On the perturbation nature of allostery: sites,mutations, and signal modulation

Highlights

• •Perturbation nature of signaling is a fundamental feature of allosteric regulation.
• •Experimental and theoretical studies of allostery using perturbation approach.
• •Structure-based statistical mechanical model of allostery.
• •Inducing and fine-tuning targeted allosteric response.
• •From current understanding of allosteric control to future tasks in its design.

1. Download : Download high-res image (314KB)
2. Download : Download full-size image

Regardless of the diversity of systems, allosteic signalling is found to be always caused by perturbations. This recurring trait of allostery serves as a foundation for developing different experimental efforts and theoretical models for the studies of allosteric mechanisms. Among computational approaches considered here particular emphasis is given to the structure-based statistical mechanical model of allostery (SBSMMA), which allows one to study the causality and energetics of allosteric communication. We argue that the reverse allosteric signaling on the basis of SBSMMA can be used for predicting latent allosteric sites and inducing a tunable allosteric response. Per-residue allosteric effects of mutations can also be explored and ‘latent drivers’ expanding the cancer mutational landscape can be predicted using SBSMMA. Most recent and important implementations of computational models in web-resources along with a brief outlook on future research directions are also discussed.     

Exploring Mechanisms of Allosteric Regulation and Communication Switching in the Multiprotein Regulatory Complexes of the Hsp90 Chaperone with Cochaperones and Client Proteins: Atomistic Insights from Integrative Biophysical Modeling and Network Analysis of Conformational Landscapes

Understanding molecular principles underlying Hsp90 chaperone functions and modulation of client activity is fundamental to dissect activation mechanisms of many proteins. In this work, we performed a computational investigation of the Hsp90-Hsp70-Hop-CR client complex to examine allosteric regulatory mechanisms underlying dynamic chaperone interactions and principles of chaperone-dependent client recognition and remodeling. Conformational dynamics analysis using high-resolution coarse-grained simulations and ensemble-based local frustration analysis suggest that the Hsp90 chaperone could recognize and recruit the GR client by invoking reciprocal dynamic exchanges near the intermolecular interfaces with the client. Using mutational scanning of the intermolecular residues in the Hsp90-Hsp70-Hop-GR complex, we identified binding energy hotspots in the regulatory complex. Perturbation-based network analysis and dynamic fluctuations-based modeling of allosteric residue potentials are employed for a detailed analysis of allosteric interaction networks and identification of conformational communication switches. We found that allosteric interactions between the Hsp90, the client-bound Hsp70 and Hop cochaperone can define two allosteric residue clusters that control client recruitment in which the intrinsic Hsp70 allostery is exploited to mediate integration of the Hsp70-bound client into the Hsp90 chaperone system. The results suggest a model of dynamics-driven allostery that enables efficient client recruitment and loading through allosteric couplings between intermolecular interfaces and communication switch centers. This study showed that the Hsp90 interactions with client proteins may operate under dynamic-based allostery in which ensembles of preexisting conformational states and intrinsic allosteric pathways present in the Hsp90 and Hsp70 chaperones can be exploited for recognition and integration of substrate proteins.     

Role of water-bridged interactions in metal ion coupled protein allostery

Allosteric communication between distant parts of proteins controls many cellular functions, in which metal ions are widely utilized as effectors to trigger the allosteric cascade. Due to the involvement of strong coordination interactions, the energy landscape dictating the metal ion binding is intrinsically rugged. How metal ions achieve fast binding by overcoming the landscape ruggedness and thereby efficiently mediate protein allostery is elusive. By performing molecular dynamics simulations for the Ca²⁺ binding mediated allostery of the calmodulin (CaM) domains, each containing two Ca²⁺ binding helix-loop-helix motifs (EF-hands), we revealed the key role of water-bridged interactions in Ca²⁺ binding and protein allostery. The bridging water molecules between Ca²⁺ and binding residue reduces the ruggedness of ligand exchange landscape by acting as a lubricant, facilitating the Ca²⁺ coupled protein allostery. Calcium-induced rotation of the helices in the EF-hands, with the hydrophobic core serving as the pivot, leads to exposure of hydrophobic sites for target binding. Intriguingly, despite being structurally similar, the response of the two symmetrically arranged EF-hands upon Ca²⁺ binding is asymmetric. Breakage of symmetry is needed for efficient allosteric communication between the EF-hands. The key roles that water molecules play in driving allosteric transitions are likely to be general in other metal ion mediated protein 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.     

