

barrel sortase fold and a His-Cys-Arg catalytic triad13,16. Each of these enzymes recognizes a C-

terminal cell wall sorting signal (CWSS) composed of a 5 residue sorting signal motif, followed

by a transmembrane helix and positively charged cytoplasmic anchoring domain. The 5 residue

sorting signal is generally of the form LPXTG, where the Pro and Thr residues are most highly

conserved, X can be any amino acid, and the first and last residue often confer specificity to the

individual sortase12,13,17. The reaction begins when the sortase binds the sorting signal and the

active site cysteine performs a nucleophilic attack on the threonine’s carbonyl carbon. This

produces a negatively charged tetrahedral intermediate which is thought to be stabilized by the

active site arginine17, and the active site histidine is thought to act as an acid to protonate the

terminal amine on the glycine of the leaving group18,19. The tetrahedral intermediate then quickly

collapses to form a semi-stable thioacyl intermediate between the active site cysteine and the

protein substrate. For the more heavily studied cell wall anchoring sortases, the next step of the

reaction requires the binding of cell wall precursor lipid II. The cross-bridge peptide of the lipid

II molecule varies from organism to organism, but will invariably contain a terminal amine

group which is deprotonated by the active site histidine before performing a nucleophilic attack

168
on the Thr carbonyl carbon, leading to a second tetrahedral intermediate which collapses to form

a new peptide bond between the sorting signal threonine and the cross-bridge peptide13. Pilin

polymerizing sortases follow the same basic mechanism but utilize the amino-nucleophile of a

lysine sidechain within their pilin protein substrate to resolve the thioacyl intermediate and

generate an isopeptide bond between the sorting signal of one monomer and the lysine of

another2,3,10. That is, the first of these two recognition motifs is the same CWSS at the C-

terminus of the protein substrate, but the other is typically a 4 residue lysine-containing pilin

motif located in the N-terminal domain of the protein3.

Several species of bacteria are known to harbor pilus gene clusters typically containing

genes for 2 or 3 pilin proteins and 1 to 3 sortases. Interestingly, these class C sortases are the

only known class of sortase to harbor an N-terminal extension known as the “lid” that

completely occludes the active site13,20–22(Fig. 1). The lid region has been found in all class C

sortases studied to date and invariably maintains a conserved DP(F/W/Y) anchor motif wherein

the aspartate residue forms a hydrogen bond with the active site arginine, and the aromatic at the

end of the motif is wedged into the active site where it forms a sulfur-aromatic interaction with

the active site cysteine22. Based on the fact that the lid completely blocks access to the sorting

signal binding site seen in all sortase-substrate complex structures reported to date17,23,24 along

with the observations that several class C sortase crystal structures are missing electron density

for regions flanking the lid anchor residues25–28 and many display elevated B-factors throughout

this region20–22, it has been proposed that the lid region of these sortases must be highly

flexible13,20–22,27.

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 Streptococcus pneumoniae is one of the most common causative agents of pneumonia,

and is also known to cause meningitis, and septicemia, and the ability of S. pneumoniae to cause

invasive disease is enhanced by the presence of pili4. S. pneumoniae can express two distinct

types of pili carried on separate genomic islands, known as pilus island 1 (PI-1) or pilus island 2

(PI-2)29. PI-1 is present in ~30% of S. pneumoniae strains and these pili have been more

thoroughly characterized. They consist of three pilus proteins, RrgA, RrgB, and RrgC, which are

polymerized by three sortases, SpnSrtC1, SpnSrtC2, and SpnSrtC3. Structures for each of these

sortases have been solved and they all display nearly identical structures, each with the

conserved lid occluding the active site20,25. Mutations to the anchor residues in the lid of

SpnSrtC1 have been shown to decrease the thermal stability of the enzyme in vitro21. These same

mutations were shown to alter the pattern of, but not abrogate, the production of polymerized

RrgB species in vitro20, but similar mutations to either Streptococcus agalactiae sortase C-1

(SagSrtC1) or Actinomyces oris sortase C-1 (AoSrtC1) had no visible effect on pilus

polymerization in vivo30,31.

Here we show that despite a number of papers previously referring to the lid region of

SrtC enzymes as flexible, the lid of SpnSrtC1 is not in fact dynamic in solution, but instead has

two dynamic regions flanking the lid which may function as hinges, as has been proposed

previously by Manzano and coworkers20. Mutations to the anchor residues of the lid make the

region highly dynamic and dramatically increase hydrolysis of a sorting signal peptide in vitro.

We also show that the lid protects the active site cysteine from oxidation, and mutations to the lid

abolish this protective effect. Based on these findings, we propose that the lid performs a

regulatory function to prevent access to the active site in the absence of additional factors, likely

170
found on the cell surface, which prepare the protein for activity by mobilizing or altering the

conformation of the lid.

