Papers by Deepanshu Choudhary
The sensory epithelium of the inner ear, found in all extant lineages of vertebrates, has been su... more The sensory epithelium of the inner ear, found in all extant lineages of vertebrates, has been subjected to over 500 million years of evolution, resulting in the complex inner ear of modern vertebrates. Inner-ear adaptations are as diverse as the species in which they are found, and such unique anatomical variations have been well studied. However, the evolutionary details of the molecular machinery that are required for hearing are less well known. Two molecules that are essential for hearing in vertebrates are cadherin-23 and protocadherin-15, proteins whose interaction with one another acts as the focal point of force transmission when converting sound waves into electrical signals that the brain can interpret. This interaction exists in every lineage of vertebrates, but little is known about the structure or mechanical properties of these proteins in most non-mammalian lineages. Here, we use various techniques to characterize the evolution of this protein interaction. Results sh...
Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) is a 140 kDa bi-functional enzyme inv... more Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) is a 140 kDa bi-functional enzyme involved in a coupled reaction, where the glutaminase active site produces ammonia that is subsequently utilized to convert FGAR to its corresponding amidine in an ATP assisted fashion. The structure of FGAR-AT has been previously determined in an inactive state and the mechanism of activation remains largely unknown. In the current study, hydrophobic cavities were used as markers to identify regions involved in domain movements that facilitate catalytic coupling and subsequent activation of the enzyme. Three internal hydrophobic cavities were located by xenon trapping experiments on FGAR-AT crystals and further, these cavities were perturbed via site-directed mutagenesis. Biophysical characterization of the mutants demonstrated that two of these three voids are crucial for stability and function of the protein, although being,20 Å from the active centers. Interestingly, correlation analys...
Nucleic Acids Research
Redβ is a single strand annealing protein from bacteriophage λ that binds loosely to ssDNA, not a... more Redβ is a single strand annealing protein from bacteriophage λ that binds loosely to ssDNA, not at all to pre-formed dsDNA, but tightly to a duplex intermediate of annealing. As viewed by electron microscopy, Redβ forms oligomeric rings on ssDNA substrate, and helical filaments on the annealed duplex intermediate. However, it is not clear if these are the functional forms of the protein in vivo. We have used size-exclusion chromatography coupled with multi-angle light scattering, analytical ultracentrifugation and native mass spectrometry (nMS) to characterize the size of the oligomers formed by Redβ in its different DNA-bound states. The nMS data, which resolve species with the highest resolution, reveal that Redβ forms an oligomer of 12 subunits in the absence of DNA, complexes ranging from 4 to 14 subunits on 38-mer ssDNA, and a much more distinct and stable complex of 11 subunits on 38-mer annealed duplex. We also measure the concentration of Redβ in cells active for recombinati...
Proceedings of the National Academy of Sciences
The vertebrate inner ear, responsible for hearing and balance, is able to sense minute mechanical... more The vertebrate inner ear, responsible for hearing and balance, is able to sense minute mechanical stimuli originating from an extraordinarily broad range of sound frequencies and intensities or from head movements. Integral to these processes is the tip-link protein complex, which conveys force to open the inner-ear transduction channels that mediate sensory perception. Protocadherin-15 and cadherin-23, two atypically large cadherins with 11 and 27 extracellular cadherin (EC) repeats, are involved in deafness and balance disorders and assemble as parallel homodimers that interact to form the tip link. Here we report the X-ray crystal structure of a protocadherin-15 + cadherin-23 heterotetrameric complex at 2.9-Å resolution, depicting a parallel homodimer of protocadherin-15 EC1-3 molecules forming an antiparallel complex with two cadherin-23 EC1-2 molecules. In addition, we report structures for 10 protocadherin-15 fragments used to build complete high-resolution models of the monom...