Exploring and exploiting allostery: Models, evolution, and drug targeting

The concept of allostery was elaborated almost 50years ago by Monod and coworkers to provide a framework for interpreting experimental studies on the regulation of protein function. In essence, binding of a ligand at an allosteric site affects the function at a distant site exploiting protein flexibility and reshaping protein energy landscape. Both monomeric and oligomeric proteins can be allosteric. In the past decades, the behavior of allosteric systems has been analyzed in many investigations while general theoretical models and variations thereof have been steadily proposed to interpret the experimental data. Allostery has been established as a fundamental mechanism of regulation in all organisms, governing a variety of processes that range from metabolic control to receptor function and from ligand transport to cell motility. A number of studies have shed light on how evolutionary pressures have favored and molded the development of allosteric features in specific macromolecular systems. The widespread occurrence of allostery has been recently exploited for the development and design of allosteric drugs that bind to either physiological or non-physiological allosteric sites leading to gain of function or loss of function. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.     

Allosteric sites can be identified based on the residue–residue interaction energy difference

Allosteric drugs act at a distance to regulate protein functions. They have several advantages over conventional orthosteric drugs, including diverse regulation types and fewer side effects. However, the rational design of allosteric ligands remains a challenge, especially when it comes to the identification allosteric binding sites. As the binding of allosteric ligands may induce changes in the pattern of residue–residue interactions, we calculated the residue–residue interaction energies within the allosteric site based on the molecular mechanics generalized Born surface area energy decomposition scheme. Using a dataset of 17 allosteric proteins with structural data for both the apo and the ligand‐bound state available, we used conformational ensembles generated by molecular dynamics simulations to compute the differences in the residue–residue interaction energies in known allosteric sites from both states. For all the known sites, distinct interaction energy differences (>25%) were observed. We then used CAVITY, a binding site detection program to identify novel putative allosteric sites in the same proteins. This yielded a total of 31 “druggable binding sites,” of which 21 exhibited >25% difference in residue interaction energies, and were hence predicted as novel allosteric sites. Three of the predicted allosteric sites were supported by recent experimental studies. All the predicted sites may serve as novel allosteric sites for allosteric ligand design. Our study provides a computational method for identifying novel allosteric sites for allosteric drug design. Proteins 2015; 83:1375–1384. © 2014 Wiley Periodicals, Inc.     

Chokepoints in Mechanical Coupling Associated with Allosteric Proteins: The Pyruvate Kinase Example

Although the critical role of allostery in controlling enzymatic processes is well appreciated, there is a current dearth in our understanding of its underlying mechanisms, including communication between binding sites. One potential key aspect of intersite communication is the mechanical coupling between residues in a protein. Here, we introduce a graph-based computational approach to investigate the mechanical coupling between distant parts of a protein, highlighting effective pathways via which protein motion can transfer energy between sites. In this method, each residue is treated as a node on a weighted, undirected graph, in which the edges are defined by locally correlated motions of those residues and weighted by the strength of the correlation. The method was validated against experimental data on allosteric regulation in the human liver pyruvate kinase as obtained from full-protein alanine-scanning mutagenesis (systematic mutation) studies, as well as computational data on two G-protein-coupled receptors. The method provides semiquantitative information on the regulatory importance of specific structural elements. It is shown that these elements are key for the mechanical coupling between distant parts of the protein by providing effective pathways for energy transfer. It is also shown that, although there are a multitude of energy transfer pathways between distant parts of a protein, these pathways share a few common nodes that represent effective “chokepoints” for the communication.     

Allostery without conformational change

A general model is presented whereby lignand-induced changes in protein dynamics could produce allosteric communication between distinct binding sites, even in the absence of a macromolecular conformational change. Theoretical analysis, based on the statistical thermodynamics of ligand binding, shows that cooperative interaction free energies amounting to several kJ · mol^-1 may be generated by this means. The effect arises out of the possible changes in frequencies and amplitudes of macromolecular thermal fluctuations in response to ligand attachment, and can involve all forms of dynamic behaviour, ranging from highly correlated, low-frequency normal mode vibrations to random local anharmonic motions of individual atoms or groups. Dynamic allostery of this form is primarily an entropy effect, and we derive approximate expressions which might allow the magnitude of the interaction in real systems to be calculated directly from experimental observations such as changes in normal mode frequencies and mean-square atomic displacements. Long-range influence of kinetic processes at different sites might also be mediated by a similar mechanism. We suggest that proteins and other biological macromolecules may have evolved to take functional advantage not only of mean conformational states but also of the inevitable thermal fluctuations about the mean.     