5.3 Results

5.3.1 Initial NMR analysis indicates SpnSrtC1 forms dimers in solution.

Soluble SpnSrtC1, residues 17-228 was uniformly labeled with 15N and 13C and purified

from E. coli. 94% of backbone resonances could be definitively assigned using standard triple

resonance assignment methods, leaving a few 1-2 residue unassigned segments, and a single 7

residue stretch of amino acids (residues Q167-E172) which could not be assigned. Interestingly,

this 7 residue stretch of amino acids forms the majority of the interface seen between the two

SpnSrtC1 monomers in the asymmetric unit of the 2008 crystal structure solved by Manzano and

coworkers20. Since residues participating in binding interactions on the intermediate exchange

timescale (i.e. binding and dissociation occurs roughly on the order of 1 ms) are frequently

broadened beyond detection, we hypothesized that these resonances could be missing due to the

existence of a transient dimerization occurring between SrtC1 monomers in solution. To

determine whether this crystallographic dimer was indeed present in solution, we performed a

series of dilutions from 2 mM to 100 μM and recorded 1H-15N HSQC spectra. As the

concentration of the protein was lowered, significant changes in the spectrum were seen in

isolated resonances, along with the appearance of several additional peaks which could only be

detected in the lowest concentration sample (Fig. 2). Although new resonances which appear in

the 100 μM spectrum could not be assigned, as this concentration is too low for the use of

significantly less sensitive triple resonance experiments that are required for backbone

assignments, the existing assigned resonances which either experienced an increase in intensity

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on dilution, or a change in chemical shift, could be identified and plotted onto the crystal

structure (Fig 2B). These residues are largely clustered in the binding interface observed in the

crystal structure, indicating that this dimer is also present in solution.

Additionally, we utilized T1 and T2 relaxation measurements to calculate the overall

tumbling time for the protein, τc, which is defined as the amount of time it takes for the protein to

rotate through 1 radian in space. τc values of known globular proteins consistently yield values of

~0.6 ns times the molecular weight (MW) of the protein in kilodaltons. The MW of soluble

SpnSrtC1 is 23.813 kDa yielding a theoretical τc of 14.3 ns. Based on measurements of the T1

and T2 relaxation constants, we calculated the τc of SpnSrtC1 in solution to be 17.8 ns at a

concentration of 1 mM. This τc is significantly higher than would be expected for a monomer of

this size. If the protein formed a stable dimer, the τc would be expected to be around 28.6 ns, and

a protein of this size would experience extremely fast T2 relaxation preventing the detection of

good quality triple resonance spectra, an effect we did not observe. The slightly elevated

observed τc value is instead likely a consequence of the intermediate exchange kinetics of

dimerization for this protein that had been suggested by the broadening of interfacial residues

discussed above. This would indicate that at any given time, only a small percentage of the

available proteins in solution would be dimeric and tumble at the slower ~28.6 ns rate, but that

these dimers are still detectable as an increase in the average τc of all the proteins in the sample,

leading to a slightly higher measured τc than expected.

5.3.2 NMR dynamics data indicate that the SpnSrtC1 lid is not dynamic in vitro.

In order to determine whether the lid of SpnSrtC1 is mobile in solution, we performed

dynamics experiments using solution NMR spectroscopy. NMR spectroscopy is a powerful

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technique for dissecting the motions of individual backbone amides across a wide range of

biologically relevant timescales. First, T1, T2, and HetNOE experiments were performed to detect

the presence of fast-motions on the ps-ns timescale. The results of T1 and T2 experiments were

used to determine the rotational correlation time, τe, for each backbone amide residue in the

protein (Fig. 3). τe represents the amount of time it takes for a single amide bond vector to rotate

through 1 radian of space. By comparing the τe to the overall rotational correlation time for the

entire protein, τc, (Fig.3A, red line) residues whose bond vectors are moving more rapidly than

the body of the protein can be detected. Any residue that rapidly samples disordered states in

solution would be expected to have a τe that is much lower than the protein’s τc, indicating that

the residue can move through space more quickly than the body of the protein. This is

comparable to a flag rapidly flapping in the wind (the mobile residues) while affixed to the bow

of a slowly rocking ship (the body of the protein). The flag moves on the same slow timescale of

the ship, but will experience faster overall motion because of additional dynamic behavior.

