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1.  FlexE: Using elastic network models to compare models of protein structure 
It is often valuable to compare protein structures to determine how similar they are. Structure comparison methods such as RMSD and GDT-TS are based solely on fixed geometry and do not take into account the intrinsic flexibility or energy landscape of the protein. We propose a method, which we call FlexE, that is based on a simple elastic network model and uses the deformation energy as measure of the similarity between two structures. FlexE can distinguish biologically relevant conformational changes from random changes, while existing geometry-based methods cannot. Additionally, FlexE incorporates the concept of thermal energy, which provides a rational way to determine when two models are “the same”. FlexE provides a unique measure of the similarity between protein structures that is complementary to existing methods.
doi:10.1021/ct300148f
PMCID: PMC4269272  PMID: 25530735
2.  Microseconds Simulations Reveal a New Sodium-binding Site and the Mechanism of Sodium-coupled Substrate Uptake by LeuT* 
The Journal of Biological Chemistry  2014;290(1):544-555.
Background: LeuT is a homologue of neurotransmitter transporters.
Results: Microseconds simulations disclose multiple translocation sites for Na+, including a new site for initial binding.
Conclusion: Coupled Na+- and substrate-binding events are accompanied by local and global (interhelical) rearrangements in the outward facing structure.
Significance: New insights are gained into Na+ and substrate uptake/efflux mechanisms of LeuT.
The bacterial sodium-coupled leucine/alanine transporter LeuT is broadly used as a model system for studying the transport mechanism of neurotransmitters because of its structural and functional homology to mammalian transporters such as serotonin, dopamine, or norepinephrine transporters, and because of the resolution of its structure in different states. Although the binding sites (S1 for substrate, and Na1 and Na2 for two co-transported sodium ions) have been resolved, we still lack a mechanistic understanding of coupled Na+- and substrate-binding events. We present here results from extensive (>20 μs) unbiased molecular dynamics simulations generated using the latest computing technology. Simulations show that sodium binds initially the Na1 site, but not Na2, and, consistently, sodium unbinding/escape to the extracellular (EC) region first takes place at Na2, succeeded by Na1. Na2 diffusion back to the EC medium requires prior dissociation of substrate from S1. Significantly, Na+ binding (and unbinding) consistently involves a transient binding to a newly discovered site, Na1″, near S1, as an intermediate state. A robust sequence of substrate uptake events coupled to sodium bindings and translocations between those sites assisted by hydration emerges from the simulations: (i) bindings of a first Na+ to Na1″, translocation to Na1, a second Na+ to vacated Na1″ and then to Na2, and substrate to S1; (ii) rotation of Phe253 aromatic group to seclude the substrate from the EC region; and (iii) concerted tilting of TM1b and TM6a toward TM3 and TM8 to close the EC vestibule.
doi:10.1074/jbc.M114.617555
PMCID: PMC4281755  PMID: 25381247
Biophysics; Membrane Transport; Neurotransmitter Transport; Sodium Transport; Transport; LeuT; NSS; Mechanism of Binding; Sodium-coupled
3.  Complete Mapping of Substrate Translocation Highlights the Role of LeuT N-terminal Segment in Regulating Transport Cycle 
PLoS Computational Biology  2014;10(10):e1003879.
Neurotransmitter: sodium symporters (NSSs) regulate neuronal signal transmission by clearing excess neurotransmitters from the synapse, assisted by the co-transport of sodium ions. Extensive structural data have been collected in recent years for several members of the NSS family, which opened the way to structure-based studies for a mechanistic understanding of substrate transport. Leucine transporter (LeuT), a bacterial orthologue, has been broadly adopted as a prototype in these studies. This goal has been elusive, however, due to the complex interplay of global and local events as well as missing structural data on LeuT N-terminal segment. We provide here for the first time a comprehensive description of the molecular events leading to substrate/Na+ release to the postsynaptic cell, including the structure and dynamics of the N-terminal segment using a combination of molecular simulations. Substrate and Na+-release follows an influx of water molecules into the substrate/Na+-binding pocket accompanied by concerted rearrangements of transmembrane helices. A redistribution of salt bridges and cation-π interactions at the N-terminal segment prompts substrate release. Significantly, substrate release is followed by the closure of the intracellular gate and a global reconfiguration back to outward-facing state to resume the transport cycle. Two minimally hydrated intermediates, not structurally resolved to date, are identified: one, substrate-bound, stabilized during the passage from outward- to inward-facing state (holo-occluded), and another, substrate-free, along the reverse transition (apo-occluded).
