Dynamic Allostery Research Articles

Dynamic allostery can drive cold adaptation in enzymes.

Adaptation of organisms to environmental niches is a hallmark of evolution. One prevalent example is that of thermal adaptation, wherein two descendants evolve at different temperature extremes1,2. Underlying the physiological differences between such organisms are changes in enzymes catalyzing essential reactions3, with orthologues from each organism undergoing adaptive mutations that preserve similar catalytic rates at their respective physiological temperatures 4,5. The sequence changes responsible for these adaptive differences, however, are often at surface exposed sites distant from the substrate binding site, leaving the active site of the enzyme structurally unperturbed6,7. How such changes are allosterically propagated to the active site, to modulate activity, is not known. Here we show that entropy-tuning changes can be engineered into distal sites of Escherichia coli adenylate kinase (AK) to quantitatively assess the role of dynamics in determining affinity, turnover, and the role in driving adaptation. The results not only reveal a dynamics-based allosteric tuning mechanism, but also uncover a spatial separation of the control of key enzymatic parameters. Fluctuations in one mobile domain (i.e. the LID) control substrate affinity, while dynamic attenuation in the other (i.e. the AMPbd) affects rate-limiting conformational changes governing enzyme turnover. Dynamics-based regulation may thus represent an elegant, widespread, and previously unrealized evolutionary adaptation mechanism that fine-tunes biological function without altering the ground state structure. Furthermore, because rigid-body conformational changes in both domains were thought to be rate limiting for turnover8,9, these adaptation studies reveal a new paradigm for understanding the relationship between dynamics and turnover in AK.

Machine learning approaches to evaluate correlation patterns in allosteric signaling: A case study of the PDZ2 domain.

Many proteins are regulated by dynamic allostery wherein regulator-induced changes in structure are comparable with thermal fluctuations. Consequently, understanding their mechanisms requires assessment of relationships between and within conformational ensembles of different states. Here we show how machine learning based approaches can be used to simplify this high-dimensional data mining task and also obtain mechanistic insight. In particular, we use these approaches to investigate two fundamental questions in dynamic allostery. First, how do regulators modify inter-site correlations in conformational fluctuations (Cij)? Second, how are regulator-induced shifts in conformational ensembles at two different sites in a protein related to each other? We address these questions in the context of the human protein tyrosine phosphatase 1E's PDZ2 domain, which is a model protein for studying dynamic allostery. We use molecular dynamics to generate conformational ensembles of the PDZ2 domain in both the regulator-bound and regulator-free states. The employed protocol reproduces methyl deuterium order parameters from NMR. Results from unsupervised clustering of Cij combined with flow analyses of weighted graphs of Cij show that regulator binding significantly alters the global signaling network in the protein; however, not by altering the spatial arrangement of strongly interacting amino acid clusters but by modifying the connectivity between clusters. Additionally, we find that regulator-induced shifts in conformational ensembles, which we evaluate by repartitioning ensembles using supervised learning, are, in fact, correlated. This correlation Δij is less extensive compared to Cij, but in contrast to Cij, Δij depends inversely on the distance from the regulator binding site. Assuming that Δij is an indicator of the transduction of the regulatory signal leads to the conclusion that the regulatory signal weakens with distance from the regulatory site. Overall, this work provides new approaches to analyze high-dimensional molecular simulation data and also presents applications that yield new insight into dynamic allostery.

An active‐like Src conformation explains its dynamic regulation

Biological signaling by protein kinases plays an essential role in cellular function. The switch between inactive and active protein kinase conformations is central to their regulation. The active conformations of protein kinases are triggered and maintained by various post‐translational modifications. The inactive conformation of Src tyrosine kinase has a non‐phosphorylated activation loop and a rolled‐out αC helix. The R‐spine at the Src kinase core is disassembled via the RS3 residue when the αC helix is disengaged. Maneuvering of the C‐spine is crucial for obtaining a pseudokinase conformation of Src that could be crucial for exploring its scaffolding functions. Our Umbrella sampling methods allow for mapping the conformational landscape of the Src kinase domain and characterizing four defining states; the inactive, active conformations and two intermediates. Mutation of the C‐spine V281F allows for the closing of the two kinase lobes in the absence of ATP. An allosteric region at the β3‐αC loop allows for engaging the αC helix and the RS3 residue of the R‐spine via the L297F mutation. The L297F/ V281F double mutant allows for stabilization of a unique catalytically dead kinase conformation in the active‐like state. These studies allow for exploration of active‐like Src kinase states that define both the underlying kinase activation switch and the dynamic allostery in the molecular communication at the kinase core.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An Allosteric Region of Src Tyrosine Kinase Allows for Stabilization of Its Active-Like Conformation