Unexpectedly, lid residues contacting the active site (D58 and W60) have similar τe values as the

entire structured core domain of the protein, 16.4 and 16.9 ns respectively, compared to a τc of

17.8 ± 1.1 ns for the structured portion of the protein. For comparison, the unstructured C-

terminus of the protein has τe values that drop as low as 10 ns. This is confirmed by HetNOE

values above 0.6 which indicates that these residues are structured in solution (Fig. 3B), as

residues are typically only considered disordered if their HetNOE is below 0.6 (Fig. 3B, red

line). Notably, residues 56-57 before and 65-70 after the lid show increased dynamics on this

timescale indicating that they are highly mobile, even though the lid residues from this N-

terminal extension that are actually engaged with the active site are not. This supports the

hypothesis that the N-terminal extension contains “hinges,” originally proposed by Manzano and

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coworkers to be present in SpnSrtC3 based on elevated B-factors for this protein being highest in

two discrete regions flanking the lid20. This hypothesis is further supported by the significant

number of SrtC structures solved to date wherein these regions either showcase the highest B-

factors in the structure, or lack density altogether20–22,25–28. Together these NMR experiments

indicate that the lid residues that contact the active site do not experience motions over the ps-ns

timescale, indicating that they are predominantly in a rigid, ordered conformation, likely akin to

that visualized in the crystal structure, whereas regions of sequence adjacent to the lid are in fact

highly mobile, and likely represent hinges for the opening of the lid and subsequent unmasking

of the active site.

As both of these experiments detect motions on the ps-ns timescale, we conducted

additional experiments to determine if the lid was instead mobile over longer μs-ms timescales.

To measure this phenomenon, we first conducted CPMG experiments32 to directly detect the Rex

component of relaxation on a per residue basis (Fig. 3C). Rex values are generally considered to

be an indicator of this slower timescale motions if they are greater than ~30% of the measured T2

for an individual residue which typically means values above 10 s-1 are considered significant.

Residues in and around the lid region do not show significantly elevated Rex values, indicating

that they are in fact not mobile on the μs-ms timescale. Additional experiments to sample within

this timescale (CEST), and out to the ms-s timescale (NZ-exchange) were also conducted, and

showed no spectral perturbations which would be indicative of longer timescale motions (data

not shown). Together, these experiments indicate that the lid of SpnSrtC1 is not dynamic in

solution as has been suggested by previous reports and is instead largely structured in the

absence of additional factors, even out to very long timescales.

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5.3.3 Mutations to the “lid” of SpnSrtC1 make the region highly dynamic in solution.

Given that the “lid” of wild-type SpnSrtC1 is not dynamic in vitro, we set out to

determine the effects of point mutations in the lid on its mobility in solution. Several previous

studies have shown that point mutations to the conserved Asp and Trp residues in the

DP(F/W/Y) motif of SpnSrtC1 lowered its thermostability21. Similar mutations were also shown

to increase the rate of hydrolysis and were even necessary to induce in vitro pilin polymerization

by SagSrtC130,33. To determine if these mutations modified the mobility of the lid, we generated

SpnSrtC1-D58A and W60A point mutants and characterized their effects on the protein’s

dynamics using the same fast-timescale T1, T2, and HetNOE experiments discussed above for the

WT enzyme. SpnSrtC1-W60A shows significantly increased mobility in the lid, and throughout

the entire region between the two hinges identified for the WT enzyme (residues 56-70). T1 and

T2 experiment derived τe values indicate that the region tumbles at 10.7 ns, 5.2 ns faster than the

average for the structured portion of this enzyme (Fig. 4). The HetNOE experiment additionally

indicates that these residues are largely disordered, with every residue that could be assigned and

characterized between the two hinges, residues 58 to 64, having reduced HetNOE values

compared to WT, with many of these being less than 0.6 indicating that they are disordered.

SpnSrtC1-D58A also shows significantly increased mobility in the lid, and especially in flanking

regions based on an analysis of the τe and HetNOE measurements (Fig. 4). Interestingly, these

experiments show decreased dynamics in a small region immediately after the conserved lid

residue, W60. To further investigate whether this less dynamic region indicates that the lid

tryptophan might be sampling a closed state similar to the WT enzyme, we performed additional

NOESY experiments and compared the spectra for the Trp indole proton. As the NOESY

experiment is able to detect the through-space coupling of nuclei within 5 Å of each other, the

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pattern of numerous peaks seen for the WT enzyme indicates that the side chain of the residue is

folded and in close proximity to several other residues (Fig. 4E). The W60 indole in the D58A

mutant on the other hand is split into 3 peaks in the HSQC, each of which shares only 1 or 2

NOESY peaks with the WT spectrum that may represent intraresidue NOEs to adjacent protons

in the sidechain. This large difference in NOE pattern suggests that the tryptophan sidechain in

the D58A mutant no longer participates in native contacts to the enzyme active site, and has

likely become dislodged from the position it maintains in the WT enzyme. This suggests that

even though several of the residues following W60 do not appear to be dynamic based on

HetNOE and τe values, the conformation of this entire region has been altered significantly, and

the native, closed state of the lid has been disrupted.

