Dynamic Allostery Research Articles

Ensemble-Based Analysis of the Dynamic Allostery in the PSD-95 PDZ3 Domain in Relation to the General Variability of PDZ Structures.

PDZ domains are abundant interaction hubs found in a number of different proteins and they exhibit characteristic differences in their structure and ligand specificity. Their internal dynamics have been proposed to contribute to their biological activity via changes in conformational entropy upon ligand binding and allosteric modulation. Here we investigate dynamic structural ensembles of PDZ3 of the postsynaptic protein PSD-95, calculated based on previously published backbone and side-chain S order parameters. We show that there are distinct but interdependent structural rearrangements in PDZ3 upon ligand binding and the presence of the intramolecular allosteric modulator helix 3. We have also compared these rearrangements in PDZ1-2 of PSD-95 and the conformational diversity of an extended set of PDZ domains available in the PDB database. We conclude that although the opening-closing rearrangement, occurring upon ligand binding, is likely a general feature for all PDZ domains, the conformer redistribution upon ligand binding along this mode is domain-dependent. Our findings suggest that the structural and functional diversity of PDZ domains is accompanied by a diversity of internal motional modes and their interdependence.

Protonation-Induced Dynamic Allostery in PDZ Domain: Evidence of Perturbation-Independent Universal Response Network.

Dynamic allostery is a relatively new paradigm where certain external perturbations may lead to modulation of conformational dynamics at a distant part of a protein without significant changes in the overall structure. While most well-characterized examples of dynamic allostery involve binding with other entities like small molecules, peptides, or nucleic acids, in this work we demonstrate that chemical modifications like protonation may lead to significant dynamical allosteric response in a PDZ domain protein. Tuning the protonation states of two histidine residues (H317 and H372), we identify the allosteric pathways responsible for the dynamic response. Interestingly, the same set of residues that constitute the allosteric response network upon ligand binding seem to be responsible for protonation-induced dynamic allostery. Thus, we propose the existence of an inherent universal response network in signaling proteins, where the same set of residues can respond to varying types of external perturbations in terms of rearrangement of hydrogen-bonded network and redistribution of electrostatic interaction energies.

Protein Allostery at Atomic Resolution

AbstractProtein allostery is a phenomenon involving the long range coupling between two distal sites in a protein. In order to elucidate allostery at atomic resoluion on the ligand‐binding WW domain of the enzyme Pin1, multistate structures were calculated from exact nuclear Overhauser effect (eNOE). In its free form, the protein undergoes a microsecond exchange between two states, one of which is predisposed to interact with its parent catalytic domain. In presence of the positive allosteric ligand, the equilibrium between the two states is shifted towards domain–domain interaction, suggesting a population shift model. In contrast, the allostery‐suppressing ligand decouples the side‐chain arrangement at the inter‐domain interface thereby reducing the inter‐domain interaction. As such, this mechanism is an example of dynamic allostery. The presented distinct modes of action highlight the power of the interplay between dynamics and function in the biological activity of proteins.

Protein Allostery at Atomic Resolution.

Protein allostery is a phenomenon involving the long range coupling between two distal sites in a protein. In order to elucidate allostery at atomic resoluion on the ligand-binding WW domain of the enzyme Pin1, multistate structures were calculated from exact nuclear Overhauser effect (eNOE). In its free form, the protein undergoes a microsecond exchange between two states, one of which is predisposed to interact with its parent catalytic domain. In presence of the positive allosteric ligand, the equilibrium between the two states is shifted towards domain-domain interaction, suggesting a population shift model. In contrast, the allostery-suppressing ligand decouples the side-chain arrangement at the inter-domain interface thereby reducing the inter-domain interaction. As such, this mechanism is an example of dynamic allostery. The presented distinct modes of action highlight the power of the interplay between dynamics and function in the biological activity of proteins.