Protein kinases play an essential role as signaling enzymes and their sensitive action in the needs of the cell requires their tight regulation. Their active conformations are exquisitely regulated and their populations controlled by various post-translational modifications. The inactive conformation of Src tyrosine kinase is characterized by its non-phosphorylated activation loop and the rolled-out αC helix in the N-lobe. The inactive conformation has the regulatory kinase spine disassembled as the RS3 residue from the αC helix is displaced. Our umbrella sampling methods to map the energy landscape of these conformations has allowed for defining four states; the inactive, active conformations and two intermediate states. In the present study we extend our umbrella sampling technique to understand the activation allostery in Src tyrosine kinase in three different mutants. The first mutant is the L297F that occupies an allosteric region in the N-terminal of the αC helix and allows for activation of Src tyrosine kinase analogous to RAFs. The second mutant is the V281F mutation in the catalytic spine that allows for the closing of the two kinase lobes in the absence of nucleotide. A double mutant of Src tyrosine kinase V281F/L297F allows for stabilization of a unique catalytically dead kinase conformation in the active-like state. This unique conformation allows for understanding of kinase switching function independent of its catalytic role. A third M314V mutation in the regulatory spine allows us to explore the maneuvering of hydrophobicity in the Src tyrosine kinase core. These studies provide a fresh perspective to understanding conformational switching in Src family of tyrosine kinases and the dynamic allostery that underlies this molecular communication. Our community map analysis of residue networks provides an additional layer of understanding to these observations.

Hinge-Shift Mechanism Modulates Allosteric Regulations in Human Pin1.

Allostery, which is regulation from distant sites, plays a major role in biology. While traditional allostery is described in terms of conformational change upon ligand binding as an underlying principle, it is possible to have allosteric regulations without significant conformational change through modulating the conformational dynamics by altering the local effective elastic modulus of the protein upon ligand binding. Pin1 utilizes this dynamic allostery to regulate its function. It is a modular protein containing a WW domain and a larger peptidyl prolyl isomerase domain (PPIase) that isomerizes phosphoserine/threonine-proline (pS/TP) motifs. The WW domain serves as a docking module, whereas catalysis solely takes place within the PPIase domain. Here, we analyze the change in the dynamic flexibility profile of the PPIase domain upon ligand binding to the WW domain. Substrate binding to the WW domain induces the formation of a new rigid hinge site around the interface of the two domains and loosens the flexibility of a rigid site existing in the Apo form around the catalytic site. This hinge-shift mechanism enhances the dynamic coupling of the catalytic positions with the PPIase domain, where the rest of the domain can cooperatively respond to the local conformational changes around the catalytic site, leading to an increase in catalytic efficiency.

Binding of anions in triply interlocked coordination catenanes and dynamic allostery for dehalogenation reactions.

By synergistic combination of multicomponent self-assembly and template-directed approaches, triply interlocked metal organic catenanes that consist of two isolated chirally identical tetrahedrons were constructed and stabilized as thermodynamic minima. In the presence of suitable template anions, the structural conversion from the isolated tetrahedral conformers into locked catenanes occurred via the cleavage of an intrinsically reversible coordination bond in each of the tetrahedrons, followed by the reengineering and interlocking of two fragments with the regeneration of the broken coordination bonds. The presence of several kinds of individual pocket that were attributed to the triply interlocked patterns enabled the possibility of encapsulating different anions, allowing the dynamic allostery between the unlocked/locked conformers to promote the dehalogenation reaction of 3-bromo-cyclohexene efficiently, as with the use of dehalogenase enzymes. The interlocked structures could be unlocked into two individual tetrahedrons through removal of the well-matched anion templates. The stability and reversibility of the locked/unlocked structures were further confirmed by the catching/releasing process that accompanied emission switching, providing opportunities for the system to be a dynamic molecular logic system.

Ensemble- and Rigidity Theory-Based Perturbation Approach To Analyze Dynamic Allostery.