5.3.4 Mutation of the lid is required to induce in vitro hydrolysis.

Knowing that the SpnSrtC1 lid is rigid in solution and becomes mobile upon mutation,

we set out to determine whether this increased mobility would affect the ability of the enzyme to

function in vitro. To test this, we performed in vitro hydrolysis experiments by incubating the

sortase with a 15 amino acid peptide derived from RrgB and containing the IPQTG sorting signal

sequence for 24 hrs at 37°C and then separating the mixture via HPLC. We then utilized MALDI

mass spectrometry to confirm the identities of the resultant peaks in the chromatogram. By

separating the substrate peptide from the cleaved product using HPLC and measuring the

absorbance at 215 nm we could monitor both the amount of cleaved product peptide formed, and

substrate peptide remaining, allowing for a thorough disambiguation of the reaction progress

which is not possible with the more commonly used FRET based assay which only reports on the

breakage of the peptide bond and not the subsequent release of the thioacyl intermediate from the

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enzyme. We conducted the experiments with WT SpnSrtC1, SpnSrtC1-D58A and W60A point

mutants used in our NMR studies, as well as an SpnSrtC1-D58A-W60A double mutant and an

additional mutant wherein the entire lid containing region, residues 55-69 inclusive, was replaced

with a 15 residue glycine-serine linker (SpnSrtC1-GSlid). We used the GSlid construct as an

approximation of a completely open state of the lid, since a construct where we completely

deleted the N-terminal extension containing the lid proved to be unstable over the timecourse of

the reaction. Interestingly, though the WT enzyme has been shown to perform a transpeptidation

reaction in vitro with a full length RrgB protein which contains both the primary IPQTG sorting

signal substrate, and secondary YPKN pilin motif substrate20, the enzyme does not catalyze the

initial cleavage step of the reaction in vitro in the absence of its secondary substrate (Fig. 5). It is

important to note that not only is the WT enzyme incapable of hydrolyzing the substrate, as

evidenced by the lack of detectable product peptide, but it is also incapable of beginning its

reaction by performing a nucleophilic attack on the IPQTG motif’s Thr carbonyl to catalyze the

creation of the thioacyl intermediate. This latter reaction could be visualized in our assay as a

reduction in the amount of substrate peptide remaining at the end of the reaction without the

appearance of a new product peak, but this was not observed. Instead, the WT SpnSrtC1 reaction

is indistinguishable from peptide alone or from a reaction with the C193A active site mutant. The

lid mutants on the other hand, which we showed by NMR to have much more mobile lid regions,

all catalyze a hydrolysis reaction to a similar extent within the error of the measurement except

for the D58A mutant which was slightly slower, perhaps due to partial occlusion of the active

site by one or more of the multiple semi-stable positions we detected for its lid Trp residue based

on NOESY data. While even the mutant enzymes only process about 15% of their substrate in 24

hrs under the conditions tested, this rate of cleavage is similar to that seen for the WT SrtB

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enzyme from Staphylococcus aureus (Jacobitz unpublished data) indicating that this could be a

fully active form of the enzyme.

5.3.5 A closed lid is important for protecting the active site cysteine from oxidation.

To further test the importance of the closed lid, we performed disulfide bonding

experiments utilizing a modified Cbz-IPQT* sorting signal substrate, where Cbz is a

carbobenzyloxy protecting group and T* is a modified threonine residue with a thiol group in

place of its C-terminus ((2R,3S)-3-amino-4-mercapto-2-butanol) which should allow it to form a

disulfide bond with the active site cysteine. This type of modified substrate has been used

previously to successfully produce stable enzyme substrate complexes for 3 other sortases17,23,24,

indicating the utility of this method. When incubated with the IPQT* substrate at room

temperature for 24 hrs, the WT SpnSrtC1 showed no detectable disulfide formation by MALDI-

MS (Fig. 6A). When the same experiment was repeated with the SpnSrtC1-W60A mutant, a

mass shift of 595 Da (theoretical mass of Cbz-IPQT* = 593.4 Da) could be detected indicating

the formation of a disulfide-bonded complex (Fig. 6B). The difference in cysteine availability

highlighted by this experiment further indicates that the lid of WT SpnSrtC1 not only prevents

unwanted catalytic activity, but actually physically limits access to the active site cysteine, which

would be able to form a disulfide bond whether or not the enzyme was in a fully catalytically

active state.

