Local motions in a benchmark of allosteric proteins

Allosteric proteins have been studied extensively in the last 40 years, but so far, no systematic analysis of conformational changes between allosteric structures has been carried out. Here, we compile a set of 51 pairs of known inactive and active allosteric protein structures from the Protein Data Bank. We calculate local conformational differences between the two structures of each protein using simple metrics, such as backbone and side-chain Cartesian displacement, and torsion angle change and rearrangement in residue-residue contacts. Thresholds for each metric arise from distributions of motions in two control sets of pairs of protein structures in the same biochemical state. Statistical analysis of motions in allosteric proteins quantifies the magnitude of allosteric effects and reveals simple structural principles about allostery. For example, allosteric proteins exhibit substantial conformational changes comprising about 20% of the residues. In addition, motions in allosteric proteins show strong bias toward weakly constrained regions such as loops and the protein surface. Correlation functions show that motions communicate through protein structures over distances averaging 10-20 residues in sequence space and 10-20 A in Cartesian space. Comparison of motions in the allosteric set and a set of 21 nonallosteric ligand-binding proteins shows that nonallosteric proteins also exhibit bias of motion toward weakly constrained regions and local correlation of motion. However, allosteric proteins exhibit twice as much percent motion on average as nonallosteric proteins with ligand-induced motion. These observations may guide efforts to design flexibility and allostery into proteins.     

Correlating allostery with rigidity

Allosteric proteins demonstrate the phenomenon of a ligand binding to a protein at a regulatory or effector site and thereby changing the chemical affinity of the catalytic site. As such, allostery is extremely important biologically as a regulatory mechanism for molecular concentrations in many cellular processes. One particularly interesting feature of allostery is that often the catalytic and effector sites are separated by a large distance. Structural comparisons of allosteric proteins resolved in both inactive and active states indicate that a variety of structural rearrangement and changes in motions may contribute to general allosteric behavior. In general it is expected that the coupling of catalytic and regulatory sites is responsible for allosteric behavior. We utilize a novel examination of allostery using rigidity analysis of the underlying graph of the protein structures. Our results indicate a general global change in rigidity associated with allosteric transitions where the R state is more rigid than the T state. A set of allosteric proteins with heterotropic interactions is used to test the hypothesis that catalytic and effector sites are structurally coupled. Observation of a rigid path connecting the effector and catalytic sites in 68.75% of the structures points to rigidity as a means by which the distal sites communicate with each other and so contribute to allosteric regulation. Thus structural rigidity is shown to be a fundamental underlying property that promotes cooperativity and non-locality seen in allostery.     

Using affinity chromatography to engineer and characterize pH‐dependent protein switches

Conformational changes play important roles in the regulation of many enzymatic reactions. Specific motions of side chains, secondary structures, or entire protein domains facilitate the precise control of substrate selection, binding, and catalysis. Likewise, the engineering of allostery into proteins is envisioned to enable unprecedented control of chemical reactions and molecular assembly processes. We here study the structural effects of engineered ionizable residues in the core of the glutathione‐S‐transferase to convert this protein into a pH‐dependent allosteric protein. The underlying rational of these substitutions is that in the neutral state, an uncharged residue is compatible with the hydrophobic environment. In the charged state, however, the residue will invoke unfavorable interactions, which are likely to induce conformational changes that will affect the function of the enzyme. To test this hypothesis, we have engineered a single aspartate, cysteine, or histidine residue at a distance from the active site into the protein. All of the mutations exhibit a dramatic effect on the protein's affinity to bind glutathione. Whereas the aspartate or histidine mutations result in permanently nonbinding or binding versions of the protein, respectively, mutant GST50C exhibits distinct pH‐dependent GSH‐binding affinity. The crystal structures of the mutant protein GST50C under ionizing and nonionizing conditions reveal the recruitment of water molecules into the hydrophobic core to produce conformational changes that influence the protein's active site. The methodology described here to create and characterize engineered allosteric proteins through affinity chromatography may lead to a general approach to engineer effector‐specific allostery into a protein structure.     

Dynamically driven protein allostery

Allosteric interactions are typically considered to proceed through a series of discrete changes in bonding interactions that alter the protein conformation. Here we show that allostery can be mediated exclusively by transmitted changes in protein motions. We have characterized the negatively cooperative binding of cAMP to the dimeric catabolite activator protein (CAP) at discrete conformational states. Binding of the first cAMP to one subunit of a CAP dimer has no effect on the conformation of the other subunit. The dynamics of the system, however, are modulated in a distinct way by the sequential ligand binding process, with the first cAMP partially enhancing and the second cAMP completely quenching protein motions. As a result, the second cAMP binding incurs a pronounced conformational entropic penalty that is entirely responsible for the observed cooperativity. The results provide strong support for the existence of purely dynamics-driven allostery.     