Identification of potential allosteric communication pathways between functional sites of the bacterial ribosome by graph and elastic network models

BackgroundAccumulated evidence indicates that bacterial ribosome employs allostery throughout its structure for protein synthesis. The nature of the allosteric communication between remote functional sites remains unclear, but the contact topology and dynamics of residues may play role in transmission of a perturbation to distant sites.Methods/resultsWe employ two computationally efficient approaches – graph and elastic network modeling to gain insights about the allosteric communication in ribosome. Using graph representation of the structure, we perform k-shortest pathways analysis between peptidyl transferase center-ribosomal tunnel, decoding center-peptidyl transferase center - previously reported functional sites having allosteric communication. Detailed analysis on intact structures points to common and alternative shortest pathways preferred by different states of translation. All shortest pathways capture drug target sites and allosterically important regions. Elastic network model further reveals that residues along all pathways have the ability of quickly establishing pair-wise communication and to help the propagation of a perturbation in long-ranges during functional motions of the complex.ConclusionsContact topology and inherent dynamics of ribosome configure potential communication pathways between functional sites in different translation states. Inter-subunit bridges B2a, B3 and P-tRNA come forward for their high potential in assisting allostery during translation. Especially B3 emerges as a potential druggable site.General significanceThis study indicates that the ribosome topology forms a basis for allosteric communication, which can be disrupted by novel drugs to kill drug-resistant bacteria. Our computationally efficient approach not only overlaps with experimental evidence on allosteric regulation in ribosome but also proposes new druggable sites.     

Thermodynamic coupling between neighboring binding sites in homo‐oligomeric ligand sensing proteins from mass resolved ligand‐dependent population distributions

Homo‐oligomeric ligand‐activated proteins are ubiquitous in biology. The functions of such molecules are commonly regulated by allosteric coupling between ligand‐binding sites. Understanding the basis for this regulation requires both quantifying the free energy ΔG transduced between sites, and the structural basis by which it is transduced. We consider allostery in three variants of the model ring‐shaped homo‐oligomeric trp RNA‐binding attenuation protein (TRAP). First, we developed a nearest‐neighbor statistical thermodynamic binding model comprising microscopic free energies for ligand binding to isolated sites ΔG ₀, and for coupling between adjacent sites, ΔG _α . Using the resulting partition function (PF) we explored the effects of these parameters on simulated population distributions for the 2 ^N possible liganded states. We then experimentally monitored ligand‐dependent population shifts using conventional spectroscopic and calorimetric methods and using native mass spectrometry (MS). By resolving species with differing numbers of bound ligands by their mass, native MS revealed striking differences in their ligand‐dependent population shifts. Fitting the populations to a binding polynomial derived from the PF yielded coupling free energy terms corresponding to orders of magnitude differences in cooperativity. Uniquely, this approach predicts which of the possible 2 ^N liganded states are populated at different ligand concentrations, providing necessary insights into regulation. The combination of statistical thermodynamic modeling with native MS may provide the thermodynamic foundation for a meaningful understanding of the structure–thermodynamic linkage that drives cooperativity.     

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.     

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.     

Protein mechanics: How force regulates molecular function

Background

Regulation of proteins is ubiquitous and vital for any organism. Protein activity can be altered chemically, by covalent modifications or non-covalent binding of co-factors. Mechanical forces are emerging as an additional way of regulating proteins, by inducing a conformational change or by partial unfolding.

Scope

We review some advances in experimental and theoretical techniques to study protein allostery driven by mechanical forces, as opposed to the more conventional ligand driven allostery. In this respect, we discuss recent single molecule pulling experiments as they have substantially augmented our view on the protein allostery by mechanical signals in recent years. Finally, we present a computational analysis technique, Force Distribution Analysis, that we developed to reveal allosteric pathways in proteins.

Major conclusions

Any kind of external perturbation, being it ligand binding or mechanical stretching, can be viewed as an external force acting on the macromolecule, rendering force-based experimental or computational techniques, a very general approach to the mechanics involved in protein allostery.