5.4 Discussion

Class C sortases are unique among sortases in that they catalyze the covalent linkage of

two protein substrates instead of attaching proteins to the cell wall peptidoglycan13. This reaction

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leads to the polymerization of long filamentous pili which are necessary for adhesion to host

cells and biofilm formation. Although the class C sortase enzymes utilize a lysine side chain

from their protein substrate as the nucleophile to conclude their reaction instead of the N-

terminus of a lipid II cross bridge peptide that other classes employ, the basic reaction is highly

similar, perhaps owing to the high degree of conservation within the core β-barrel of the sortase

motif. All sortases studied to date utilize the same conserved His-Cys-Arg catalytic triad, and are

thought to recognize their substrates using the same binding groove beginning at the β6/β7 loop

and moving towards the active site. Given the number of similarities shared between members of

this family it is perhaps unsurprising that we observed the dimerization of SpnSrtC1 in solution

(Fig. 2), as dimerization in solution has already been detected for several other members of the

family34–36. Interestingly, while the details of this dimerization event are not fully understood,

and each of the proposed binding interfaces is somewhat variable, all of these proteins have

utilized residues from the β4-β5 loop, on the “back” face of the enzyme, opposite the active site.

This dimerization was recently shown to alter the rate of catalysis for S. aureus SrtA in vivo36. It

is interesting to note that the vast majority of sequences deposited for SpnSrtC1 that can be

identified from a BLAST search with the 279 residue UniProt sequence, Q97SB9, referenced in

previous studies contain exactly 21 additional N-terminal residues, and analysis of this full 300

residue sequence with the TMHMM server37 predicts an additional N-terminal transmembrane

(TM) helix with >99.9% probability that could not have been predicted from the shortened

sequence referenced previously. This predicted N-terminal TM-helix has been shown to be

commonplace among other class C sortases and is known to be necessary for determining

appropriate membrane localization30. Surprisingly, both N and C-termini from both structures in

the proposed dimer visualized in the SpnSrtC1 crystal structure all localize to a single face of the

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dimeric assembly which puts the two active sites on opposite ends, each facing solvent. It is

possible that this dimer is significant in vivo when faced with a full length RrgB substrate that

contains both the primary and secondary recognition motifs as these two motifs on the RrgB

protein are separated from each other by 95 Å which is significantly longer than the 35 Å that

separate the two sortase active sites in the dimer. This indicates that a single tri-domain RrgB

protein could feasibly interact with both active sites of the dimer simultaneously, potentially

allowing a single sortase dimer to polymerize an entire pilus by simply adding monomers

iteratively at one active site then the next without the need for sortases to “line up” to carry out a

concerted pilus polymerization as has been suggested previously2,10.

In addition to their functional distinction, class C sortases are also structurally unique in

that they exhibit an N-terminal extension which occludes the active site and has thus been termed

the lid13,20–22. Numerous publications have referred to this lid as “flexible” based on elevated B-

factors or missing density in adjacent regions of crystal structures, or based on the fact that

removal of the lid would be necessary for substrates to access the active site13,20–22,27. NMR

dynamics data indicates to the contrary that in solution the key lid residues that actually contact

the active site are not dynamic, but are instead flanked by dynamic regions which potentially act

as hinges for the removal of the lid and unmasking of the active site under the appropriate

circumstances (Fig. 3). Additional analysis of the ability of the WT enzyme to perform the initial

cleavage step of its reaction in vitro with purified peptide containing its primary IPQTG sorting

signal substrate indicates that this closed conformation of the lid actually prevents catalysis. Only

when mutations to the lid are introduced which increase the flexibility of this region, as indicated

by reduction in both the τe and HetNOE values for these residues (Fig. 4), does the enzyme

catalyze the initial step of its reaction in vitro (Fig. 5). The same motion-inducing mutations are

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also required to grant access to the cysteine even for the simple production of a disulfide bond,

the formation of which does not require access to the rest of the binding site or participation from

any of the other active site residues (Fig. 6). This indicates that not only does the rigidity of the

lid in the WT enzyme prevent the hydrolysis of the enzyme’s native substrate in solution, but

that it does so by completely preventing access to the active site cysteine instead of simply

inactivating the enzyme via some form of conformational manipulation, or obstruction of

individual recognition pockets in the binding site.

Numerous publications have confirmed the enzyme’s ability to perform this reaction

under native conditions in vivo25,38,39, and it has even been shown to perform the native

transpeptidation reaction in vitro when presented with full length substrates containing both its

primary, IPQTG sorting signal, and secondary, YPKN pilin motif substrates20,21. All other

classes of sortase enzymes studied to date have been shown to catalyze the hydrolysis of their

primary substrate in vitro when deprived of a secondary substrate with which to perform their

native transpeptidation reaction28,30,35,40–43. Since SpnSrtC1 cannot perform the initial cleavage of

its primary binding motif in vitro in the absence of secondary substrate as numerous other

sortases have been shown to do (without the artificial mobilization of its lid through the

incorporation of destabilizing mutations) it follows that the recognition of an additional factor

must be required to remove the lid and allow for catalysis. Interestingly, our in vitro data also

suggests that in the absence of a secondary substrate or other additional factors, WT SpnSrtC1

does not even form a thioacyl intermediate, as this would be detectable as a loss of substrate,

without an accompanying formation of hydrolysis product (Fig. 5). This indicates that the

thioacyl intermediate reported previously from in vitro reactions with full length protein

substrates can only form when the lid is opened by additional interactions with the substrate that

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are outside of the sorting signal21. It is tempting to assume that YPKN pilin motif would fulfill

the role of this additional factor that unhinges the lid, but simply based on the fact that this

enzyme has been shown to catalyze the transpeptidation in vitro when provided with a full length

substrate, it is only possible to conclude that there is another region of the RrgB substrate protein

outside of the IPQTG sorting signal that activates the sortase, presumably by opening the lid.