The αβTCR mechanosensor exploits dynamic ectodomain allostery to optimize its ligand recognition site

Each [Formula: see text]T cell receptor (TCR) functions as a mechanosensor. The TCR is comprised of a clonotypic TCR[Formula: see text] ligand-binding heterodimer and the noncovalently associated CD3 signaling subunits. When bound by ligand, an antigenic peptide arrayed by a major histocompatibility complex molecule (pMHC), the TCR[Formula: see text] has a longer bond lifetime under piconewton-level loads. The atomistic mechanism of this "catch bond" behavior is unknown. Here, we perform molecular dynamics simulation of a TCR[Formula: see text]-pMHC complex and its variants under physiologic loads to identify this mechanism and any attendant TCR[Formula: see text] domain allostery. The TCR[Formula: see text]-pMHC interface is dynamically maintained by contacts with a spectrum of occupancies, introducing a level of control via relative motion between Vα and Vβ variable domains containing the pMHC-binding complementarity-determining region (CDR) loops. Without adequate load, the interfacial contacts are unstable, whereas applying sufficient load suppresses Vα-Vβ motion, stabilizing the interface. A second level of control is exerted by Cα and Cβ constant domains, especially Cβ and its protruding FG-loop, that create mismatching interfaces among the four TCR[Formula: see text] domains and with a pMHC ligand. Applied load enhances fit through deformation of the TCR[Formula: see text] molecule. Thus, the catch bond involves the entire TCR[Formula: see text] conformation, interdomain motion, and interfacial contact dynamics, collectively. This multilayered architecture of the machinery fosters fine-tuning of cellular response to load and pMHC recognition. Since the germline-derived TCR[Formula: see text] ectodomain is structurally conserved, the proposed mechanism can be universally adopted to operate under load during immune surveillance by diverse [Formula: see text]TCRs constituting the T cell repertoire.

A single amino acid substitution uncouples catalysis and allostery in an essential biosynthetic enzyme in Mycobacterium tuberculosis

Allostery exploits the conformational dynamics of enzymes by triggering a shift in population ensembles toward functionally distinct conformational or dynamic states. Allostery extensively regulates the activities of key enzymes within biosynthetic pathways to meet metabolic demand for their end products. Here, we have examined a critical enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), at the gateway to aromatic amino acid biosynthesis in Mycobacterium tuberculosis, which shows extremely complex dynamic allostery: three distinct aromatic amino acids jointly communicate occupancy to the active site via subtle changes in dynamics, enabling exquisite fine-tuning of delivery of these essential metabolites. Furthermore, this allosteric mechanism is co-opted by pathway branchpoint enzyme chorismate mutase upon complex formation. In this study, using statistical coupling analysis, site-directed mutagenesis, isothermal calorimetry, small-angle X-ray scattering, and X-ray crystallography analyses, we have pinpointed a critical node within the complex dynamic communication network responsible for this sophisticated allosteric machinery. Through a facile Gly to Pro substitution, we have altered backbone dynamics, completely severing the allosteric signal yet remarkably, generating a nonallosteric enzyme that retains full catalytic activity. We also identified a second residue of prime importance to the inter-enzyme communication with chorismate mutase. Our results reveal that highly complex dynamic allostery is surprisingly vulnerable and provide further insights into the intimate link between catalysis and allostery.

The Complete Molecular Analysis of PTP1B Function

Protein function originates from a cooperation of structural rigidity, dynamics at different timescales and allostery. Recently, we showed that PTP1B, a drug target for diabetes and cancer that catalyzes the dephosphorylation of numerous substrates in essential signaling pathways, uses both conformational and dynamic allostery to communicate between its allosteric and active sites. Further, we also discovered that the rigidity of the catalytically essential WPD loop is indispensable for PTP1B activity, demonstrating that both conformational rigidity and dynamics are essential for controlling protein activity. To determine if additional pockets distal from the active site may also influence PTP1B activity, we integrated bioinformatics analysis, activity assays, X‐ray crystallography and a broad variety of different NMR dynamics measurements. Our data revealed the presence of additional sites, distinct from the established allosteric pocket, which can potently alter PTP1B activity when perturbed. Our data suggest that that are a variety of ways to modulate the catalytic activity of PTP1B, and, in turn, may mitigate disorders due to PTP1B dysregulation, such as cancer.Support or Funding InformationT32 GM008804