Author Summary
Bacterial leucine transporter (LeuT) belongs to neurotransmitter:sodium symporter (NSS) family. Its human orthologs include dopamine transporter and serotonin transporter. Malfunction of NSS members has been implicated in neurological diseases, hence the significance of elucidating their mechanism of function as clinically relevant drug targets. NSSs co-transport substrates (neurotransmitters or amino acids) and sodium ions across the cell membrane via alternating access to extracellular and intracellular media, which enables the uptake of substrate and ions from the extracellular region and their release to the intracellular region. Despite significant progress in elucidating the structure and function of NSS family members, their mechanism of function and the role of their N-terminal segment exposed to the cell interior remain elusive. Here, we provide for the first time a full-atomic time-resolved description of the complete transport cycle of LeuT using multiscale simulations. Two major findings are (i) elucidation of the structure and dynamics of the N-terminal segment which helps in mediating substrate and cation release and resuming the transport cycle, and (ii) determination of the structures of two minimally hydrated intermediates occluded to both extracellular and intracellular environments.
doi:10.1371/journal.pcbi.1003879
PMCID: PMC4191883  PMID: 25299050
4.  BalestraWeb: efficient online evaluation of drug–target interactions 
Bioinformatics  2014;31(1):131-133.
Summary: BalestraWeb is an online server that allows users to instantly make predictions about the potential occurrence of interactions between any given drug–target pair, or predict the most likely interaction partners of any drug or target listed in the DrugBank. It also permits users to identify most similar drugs or most similar targets based on their interaction patterns. Outputs help to develop hypotheses about drug repurposing as well as potential side effects.
Availability and implementation: BalestraWeb is accessible at http://balestra.csb.pitt.edu/. The tool is built using a probabilistic matrix factorization method and DrugBank v3, and the latent variable models are trained using the GraphLab collaborative filtering toolkit. The server is implemented using Python, Flask, NumPy and SciPy.
Contact: bahar@pitt.edu
doi:10.1093/bioinformatics/btu599
PMCID: PMC4271144  PMID: 25192741
5.  Significance of p53 dynamics in regulating apoptosis in response to ionizing radiation, and polypharmacological strategies 
Scientific Reports  2014;4:6245.
Developing pharmacological strategies for controlling ionizing radiation (IR)-induced cell death is important for both mitigating radiation damage and alleviating the side effects of anti-cancer radiotherapy manifested in surrounding tissue morbidity. Exposure to IR often triggers the onset of p53-dependent apoptotic pathways. Here we build a stochastic model of p53 induced apoptosis comprised of coupled modules of nuclear p53 activation, mitochondrial cytochrome c release and cytosolic caspase activation that also takes into account cellular heterogeneity. Our simulations show that the strength of p53 transcriptional activity and its coupling (or timing with respect) to mitochondrial pore opening are major determinants of cell fate: for systems where apoptosis is elicited via a p53-transcription-independent mechanism, direct activation of Bax by p53 becomes critical to IR-induced-damage initiation. We further show that immediate administration of PUMA inhibitors following IR exposure effectively suppresses excessive cell death, provided that there is a strong caspase/Bid feedback loop; however, the efficacy of the treatment diminishes with increasing delay in treatment implementation. In contrast, the combined inhibition of Bid and Bax elicits an anti-apoptotic response that is effective over a range of time delays.
doi:10.1038/srep06245
PMCID: PMC4150106  PMID: 25175563
6.  Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis 
PLoS Genetics  2014;10(7):e1004376.
CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms. CFTRBD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system.
Author Summary
Genetic disorders of ion channels can affect the body's ability to function properly in many ways. CFTR, an ion channel regulating movement of chloride and bicarbonate across cell membranes, is important for absorbing and secreting fluids. If the gene responsible for the CFTR channel is mutated severely, the result is cystic fibrosis, a hereditary disorder in which the patient develops thick mucus, especially in the lungs, as well as scarring (fibrosis) in the pancreas. Cystic fibrosis also affects the sweat glands, nasal sinuses, intestines, liver, and male reproductive system. Mutations to the CFTR gene that do not cause cystic fibrosis have been considered benign. However, we discovered 9 CFTR mutations that do not cause cystic fibrosis but do cause inflammation and scarring of the pancreas (chronic pancreatitis). These mutant CFTR channels secrete chloride, which is important in the sweat glands, lungs, and intestines, but not bicarbonate, which is important in the pancreas, sinuses, and male reproductive tract. We found patients with any of these 9 mutations had chronic pancreatitis, and often sinus infections, and male infertility, but not other symptoms of cystic fibrosis. Our computer models and data will help researchers develop better drugs and help physicians treating patients with chronic pancreatitis.
doi:10.1371/journal.pgen.1004376
PMCID: PMC4102440  PMID: 25033378
7.  ATPase Subdomain IA Is a Mediator of Interdomain Allostery in Hsp70 Molecular Chaperones 
PLoS Computational Biology  2014;10(5):e1003624.