Unfortunately even this conclusion is not infallible, as the data for the in vitro reaction is not

entirely convincing given that we, and others, have observed soluble pilin proteins to associate

into stable higher molecular weight species which closely mimic product of the sortase catalyzed
15
polymerization reaction (unpublished data, and ). Additionally, the WT S. agalactiae SrtC1

protein was shown to cleave its primary substrate in vitro, but still does not perform the

transpeptidation reaction in the presence of a second pilin motif containing peptide. Mutations to

the lid of this protein were shown to increase the rate of peptide hydrolysis in vitro by ~2.5 fold,

and had the additional activating effect of permitting transpeptidation in solution30,33. The fact

that both the S. pneumoniae system studied in this work and the S. agalactiae system studied

previously are not fully active in vitro with an intact lid reiterates the necessity for additional

factors, outside of the two well-known substrate motifs, to open the lid of the enzyme and allow

catalysis to proceed as it does in vivo. Sortase enzymes have been observed to colocalize with a

number of factors on the cell surface, including the SEC translocon, cell wall synthesis

machinery, and their substrates13,44–46, and we postulate that either additional regions within the

pilin proteins themselves, or one of these additional surface factors must be necessary for

removing the lid and activating the enzyme in vivo.

From this evidence it is clear that the lid present in class C enzymes is competent in

preventing the reaction from taking place. But why would an entire class of enzymes have

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evolved a conserved feature to prevent or retard their primary function? First and foremost, the

hydrolysis and subsequent loss of protein substrates to solvent, which are intended to instead be

polymerized and attached to the cell surface, is inherently wasteful for the cell. While accidental

cleavage and loss of a few proteins here and there (as may be common for cell wall anchoring

sortases) might be acceptable to the cell, the energetic cost of building a 100 protein, 1 μm long

pilus, and then subsequently cleaving its C-terminus and releasing it into solvent could be

crippling especially if this was allowed to happen at regular intervals. It’s also possible that

preventing the functionality of the enzyme is simply a byproduct of protecting the active site

from inactivation by oxidation of its cysteine. It has been shown previously that the cysteines of

several other sortase enzymes are susceptible to oxidation as evidenced by both an increase in

catalytic activity reported in the presence of reducing agents, and structures of Streptococcus

pyogenes SrtA solved with its active site cysteine in multiple oxidation states47,48. It has even

been proposed that certain host organisms may create an oxidizing environment surrounding a

site of infection to deactivate sortase enzymes at the cell surface and render those cells

avirulent48,49. The fact that we observed rapid formation of a disulfide linked complex between

an IPQT* sorting signal mimic and the W60A lid mutant of SpnSrtC1, but not between the same

mimic and the WT protein suggests that the lid indeed functions to prevent oxidation of the

active site cysteine (Fig. 6). When tested in vivo, SrtC1 lid mutants showed no discernable effect

on transpeptidation30,31. This is in agreement with our proposal that the lid is necessary largely to

avoid the energetic penalties associated with cleavage and loss of large numbers of pilus proteins

or oxidative inactivation of the sortase itself, as these energetic penalties would likely only

provide a fitness advantage in a host setting where slight improvements in efficiency could be

pivotal in determining the cell’s ability to maintain a successful infection. We thus propose that

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the lid functions as a regulatory mechanism for the class C sortase that prevents the unwanted

cleavage of substrates, and protects the active site from oxidative inactivation.

5.5 Experimental Procedures

5.5.1 Cloning, protein expression and purification.

DNA encoding codon optimized soluble SpnSrtC1, residues 17-228 based on uniprot

sequence Q97SB9, was generated by recursive PCR from overlapping primers50. Codon

optimization was performed with OPTIMIZER51. DNA encoding SpnSrtC1 was then cloned into

the pESUMO vector (LifeSensors) and transformed into Escherichia coli BL21 (DE3) cells

(Stratagene). Expression was induced at an OD600 of 0.5 by addition of 1 mM isopropyl β-D-1-

thiogalactopyranoside (IPTG) for 5 hrs at 37 °C. Protein was purified by affinity purification

using HisPur Co2+ resin (Thermo) as per the manufacturer’s instructions. The His6-SUMO tag

was cleaved by incubating the protein overnight at 4 °C with recombinant ULP1 protease and

subsequently removed by reapplying the protein mixture to HisPur Co2+ resin. D58A, W60A,

D58A-W60A double mutant, and GSlid mutants of SpnSrtC1 were generated by site directed
15 13
mutagenesis using standard procedures. N and C labeled variants were produced by

expressing the proteins in M9-minimal media supplemented with 15N NH4Cl and/or 13C glucose.

