Coupling between global dynamics and signal transduction pathways: a mechanism of allostery for chaperonin GroEL

Despite significant efforts toward understanding the molecular basis of allosteric communication, the mechanisms by which local energetic and conformational changes cooperatively diffuse from ligand-binding sites to distal regions across the 3-dimensional structure of allosteric proteins remain to be established. Recent experimental and theoretical evidence supports the view that allosteric communication is facilitated by the intrinsic ability of the biomolecules to undergo collective changes in structure, triggered by ligand binding. Two groups of studies recently proved to provide insights into such intrinsic, structure-induced effects: elastic network models that permit us to visualize the cooperative changes in conformation that are most readily accessible near native state conditions, and information-theoretic approaches that elucidate the most efficient pathways of signal transmission favored by the overall architecture. Using a combination of these two approaches, we highlight, by way of application to the bacterial chaperonin complex GroEL-GroES, how the most cooperative modes of motion play a role in mediating the propagation of allosteric signals. A functional coupling between the global dynamics sampled under equilibrium conditions and the signal transduction pathways inherently favored by network topology appears to control allosteric effects.     

Interesting structural and dynamical behaviors exhibited by the AF-6 PDZ domain upon Bcr peptide binding

PDZ (postsynaptic density-95, disks large, zonula occludens-1) domains are small, protein-protein interaction modules that have multiple binding surfaces for the docking of diverse molecules. These domains can propagate signals from ligand-binding site to distal regions of the structure through allosteric communication. Recent works have revealed that picosecond to nanosecond time scale dynamics play a potential role in propagating long-range signals within a protein. Comparison of AF-6 PDZ domain structures in free and complex forms shows a conformation rearrangement of distal surface 2, which is far from the peptide binding groove. The relaxation dispersion experiments detected that the free AF-6 PDZ domain was sampling multiple conformations; millisecond dynamics mapped a network for allostery signal transmission throughout the AF-6 PDZ domain in the weak saturation state, and intramolecular motions were observed in distal surface 1 when the protein was saturated. These results provide evidence that the allosteric process in the AF-6 PDZ domain is not two-state; instead, the millisecond dynamic network provides a mechanism for the transmission of allosteric signals throughout a protein. Interestingly, the two distal surfaces of the AF-6 PDZ domain respond differently to peptide binding; distal surface 1 changes in millisecond dynamics, whereas distal surface 2 undergoes structural rearrangement. The significance of the different response patterns in the signaling pathway and its relevance to the function of the AF-6 PDZ domain should be studied further.     

Regulation of signal transduction at M2 muscarinic receptor

Muscarinic acetylcholine receptors mediate transmission of an extracellular signal represented by released acetylcholine to neuronal or effector cells. There are five subtypes of closely homologous muscarinic receptors which are coupled by means of heterotrimeric G-proteins to a variety of signaling pathways resulting in a multitude of target cell effects. Endogenous agonist acetylcholine does not discriminate among individual subtypes and due to the close homology of the orthosteric binding site the same holds true for most of exogenous agonists. In addition to the classical binding site muscarinic receptors have one or more allosteric binding sites at extracellular domains. Binding of allosteric modulators induces conformational changes in the receptor that result in subtype-specific changes in orthosteric binding site affinity for both muscarinic agonists and antagonists. This overview summarizes our recent experimental effort in investigating certain aspects of M2 muscarinic receptor functioning concerning i) the molecular determinants that contribute to the binding of allosteric modulators, ii) G-protein coupling specificity and subsequent cellular responses and iii) possible functional assays that exploit the unique properties of allosteric modulators for characterization of muscarinic receptor subtypes in intact tissue. A detailed knowledge of allosteric properties of muscarinic receptors is required to permit drug design that will modulate signal transmission strength of specific muscarinic receptor subtypes. Furthermore, allosteric modulation of signal transmission strength is determined by cooperativity rather than concentration of allosteric modulator and thus reduces the danger of overdose.     