The versatile functions of the heat shock protein 70 (Hsp70) family of molecular chaperones rely on allosteric interactions between their nucleotide-binding and substrate-binding domains, NBD and SBD. Understanding the mechanism of interdomain allostery is essential to rational design of Hsp70 modulators. Yet, despite significant progress in recent years, how the two Hsp70 domains regulate each other's activity remains elusive. Covariance data from experiments and computations emerged in recent years as valuable sources of information towards gaining insights into the molecular events that mediate allostery. In the present study, conservation and covariance properties derived from both sequence and structural dynamics data are integrated with results from Perturbation Response Scanning and in vivo functional assays, so as to establish the dynamical basis of interdomain signal transduction in Hsp70s. Our study highlights the critical roles of SBD residues D481 and T417 in mediating the coupled motions of the two domains, as well as that of G506 in enabling the movements of the α-helical lid with respect to the β-sandwich. It also draws attention to the distinctive role of the NBD subdomains: Subdomain IA acts as a key mediator of signal transduction between the ATP- and substrate-binding sites, this function being achieved by a cascade of interactions predominantly involving conserved residues such as V139, D148, R167 and K155. Subdomain IIA, on the other hand, is distinguished by strong coevolutionary signals (with the SBD) exhibited by a series of residues (D211, E217, L219, T383) implicated in DnaJ recognition. The occurrence of coevolving residues at the DnaJ recognition region parallels the behavior recently observed at the nucleotide-exchange-factor recognition region of subdomain IIB. These findings suggest that Hsp70 tends to adapt to co-chaperone recognition and activity via coevolving residues, whereas interdomain allostery, critical to chaperoning, is robustly enabled by conserved interactions.
Author Summary
The Hsp70 family of molecular chaperones assists in protein folding, degradation, assembly/disassembly of some complexes, and intracellular trafficking. These activities in the cell are accomplished by coupled conformational switches/signals between their nucleotide-binding and substrate-binding domains (NBD and SBD), assisted by cognate co-chaperones. Despite significant progress in the field, the molecular basis of Hsp70 machinery and the key interactions that regulate interdomain communication are not fully understood. Using a combination of experimental and computational methods, including in vivo functional assays, sequence- and structure-based analyses and perturbation response scanning, we identified a network of conserved interactions in subdomain IA of the NBD, which plays a key (effector) role in propagating signals between the ATP-binding and substrate-binding sites. Subdomain IIA, on the other hand, appears to adapt to J-domain co-chaperone binding by virtue of a broadly distributed cluster of co-evolving residues on the recognition surface.
doi:10.1371/journal.pcbi.1003624
PMCID: PMC4022485  PMID: 24831085
8.  Exploring the Conformational Transitions of Biomolecular Systems Using a Simple Two-State Anisotropic Network Model 
PLoS Computational Biology  2014;10(4):e1003521.
Biomolecular conformational transitions are essential to biological functions. Most experimental methods report on the long-lived functional states of biomolecules, but information about the transition pathways between these stable states is generally scarce. Such transitions involve short-lived conformational states that are difficult to detect experimentally. For this reason, computational methods are needed to produce plausible hypothetical transition pathways that can then be probed experimentally. Here we propose a simple and computationally efficient method, called ANMPathway, for constructing a physically reasonable pathway between two endpoints of a conformational transition. We adopt a coarse-grained representation of the protein and construct a two-state potential by combining two elastic network models (ENMs) representative of the experimental structures resolved for the endpoints. The two-state potential has a cusp hypersurface in the configuration space where the energies from both the ENMs are equal. We first search for the minimum energy structure on the cusp hypersurface and then treat it as the transition state. The continuous pathway is subsequently constructed by following the steepest descent energy minimization trajectories starting from the transition state on each side of the cusp hypersurface. Application to several systems of broad biological interest such as adenylate kinase, ATP-driven calcium pump SERCA, leucine transporter and glutamate transporter shows that ANMPathway yields results in good agreement with those from other similar methods and with data obtained from all-atom molecular dynamics simulations, in support of the utility of this simple and efficient approach. Notably the method provides experimentally testable predictions, including the formation of non-native contacts during the transition which we were able to detect in two of the systems we studied. An open-access web server has been created to deliver ANMPathway results.
Author Summary
Many biomolecules are like tiny molecular machines that need to change their shapes and visit many states to perform their biological functions. For a complete molecular understanding of a biological process, one needs to have information on the relevant stable states of the system in question, as well as the pathways by which the system travels from one state to another. We report here an efficient computational method that uses the knowledge of experimental structures of a pair of stable states in order to construct an energetically favoravle pathway between them. We adopt a simple representation of the molecular system by replacing the atoms with beads connected by springs and constructing an energy function with two minima around the end-states. We searched for the structure with highest energy that the system is most likely to visit during the transition and created two paths starting from this structure and proceeding toward the end-states. The combined result of these two paths is the minimum energy pathway between the two stable states. We apply this method to study important structural changes in one enzyme and three large proteins that transport small molecules and ions across the cell membrane.
doi:10.1371/journal.pcbi.1003521
PMCID: PMC3974643  PMID: 24699246
9.  Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells 
Nature cell biology  2013;15(10):1197-1205.