For these expressions, cells were grown to an OD600 of 0.5 at 37 °C, then transferred to an 18 °C

incubator and induced with 1 mM IPTG for 16 hrs. Labeled proteins were purified as specified

above.

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5.5.2 NMR spectroscopy.

All protein samples used for NMR experiments were concentrated and dialyzed into

NMR buffer (50 mM sodium phosphate pH 6.5, 50 mM NaCl, 10% D2O). NMR spectra were

recorded on Bruker 500, 600, and 800 MHZ spectrometers equipped with triple resonance

cryogenic probes and processed using NMRPipe52. Chemical shift assignments were obtained

through the analysis of HNCO, HN(CA)CO, HNCA, HNCACB, CBCA(CO)NH, CC(CO)NH,

and HBHANH experiments in CARA 1.9.053.

Dilution experiments to investigate the potential for SpnSrtC1 to dimerize were

performed by collecting 15N-HSQC spectra beginning at 2 mM, and diluting the protein serially

with NMR buffer + 10% D2O to 1.5, 1, 1.5, 0.5, 0.25, 0.2, and 0.1 mM. The number of scans was

increased to account for a loss of signal from a reduction of concentration based on the following

relationship:

NS = NS0(C1/C2)2

Analysis of signal intensities and chemical shift changes was performed in SPARKY54, and

structure figures were generated in PyMOL55.

15
Dynamics experiments were conducted on 1 mM N labeled samples in NMR buffer

described above. Heteronuclear NOE, T1 and T2 experiments were conducted at 298 K and

analyzed in SPARKY54. The residue specific tumbling time, τe was calculated using a spherical

approximation based on the following equation:

1
= 6 −7
4

where νN is the 15N resonance frequency in Hz. The molecular tumbling time, τc, was calculated

as the average of all τe values for the protein. CPMG experiments were conducted using a

185
modified approach based on the original method developed by Palmer et. al.56. Here, full T2

datasets were collected for 3 different CPMG pulse delays, 0.0023 s, 0.0048 s, and 0.000514 s.

R2 values (1/T2) from these measurements were used to calculate Rex by subtracting the

maximum calculated R2 (either the 0.0023 s or 0.0048 s delay, as R2 values calculated at these

longer delays are known to oscillate slightly), from the minimum R2 determined at the shortest

delay (0.00514 s).

5.5.3 In vitro cleavage assay.

SpnSrtC1 and mutants were dialyzed into assay buffer (25 mM TRIS-HCl pH 7.5, 100

mM NaCl). Sorting signal containing peptide: NH2-VNKKITIPQTGGIGT-CO2 was synthesized

by LifeTein and used without further purification. Lyophilized peptide was dissolved in assay

buffer to 2 mM and final concentration was verified using a BCA assay kit (Thermo) per

manufacturer’s instructions. Reactions were conducted with 100 μM enzyme and 200 μM

peptide in a total volume of 100 μL at 37 °C. After 24 hrs, reactions were quenched by addition

of 50 μL 1 M HCl and 100 μL of the reaction mixture was injected onto a Waters XSelect HSS

C18 reversed phase HPLC column. Peptides were eluted with a gradient from 5-30% acetonitrile,

0.1% trifluoroacetic acid over 20 mins. Elution and quantification of peptides was conducted by

monitoring absorbance of the peptide bond at 215 nm. Fractions from peaks corresponding to

substrate and product were collected and their identities confirmed by matrix assisted laser

desorption ionization-mass spectrometry (MALDI-MS).

186
5.5.4 In vitro oxidation assay.

The IPQT* (where T* is (2R,3S)-3-amino-4-mercapto-2-butanol) substrate mimic was

synthesized as in 57. The compound was mixed with SpnSrtC1 WT or SpnSrtC1-W60A mutant at

a 10:1 molar ratio of mimic to protein and allowed to mix on a rotisserie at room temperature for

24 hrs. Reactions were then applied to zip-tip C18 pipette tips (Millipore) and eluted as per

manufacturer’s instructions. 1 μL eluent was then spotted onto a MALDI plate in triplicate and

crystallized by the addition of 0.5 μL 2,5-dihydroxybenzoic acid (DHB) matrix dissolved in 50%

ethanol, 0.1% TFA. Formation of a disulfide bond with the mimic was detected via MALDI-MS

by a mass increase of 592 Da.