Structure-Based Network Analysis of Activation Mechanisms in the ErbB Family of Receptor Tyrosine Kinases: The Regulatory Spine Residues Are Global Mediators of Structural Stability and Allosteric Interactions

The ErbB protein tyrosine kinases are among the most important cell signaling families and mutation-induced modulation of their activity is associated with diverse functions in biological networks and human disease. We have combined molecular dynamics simulations of the ErbB kinases with the protein structure network modeling to characterize the reorganization of the residue interaction networks during conformational equilibrium changes in the normal and oncogenic forms. Structural stability and network analyses have identified local communities integrated around high centrality sites that correspond to the regulatory spine residues. This analysis has provided a quantitative insight to the mechanism of mutation-induced “superacceptor” activity in oncogenic EGFR dimers. We have found that kinase activation may be determined by allosteric interactions between modules of structurally stable residues that synchronize the dynamics in the nucleotide binding site and the αC-helix with the collective motions of the integrating αF-helix and the substrate binding site. The results of this study have pointed to a central role of the conserved His-Arg-Asp (HRD) motif in the catalytic loop and the Asp-Phe-Gly (DFG) motif as key mediators of structural stability and allosteric communications in the ErbB kinases. We have determined that residues that are indispensable for kinase regulation and catalysis often corresponded to the high centrality nodes within the protein structure network and could be distinguished by their unique network signatures. The optimal communication pathways are also controlled by these nodes and may ensure efficient allosteric signaling in the functional kinase state. Structure-based network analysis has quantified subtle effects of ATP binding on conformational dynamics and stability of the EGFR structures. Consistent with the NMR studies, we have found that nucleotide-induced modulation of the residue interaction networks is not limited to the ATP site, and may enhance allosteric cooperativity with the substrate binding region by increasing communication capabilities of mediating residues.     

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.     

Allosteric regulation and catalysis emerge via a common route

Allosteric regulation of protein function is a mechanism by which an event in one place of a protein structure causes an effect at another site, much like the behavior of a telecommunications network in which a collection of transmitters, receivers and transceivers communicate with each other across long distances. For example, ligand binding or an amino acid mutation at an allosteric site can alter enzymatic activity or binding affinity in a distal region such as the active site or a second binding site. The mechanism of this site-to-site communication is of great interest, especially since allosteric effects must be considered in drug design and protein engineering. In this review, conformational mobility as the common route between allosteric regulation and catalysis is discussed. We summarize recent experimental data and the resulting insights into allostery within proteins, and we discuss the nature of future studies and the new applications that may result from increased understanding of this regulatory mechanism.     

Validation of an Allosteric Binding Site of Src Kinase Identified by Unbiased Ligand Binding Simulations

Allostery plays a primary role in regulating protein activity, making it an important mechanism in human disease and drug discovery. Identifying allosteric regulatory sites to explore their biological significance and therapeutic potential is invaluable to drug discovery; however, identification remains a challenge. Allosteric sites are often “cryptic” without clear geometric or chemical features. Since allosteric regulatory sites are often less conserved in protein kinases than the orthosteric ATP binding site, allosteric ligands are commonly more specific than ATP competitive inhibitors. We present a generalizable computational protocol to predict allosteric ligand binding sites based on unbiased ligand binding simulation trajectories. We demonstrate the feasibility of this protocol by revisiting our previously published ligand binding simulations using the first identified viral proto-oncogene, Src kinase, as a model system. The binding paths for kinase inhibitor PP1 uncovered three metastable intermediate states before binding the high-affinity ATP-binding pocket, revealing two previously known allosteric sites and one novel site. Herein, we validate the novel site using a combination of virtual screening and experimental assays to identify a V-type allosteric small-molecule inhibitor that targets this novel site with specificity for Src over closely related kinases. This study provides a proof-of-concept for employing unbiased ligand binding simulations to identify cryptic allosteric binding sites and is widely applicable to other protein–ligand systems.     