Allostery and conformational free energy changes in human tryptophanyl‐tRNA synthetase from essential dynamics and structure networks

The interdependence of the concept of allostery and enzymatic catalysis, and they being guided by conformational mobility is gaining increased prominence. However, to gain a molecular level understanding of allostery and hence of enzymatic catalysis, it is of utter importance that the networks of amino acids participating in allostery be deciphered. Our lab has been exploring the methods of network analysis combined with molecular dynamics simulations to understand allostery at molecular level. Earlier we had outlined methods to obtain communication paths and then to map the rigid/flexible regions of proteins through network parameters like the shortest correlated paths, cliques, and communities. In this article, we advance the methodology to estimate the conformational populations in terms of cliques/communities formed by interactions including the side‐chains and then to compute the ligand‐induced population shift. Finally, we obtain the free‐energy landscape of the protein in equilibrium, characterizing the free‐energy minima accessed by the protein complexes. We have chosen human tryptophanyl‐tRNA synthetase (hTrpRS), a protein responsible for charging tryptophan to its cognate tRNA during protein biosynthesis for this investigation. This is a multidomain protein exhibiting excellent allosteric communication. Our approach has provided valuable structural as well as functional insights into the protein. The methodology adopted here is highly generalized to illuminate the linkage between protein structure networks and conformational mobility involved in the allosteric mechanism in any protein with known structure. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Is allostery an intrinsic property of all dynamic proteins?

Allostery involves coupling of conformational changes between two widely separated binding sites. The common view holds that allosteric proteins are symmetric oligomers, with each subunit existing in "at least" two conformational states with a different affinity for ligands. Recent observations such as the allosteric behavior of myoglobin, a classical example of a nonallosteric protein, call into question the existing allosteric dogma. Here we argue that all (nonfibrous) proteins are potentially allosteric. Allostery is a consequence of re-distributions of protein conformational ensembles. In a nonallosteric protein, the binding site shape may not show a concerted second-site change and enzyme kinetics may not reflect an allosteric transition. Nevertheless, appropriate ligands, point mutations, or external conditions may facilitate a population shift, leading a presumably nonallosteric protein to behave allosterically. In principle, practically any potential drug binding to the protein surface can alter the conformational redistribution. The question is its effectiveness in the redistribution of the ensemble, affecting the protein binding sites and its function. Here, we review experimental observations validating this view of protein allostery.     

The role of slow and fast protein motions in allosteric interactions

Allostery is fundamentally thermodynamic in nature. Long-range communication in proteins may be mediated not only by changes in the mean conformation with enthalpic contribution but also by changes in dynamic fluctuations with entropic contribution. The important role of protein motions in mediating allosteric interactions has been established by NMR spectroscopy. By using CAP as a model system, we have shown how changes in protein structure and internal dynamics can allosterically regulate protein function and activity. The results indicate that changes in conformational entropy can give rise to binding enhancement, binding inhibition, or have no effect in the expected affinity, depending on the magnitude and sign of enthalpy–entropy compensation. Moreover, allosteric interactions can be regulated by the modulation a low-populated conformation states that serve as on-pathway intermediates for ligand binding. Taken together, the interplay between fast internal motions, which are intimately related to conformational entropy, and slow internal motions, which are related to poorly populated conformational states, can regulate protein activity in a way that cannot be predicted on the basis of the protein’s ground-state structure.     

Contrasting roles of dynamics in protein allostery: NMR and structural studies of CheY and the third PDZ domain from PSD-95

Allosteric regulation is a ubiquitous phenomenon exploited in biological processes to control cells in a myriad of ways. It is also of emerging interest in the design of functional proteins and therapeutics. Even though allostery was proposed over 50 years ago and has been studied intensively from a structural perspective, many key details of allosteric mechanisms remain mysterious. Over the last decade significant attention has been paid to the “dynamic component” of allostery, as opposed to the analysis of rigid structures. Nuclear magnetic resonance spectroscopy and its ability to detect conformationally dynamic processes at atomic resolution have played an important role in expanding our understanding of allosteric mechanisms and opening up new questions. This article focuses on work that highlights how protein dynamics can factor into allosteric processes in distinct ways. Two cases are contrasted. The first considers the “traditionally allosteric” protein CheY, which undergoes a conformational change as a key element of its allostery. The second considers the more rarely observed “dynamic allostery” in a PDZ domain, in which allosteric behavior arises from changes in internal structural dynamics. Interestingly, the dynamic processes in these two contrasting examples occur on different timescales. In the case of the PDZ domain, subsequent experimental and computational work is reviewed to reveal a more complete picture of this interesting case of allostery.     