Recognition of injured mitochondria for degradation by macroautophagy is essential for cellular health, but the mechanisms remain poorly understood. Cardiolipin is an inner mitochondrial membrane phospholipid. We found that rotenone, staurosporine, 6-hydroxydopamine and other pro-mitophagy stimuli caused externalization of cardiolipin to the mitochondrial surface in primary cortical neurons and SH-SY5Y cells. RNAi knockdown of cardiolipin synthase or of phospholipid scramblase-3, which transports cardiolipin to the outer mitochondrial membrane, decreased mitochondrial delivery to autophagosomes. Furthermore, we found that the autophagy protein microtubule-associated-protein-1-light chain-3 (LC3), which mediates both autophagosome formation and cargo recognition, contains cardiolipin-binding sites important for the engulfment of mitochondria by the autophagic system. Mutation of LC3 residues predicted as cardiolipin-interaction sites by computational modeling inhibited its participation in mitophagy. These data indicate that redistribution of cardiolipin serves as an “eat-me” signal for the elimination of damaged mitochondria from neuronal cells.
doi:10.1038/ncb2837
PMCID: PMC3806088  PMID: 24036476
10.  Predicting Drug–Target Interactions Using Probabilistic Matrix Factorization 
Quantitative analysis of known drug–target interactions emerged in recent years as a useful approach for drug repurposing and assessing side effects. In the present study, we present a method that uses probabilistic matrix factorization (PMF) for this purpose, which is particularly useful for analyzing large interaction networks. DrugBank drugs clustered based on PMF latent variables show phenotypic similarity even in the absence of 3D shape similarity. Benchmarking computations show that the method outperforms those recently introduced provided that the input data set of known interactions is sufficiently large—which is the case for enzymes and ion channels, but not for G-protein coupled receptors (GPCRs) and nuclear receptors. Runs performed on DrugBank after hiding 70% of known interactions show that, on average, 88 of the top 100 predictions hit the hidden interactions. De novo predictions permit us to identify new potential interactions. Drug–target pairs implicated in neurobiological disorders are overrepresented among de novo predictions.
doi:10.1021/ci400219z
PMCID: PMC3871285  PMID: 24289468
11.  Longer simulations sample larger subspaces of conformations while maintaining robust mechanisms of motion 
Proteins  2011;10.1002/prot.23225.
Recent studies suggest that protein motions observed in molecular simulations are related to biochemical activities, although the computed time scales do not necessarily match those of the experimentally observed processes. The molecular origin of this conflicting observation is explored here for a test protein, cyanovirin-N (CV-N), through a series of molecular dynamics simulations that span a time range of three orders of magnitude up to 0.4 microseconds. Strikingly, increasing the simulation time leads to an approximately uniform amplification of the motional sizes, while maintaining the same conformational mechanics. Residue fluctuations exhibit amplitudes of 1–2 Å in the nanosecond simulations, while their average sizes increase by a factor of 4–5 in the microsecond regime. The mean-square displacements averaged over all residues (y) exhibit a power law dependence of the form y ∝ x0.26 on the simulation time (x). Essential dynamics analysis of the trajectories, on the other hand, demonstrates that CV-N has robust preferences to undergo specific types of motions that already can be detected at short simulation times, provided that multiple runs are performed and carefully analyzed.
doi:10.1002/prot.23225
PMCID: PMC3290687  PMID: 22105881
structure-encoded dynamics; molecular dynamics simulations; power law; global motions; equilibrium fluctuations of cyanovirin-N
12.  Domain Swapping Proceeds via Complete Unfolding: A 19F- and 1H- NMR Study of the Cyanovirin-N Protein 
Domain swapping creates protein oligomers by exchange of structural units between identical monomers. At present, no unifying molecular mechanism of domain swapping has emerged. Here we used the protein Cyanovirin-N and 19F-NMR to investigate the process of domain swapping. CV-N is an HIV inactivating protein that can exist as a monomer or a domain-swapped dimer. We measured thermodynamic and kinetic parameters of the conversion process and determined the size of the energy barrier between the two species. The barrier is very large and of similar magnitude to that for equilibrium unfolding of the protein. Therefore, for CV-N, overall unfolding of the polypeptide is required for domain swapping.
doi:10.1021/ja210118w
PMCID: PMC3326405  PMID: 22296296
13.  Intracellular Gating in an Inward-facing State of Aspartate Transporter GltPh Is Regulated by the Movements of the Helical Hairpin HP2* 
The Journal of Biological Chemistry  2013;288(12):8231-8237.
Background: Mechanisms of sodium-coupled substrate binding/release are largely unknown in the family of glutamate transporters.
Results: Opening of helical hairpin HP2 leads to spontaneous substrate release from an aspartate transporter in the inward-facing state.
Conclusion: Substrate dissociation is preceded by the release of a sodium ion.
Significance: Exploring the dynamics of substrate gating is crucial for understanding the mechanisms of the full transport cycle.
Sodium-coupled neurotransmitter transporters play a key role in neuronal signaling by clearing excess transmitter from the synapse. Structural data on a trimeric archaeal aspartate transporter, GltPh, have provided valuable insights into structural features of human excitatory amino acid transporters. However, the time-resolved mechanisms of substrate binding and release, as well as that of coupling to sodium co-transport, remain largely unknown for this important family. We present here the results of the most extensive simulations performed to date for GltPh in both outward-facing and inward-facing states by taking advantage of significant advances made in recent years in molecular simulation technology. The generated multiple microsecond trajectories consistently show that the helical hairpin HP2, not HP1, serves as an intracellular gate (in addition to its extracellular gating role). In contrast to previous proposals, HP1 can neither initiate nor accommodate neurotransmitter release without prior opening of HP2 by at least 4.0 Å. Aspartate release invariably follows that of a sodium ion located near the HP2 gate entrance. Asp-394 on TM8 and Arg-276 on HP1 emerge as key residues that promote the reorientation and diffusion of substrate toward the cell interior. These findings underscore the significance of examining structural dynamics, as opposed to static structure(s), to make inferences on the mechanisms of transport and key interactions.
doi:10.1074/jbc.M112.438432
PMCID: PMC3605641  PMID: 23386619
Aspartate; Membrane Transport; Neurotransmitter Transport; Sodium Transport; Transporters; Inward-facing; Sodium-coupled; Substrate Release
14.  Comparative Dynamics of NMDA- and AMPA-Glutamate Receptor N-Terminal Domains 
Structure(London, England:1993)  2012;20(11):1838-1849.