Allostery describes the functional coupling between sites in biomolecules. Recently, the role of changes in protein dynamics for allosteric communication has been highlighted. A quantitative and predictive description of allostery is fundamental for understanding biological processes. Here, we integrate an ensemble-based perturbation approach with the analysis of biomolecular rigidity and flexibility to construct a model of dynamic allostery. Our model, by definition, excludes the possibility of conformational changes, evaluates static, not dynamic, properties of molecular systems, and describes allosteric effects due to ligand binding in terms of a novel free-energy measure. We validated our model on three distinct biomolecular systems: eglin c, protein tyrosine phosphatase 1B, and the lymphocyte function-associated antigen 1 domain. In all cases, it successfully identified key residues for signal transmission in very good agreement with the experiment. It correctly and quantitatively discriminated between positively or negatively cooperative effects for one of the systems. Our model should be a promising tool for the rational discovery of novel allosteric drugs.

Decomposing Dynamical Couplings in Mutated scFv Antibody Fragments into Stabilizing and Destabilizing Effects

Conformational fluctuations within scFv antibodies are characterized by a novel perturbation-response decomposition of molecular dynamics trajectories. Both perturbation and response profiles are stratified into stabilizing and destabilizing conditions. The linker between the VH and VL domains exhibits the dominant dynamical response by being coupled to nearly the entire protein, responding to both stabilizing and destabilizing perturbations. Perturbations within complementarity-determining regions (CDR) induce rich behavior in dynamic response. Among many effects, stabilizing any CDR loop in the VH domain triggers a destabilizing response in all CDR loops in the VL domain and vice versa. Destabilizing residues within the VL domain are likely to stabilize all CDR loops in the VH domain, and, when these residues are not buried, the CDR loops in the VL domain are also likely to be stabilized. These effects, described by shifts in normal mode characteristics, initiate a propensity for dynamic allostery with possible functional implications in bispecific antibodies.

Hidden electrostatic basis of dynamic allostery in a PDZ domain

Allosteric effect implies ligand binding at one site leading to structural and/or dynamical changes at a distant site. PDZ domains are classic examples of dynamic allostery without conformational changes, where distal side-chain dynamics is modulated on ligand binding and the origin has been attributed to entropic effects. In this work, we unearth the energetic basis of the observed dynamic allostery in a PDZ3 domain protein using molecular dynamics simulations. We demonstrate that electrostatic interaction provides a highly sensitive yardstick to probe the allosteric modulation in contrast to the traditionally used structure-based parameters. There is a significant population shift in the hydrogen-bonded network and salt bridges involving side chains on ligand binding. The ligand creates a local energetic perturbation that propagates in the form of dominolike changes in interresidue interaction pattern. There are significant changes in the nature of specific interactions (nonpolar/polar) between interresidue contacts and accompanied side-chain reorientations that drive the major redistribution of energy. Interestingly, this internal redistribution and rewiring of side-chain interactions led to large cancellations resulting in small change in the overall enthalpy of the protein, thus making it difficult to detect experimentally. In contrast to the prevailing focus on the entropic or dynamic effects, we show that the internal redistribution and population shift in specific electrostatic interactions drive the allosteric modulation in the PDZ3 domain protein.

Dynamic Allostery Modulates Catalytic Activity by Modifying the Hydrogen Bonding Network in the Catalytic Site of Human Pin1.

Allosteric communication among domains in modular proteins consisting of flexibly linked domains with complimentary roles remains poorly understood. To understand how complementary domains communicate, we have studied human Pin1, a representative modular protein with two domains mutually tethered by a flexible linker: a WW domain for substrate recognition and a peptidyl-prolyl isomerase (PPIase) domain. Previous studies of Pin1 showed that physical contact between the domains causes dynamic allostery by reducing conformation dynamics in the catalytic domain, which compensates for the entropy costs of substrate binding to the catalytic site and thus increases catalytic activity. In this study, the S138A mutant PPIase domain, a mutation that mimics the structural impact of the interdomain contact, was demonstrated to display dynamic allostery by rigidification of the α2-α3 loop that harbors the key catalytic residue C113. The reduced dynamics of the α2-α3 loop stabilizes the C113–H59 hydrogen bond in the hydrogen-bonding network of the catalytic site. The stabilized hydrogen bond between C113 and H59 retards initiation of isomerization, which explains the reduced isomerization rate by ~20% caused by the S138A mutation. These results provide new insight into the interdomain allosteric communication of Pin1.

Molecular Dynamics-Markov State Model of Protein Ligand Binding and Allostery in CRIB-PDZ: Conformational Selection and Induced Fit.