Dynamically Driven Protein Allostery Exhibits Disparate Responses for Fast and Slow Motions

There is considerable interest in the dynamic aspect of allosteric action, and in a growing list of proteins allostery has been characterized as being mediated predominantly by a change in dynamics, not a transition in conformation. For considering conformational dynamics, a protein molecule can be simplified into a number of relatively rigid microdomains connected by joints, corresponding to, e.g., communities and edges from a community network analysis. Binding of an allosteric activator strengthens intermicrodomain coupling, thereby quenching fast (e.g., picosecond to nanosecond) local motions but initiating slow (e.g., microsecond to millisecond), cross-microdomain correlated motions that are potentially of functional importance. This scenario explains allosteric effects observed in many unrelated proteins.     

The role of protein conformational fluctuations in allostery, function, and evolution

It is now well-known that proteins exist at equilibrium as ensembles of conformational states rather than as unique static structures. Here we review from an ensemble perspective important biological effects of such spontaneous fluctuations on protein allostery, function, and evolution. However, rather than present a thorough literature review on each subject, we focus instead on connecting these phenomena through the ensemble-based experimental, theoretical, and computational investigations from our laboratory over the past decade. Special emphasis is given to insights that run counter to some of the prevailing ideas that have emerged over the past 40 years of structural biology research. For instance, when proteins are viewed as conformational ensembles rather than as single structures, the commonly held notion of an allosteric pathway as an obligate series of individual structural distortions loses its meaning. Instead, allostery can result from energetic linkage between distal sites as one Boltzmann distribution of states transitions to another. Additionally, the emerging principles from this ensemble view of proteins have proven surprisingly useful in describing the role of intrinsic disorder in inter-domain communication, functional adaptation mediated by mutational control of fluctuations, and evolutionary conservation of the energetics of protein stability.     

Allostery in pharmacology: thermodynamics, evolution and design

This review focuses on basic models of allostery, the ambiguous application of the allosteric term in pharmacology illustrated by receptors, the role of thermodynamics in allosteric mechanisms, evolution and design of allostery. The initial step of ligand activation is closure of the agonist-binding cavity. Large entropy increases accompany the agonist-elicited conformational changes of pentameric ligand-gated ion channels due to cavity closure and rearrangement of transmembrane helices. The effects of point mutations on thermodynamic parameters of binding and function can reveal energetic coupling of neighbouring (and distant) amino acid residues in activation. High-order double-mutant cycle analysis and rate-equilibrium linear free-energy relationships can identify the trajectory and conformational spread of activation.Protein assembly and allostery can be deduced from colocalization and physicochemical principles. Molecular evolution has led from homooligomerization of protomers to heterotropic cooperativity and to allosteric regulation. Examples are discussed such as similar paths of protein (dis)assembly and evolution, irreversible evolution, statistical analysis of sequence homology revealing coevolution, different impacts of adaptation and evolution on hemoglobin, and the flagellar motor switch of bacteria. The driving force of dynamic allostery is associated with funnel-like free energy landscapes of protein binding and shifts in conformational fluctuations upon binding. Allostery can be designed based on our increasing knowledge of natural allosteric mechanisms and evolution. The allosteric principle has been applied for various bio/macro/molecular and signal transduction systems as well as in cognitive sciences.     

Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design

A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, K_D, in the μM range or lower). Amide hydrogen deuterium Exchange mass spectrometry (HDXMS) is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (K_D ~ nM) and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD).     

An ensemble view of thrombin allostery

Abstract Thrombin is the central protease of the coagulation cascade. Its activity is tightly regulated to ensure rapid blood clotting while preventing uncontrolled thrombosis. Thrombin interacts with multiple substrates and cofactors and is critically involved in both pro- and anticoagulant pathways of the coagulation network. Its allosteric regulation, especially by the monovalent cation Na+, has been the focus of research for more than 30 years. It is believed that thrombin can adopt an anticoagulant ('slow') conformation and, after Na+ binding, a structurally distinct procoagulant ('fast') state. In the past few years, however, the general view of allostery has evolved from one of rigid structural changes towards thermodynamic ensembles of conformational states. With this background, the view of the allosteric regulation of thrombin has also changed. The static view of the two-state model has been dismissed in favor of a more dynamic view of thrombin allostery. Herein, we review recent data that demonstrate that apo-thrombin is zymogen-like and exists as an ensemble of conformations. Furthermore, we describe how ligand binding to thrombin allosterically stabilizes conformations on the continuum from zymogen to protease.     