Allosteric Activation of PI3Kα Results in Dynamic Access to Catalytically Competent Conformations

Class I phosphoinositide-3-kinases (PI3Ks) phosphorylate PIP2 at its 3' inositol position to generate PIP3, a second messenger that influences signaling cascades regulating cellular growth, survival, and proliferation. Previous studies have suggested that PI3Kα activation involves dislodging the p85α nSH2 domain from the p110α catalytic subunit by binding activated receptor tyrosine kinases. We carried out molecular dynamics simulations to determine, mechanistically and structurally, how PI3Kα conformations are influenced by physiological effectors and the nSH2 domain. We demonstrate that changes in protein dynamics mediated by allosteric regulation significantly increase the population of catalytically competent states without changing the enzyme ground-state structure. Furthermore, we demonstrate that modulation of active-site residue interactions with enzyme substrates can reciprocally influence nSH2 domain dynamics. Together, these results suggest that dynamic allostery plays a role in populating the catalytically competent conformation of PI3Kα, and provide a key platform for the design of novel chemotherapeutic PI3Kα inhibitors.

Investigating the Residue-Level Dynamics of the Thrombin-Thrombomodulin Interaction

The serine protease, thrombin catalyzes the final step in the blood clotting cascade by cleaving, and thereby activating, proteins in the blood that consequently form a blood clot. However, thrombin has also demonstrated an anticoagulative role as well, which is initiated by the binding of the cofactor thrombomodulin to thrombin, and is characterized by a change in thrombin substrate affinity away from procoagulative substrates and towards activation of protein C instead. Despite decades of study, the mechanism behind this shift in the substrate preference of thrombin remains unclear. However, evidence from previous studies suggests that this change in activity results from changes in the intermolecular protein dynamics of thrombin that are induced by the binding of thrombomodulin. As a result, the thrombin-thrombomodulin interaction has been suggested to undergo dynamic allostery. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) experiments have been used to identify the solvent-exposed regions of thrombin that undergo changes in protein dynamics when thrombomodulin is present, yet the limits of the HDX-MS technique prevent the identification of changes at residue-level resolution, as well as any changes in motion of residues buried in the protein core of thrombin. In order to supplement the HDX-MS data collected, Curr-Purcell-Meibloom-Gill (CPMG) NMR experiments have been conducted on S195M mutant thrombin in the presence of thrombomodulin. The results of these experiments will be compared to previous CPMG results published on S195M Mutant thrombin in the absence of thrombomodulin in order to identify which residues are affected by the binding of thrombomodulin to thrombin. Thus, by comparing the rates of transitions to rare states determined from the CPMG experiments, new insights regarding the allosteric transition between thrombin's procoagulative and anticoagulative states will be obtained.

DECA, A Comprehensive, Automatic Post-processing Program for HDX-MS Data*

Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) has become widely popular for mapping protein-ligand interfaces, for understanding protein-protein interactions, and for discovering dynamic allostery. Several platforms are now available which provide large data sets of amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) data. Although many of these platforms provide some down-stream processing, a comprehensive software that provides the most commonly used down-stream processing tools such as automatic back-exchange correction options, analysis of overlapping peptides, calculations of relative deuterium uptake into regions of the protein after such corrections, rigorous statistical analysis of the significance of uptake differences, and generation of high quality figures for data presentation is not yet available. Here we describe the Deuterium Exchange Correction and Analysis (DECA) software package, which provides all these downstream processing options for data from the most popular mass spectrometry platforms. The major functions of the software are demonstrated on sample data.

Dynamic regulatory features of the protein tyrosine kinases.