Summary
Ionotropic glutamate receptors (iGluRs) harbor two extracellular domains: the membrane-proximal ligand-binding domain (LBD) and the distal N-terminal domain (NTD). These are involved in signal sensing: the LBD binds L-glutamate, which activates the receptor channel. Ligand binding to the NTD modulates channel function in the NMDA receptor subfamily of iGluRs, which has not been observed for the AMPAR subfamily to date. Structural data suggest that AMPAR NTDs are packed into tight dimers and have lost their signaling potential. Here, we assess NTD dynamics from both subfamilies, using a variety of computational tools. We describe the conformational motions that underly NMDAR NTD allosteric signaling. Unexpectedly, AMPAR NTDs are capable of undergoing similar dynamics; although dimerization imposes restrictions, the two subfamilies sample similar, interconvertible conformational subspaces. Finally, we solve the crystal structure of AMPAR GluA4 NTD, and combined with molecular dynamics simulations, we characterize regions pivotal for an as-yet-unexplored dynamic spectrum of AMPAR NTDs.
Highlights
► Crystal structure newly resolved for AMPAR GluA4 N-terminal domain (NTD) ► Dynamics underlying NMDA NR2B NTD allostery ► Similar global dynamics between AMPA- and NMDA-receptor NTDs ► Critical role of dimer interface contacts for GluA3 NTD allostery
Dutta et al. examine the dynamics and allostery of ionotropic glutamate receptors N-terminal domains (NTDs). Systematic comparison of the members of the AMPAR subfamily, and across the AMPAR and NMDAR subfamilies, reveals mechanisms of global and local motions favored by the NTDs and allosteric potential in AMPARs.
doi:10.1016/j.str.2012.08.012
PMCID: PMC3496038  PMID: 22959625
15.  Coupling between Catalytic Loop Motions and Enzyme Global Dynamics 
PLoS Computational Biology  2012;8(9):e1002705.
Catalytic loop motions facilitate substrate recognition and binding in many enzymes. While these motions appear to be highly flexible, their functional significance suggests that structure-encoded preferences may play a role in selecting particular mechanisms of motions. We performed an extensive study on a set of enzymes to assess whether the collective/global dynamics, as predicted by elastic network models (ENMs), facilitates or even defines the local motions undergone by functional loops. Our dataset includes a total of 117 crystal structures for ten enzymes of different sizes and oligomerization states. Each enzyme contains a specific functional/catalytic loop (10–21 residues long) that closes over the active site during catalysis. Principal component analysis (PCA) of the available crystal structures (including apo and ligand-bound forms) for each enzyme revealed the dominant conformational changes taking place in these loops upon substrate binding. These experimentally observed loop reconfigurations are shown to be predominantly driven by energetically favored modes of motion intrinsically accessible to the enzyme in the absence of its substrate. The analysis suggests that robust global modes cooperatively defined by the overall enzyme architecture also entail local components that assist in suitable opening/closure of the catalytic loop over the active site.
Author Summary
Protein loops have critical roles in ligand binding and catalysis. An unresolved issue in this context is the extent to which the intrinsic dynamics of proteins predispose loops to perform their molecular function. In this work, we (i) critically examine the structural changes undergone by functional/catalytic loops based on a set of enzyme crystal structures in the presence/absence of a ligand, and (ii) examine to what extent those motions are correlated with, or driven by, the global modes that are predictable using simplified, physics-based models. Using a dataset of 117 structures for ten enzymes of different sizes and oligomerization states, we show that the collective modes defined by the protein topology favor loop rearrangements in reasonable agreement with those experimentally observed upon activation. These results suggest that simple but robust motions encoded by the entire architecture, not the local binding site only, assist in binding of the ligand, positioning of the catalytic loop, and/or sequestration of the catalytic site, which in turn, enable efficient catalysis.
doi:10.1371/journal.pcbi.1002705
PMCID: PMC3459879  PMID: 23028297
16.  A Conformational Switch in a Partially Unwound Helix Selectively Determines the Pathway for Substrate Release from the Carnitine/γ-Butyrobetaine Antiporter CaiT* 
The Journal of Biological Chemistry  2012;287(38):31823-31832.
Background: Crystal structures of an inward-facing CaiT conformation with four substrate sites are reported.
Results: This conformation is substrate-releasing. Arg262 facilitates release from the primary site.