RIalpha subunit of PKA: a cAMP-free structure reveals a hydrophobic capping mechanism for docking cAMP into site B

In eukaryotes the primary target for cAMP, a ubiquitous second messenger, is cAMP-dependent protein kinase (PKA). Understanding how binding and release of cAMP changes the cAMP binding domains and then triggers long-range allosteric responses is an important challenge. This conformational switching requires structure solutions of cAMP binding domains in cAMP-bound and cAMP-free states. We describe for the first time a crystal structure of the cAMP binding domains of PKA type Ialpha regulatory subunit where site A is occupied by cGMP and site B is unoccupied. The structure reveals that the carboxyl terminus of domain B serves as a hydrophobic cap, locking the cyclic nucleotide via its adenine ring into the beta-barrel. In the absence of cAMP, the "cap" is released via an extension of the C-terminal helix. This simple hinge mechanism for binding and release of cAMP also provides a mechanism for allosteric communication between sites A and B.     

Detection of allosteric signal transmission by information-theoretic analysis of protein dynamics

Allostery offers a highly specific way to modulate protein function. Therefore, understanding this mechanism is of increasing interest for protein science and drug discovery. However, allosteric signal transmission is difficult to detect experimentally and to model because it is often mediated by local structural changes propagating along multiple pathways. To address this, we developed a method to identify communication pathways by an information-theoretical analysis of molecular dynamics simulations. Signal propagation was described as information exchange through a network of correlated local motions, modeled as transitions between canonical states of protein fragments. The method was used to describe allostery in two-component regulatory systems. In particular, the transmission from the allosteric site to the signaling surface of the receiver domain NtrC was shown to be mediated by a layer of hub residues. The location of hubs preferentially connected to the allosteric site was found in close agreement with key residues experimentally identified as involved in the signal transmission. The comparison with the networks of the homologues CheY and FixJ highlighted similarities in their dynamics. In particular, we showed that a preorganized network of fragment connections between the allosteric and functional sites exists already in the inactive state of all three proteins.     

Ligand-dependent dynamics and intramolecular signaling in a PDZ domain

Allosteric communication is a fundamental process that proteins use to propagate signals from one site to functionally important distal sites. Although allostery is usually associated with multimeric proteins and enzymes, “long-range” communication may be a fundamental property of proteins. In some cases, communication occurs with minimal structural change. PDZ (post-synaptic density-95/discs large/zonula occludens-1) domains are small, protein-protein binding modules that can use multiple surfaces for docking diverse molecules. Furthermore, these domains have long-range energetic couplings that link the ligand-binding site to distal regions of the structure. Here, we show that allosteric behavior in a representative member of the PDZ domain family may be directly detected using side-chain methyl dynamics measurements. The changes in side-chain dynamics parameters in the second PDZ domain from the human tyrosine phosphatase 1E (hPTP1E) were determined upon binding a peptide target. Long-range dynamic effects were detected that correspond to previously observed pair-wise energetic couplings. These results provide one of the first experimental examples for the potential role of ps-ns timescale dynamics in propagating long-range signals within a protein, and reinforce the idea that dynamic fluctuations in proteins contribute to allosteric signal transduction.     

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.     

基于弹性网络模型的蛋白质变构路径与关键残基识别研究

目的 变构效应在蛋白质生物学功能执行过程中发挥着重要的调控作用，如何基于蛋白质空间结构，有效识别变构信号的传播路径和关键的残基位点是蛋白质结构-功能关系研究领域的热点科学问题。方法 本研究利用基于弹性网络模型（elastic network model，ENM）的力分布计算方法，通过分析蛋白质对外力的响应过程，来识别体系的变构路径以及变构过程中的关键残基。在该方法中，对蛋白质的关键变构位点施加外力，通过对体系形变以及内力分布情况的分析，有效识别与外力承载区域形变相耦合的关键残基，从而得到力信号在蛋白质结构内的传播路径。结果 利用该方法研究了人类磷酸甘油酸激酶（human phosphoglycerate kinase，hPGK）和蛋白质酪氨酸磷酸酶（protein tyrosine phosphatase，PTP）PDZ2结构域的变构调控路径和关键残基。对于hPGK，识别出从底物结合位点到铰链区的两条变构信号传导路径。对于PTP PDZ2，也成功识别出从配体结合位点传递到蛋白质远端的两条长程变构调控路径。计算结果与实验和分子动力学（molecular dynamics，MD）模拟得到的结果一致。结论 本研究为蛋白质体系关键残基识别及变构路径研究提供了有效的分析方法。     