The SRC, Abelson murine leukemia viral oncogene homolog 1, TEC and C-terminal SRC Kinase families of non-receptor tyrosine kinases (collectively the Src module kinases) mediate an array of cellular signaling processes and are therapeutic targets in many disease states. Crystal structures of Src modules kinases provide valuable insights into the regulatory mechanisms that control activation and generate a framework from which drug discovery can advance. The conformational ensembles visited by these multidomain kinases in solution are also key features of the regulatory machinery controlling catalytic activity. Measurement of dynamic motions within kinases substantially augments information derived from crystal structures. In this review, we focus on a body of work that has transformed our understanding of non-receptor tyrosine kinase regulation from a static view to one that incorporates how fluctuations in conformational ensembles and dynamic motions influence activation status. Regulatory dynamic networks are often shared across and between kinase families while specific dynamic behavior distinguishes unique regulatory mechanisms for select kinases. Moreover, intrinsically dynamic regions of kinases likely play important regulatory roles that have only been partially explored. Since there is clear precedence that kinase inhibitors can exploit specific dynamic features, continued efforts to define conformational ensembles and dynamic allostery will be key to combating drug resistance and devising alternate treatments for kinase-associated diseases.

Autoencoder-Based Detection of Dynamic Allostery Triggered by Ligand Binding Based on Molecular Dynamics.

Dynamic allostery on proteins, triggered by regulator binding or chemical modifications, transmits information from the binding site to distant regions, dramatically altering protein function. It is accompanied by subtle changes in side-chain conformations of the protein, indicating that the changes in dynamics, and not rigid or large conformational changes, are essential to understand regulation of protein function. Although a lot of experimental and theoretical studies have been dedicated to investigate this issue, the regulation mechanism of protein function is still being debated. Here, we propose an autoencoder-based method that can detect dynamic allostery. The method is based on the comparison of time fluctuations of protein structures, in the form of distance matrices, obtained from molecular dynamics simulations in ligand-bound and -unbound forms. Our method detected that the changes in dynamics by ligand binding in the PDZ2 domain led to the reorganization of correlative fluctuation motions among residue pairs, which revealed a different view of the correlated motions from the PCA and DCCM. In addition, other correlative motions were also found as a result of the dynamic perturbation from the ligand binding, which may lead to dynamic allostery. This autoencoder-based method would be usefully applied to the signal transduction and mutagenesis systems involved in protein functions and severe diseases.

Single Point Mutations Modulate Dynamical Allostery in IgG4 Monoclonal Antibodies

A novel computational method [1] is employed to investigate dynamical allostery within a set of IgG4 antibody mutants. Single point mutations in the CH3-CH3 interfacial region are known to decrease stability of the bivalent form [2,3]. We analyze long-range dynamical couplings that appear in the native protein and in mutants: F405R, T394D, Y349D and L368W. A covariance matrix is constructed from 2μs molecular dynamics trajectories from coarse-grained simulations using the Martini force field. An effective elastic network model is constructed based on inverting the covariance matrix at the residue level. The effective elastic network is subsequently perturbed at targeted residues, which increases or decreases the elastic strength between all residue pairs for residues within 10 Angstroms from the perturbation site. Upon perturbation, a response in the mean square fluctuation (MSF) is monitored at all distal residues. A stabilizing or destabilizing response is respectively identified when MSF decreases or increases by a statistically significant amount, and all IgG4 proteins exhibit long-range couplings. Destabilizing response occurs in CDR loops based on perturbations in the constant CH2 region. In the reverse direction, stabilizing perturbations in CDR loops produce destabilizing responses in the CH2 regions. Both of these regions are critical for function in antibody-dependent cell-mediated cytotoxicity, suggesting the presence of dynamical allostery. Similar response profiles group in two pairs of mutants in corroboration with the experiments [3], where the mutations F405R and T394D increase the half-antibody population, while Y349D and L368W establish monomer-dimer equilibrium. Furthermore, the allosteric pathways are modulated by the mutations, which has important ramifications for protein design and formulation. [1] Azhagiya Singam E.R., et. al. J. Am. Chem. Soc. 139(48) 17508-17517 (2017). [2] Shan, L., et. al. PLoS ONE 11:e0160345 (2016). [3] Wilkinson, I.C., et. al. MAbs 5:406-417 (2013).