Conformational selection and induced fit are well-known contributors to ligand binding and allosteric effects in proteins. Molecular dynamics (MD) simulations now enable the theoretical study of protein-ligand binding in terms of ensembles of interconverting microstates and the population shifts characteristic of "dynamical allostery." Here we investigate protein-ligand binding and allostery based on a Markov state model (MSM) with states and rates obtained from all-atom MD simulations. As an exemplary case, we consider the single domain protein par-6 PDZ with and without ligand and allosteric effector. This is one of the smallest proteins in which allostery has been experimentally observed. In spite of the increased complexity intrinsic to a statistical ensemble perspective, we find that conformational selection and induced fit mechanisms can be readily identified in the analysis. In the nonallosteric pathway, MD-MSM shows that PDZ binds ligand via conformational selection. However, the allosteric pathway requires an activation step that involves a conformational change induced by the allosteric effector Cdc42. Once in the allosterically activated state, we find that ligand binding can proceed by conformational selection. Our MD-MSM model predicts that allostery in this and possibly other systems involves both induced fit and conformational selection, not just one or the other.

Dynamic Allostery in Regulated Entry of Newcastle Disease Virus into Host Cells

Newcastle disease virus (NDV) belongs to the family of paramyxoviruses that cause numerous fatal diseases in humans and farm animals. Here we focus on mechanisms that underlie its regulated entry into host cells. To gain entry, NDV utilizes two of its membrane proteins: Hemagglutinin-Neuraminidase (HN) and the fusion (F) protein. HN binds to sialic acids expressed on the host cell, and this binding stimulates HN to activate F, which, in turn, mediates virus-host membrane fusion. HN interacts with sialic acid and F through separate sites located on two different domains, however, no models explain this allosteric coupling. In fact, the analogous mechanisms in other paramyxoviruses also remain undetermined. Starting with X-ray structures of HN‘s receptor binding domain (bound to sialic acid derivatives), we examine using molecular dynamics how sialic acid affects HN's receptor binding domain (RBD) as well as HN's RBD-RBD dimeric interface. We note first that sialic acid induces only minor structural changes in individual RBDs - an observation similar to proteins like GPCRs that are regulated by dynamic allostery. Despite inducing minor changes in individual RBDs, we find that sialic acid reorients the RBD-RBD interface, and that this reorientation contributes to HN stimulation. Finally, we note that the induced RBD-RBD reorientation is unlike what we observed in the case Nipah's HN homolog (Dutta et al. Biophys. J. 2016), suggesting that fusion stimulation mechanisms are not conserved across paramyxoviruses.

Incorporation of Multi-State Information Improves Molecular Modelling of Dynamic Allostery: A Case Study of PDZ Domains Computational Quantitative Characterization of Entropic Signaling Pathways in Proteins: A Case Study on Human PDZ2 Domain of PTP1E

Incorporation of Multi-State Information Improves Molecular Modelling of Dynamic Allostery: A Case Study of PDZ Domains Computational Quantitative Characterization of Entropic Signaling Pathways in Proteins: A Case Study on Human PDZ2 Domain of PTP1E

Conformational Rigidity and Protein Dynamics at Distinct Timescales Regulate PTP1B Activity and Allostery

Protein function originates from a cooperation of structural rigidity, dynamics at different timescales, and allostery. However, how these three pillars of protein function are integrated is still only poorly understood. Here we show how these pillars are connected in Protein Tyrosine Phosphatase 1B (PTP1B), a drug target for diabetes and cancer that catalyzesthe dephosphorylation of numerous substrates in essential signaling pathways. By combining new experimental and computational data on WT-PTP1B and ≥10 PTP1B variants in multiple states, we discovered a fundamental and evolutionarily conserved CH/π switch that is critical for positioning the catalytically important WPD loop. Furthermore, our data show that PTP1B uses conformational and dynamic allostery to regulate its activity. This shows that both conformational rigidity and dynamics are essential for controlling protein activity. This connection between rigidity and dynamics at different timescales is likely a hallmark of all enzyme function.

Native State Volume Fluctuations in Proteins as a Mechanism for Dynamic Allostery.

Allostery enables tight regulation of protein function in the cellular environment. Although existing models of allostery are firmly rooted in the current structure-function paradigm, the mechanistic basis for allostery in the absence of structural change remains unclear. In this study, we show that a typical globular protein is able to undergo significant changes in volume under native conditions while exhibiting no additional changes in protein structure. These native state volume fluctuations were found to correlate with changes in internal motions that were previously recognized as a source of allosteric entropy. This finding offers a novel mechanistic basis for allostery in the absence of canonical structural change. The unexpected observation that function can be derived from expanded, low density protein states has broad implications for our understanding of allostery and suggests that the general concept of the native state be expanded to allow for more variable physical dimensions with looser packing.