Thermal repair of tryptophan synthase mutations in a regulatory intersubunit salt bridge. Evidence from arrhenius plots, absorption spectra, and primary kinetic isotope effects

This work is aimed at understanding how protein structure and conformation regulate activity and allosteric communication in the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium. Previous crystallographic and kinetic results suggest that both monovalent cations and a salt bridge between alpha subunit Asp(56) and beta subunit Lys(167) play allosteric roles. Here we show that mutation of either of these salt bridging residues produced deleterious effects that could be repaired by increased temperature in combination with CsCl or with NaCl plus an alpha subunit ligand, alpha-glycerol 3-phosphate. Arrhenius plots of the activity data under these conditions were nonlinear. The same conditions yielded temperature-dependent changes in the equilibrium distribution of enzyme-substrate intermediates and in primary kinetic isotope effects. We correlate the results with a model in which the mutant enzymes are converted by increased temperature from a low activity, "open" conformation to a high activity, "closed" conformation under certain conditions. The allosteric ligand and different monovalent cations affected the equilibrium between the open and closed forms. The results suggest that alpha subunit Asp(56) and beta subunit Lys(167) are not essential for catalysis and for allosteric communication between the alpha and beta subunits but that their mutual interaction is important in stabilization of the active, closed form of the alpha(2)beta(2) complex.     

Coupling dynamics and evolutionary information with structure to identify protein regulatory and functional binding sites

Binding sites in proteins can be either specifically functional binding sites (active sites) that bind specific substrates with high affinity or regulatory binding sites (allosteric sites), that modulate the activity of functional binding sites through effector molecules. Owing to their significance in determining protein function, the identification of protein functional and regulatory binding sites is widely acknowledged as an important biological problem. In this work, we present a novel binding site prediction method, Active and Regulatory site Prediction (AR-Pred), which supplements protein geometry, evolutionary, and physicochemical features with information about protein dynamics to predict putative active and allosteric site residues. As the intrinsic dynamics of globular proteins plays an essential role in controlling binding events, we find it to be an important feature for the identification of protein binding sites. We train and validate our predictive models on multiple balanced training and validation sets with random forest machine learning and obtain an ensemble of discrete models for each prediction type. Our models for active site prediction yield a median area under the curve (AUC) of 91% and Matthews correlation coefficient (MCC) of 0.68, whereas the less well-defined allosteric sites are predicted at a lower level with a median AUC of 80% and MCC of 0.48. When tested on an independent set of proteins, our models for active site prediction show comparable performance to two existing methods and gains compared to two others, while the allosteric site models show gains when tested against three existing prediction methods. AR-Pred is available as a free downloadable package at https://github.com/sambitmishra0628/AR-PRED_source .     

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.     

Structure of Sla1p homology domain 1 and interaction with the NPFxD endocytic internalization motif

Adaptor proteins play important endocytic roles including recognition of internalization signals in transmembrane cargo. Sla1p serves as the adaptor for uptake of transmembrane proteins containing the NPFxD internalization signal, and is essential for normal functioning of the actin cytoskeleton during endocytosis. The Sla1p homology domain 1 (SHD1) within Sla1p is responsible for recognition of the NPFxD signal. This study presents the NMR structure of the NPFxD-bound state of SHD1 and a model for the protein-ligand complex. The alpha+beta structure of the protein reveals an SH3-like topology with a solvent-exposed hydrophobic ligand binding site. NMR chemical shift perturbations and effects of structure-based mutations on ligand binding in vitro define residues that are key for NPFxD binding. Mutations that abolish ligand recognition in vitro also abolish NPFxD-mediated receptor internalization in vivo. Thus, SHD1 is a novel functional domain based on SH3-like topology, which employs a unique binding site to recognize the NPFxD endocytic internalization signal. Its distant relationship with the SH3 fold endows this superfamily with a new role in endocytosis.     