Cold Adaptation in an Enzyme Can Be Driven by Dynamic Allostery

The ability of organisms to diverge and adapt to different environmental niches is a hallmark of evolution, and one of the most prevalent examples is that of thermal adaptation, where two similar organisms can evolve to survive at different extremes of temperature. Underlying the physiological differences between such organisms are changes in the enzymes that catalyze the various reactions, and these changes are often located at surface-exposed amino acids distant from the active site. How such changes mediate the observed differences is activity is not clear. Here we examine surface-exposed GLY mutations to LID and AMPbd domains of E. Coli. adenylate kinase (AK), two domains that are purported to be responsible for mediating enzyme turnover. We show using advanced NMR techniques as well a both isothermal titration (ITC) and differential scanning calorimetry (DSC) that Km (or substrate affinity for ligand), and kcat can be independently and rationally tuned, by making specific surface-exposed mutations in different regions of the molecule. These studies provide insight into how enzymes can evolve to utilize local fluctuations to control catalysis and how evolution can precisely and independently tune these parameters.

A Thermodynamic View of Dynamic Allostery in a PDZ Domain Protein

A Thermodynamic View of Dynamic Allostery in a PDZ Domain Protein

Integrating Experiment and Theory to Understand TCR-pMHC Dynamics.

The conformational dynamism of proteins is well established. Rather than having a single structure, proteins are more accurately described as a conformational ensemble that exists across a rugged energy landscape, where different conformational sub-states interconvert. The interaction between αβ T cell receptors (TCR) and cognate peptide-MHC (pMHC) is no exception, and is a dynamic process that involves substantial conformational change. This review focuses on technological advances that have begun to establish the role of conformational dynamics and dynamic allostery in TCR recognition of the pMHC and the early stages of signaling. We discuss how the marriage of molecular dynamics (MD) simulations with experimental techniques provides us with new ways to dissect and interpret the process of TCR ligation. Notably, application of simulation techniques lags behind other fields, but is predicted to make substantial contributions. Finally, we highlight integrated approaches that are being used to shed light on some of the key outstanding questions in the early events leading to TCR signaling.

Mutations Utilize Dynamic Allostery to Confer Resistance in TEM-1 β-lactamase.

β-lactamases are enzymes produced by bacteria to hydrolyze β-lactam antibiotics as a common mechanism of resistance. Evolution in such enzymes has been rendering a wide variety of antibiotics impotent, therefore posing a major threat. Clinical and in vitro studies of evolution in TEM-1 β-lactamase have revealed a large number of single point mutations that are responsible for driving resistance to antibiotics and/or inhibitors. The distal locations of these mutations from the active sites suggest that these allosterically modulate the antibiotic resistance. We investigated the effects of resistance driver mutations on the conformational dynamics of the enzyme to provide insights about the mechanism of their long-distance interactions. Through all-atom molecular dynamics (MD) simulations, we obtained the dynamic flexibility profiles of the variants and compared those with that of the wild type TEM-1. While the mutational sites in the variants did not have any direct van der Waals interactions with the active site position S70 and E166, we observed a change in the flexibility of these sites, which play a very critical role in hydrolysis. Such long distance dynamic interactions were further confirmed by dynamic coupling index (DCI) analysis as the sites involved in resistance driving mutations exhibited high dynamic coupling with the active sites. A more exhaustive dynamic analysis, using a selection pressure for ampicillin and cefotaxime resistance on all possible types of substitutions in the amino acid sequence of TEM-1, further demonstrated the observed mechanism. Mutational positions that play a crucial role for the emergence of resistance to new antibiotics exhibited high dynamic coupling with the active site irrespective of their locations. These dynamically coupled positions were neither particularly rigid nor particularly flexible, making them more evolvable positions. Nature utilizes these sites to modulate the dynamics of the catalytic sites instead of mutating the highly rigid positions around the catalytic site.

Gears-In-Motion: The Interplay of WW and PPIase Domains in Pin1.