187
5.6 Figures

A B C

Figure 5.1 S. pneumoniae SrtC1 structure and lid motif.

(A) Cartoon representation of SpnSrtC1 from PDB 2W1J20. Active site His, Cys, Arg, and lid Trp

residue shown as sticks. N-terminal extension preceding the sortase β-barrel core is colored red.

(B) Transparent surface representation of SpnSrtC1, showing the lid occluding the active site.

(C) Zoom in of SpnSrtC1 active site, showing conserved DP(F/W/Y) lid motif in active site as

sticks.

188
 A

Figure 5.2 NMR analysis shows SpnSrtC1 is dimeric in vitro.

(A) HSQC of 300 μM SpnSrtC1 (green) overlayed over an HSQC of 100 μM SpnSrtC1 (red),

showing the appearance of several peaks along with the movement or increase in intensity of

several others. (B) The two monomers in the asymmetric unit of the crystal structure of

SpnSrtC1, PDB ID 2W1J20, are shown as a cartoon representation. Residues with increases in

peak height or changes in chemical shift of greater than 1 stdev from the mean are displayed on

the structure in red, unassigned residues are shown in beige, and both are displayed as sticks.

189
 A 30
25
20

Te (ns)
15
10
5
0

B 1.5
1
0.5
HetNOE

0
-0.5
-1
-1.5
-2

C 50
40
Rex (s-1)

30
20
10
0

Residue

Figure 5.3 NMR dynamics of SpnSrtC1.

A diagram of secondary structure elements is shown at the top of the figure where helices are

represented as cylinders and β-strands as arrows. (A) Residue specific tumbling time (τe) for

each assigned residue in SpnSrtC1. Average for the structured region of the protein plotted as a

blue line. (B) Heteronuclear NOE for each residue in SpnSrtC1. A red line is shown at a HetNOE

value of 0.6. A HetNOE value below this line indicates that the residue can be considered

190
dynamic. (C) Rex component of relaxation as determined by direct CPMG detection, plotted for

each residue.

191
Figure 5.4 NMR shows SpnSrtC1 mutants have more dynamic lids.

A diagram of secondary structure elements is shown at the top where helices are represented as

cylinders and β-strands as arrows. (A) Residue specific tumbling time (τe) for each assigned

192
residue in SrtC1-W60A. (B) Heteronuclear NOE for each residue in SpnSrtC1-W60A. Red line

at 0.6 indicates the cutoff below which residues are considered disordered (C) Residue specific

tumbling time (τe) for each assigned residue in SrtC1-D58A. (D) Heteronuclear NOE for each

residue in SpnSrtC1-D58A. Red line at 0.6 indicates the cutoff below which residues are

considered disordered. (E) Comparison of NOESY spectra for the W60 indole proton for the WT

SpnSrtC1 protein, left, or three possible indole configurations seen in the D58A mutant,

represented by the right 3 strips.

193
 A

Product formed (µM)

35
30
25
20
15
10
5
0 * *

B
IPQTG Remaining (µM)

250
200
150
100
50
0

Figure 5.5 SpnSrtC1 lid mutants are more active in vitro.

(A) Product formed after 24 hr reactions with IPQTG containing peptide is shown for WT, and

various SpnSrtC1 mutant constructs. (B) The amount of substrate remaining after 24 hr

incubation for the same reaction detailed above.

194
A 100
23689.60
B 100
23607.04
90

% Intensity
90
% Intensity

80
80 70
70 60
60 50
50 40
40 30
30 20
20 10
10 0
0 15000 19000 23000 27000 31000 35000
15000 19000 23000 27000 31000 35000
Mass (m/z) Mass (m/z)

23691.96 24202.25
100 100
90
% Intensity

90

% Intensity
80 80
70 70
60 60
50 50
40 40
30 30
20 20
10 10
0 0
15000 19000 23000 27000 31000 35000 15000 19000 23000 27000 31000 35000
Mass (m/z) Mass (m/z)

Figure 5.6 The lid of SpnSrtC1 protects the active site cysteine from modification by a

sulfhydryl modifier.

(A) SrtC1 before (top) and after (bottom) 24 hr incubation with IPQT* disulfide bond forming

substrate mimic showing no significant formation of the disulfide complex. Theoretical mass of

SrtC1-WT = 23,813 Da, SrtC1-IPQT* complex = 24,405 Da (B) SrtC1-W60A mutant before

(top) and after (bottom) 24 hr incubation with IPQT* disulfide forming peptide showing

conversion to the disulfide bonded complex. Theoretical mass of SrtC1-W60A = 23,698 Da,

SrtC1-W60A-IPQT* complex = 24,290 Da.

195
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