Conclusion: Unbinding pathway from the primary site is determined by a broken helical portion of a transmembrane domain.
Significance: Mechanism of substrate release may be relevant to structural homologues such as neurotransmitter transporters.
CaiT is a homotrimeric antiporter that exchanges l-carnitine (CRN) with γ-butyrobetaine (GBB) across the bacterial membrane. Three structures have been resolved to date for CaiT, all in the inward-facing state: CRN-bound (with four CRNs per subunit), GBB-bound (two GBBs per subunit), and apo. One of the reported binding sites is the counterpart of the primary site observed in structurally similar transporters. However, the mechanism and pathway(s) of CRN/GBB unbinding and translocation, or even the ability of the substrates to dislodge from the reported binding sites, are yet to be determined. To shed light on these issues, we performed a total of 1.3 μs of molecular dynamics simulations and examined the dynamics of substrate-bound CaiT structures under different conditions. We find that both CRN and GBB are able to dissociate completely from their primary site into the cytoplasm. Substrate molecules initially located at the secondary sites dissociate even faster (within tens of nanoseconds) into the extra- or intracellular regions. Interestingly, the unbinding pathway from the primary site appears to be dictated by the geometry of the unwound part of the transmembrane (TM) helix 3, mostly around Thr100 therein. Arg262 on TM7, which apparently mimics the role of Na+ in CaiT structural homologues, plays a key role in triggering the dissociation of the substrate away from the primary site and guiding its release to the cytoplasm provided that the unwound part of TM3 switches from a shielding to a yielding pose.
doi:10.1074/jbc.M112.397364
PMCID: PMC3442516  PMID: 22843728
Biophysics; Carnitine; Membrane Proteins; Molecular Dynamics; Transport
17.  Sequence Evolution Correlates with Structural Dynamics 
Molecular Biology and Evolution  2012;29(9):2253-2263.
Biochemical activity and core stability are essential properties of proteins, maintained usually by conserved amino acids. Structural dynamics emerged in recent years as another essential aspect of protein functionality. Structural dynamics enable the adaptation of the protein to binding substrates and to undergo allosteric transitions, while maintaining the native fold. Key residues that mediate structural dynamics would thus be expected to be conserved or exhibit coevolutionary patterns at least. Yet, the correlation between sequence evolution and structural dynamics is yet to be established. With recent advances in efficient characterization of structural dynamics, we are now in a position to perform a systematic analysis. In the present study, a set of 34 enzymes representing various folds and functional classes is analyzed using information theory and elastic network models. Our analysis shows that the structural regions distinguished by their coevolution propensity as well as high mobility are predisposed to serve as substrate recognition sites, whereas residues acting as global hinges during collective dynamics are often supported by conserved residues. We propose a mobility scale for different types of amino acids, which tends to vary inversely with amino acid conservation. Our findings suggest the balance between physical adaptability (enabled by structure-encoded motions) and chemical specificity (conferred by correlated amino acid substitutions) underlies the selection of a relatively small set of versatile folds by proteins.
doi:10.1093/molbev/mss097
PMCID: PMC3424413  PMID: 22427707
protein dynamics; sequence evolution; Gaussian network model; specificity and adaptability
18.  Development of Small-Molecule PUMA Inhibitors for Mitigating Radiation-Induced Cell Death 
PUMA (p53 upregulated modulator of apoptosis) is a Bcl-2 homology 3 (BH3)-only Bcl-2 family member and a key mediator of apoptosis induced by a wide variety of stimuli. PUMA is particularly important in initiating radiation-induced apoptosis and damage in the gastrointestinal and hematopoietic systems. Unlike most BH3-only proteins, PUMA neutralizes all five known antiapoptotic Bcl-2 members through high affinity interactions with its BH3 domain to initiate mitochondria-dependent cell death. Using structural data on the conserved interactions of PUMA with Bcl-2-like proteins, we developed a pharmacophore model that mimics these interactions. In silico screening of the ZINC 8.0 database with this pharmacophore model yielded 142 compounds that could potentially disrupt these interactions. Thirteen structurally diverse compounds with favorable in silico ADME/Toxicity profiles have been retrieved from this set. Extensive testing of these compounds using cell-based and cell-free systems identified lead compounds that confer considerable protection against PUMA-dependent and radiation-induced apoptosis, and inhibit the interaction between PUMA and Bcl-xL.
PMCID: PMC3086011  PMID: 21320058
Inhibition of PUMA-induced apoptosis; Bcl-2 protein family; BH3 domain; protein-protein interactions; pharmacophore modeling; druggability; virtual screening of libraries of small compounds
19.  The mechanism of substrate release by the aspartate transporter GltPh: insights from simulations† 
Molecular bioSystems  2010;7(3):832-842.