Pin1 belongs to the family of the peptidyl-prolyl cis-trans isomerase (PPIase), which is a class of enzymes that catalyze the cis/trans isomerization of the Proline residue. Pin1 is unique and only catalyzes the phosphorylated Serine/Threonine-Proline (S/T-P) motifs of a subset of proteins. Since the discovery of Pin1 as a key protein in cell cycle regulation, it has been implicated in numerous diseases, ranging from cancer to neurodegenerative diseases. The main features of Pin1 lies in its two main domains: the WW (two conserved tryptophan) domain and the PPIase domain. Despite extensive studies trying to understand the mechanisms of Pin1 functions, how these two domains contribute to the biological roles of Pin1 in cellular signaling requires more investigations. The WW domain of Pin1 is known to have a higher affinity to its substrate than that of the PPIase domain. Yet, the WW domain seems to prefer the trans configuration of phosphorylated S/T-P motif, while the PPIase catalyzes the cis to trans isomerasion. Such contradicting information has generated much confusion as to the actual mechanism of Pin1 function. In addition, dynamic allostery has been suggested to be important for Pin1 function. Henceforth, in this review, we will be looking at the progress made in understanding the function of Pin1, and how these understandings can aid us in overcoming the diseases implicated by Pin1 such as cancer during drug development.

Dynamic regulation of HIV-1 capsid interaction with the restriction factor TRIM5α identified by magic-angle spinning NMR and molecular dynamics simulations

The host factor protein TRIM5α plays an important role in restricting the host range of HIV-1, interfering with the integrity of the HIV-1 capsid. TRIM5 triggers an antiviral innate immune response by functioning as a capsid pattern recognition receptor, although the precise mechanism by which the restriction is imposed is not completely understood. Here we used an integrated magic-angle spinning nuclear magnetic resonance and molecular dynamics simulations approach to characterize, at atomic resolution, the dynamics of the capsid's hexameric and pentameric building blocks, and the interactions with TRIM5α in the assembled capsid. Our data indicate that assemblies in the presence of the pentameric subunits are more rigid on the microsecond to millisecond timescales than tubes containing only hexamers. This feature may be of key importance for controlling the capsid's morphology and stability. In addition, we found that TRIM5α binding to capsid induces global rigidification and perturbs key intermolecular interfaces essential for higher-order capsid assembly, with structural and dynamic changes occurring throughout the entire CA polypeptide chain in the assembly, rather than being limited to a specific protein-protein interface. Taken together, our results suggest that TRIM5α uses several mechanisms to destabilize the capsid lattice, ultimately inducing its disassembly. Our findings add to a growing body of work indicating that dynamic allostery plays a pivotal role in capsid assembly and HIV-1 infectivity.

Dynamic Allostery in PLCγ1 and Its Modulation by a Cancer Mutation Revealed by MD Simulation and NMR

Phosphatidylinositol phospholipase Cγ (PLCγ) is an intracellular membrane-associated second-messenger signaling protein activated by tyrosine kinases such as fibroblast growth factor receptor 1. PLCγ contains the regulatory γ-specific array (γSA) comprising a tandem Src homology 2 (SH2) pair, an SH3 domain, and a split pleckstrin homology domain. Binding of an activated growth factor receptor to γSA leads to Tyr783 phosphorylation and consequent PLCγ activation. Several disease-relevant mutations in γSA have been identified; all lead to elevated phospholipase activity. In this work, we describe an allosteric mechanism that connects the Tyr783 phosphorylation site to the nSH2-cSH2 junction and involves dynamic interactions between the cSH2-SH3 linker and cSH2. Molecular dynamics simulations of the tandem SH2 protein suggest that Tyr783 phosphorylation is communicated to the nSH2-cSH2 junction by modulating cSH2 binding to sections of the cSH2-SH3 linker. NMR chemical shift perturbation analyses for designed tandem SH2 constructs reveal combined fast and slow dynamic processes that can be attributed to allosteric communication involving these regions of the protein, establishing an example in which complex N-site exchange can be directly inferred from 1H,15N-HSQC spectra. Furthermore, in tandem SH2 and γSA constructs, molecular dynamics and NMR results show that the Arg687Trp mutant in PLCγ1 (equivalent to the cancer mutation Arg665Trp in PLCγ2) perturbs the dynamic allosteric pathway. This combined experimental and computational study reveals a rare example of multistate kinetics involved in a dynamic allosteric process that is modulated in the context of a disease-relevant mutation. The allosteric influences and the weakened binding of the cSH2-SH3 linker to cSH2 should be taken into account in any more holistic investigation of PLCγ regulation.