Networks of Dynamic Allostery Regulate Enzyme Function

Many protein systems rely on coupled dynamic networks to allosterically regulate function. However, the broad conformational space sampled by non-coherently dynamic systems has precluded detailed analysis of their communication mechanisms. Here, we have developed a methodology that combines the high sensitivity afforded by nuclear magnetic resonance relaxation techniques and single-site multiple mutations, termed RASSMM, to identify two allosterically coupled dynamic networks within the non-coherently dynamic enzyme cyclophilin A. Using this methodology, we discovered two key hotspot residues, Val6 and Val29, that communicate through these networks, the mutation of which altered active-site dynamics, modulating enzymatic turnover of multiple substrates. Finally, we utilized molecular dynamics simulations to identify the mechanism by which one of these hotspots is coupled to the larger dynamic networks. These studies confirm a link between enzyme dynamics and the catalytic cycle of cyclophilin A and demonstrate how dynamic allostery may be engineered to tune enzyme function.

Timing Correlations in Proteins Predict Functional Modules and Dynamic Allostery.

How protein structure encodes functionality is not fully understood. For example, long-range intraprotein communication can occur without measurable conformational change and is often not captured by existing structural correlation functions. It is shown here that important functional information is encoded in the timing of protein motions, rather than motion itself. I introduce the conditional activity function to quantify such timing correlations among the degrees of freedom within proteins. For three proteins, the conditional activities between side-chain dihedral angles were computed using the output of microseconds-long atomistic simulations. The new approach demonstrates that a sparse fraction of side-chain pairs are dynamically correlated over long distances (spanning protein lengths up to 7 nm), in sharp contrast to structural correlations, which are short-ranged (<1 nm). Regions of high self- and inter-side-chain dynamical correlations are found, corresponding to experimentally determined functional modules and allosteric connections, respectively.

Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases.

Bruton’s tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA). Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site.

Signaling and Adaptation Modulate the Dynamics of the Photosensoric Complex of Natronomonas pharaonis.

Motile bacteria and archaea respond to chemical and physical stimuli seeking optimal conditions for survival. To this end transmembrane chemo- and photoreceptors organized in large arrays initiate signaling cascades and ultimately regulate the rotation of flagellar motors. To unravel the molecular mechanism of signaling in an archaeal phototaxis complex we performed coarse-grained molecular dynamics simulations of a trimer of receptor/transducer dimers, namely NpSRII/NpHtrII from Natronomonas pharaonis. Signaling is regulated by a reversible methylation mechanism called adaptation, which also influences the level of basal receptor activation. Mimicking two extreme methylation states in our simulations we found conformational changes for the transmembrane region of NpSRII/NpHtrII which resemble experimentally observed light-induced changes. Further downstream in the cytoplasmic domain of the transducer the signal propagates via distinct changes in the dynamics of HAMP1, HAMP2, the adaptation domain and the binding region for the kinase CheA, where conformational rearrangements were found to be subtle. Overall these observations suggest a signaling mechanism based on dynamic allostery resembling models previously proposed for E. coli chemoreceptors, indicating similar properties of signal transduction for archaeal photoreceptors and bacterial chemoreceptors.

Dynamic Allostery of the Catabolite Activator Protein Revealed by Interatomic Forces.

The Catabolite Activator Protein (CAP) is a showcase example for entropic allostery. For full activation and DNA binding, the homodimeric protein requires the binding of two cyclic AMP (cAMP) molecules in an anti-cooperative manner, the source of which appears to be largely of entropic nature according to previous experimental studies. We here study at atomic detail the allosteric regulation of CAP with Molecular dynamics (MD) simulations. We recover the experimentally observed entropic penalty for the second cAMP binding event with our recently developed force covariance entropy estimator and reveal allosteric communication pathways with Force Distribution Analyses (FDA). Our observations show that CAP binding results in characteristic changes in the interaction pathways connecting the two cAMP allosteric binding sites with each other, as well as with the DNA binding domains. We identified crucial relays in the mostly symmetric allosteric activation network, and suggest point mutants to test this mechanism. Our study suggests inter-residue forces, as opposed to coordinates, as a highly sensitive measure for structural adaptations that, even though minute, can very effectively propagate allosteric signals.