Glutamate transporters regulate excitatory amino acid neurotransmission across neuronal and glial cell membranes by coupling the translocation of their substrate (aspartate or glutamate) into the intracellular (IC) medium to the energetically favorable transport of sodium ions or other cations. The first crystallographically resolved structure of this family, the archaeal aspartate transporter, GltPh, has served as a structural paradigm for elucidating the mechanism of substrate translocation by these transporters. Two helical hairpins, HP2 and HP1, at the core domains of the three subunits that form this membrane protein have been proposed to act as the respective extracellular and IC gates for substrate intake and release. Molecular dynamics simulations using the outward-facing structure have confirmed that the HP2 loop acts as an EC gate. The mechanism of substrate release at atomic scale, however, remained unknown due to the lack of structural data until the recent determination of the inward-facing structure of GltPh. In the present study, we use this recently resolved structure to simulate the release of substrate to the cytoplasm and the roles of HP1 and HP2 in this process. The highly flexible HP2 loop is observed to serve as an activator (or initiator) prompting the release of a gatekeeper Na+ to the cytoplasm and promoting the influx of water molecules from the cytoplasm, which effectively disrupt substrate–protein interactions and drive the dislodging of the substrate from its binding site. The completion of substrate release and exit, however, entails the opening of the highly stable HP1 loop as well. Overall, the unique conformational flexibility of the HP2 loop, the dissociation of a Na+, the hydration of binding pocket, and final yielding of the HP1 loop 3-Ser motif emerge as the successive events controlling the release of the bound substrate to the cell interior by glutamate transporters.
doi:10.1039/c0mb00175a
PMCID: PMC3227142  PMID: 21161089
20.  Changes in Dynamics upon Oligomerization Regulate Substrate Binding and Allostery in Amino Acid Kinase Family Members 
PLoS Computational Biology  2011;7(9):e1002201.
Oligomerization is a functional requirement for many proteins. The interfacial interactions and the overall packing geometry of the individual monomers are viewed as important determinants of the thermodynamic stability and allosteric regulation of oligomers. The present study focuses on the role of the interfacial interactions and overall contact topology in the dynamic features acquired in the oligomeric state. To this aim, the collective dynamics of enzymes belonging to the amino acid kinase family both in dimeric and hexameric forms are examined by means of an elastic network model, and the softest collective motions (i.e., lowest frequency or global modes of motions) favored by the overall architecture are analyzed. Notably, the lowest-frequency modes accessible to the individual subunits in the absence of multimerization are conserved to a large extent in the oligomer, suggesting that the oligomer takes advantage of the intrinsic dynamics of the individual monomers. At the same time, oligomerization stiffens the interfacial regions of the monomers and confers new cooperative modes that exploit the rigid-body translational and rotational degrees of freedom of the intact monomers. The present study sheds light on the mechanism of cooperative inhibition of hexameric N-acetyl-L-glutamate kinase by arginine and on the allosteric regulation of UMP kinases. It also highlights the significance of the particular quaternary design in selectively determining the oligomer dynamics congruent with required ligand-binding and allosteric activities.
Author Summary
Protein function requires a three-dimensional structure with specific dynamic features for catalytic and binding events, and, in many cases, the structure results from the assembly of more than one polypeptide chain (also called monomer or subunit) to form an oligomer or multimer. Proteins such as hemoglobin or chaperonin GroEL are oligomers formed by 2 and 14 subunits, respectively, whereas virus capsids are multimers composed of hundreds of monomers. In these cases, the architecture of the interface between the subunits and the overall assembly geometry are essential in determining the functional motions that these sophisticated structures are able to perform under physiological conditions. Here we present results from our computational study of the large-amplitude motions of dimeric and hexameric proteins that belong to the Amino Acid Kinase family. Our study reveals that the monomers in these oligomeric proteins are arranged in such a way that the oligomer inherits the intrinsic dynamic features of its components. The packing geometry additionally confers the ability to perform highly cooperative conformational changes that involve all monomers and enable the biological activity of the multimer. The study highlights the significance of the quaternary design in favoring the oligomer dynamics that enables ligand-binding and allosteric regulation functions.
doi:10.1371/journal.pcbi.1002201
PMCID: PMC3182869  PMID: 21980279
21.  Metal-binding sites are designed to achieve optimal mechanical and signaling properties 
Structure (London, England : 1993)  2010;18(9):1140-1148.
Many proteins require bound metals to achieve their function. We take advantage of increasing structural data on metal-binding proteins to elucidate three properties: the involvement of metal-binding sites in the global dynamics of the protein, predicted by elastic network models, their exposure/burial to solvent, and their signal-processing properties indicated by Markovian stochastics analysis. Systematic analysis of a dataset of 145 structures reveals that the residues that coordinate metal ions enjoy remarkably efficient and precise signal transduction properties. These properties are rationalized in terms of their physical properties: participation in hinge sites that control the softest modes collectively accessible to the protein and occupancy of central positions minimally exposed to solvent. Our observations suggest that metal-binding sites may have been evolutionary selected to achieve optimum allosteric communication. They also provide insights into basic principles for designing metal-binding sites, which are verified to be met by recently designed de novo metal-binding proteins.
doi:10.1016/j.str.2010.06.013
PMCID: PMC2937013  PMID: 20826340
metal-binding sites; elastic network models; GNM; signal propagation; allosteric communication
22.  ProDy: Protein Dynamics Inferred from Theory and Experiments 
Bioinformatics  2011;27(11):1575-1577.
