Papers by Christopher Tate
The importance of interactions with helix 5 in determining the efficacy of β-adrenoceptor ligands
MOL#101030 2 Structure of 7-methylcyanopindolol-bound β 1 AR
33 34 The adenosine A2A receptor (A2AR) is a prototypical G protein-coupled receptor (GPCR) that ... more 33 34 The adenosine A2A receptor (A2AR) is a prototypical G protein-coupled receptor (GPCR) that 35 couples to the heterotrimeric G protein GS. Here we determine the structure by electron cryo36 microscopy (cryo-EM) of A2AR at pH 7.5 bound to the small molecule agonist NECA and 37 coupled to an engineered heterotrimeric G protein, which contains mini-GS, the subunits 38 and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 Å or better. 39 Comparison with the 3.4 Å resolution crystal structure shows that the receptor and mini-GS 40 are virtually identical and that the density of the side chains and ligand are of comparable 41 quality. However, the cryo-EM density map also indicates regions that are flexible in 42 comparison to the crystal structures, which unexpectedly includes regions in the ligand 43 binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the 44 subunit of the G protein was observed. 45
Biochemical Society Transactions
G protein-coupled receptors (GPCRs) are the largest single family of cell surface receptors encod... more G protein-coupled receptors (GPCRs) are the largest single family of cell surface receptors encoded by the human genome and they play pivotal roles in co-ordinating cellular systems throughout the human body, making them ideal drug targets. Structural biology has played a key role in defining how receptors are activated and signal through G proteins and β-arrestins. The application of structure-based drug design (SBDD) is now yielding novel compounds targeting GPCRs. There is thus significant interest from both academia and the pharmaceutical industry in the structural biology of GPCRs as currently only about one quarter of human non-odorant receptors have had their structure determined. Initially, all the structures were determined by X-ray crystallography, but recent advances in electron cryo-microscopy (cryo-EM) now make GPCRs tractable targets for single-particle cryo-EM with comparable resolution to X-ray crystallography. So far this year, 78% of the 99 GPCR structures deposite...
Annual Review of Pharmacology and Toxicology
Electron cryo-microscopy (cryo-EM) has revolutionized structure determination of membrane protein... more Electron cryo-microscopy (cryo-EM) has revolutionized structure determination of membrane proteins and holds great potential for structure-based drug discovery. Here we discuss the potential of cryo-EM in the rational design of therapeutics for membrane proteins compared to X-ray crystallography. We also detail recent progress in the field of drug receptors, focusing on cryo-EM of two protein families with established therapeutic value, the γ-aminobutyric acid A receptors (GABAARs) and G protein–coupled receptors (GPCRs). GABAARs are pentameric ion channels, and cryo-EM structures of physiological heteromeric receptors in a lipid environment have uncovered the molecular basis of receptor modulation by drugs such as diazepam. The structures of ten GPCR–G protein complexes from three different classes of GPCRs have now been determined by cryo-EM. These structures give detailed insights into molecular interactions with drugs, GPCR–G protein selectivity, how accessory membrane proteins ...
eLife
The adenosine A2A receptor (A2AR) is a prototypical G protein-coupled receptor (GPCR) that couple... more The adenosine A2A receptor (A2AR) is a prototypical G protein-coupled receptor (GPCR) that couples to the heterotrimeric G protein GS. Here, we determine the structure by electron cryo-microscopy (cryo-EM) of A2AR at pH 7.5 bound to the small molecule agonist NECA and coupled to an engineered heterotrimeric G protein, which contains mini-GS, the βγ subunits and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 Å or better. Comparison with the 3.4 Å resolution crystal structure shows that the receptor and mini-GS are virtually identical and that the density of the side chains and ligand are of comparable quality. However, the cryo-EM density map also indicates regions that are flexible in comparison to the crystal structures, which unexpectedly includes regions in the ligand binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the β subunit of the G protein was observed.
Protein engineering, design & selection : PEDS, 2016
G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling in response to extracellular ... more G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling in response to extracellular stimuli, and are important therapeutic targets in a wide range of diseases. Structure determination of GPCRs in all activation states is important to elucidate the precise mechanism of signal transduction and to facilitate optimal drug design. However, due to their inherent instability, crystallisation of GPCRs in complex with cytoplasmic signalling proteins, such as heterotrimeric G proteins and β-arrestins, has proved challenging. Here, we describe the design of a minimal G protein, mini-Gs, which is composed solely of the GTPase domain from the adenylate cyclase stimulating G protein Gs Mini-Gs is a small, soluble protein, which efficiently couples GPCRs in the absence of Gβγ subunits. We engineered mini-Gs, using rational design mutagenesis, to form a stable complex with detergent-solubilised β1-adrenergic receptor (β1AR). Mini G proteins induce similar pharmacological and structural...
Bio-protocol, Jan 20, 2017
Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell ... more Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR-G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are unstable and conformationally dynamic when coupled to a receptor. We therefore developed an engineered minimal G protein, mini-Gs, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A2A receptor (A2AR) in its active conformation. Mini G proteins are potentially useful tools in a variety of applications, including characterising GPCR pharmacology, binding affinity and kinetic experiments, agonist drug discovery, and structure determination of GPCR-G protein complexes. Here, we describe a detailed protocol for the expression and purification of mini-Gs.