Evolutionarily conserved allosteric network in the Cys loop family of ligand-gated ion channels revealed by statistical covariance analyses

The Cys loop family of ligand-gated ion channels mediate fast synaptic transmission for communication between neurons. They are allosteric proteins, in which binding of a neurotransmitter to its binding site in the extracellular amino-terminal domain triggers structural changes in distant transmembrane domains to open a channel for ion flow. Although the locations of binding site and channel gating machinery are well defined, the structural basis of the activation pathway coupling binding and channel opening remains to be determined. In this paper, by analyzing amino acid covariance in a multiple sequence alignment, we have identified an energetically interconnected network in the Cys loop family of ligand-gated ion channels. Statistical coupling and correlated mutational analyses along with clustering revealed a highly coupled cluster. Mapping the positions in the cluster onto a three-dimensional structural model demonstrated that these highly coupled positions form an interconnected network linking experimentally identified binding domains through the coupling region to the gating machinery. In addition, these highly coupled positions are also condensed in the transmembrane domains, which are a recent focus for the sites of action of many allosteric modulators. Thus, our results revealed a genetically interconnected network that potentially plays an important role in the allosteric activation and modulation of the Cys loop family of ligand-gated ion channels.     

Coupling of global and local vibrational modes in dynamic allostery of proteins

It is now recognized that internal global protein dynamics play an important role in the allosteric function of many proteins. Alterations of protein flexibility on effector binding affect the entropic cost of binding at a distant site. We present a coarse-grained model for a potential amplification of such entropic allostery due to coupling of fast, localized modes to the slow, global modes. We show how such coupling can give rise to large compensating entropic and enthalpic terms. The model corresponds to the pattern of calorimetry and NMR data from experiments on the Met repressor.     

Multiple allosteric sites on muscarinic receptors

Proteins and small molecules are capable of regulating the agonist binding and function of G-protein coupled receptors by multiple allosteric mechanisms. In the case of muscarinic receptors, there is the well-characterised allosteric site that binds, for example, gallamine and brucine. The protein kinase inhibitor, KT5720, has now been shown to bind to a second allosteric site and to regulate agonist and antagonist binding. The binding of brucine and gallamine does not affect KT5720 binding nor its effects on the dissociation of [3H]-N-methylscopolamine from M1 receptors. Therefore it is possible to have a muscarinic receptor with three small ligands bound simultaneously. A model of the M1 receptor, based on the recently determined structure of rhodopsin, has the residues that have been shown to be important for gallamine binding clustered within and to one side of a cleft in the extracellular face of the receptor. This cleft may represent the access route of acetylcholine to its binding site.     

Ionic interactions at both inter-ring contact sites of GroEL are involved in transmission of the allosteric signal: a time-resolved infrared difference study

The biological activity of the double-ring chaperonin GroEL is regulated by complex allosteric interactions, which include positive intra-ring and negative inter-ring cooperativity. To further characterize inter-ring communication, the nucleotide-induced absorbance changes in the vibrational spectrum of the chaperonin GroEL, of two single-point mutants suppressing one inter-ring ionic contact (E461K and E434K) and of a single-ring version of this protein, were investigated by time-resolved infrared difference spectroscopy. Interaction of the nucleotide with the proteins was triggered by its photochemical release from a biologically inactive caged precursor [P3-1-(2-nitro) phenylethyl nucleotide]. The results indicate that (1) ATP binding to the protein induces a conformational change that affects concomitantly both intra-ring and inter-ring communication, and (2) the experimental absorbance changes are sensitive to the double-ring structure of the protein. The characterization of the single-point, inter-ring mutants demonstrates that ionic interactions at both contact sites are involved in the transmission of the allosteric signal. However, both mutations have different effects on the inter-ring interface. While that of E461K still retains ionic contacts sensitive to ATP binding, E434K shows spectroscopic features similar to those of the single-ring version of the protein, therefore suggesting that electrostatic interactions at these contact sites contribute differently to the stability of the inter-ring interface.