Summary: We developed a Python package, ProDy, for structure-based analysis of protein dynamics. ProDy allows for quantitative characterization of structural variations in heterogeneous datasets of structures experimentally resolved for a given biomolecular system, and for comparison of these variations with the theoretically predicted equilibrium dynamics. Datasets include structural ensembles for a given family or subfamily of proteins, their mutants and sequence homologues, in the presence/absence of their substrates, ligands or inhibitors. Numerous helper functions enable comparative analysis of experimental and theoretical data, and visualization of the principal changes in conformations that are accessible in different functional states. ProDy application programming interface (API) has been designed so that users can easily extend the software and implement new methods.
Availability: ProDy is open source and freely available under GNU General Public License from http://www.csb.pitt.edu/ProDy/.
Contact: ahb12@pitt.edu; bahar@pitt.edu
doi:10.1093/bioinformatics/btr168
PMCID: PMC3102222  PMID: 21471012
23.  A Comparative Analysis of the Equilibrium Dynamics of a Designed Protein Inferred from NMR, X-ray and Computations 
Proteins  2009;77(4):927-939.
A detailed analysis of high-resolution structural data and computationally predicted dynamics was carried out for a designed sugar binding protein. The mean-square-deviations in the positions of residues derived from Nuclear Magnetic Resonance (NMR) models, and those inferred from X-ray crystallographic B-factors for two different crystal forms were compared with the predictions based on the Gaussian Network Model (GNM), and the results from molecular dynamics (MD) simulations. GNM systematically yielded a higher correlation than MD, with experimental data, suggesting that the lack of atomistic details in the coarse-grained GNM is more than compensated for by the mathematically exact evaluation of fluctuations using the native contacts topology. Evidence is provided that particular loop motions are curtailed by intermolecular contacts in the crystal environment causing a discrepancy between theory and experiments. Interestingly, the information conveyed by X-ray crystallography becomes more consistent with NMR models and computational predictions when ensembles of X-ray models are considered. Less precise (broadly distributed) ensembles indeed appear to describe the accessible conformational space under native state conditions better than B-factors. Our results highlight the importance of utilizing multiple conformations obtained by alternative experimental methods, and analyzing results from both coarse-grained models and atomic simulations, for accurate assessment of motions accessible to proteins under native state conditions.
doi:10.1002/prot.22518
PMCID: PMC2767477  PMID: 19688820
equilibrium dynamics; ensemble of conformations; inter-residue contact topology; crystal contacts; elastic network model; sugar-binding protein
25.  Role of Hsp70 ATPase Domain Intrinsic Dynamics and Sequence Evolution in Enabling its Functional Interactions with NEFs 
PLoS Computational Biology  2010;6(9):e1000931.
Catalysis of ADP-ATP exchange by nucleotide exchange factors (NEFs) is central to the activity of Hsp70 molecular chaperones. Yet, the mechanism of interaction of this family of chaperones with NEFs is not well understood in the context of the sequence evolution and structural dynamics of Hsp70 ATPase domains. We studied the interactions of Hsp70 ATPase domains with four different NEFs on the basis of the evolutionary trace and co-evolution of the ATPase domain sequence, combined with elastic network modeling of the collective dynamics of the complexes. Our study reveals a subtle balance between the intrinsic (to the ATPase domain) and specific (to interactions with NEFs) mechanisms shared by the four complexes. Two classes of key residues are distinguished in the Hsp70 ATPase domain: (i) highly conserved residues, involved in nucleotide binding, which mediate, via a global hinge-bending, the ATPase domain opening irrespective of NEF binding, and (ii) not-conserved but co-evolved and highly mobile residues, engaged in specific interactions with NEFs (e.g., N57, R258, R262, E283, D285). The observed interplay between these respective intrinsic (pre-existing, structure-encoded) and specific (co-evolved, sequence-dependent) interactions provides us with insights into the allosteric dynamics and functional evolution of the modular Hsp70 ATPase domain.
Author Summary
The heat shock protein 70 (Hsp70) serves as a housekeeper in the cell, assisting in the correct folding, trafficking, and degradation of many proteins. The ATPase domain is the control unit of this molecular machine and its efficient functioning requires interactions with co-chaperones, including, in particular, the nucleotide exchange factors (NEFs). We examined the molecular motions of the ATPase domain in both NEF-bound and -unbound forms. We found that the NEF-binding surface enjoys large global movements prior to NEF binding, which presumably facilitates NEF recognition and binding. NEF binding stabilizes the ATPase domain in an open form and thereby facilitates the nucleotide exchange step of the chaperone cycle. A series of highly correlated amino acids were distinguished at the NEF-binding sites of the Hsp70 ATPase domain, which highlights the adaptability of the ATPase domain, both structurally and sequentially, to recognize NEFs. In contrast, the nucleotide-binding residues are tightly held near a global hinge center and are highly conserved. The contrasting properties of these two groups of residues point to an evolutionarily optimized balance between conserved/constrained and co-evolved/mobile amino acids, which enables the functional interactions of the modular Hps70 ATPase domains with NEFs.
doi:10.1371/journal.pcbi.1000931
PMCID: PMC2940730  PMID: 20862304

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