Journal of the American Chemical Society, Jan 30, 2016
Stability of detergent-solubilized G-protein-coupled receptors (GPCRs) is crucial for their purif... more Stability of detergent-solubilized G-protein-coupled receptors (GPCRs) is crucial for their purification in a biologically relevant state, and it is well-known that short chain detergents such as octylglucoside are more denaturing than long chain detergents such as dodecylmaltoside. However, the molecular basis for this phenomenon is poorly understood. To gain insights into the mechanism of detergent destabilization of GPCRs, we used atomistic molecular dynamics simulations of thermostabilized adenosine receptor (A2AR) mutants embedded in either a lipid bilayer or detergent micelles of alkylmaltosides and alkylglucosides. A2AR mutants in dodecylmaltoside or phospholipid showed low flexibility and good interhelical packing. In contrast, A2AR mutants in either octylglucoside or nonylglucoside showed decreased α-helicity in transmembrane regions, decreased α-helical packing, and the interpenetration of detergent molecules between transmembrane α-helices. This was not observed in octylg...
Nature, 2008
G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and ... more G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 Å resolution crystal structure of a β1-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane α-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the β1-adrenergic receptor and binding of carazolol to the β2-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the β2-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.
Biotechnology Journal, 2016
The production of recombinant proteins for biotherapeutic use is a multibillion dollar industry, ... more The production of recombinant proteins for biotherapeutic use is a multibillion dollar industry, which has seen continual growth in recent years. In order to produce the best protein with minimal cost and time, selection methods are utilized during the cell line development process in order to select for the most desirable clonal cell line from a heterogeneous transfectant pool. Today, there is a vast array of potential selection methods available, which vary in cost, complexity and efficacy. This review aims to highlight cell line selection methods that exist for the isolation of high-producing clones, and also reviews techniques that can be used to predict, at a small scale, the performance of clones at large, industrially-relevant scales.
Biophysical Journal, 2015
Biochimica et biophysica acta, Jan 2, 2015
The tetracycline antiporter TetA(B) is a member of the Major Facilitator Superfamily which confer... more The tetracycline antiporter TetA(B) is a member of the Major Facilitator Superfamily which confers tetracycline resistance to cells by coupling the efflux of tetracycline to the influx of protons down their chemical potential gradient. Although it is a medically important transporter, its structure has yet to be determined. One possibility for why this has proven difficult is that the transporter may be conformationally heterogeneous in the purified state. To overcome this, we developed two strategies to rapidly identify TetA(B) mutants that were transport-defective and that could still bind tetracycline. Up to 9 amino acid residues could be deleted from the loop between transmembrane α-helices 6 and 7 with only a slight decrease in affinity of tetracycline binding as measured by isothermal titration calorimetry, although the mutant was transport-defective. Scanning mutagenesis where all the residues between 2 and 389 were mutated to either valine, alanine or glycine (VAG scan) iden...
International Review of Cytology, 1992
Sugar-Cation Symport Systems in Bacteria* Peter JF Henderson, Stephen A. Baldwin, Michael T. Cair... more Sugar-Cation Symport Systems in Bacteria* Peter JF Henderson, Stephen A. Baldwin, Michael T. Cairns, Bambos M. Charalambous, H. Claire Dent, Frank Gunn, Wei-Jun Liang, Valerie A. Lucas, Giles E. Martin, Terry P. McDonald, Brian J. McKeown, Jennifer AR Muiry, ...
Small multidrug resistance (SMR) transporters contribute to bacterial resistance by coupling the ... more Small multidrug resistance (SMR) transporters contribute to bacterial resistance by coupling the efflux of a wide range of toxic aromatic cations, some of which are commonly used as antibiotics and antiseptics, to proton influx. EmrE is a prototypical small multidrug resistance transporter comprising four transmembrane segments (M1-M4) that forms dimers. It was suggested recently that EmrE molecules in the dimer have different topologies, i.e. monomers have opposite orientations with respect to the membrane plane. A 3-D structure of EmrE acquired by electron cryo-microscopy (cryo-EM) at 7.5 Å resolution in the membrane plane showed that parts of the structure are related by quasi-symmetry. We used this symmetry relationship, combined with sequence conserva- tion data, to assign the transmembrane segments in EmrE to the densities seen in the cryo-EM structure. A Cα model of the transmembrane region was constructed by considering the evolutionary conservation pattern of each helix. Th...
Current Opinion in Structural Biology, 2012
G protein-coupled receptors (GPCRs) play a major role in intercellular communication by binding s... more G protein-coupled receptors (GPCRs) play a major role in intercellular communication by binding small diffusible ligands (agonists) at the extracellular surface. Agonist-binding induces a conformational change in the receptor, which results in the binding and activation of heterotrimeric G proteins within the cell. Ten agonist-bound structures of non-rhodopsin GPCRs published last year defined for the first time the molecular details of receptor activated states and how inverse agonists, partial agonists and full agonists bind to produce different effects on the receptor. In addition, the structure of the b 2adrenoceptor coupled to a heterotrimeric G protein showed how the opening of a cleft in the cytoplasmic face of the receptor as a consequence of agonist binding results in G protein coupling and activation of the G protein.
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Papers by Christopher Tate