General significance

This unifying view might aid to decipher how complex allosteric protein machineries are regulated on the single molecular level.     

Allosteric Communication Occurs via Networks of Tertiary and Quaternary Motions in Proteins

Allosteric proteins bind an effector molecule at one site resulting in a functional change at a second site. We hypothesize that allosteric communication in proteins relies upon networks of quaternary (collective, rigid-body) and tertiary (residue–residue contact) motions. We argue that cyclic topology of these networks is necessary for allosteric communication. An automated algorithm identifies rigid bodies from the displacement between the inactive and the active structures and constructs “quaternary networks” from these rigid bodies and the substrate and effector ligands. We then integrate quaternary networks with a coarse-grained representation of contact rearrangements to form “global communication networks” (GCNs). The GCN reveals allosteric communication among all substrate and effector sites in 15 of 18 multidomain and multimeric proteins, while tertiary and quaternary networks exhibit such communication in only 4 and 3 of these proteins, respectively. Furthermore, in 7 of the 15 proteins connected by the GCN, 50% or more of the substrate-effector paths via the GCN are “interdependent” paths that do not exist via either the tertiary or the quaternary network. Substrate-effector “pathways” typically are not linear but rather consist of polycyclic networks of rigid bodies and clusters of rearranging residue contacts. These results argue for broad applicability of allosteric communication based on structural changes and demonstrate the utility of the GCN. Global communication networks may inform a variety of experiments on allosteric proteins as well as the design of allostery into non-allosteric proteins.     

The changing landscape of protein allostery

It is becoming increasingly clear that the fundamental capacity to undergo conformational change in response to ligand binding is intrinsic to proteins. This property confers on proteins the ability to be allosterically modulated in order to shift substrate binding affinities, alter enzymatic activity or regulate protein-protein interaction. How this allosteric modulation occurs--the pathways of communication, the shifting of conformational ensembles and the altered molecular dynamics--has received considerable attention during the past two years. Recent progress has helped outline the molecular origins of allostery in proteins as diverse as Hsp70 molecular chaperones and signal integrating proteins, such as WASP. In addition, allosteric properties have been successfully engineered into proteins for drug design or the development of novel biosensors. Methodological advances have provided exciting prospects for new insights and new biological roles of allosteric systems have been uncovered.     

Design and engineering of allosteric communications in proteins

Allostery in proteins plays an important role in regulating protein activities and influencing many biological processes such as gene expression, enzyme catalysis, and cell signaling. The process of allostery takes place when a signal detected at a site on a protein is transmitted via a mechanical pathway to a functional site and, thus, influences its activity. The pathway of allosteric communication consists of amino acids that form a network with covalent and non-covalent bonds. By mutating residues in this allosteric network, protein engineers have successfully established novel allosteric pathways to achieve desired properties in the target protein. In this review, we highlight the most recent and state-of-the-art techniques for allosteric communication engineering. We also discuss the challenges that need to be overcome and future directions for engineering protein allostery.     

A new concept to reveal protein dynamics based on energy dissipation

Protein dynamics is essential for its function, especially for intramolecular signal transduction. In this work we propose a new concept, energy dissipation model, to systematically reveal protein dynamics upon effector binding and energy perturbation. The concept is applied to better understand the intramolecular signal transduction during allostery of enzymes. The E. coli allosteric enzyme, aspartokinase III, is used as a model system and special molecular dynamics simulations are designed and carried out. Computational results indicate that the number of residues affected by external energy perturbation (i.e. caused by a ligand binding) during the energy dissipation process shows a sigmoid pattern. Using the two-state Boltzmann equation, we define two parameters, the half response time and the dissipation rate constant, which can be used to well characterize the energy dissipation process. For the allostery of aspartokinase III, the residue response time indicates that besides the ACT2 signal transduction pathway, there is another pathway between the regulatory site and the catalytic site, which is suggested to be the β15-αK loop of ACT1. We further introduce the term "protein dynamical modules" based on the residue response time. Different from the protein structural modules which merely provide information about the structural stability of proteins, protein dynamical modules could reveal protein characteristics from the perspective of dynamics. Finally, the energy dissipation model is applied to investigate E. coli aspartokinase III mutations to better understand the desensitization of product feedback inhibition via allostery. In conclusion, the new concept proposed in this paper gives a novel holistic view of protein dynamics, a key question in biology with high impacts for both biotechnology and biomedicine.