Differential Modulation of Functional Dynamics and Allosteric Interactions in the Hsp90-Cochaperone Complexes with p23 and Aha1: A Computational Study

Allosteric interactions of the molecular chaperone Hsp90 with a large cohort of cochaperones and client proteins allow for molecular communication and event coupling in signal transduction networks. The integration of cochaperones into the Hsp90 system is driven by the regulatory mechanisms that modulate the progression of the ATPase cycle and control the recruitment of the Hsp90 clientele. In this work, we report the results of computational modeling of allosteric regulation in the Hsp90 complexes with the cochaperones p23 and Aha1. By integrating protein docking, biophysical simulations, modeling of allosteric communications, protein structure network analysis and the energy landscape theory we have investigated dynamics and stability of the Hsp90-p23 and Hsp90-Aha1 interactions in direct comparison with the extensive body of structural and functional experiments. The results have revealed that functional dynamics and allosteric interactions of Hsp90 can be selectively modulated by these cochaperones via specific targeting of the regulatory hinge regions that could restrict collective motions and stabilize specific chaperone conformations. The protein structure network parameters have quantified the effects of cochaperones on conformational stability of the Hsp90 complexes and identified dynamically stable communities of residues that can contribute to the strengthening of allosteric interactions. According to our results, p23-mediated changes in the Hsp90 interactions may provide “molecular brakes” that could slow down an efficient transmission of the inter-domain allosteric signals, consistent with the functional role of p23 in partially inhibiting the ATPase cycle. Unlike p23, Aha1-mediated acceleration of the Hsp90-ATPase cycle may be achieved via modulation of the equilibrium motions that facilitate allosteric changes favoring a closed dimerized form of Hsp90. The results of our study have shown that Aha1 and p23 can modulate the Hsp90-ATPase activity and direct the chaperone cycle by exerting the precise control over structural stability, global movements and allosteric communications in Hsp90.     

Engineered control of enzyme structural dynamics and function

Enzymes undergo a range of internal motions from local, active site fluctuations to large‐scale, global conformational changes. These motions are often important for enzyme function, including in ligand binding and dissociation and even preparing the active site for chemical catalysis. Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions. Post‐translational covalent modification can provide an additional level of enzyme control. These studies have not only provided insights into the functional role of protein motions, but they offer opportunities to create stimulus‐responsive enzymes. These enzymes can be engineered to respond to a number of external stimuli, including light, pH, and the presence of novel allosteric modulators. Altogether, the ability to engineer and control enzyme structural dynamics can provide new tools for biotechnology and medicine.     

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring.     

Role of packing defects in the evolution of allostery and induced fit in human UDP-glucose dehydrogenase

Allosteric feedback inhibition is the mechanism by which metabolic end products regulate their own biosynthesis by binding to an upstream enzyme. Despite its importance in controlling metabolism, there are relatively few allosteric mechanisms understood in detail. This is because allostery does not have an identifiable structural motif, making the discovery of new allosteric enzymes a difficult process. The lack of a conserved motif implies that the evolution of each allosteric mechanism is unique. Here we describe an atypical allosteric mechanism in human UDP-α-d-glucose 6-dehydrogenase (hUGDH) based on an easily acquired and identifiable structural attribute: packing defects in the protein core. In contrast to classic allostery, the active and allosteric sites in hUGDH are present as a single, bifunctional site. Using two new crystal structures, we show that binding of the feedback inhibitor, UDP-α-d-xylose, elicits a distinct induced-fit response; a buried loop translates ～4 ? along and rotates ～180° about the main chain axis, requiring surrounding side chains to repack. This allosteric transition is facilitated by packing defects, which negate the steric conformational restraints normally imposed by the protein core. Sedimentation velocity studies show that this repacking favors the formation of an inactive hexameric complex with unusual symmetry. We present evidence that hUGDH and the unrelated enzyme dCTP deaminase have converged to very similar atypical allosteric mechanisms using the same adaptive strategy, the selection for packing defects. Thus, the selection for packing defects is a robust mechanism for the evolution of allostery and induced fit.     

Modulation of allostery of pyruvate kinase by shifting of an ensemble of microstates

Since the introduction of the concepts of allostery about four decades ago, much advancement has been made in elucidating the structure-function correlation in allostery. However, there are still a number of issues that remain unresolved. In this review we used mammalian pyruvate kinase (PK) as a model system to understand the role of protein dynamics in modulating cooperativity. PK has a triosephosphate isomerase (TIM)(α/β)₈ barrel structural motif. PK is an ideal system to address basic questions regarding regulatory mechanisms about this common (α/β)₈ structural motif. The simplest model accounting for all of the solution thermodynamic and kinetic data on ligand-enzyme interactions involves two conformational states, inactive E^T and active E^R. These conformational states are represented by domain movements. Further studies provide the first evidence for a differential effect of ligand binding on the dynamics of the structural elements, not major secondary structural changes. These data are consistent with our model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the inactive E^T and active E^R represent the two extreme end states. Sequence differences and ligands can modulate the distribution of states leading to alterations of functions. The future work includes: defining the network of functionally connected residues; elucidating the chemical principles governing the sequence differences which affect functions; and probing the nature of mutations on the stability of the secondary structural elements, which in turn modulate allostery.