PLOS ONE
Protein-protein interactions play a crucial role in biological processes such as cell-cell adhesi... more Protein-protein interactions play a crucial role in biological processes such as cell-cell adhesion, immune system-pathogen interactions, and sensory perception. Understanding the structural determinants of protein-protein complex formation and obtaining quantitative estimates of their dissociation constant (K D) are essential for the study of these interactions and for the discovery of new therapeutics. At the same time, it is equally important to characterize protein-protein interactions in a high-throughput fashion. Here, we use a modified thermal scanning assay to test interactions of wild type (WT) and mutant variants of N-terminal fragments (EC1+2) of cadherin-23 and protocadherin-15, two proteins essential for innerear mechanotransduction. An environmentally sensitive fluorescent dye (SYPRO orange) is used to monitor melting temperature (T m) shifts of protocadherin-15 EC1+2 (pcdh15) in the presence of increasing concentrations of cadherin-23 EC1+2 (cdh23). These T m shifts are absent when we use proteins containing deafness-related missense mutations known to disrupt cdh23 binding to pcdh15, and are increased for some rationally designed mutants expected to enhance binding. In addition, surface plasmon resonance binding experiments were used to test if the T m shifts correlated with changes in binding affinity. We used this approach to find a double mutation (cdh23(T15E)-pcdh15(G16D)) that enhances binding affinity of the cadherin complex by 1.98 kJ/mol, roughly twofold that of the WT complex. We suggest that the thermal scanning methodology can be used in high-throughput format to quickly compare binding affinities (K D from nM up to 100 μM) for some heterodimeric protein complexes and to screen small molecule libraries to find protein-protein interaction inhibitors and enhancers.
Biophysical Journal
Protein-peptide interactions play essential roles in many cellular processes and their structural... more Protein-peptide interactions play essential roles in many cellular processes and their structural characterization is the major focus of current experimental and theoretical research. Two decades ago, it was proposed to employ the steered molecular dynamics to assess the strength of protein-peptide interactions 1. The idea behind using steered molecular dynamics simulations is that the mechanical stability can be used as an efficient alternative to computationally highly demanding estimation of binding affinity and aggregation rate 2,3. However, mechanical stability defined as a peak in force-extension profile depends on the choice of the pulling direction. Here we propose an uncommon choice of the pulling direction along resultant dipole moment vector, which has not been explored in simulations so far. Using explicit solvent all-atom MD simulations, we apply steered molecular dynamics technique to probe mechanical resistance of protein-peptide system pulled along two different vectors 4. A novel pulling direction, along the resultant dipole moment vector, results in stronger forces compared to commonly used peptide unbinding along center of masses vector. Our results demonstrate that resultant dipole moment is one of the factors influencing the mechanical stability of protein-peptide complex.
The cadherin superfamily of proteins is defined by the presence of extracellular cadherin (EC) re... more The cadherin superfamily of proteins is defined by the presence of extracellular cadherin (EC) repeats that engage in protein-protein interactions to mediate cell-cell adhesion, cell signaling, and mechanotransduction. The extracellular domains of non-classical cadherins often have a large number of EC repeats along with other subdomains of various folds. Protocadherin-15 (PCDH15), a protein component of the inner-ear tip link filament essential for mechanotransduction, has eleven EC repeats and a membrane adjacent domain (MAD12) of atypical fold. Here we report the crystal structure of a pig PCDH15 fragment including EC10, EC11, and MAD12 in a parallel dimeric arrangement. MAD12 has a unique molecular architecture and folds as a ferredoxin-like domain similar to that found in the nucleoporin protein Nup54. Analytical ultracentrifugation experiments along with size exclusion chromatography coupled to multi-angle laser light scattering and small-angle X-ray scattering corroborate the...
Biophysical Journal, 2016
High-throughput thermal screening (HTTS) has generally been used to analyze the stability of libr... more High-throughput thermal screening (HTTS) has generally been used to analyze the stability of libraries of mutant proteins, as well as stability changes for proteins with a battery of ligands or a plethora of buffer conditions. In all cases, an environmentally sensitive fluorescent dye (SYPRO orange) is used to monitor melting temperature shifts under various conditions. HTTS has also been used to characterize protein-protein interactions, but its general applicability to protein complexes remains to be tested. Here, we use the complex formed by the N-terminal fragments of Protocadherin-15 (pcdh15) and Cadherin-23 (cdh23), essential for inner-ear mechanotransduction, to test modified HTTS assays that evaluate protein-protein binding affinities. By mixing cdh23 and pcdh15 at different ratios, we observed increased thermal stability of pcdh15 caused by shifts in chemical equilibrium towards the complex. In addition, protein fragments carrying missense mutations known to cause deafness and reduce binding affinity lack these shifts. These results suggest that HTTS can be used to qualitatively evaluate cadherin protein-protein interactions. We further validated our HTTS results using surface plasmon resonance-based affinity measurements. Surprisingly, we found that in some cases thermal stability shifts correlate well with koff rather than Kd. Our modified protocols might be applicable to any heterodimeric protein complex with affinities ranging from ∼ 1 nanoM to 100 microM.
The Journal of Physical Chemistry A, 2012
If the Born-Oppenheimer approximation is invoked for the description of chemical reactions, the e... more If the Born-Oppenheimer approximation is invoked for the description of chemical reactions, the electron density rearranges following the motion of the nuclei. Even though this approach is central to theoretical chemistry, the explicit time dependence of the electron density is rarely studied, especially if the nuclei are treated quantum mechanically. In this article, we model the motion of benzene along the Kekulé vibrational coordinate to simulate the nuclear dynamics and electron density dynamics in the electronic ground state. Details of the change of core, valence, and π electrons are determined and analyzed. We show how the pictures anticipated by drawing Lewis structures of the rearrangement correlate with the time-dependent quantum description of the process.
Protocadherin-15 (PCDH15), an atypical member of the cadherin superfamily, is essential for verte... more Protocadherin-15 (PCDH15), an atypical member of the cadherin superfamily, is essential for vertebrate hearing and its dysfunction has been associated with deafness and progressive blindness. The PCDH15 ectodomain, made of eleven extracellular cadherin (EC1-11) repeats and a membrane adjacent domain (MAD12), assembles as a parallel homodimer that interacts with cadherin-23 (CDH23) to form the tip link, a fine filament necessary for inner-ear mechanotransduction. Here we report X-ray crystal structures of a PCDH15 + CDH23 heterotetrameric complex and ten PCDH15 fragments that were used to build complete high-resolution models of the monomeric PCDH15 ectodomain. Using molecular dynamics (MD) simulations and validated crystal contacts we propose models for complete PCDH15 parallel homodimers and the tip-link bond. Steered MD simulations of these models predict their strength and suggest conditions in which a multimodal PCDH15 ectodomain can act as a stiff or soft gating spring. These r...
PLoS ONE, 2013
Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) is a 140 kDa bi-functional enzyme inv... more Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) is a 140 kDa bi-functional enzyme involved in a coupled reaction, where the glutaminase active site produces ammonia that is subsequently utilized to convert FGAR to its corresponding amidine in an ATP assisted fashion. The structure of FGAR-AT has been previously determined in an inactive state and the mechanism of activation remains largely unknown. In the current study, hydrophobic cavities were used as markers to identify regions involved in domain movements that facilitate catalytic coupling and subsequent activation of the enzyme. Three internal hydrophobic cavities were located by xenon trapping experiments on FGAR-AT crystals and further, these cavities were perturbed via site-directed mutagenesis. Biophysical characterization of the mutants demonstrated that two of these three voids are crucial for stability and function of the protein, although being ,20 Å from the active centers. Interestingly, correlation analysis corroborated the experimental findings, and revealed that amino acids lining the functionally important cavities form correlated sets (co-evolving residues) that connect these regions to the amidotransferase active center. It was further proposed that the first cavity is transient and allows for breathing motion to occur and thereby serves as an allosteric hotspot. In contrast, the third cavity which lacks correlated residues was found to be highly plastic and accommodated steric congestion by local adjustment of the structure without affecting either stability or activity.
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Papers by Deepanshu Choudhary