Protein Domain Motions Research Articles

Mechanistic insights into the allosteric inactivation mechanism of ZAP-70 induced by the hot spot W165C mutation

Zeta chain-associated protein kinase 70 (ZAP-70) is a non-receptor tyrosine kinase that interacts with the activated T-cell receptor to transduce downstream signals, and thus plays an important role in the adaptive immune system. The biphosphorylated immunotyrosine-based activation motifs (ITAM-Y2P) binds to the N-SH2 and C-SH2 domains of ZAP-70 to promote the activation of ZAP-70. The present study explores molecular mechanisms of allosteric inactivation of ZAP-70 induced by the hot spot W165C mutation through atomically detailed molecular dynamics simulation approaches. We report microsecond-length simulations of two states of the tandem SH2 domains of ZAP-70 in complex with the ITAM-Y2P motif, including the wild-type and W165C mutant. Extensive analysis of local flexibility and dynamical correlated motions show that W165C mutation changes coupled motions of protein domains and community networks. The binding affinities of the ITAM-Y2P motif to the wild-type and W165C mutant of ZAP-70 are predicted using binding free energy calculations. The results suggest that the driving force to decrease the binding affinity in the W165C mutant derives from the difference in the protein-protein electrostatic interactions. Moreover, the per-residue free energy decomposition unravels that the contributions from residues in the phosphorylated Tyr315 (pY315) binding site, in particular pY315 of ITAM-Y2P, and Arg43, Tyr240 of ZAP-70, are the key determinants for the loss of binding affinity. This study may insights into our understanding of the pathological mechanism of ZAP-70. Communicated by Ramaswamy H. Sarma

Collective Domain Motion Facilitates Water Transport in SGLT1.

The human sodium-glucose cotransporter protein (SGLT1) is an important representative of the sodium solute symporters belonging to the secondary active transporters that are critical to the homeostasis of sugar, sodium, and water in the cell. The underlying transport mechanism of SGLT1 is based on switching between inward- and outward-facing conformations, known as the alternating access model, which is crucial for substrate transport, and has also been postulated for water permeation. However, the nature of water transport remains unclear and is disputed along the passive and active transport, with the latter postulating the presence of the pumping effect. To better examine the water transport in SGLT1, we performed a series of equilibrium all-atom molecular dynamics simulations, totaling over 6 μs of sample representative conformational states of SGLT1 and its complexes, with the natural substrates, ions, and inhibitors. In addition to elucidating the basic physical factors influencing water permeation, such as channel openings and energetics, we focus on dynamic flexibility and its relationship with domain motion. Our results clearly demonstrate a dependence of instantaneous water flux on the channel opening and local water diffusion in the channel, strongly supporting the existence of a passive water transport in SGLT1. In addition, a strong correlation found between the local water diffusion and protein domain motion, resembling the "rocking-bundle" motion, reveals its facilitating role in the water transport.

Laws: Local alignment of water sites-Describing allosteric water networks in enzymes.

Water is an essential component of biological systems; it affects the structure and stability of biological machinery. Highly conserved water networks have been shown to play a crucial role in the motion of protein domains and affect protein function by mediating allosteric communication. Perturbations to these networks have also been implicated in human diseases. Data from x-ray crystallography provide the locations of crystallographic water sites (CWS) coordinated by the protein which can be potentially used to study the conserved water networks. Here, we propose a computational method called local alignment for water sites (LAWS) to identify conserved water networks in molecular dynamics (MD) simulations. This method is based on multilateration — an algorithm widely used in GPS tracking. LAWS considers the contacts formed by CWS and protein atoms in the crystal structure and uses these interaction distances to track ordered waters in a simulation. We apply our method to simulations of an enzyme, fluoroacetate dehalogenase, and analyze the occupancy, residence times and correlated motions of individual water molecules coupled to the protein. Our results help to determine a highly conserved water network involved in allosteric regulation.

Conformational motions and ligand-binding underlying gating and regulation in IP3R channel

Inositol-1,4,5-trisphosphate receptors (IP3Rs) are activated by IP3 and Ca2+ and their gating is regulated by various intracellular messengers that finely tune the channel activity. Here, using single particle cryo-EM analysis we determined 3D structures of the nanodisc-reconstituted IP3R1 channel in two ligand-bound states. These structures provide unprecedented details governing binding of IP3, Ca2+ and ATP, revealing conformational changes that couple ligand-binding to channel opening. Using a deep-learning approach and 3D variability analysis we extracted molecular motions of the key protein domains from cryo-EM density data. We find that IP3 binding relies upon intrinsic flexibility of the ARM2 domain in the tetrameric channel. Our results highlight a key role of dynamic side chains in regulating gating behavior of IP3R channels. This work represents a stepping-stone to developing mechanistic understanding of conformational pathways underlying ligand-binding, activation and regulation of the channel.

Tracking protein domain movements by EPR distance determination and multilateration.

Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful approach to supplement the large toolbox of methods for protein structure determination. The strength of EPR spectroscopy is the ability to map flexibility of protein domains and conformational ensembles. EPR distance determination in the nanometer range and subsequent multilateration enables the three-dimensional visualization of the localization of a spin label in a protein domain. Therefore, multilateration can not only be used to represent the degree of flexibility of protein structural elements, but also track movements of whole domains. We report a detailed protocol for all needed steps, beginning with the choice of the labeling sites, the spin labeling reaction, the EPR distance measurement by double electron-electron resonance (DEER) and finally the multilateration exploiting the EPR distance restraints.

Normal-mode driven exploration of protein domain motions.

Domain motions involved in the function of proteins can often be well described as a combination of motions along a handfull of low-frequency modes, that is, with the values of a few normal coordinates. This means that, when the functional motion of a protein is unknown, it should prove possible to predict it, since it amounts to guess a few values. However, without the help of additional experimental data, using normal coordinates for generating accurate conformers far away from the initial one is not so straightforward. To do so, a new approach is proposed: instead of building conformers directly with the values of a subset of normal coordinates, they are built in two steps, the conformer built with normal coordinates being just used for defining a set of distance constraints, the final conformer being built so as to match them. Note that this approach amounts to transform the problem of generating accurate protein conformers using normal coordinates into a better known one: the distance-geometry problem, which is herein solved with the help of the ROSETTA software. In the present study, this approach allowed to rebuild accurately six large amplitude conformational changes, using at most six low-frequency normal coordinates. As a consequence of the low-dimensionality of the corresponding subspace, random exploration also proved enough for generating low-energy conformers close to the known end-point of the conformational change of the LAO binding protein, lysozyme T4 and adenylate kinase.

Anomalous Diffusion of Peripheral Membrane Signaling Proteins from All-Atom Molecular Dynamics Simulations.

Peripheral membrane proteins bind transiently to membrane surfaces as part of many signaling pathways. The bound proteins perform two-dimensional (2-D) diffusion on the membrane surface during the recruitment function. To better understand the interplay between the 2-D diffusion of these protein domains and their membrane binding modes, we performed multimicrosecond all-atom molecular dynamics simulations of two regulatory domains, a C2 domain and a pleckstrin homology (PH) domain, in their experimentally determined bound configuration to a lipid bilayer. The protein bound configurations are preserved throughout the simulation trajectories. Both protein domains exhibit anomalous diffusion with distinct features in their dynamics that reflect their different modes of binding. An analysis of their diffusive behavior reveals common features with the diffusion of lipid molecules in lipid bilayers, suggesting that the 2-D motion of protein domains bound to the membrane surface is modulated by the viscoelastic nature of the lipid bilayer.

Clutch mechanism of chemomechanical coupling in a DNA resecting motor nuclease

Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The N-terminal motor domain of the AdnB subunit hydrolyzes ATP to drive rapid and processive 3' to 5' translocation of AdnAB on the tracking DNA strand. ATP hydrolysis is mechanically productive when oscillating protein domain motions synchronized with the ATPase cycle propel the DNA tracking strand forward by a single-nucleotide step, in what is thought to entail a pawl-and-ratchet-like fashion. By gauging the effects of alanine mutations of the 16 amino acids at the AdnB-DNA interface on DNA-dependent ATP hydrolysis, DNA translocation, and DSB resection in ensemble and single-molecule assays, we gained key insights into which DNA contacts couple ATP hydrolysis to motor activity. The results implicate AdnB Trp325, which intercalates into the tracking strand and stacks on a nucleobase, as the singular essential constituent of the ratchet pawl, without which ATP hydrolysis on ssDNA is mechanically futile. Loss of Thr663 and Thr118 contacts with tracking strand phosphates and of His665 with a nucleobase drastically slows the AdnAB motor during DSB resection. Our findings for AdnAB prompt us to analogize its mechanism to that of an automobile clutch.

Intermediate scattering functions of a rigid body monoclonal antibody protein in solution studied by dissipative particle dynamic simulation.

In the past decade, there was increased research interest in studying internal motions of flexible proteins in solution using Neutron Spin Echo (NSE) as NSE can simultaneously probe the dynamics at the length and time scales comparable to protein domain motions. However, the collective intermediate scattering function (ISF) measured by NSE has the contributions from translational, rotational, and internal motions, which are rather complicated to be separated. Widely used NSE theories to interpret experimental data usually assume that the translational and rotational motions of a rigid particle are decoupled and independent to each other. To evaluate the accuracy of this approximation for monoclonal antibody (mAb) proteins in solution, dissipative particle dynamic computer simulation is used here to simulate a rigid-body mAb for up to about 200 ns. The total ISF together with the ISFs due to only the translational and rotational motions as well as their corresponding effective diffusion coefficients is calculated. The aforementioned approximation introduces appreciable errors to the calculated effective diffusion coefficients and the ISFs. For the effective diffusion coefficient, the error introduced by this approximation can be as large as about 10% even though the overall agreement is considered reasonable. Thus, we need to be cautious when interpreting the data with a small signal change. In addition, the accuracy of the calculated ISFs due to the finite computer simulation time is also discussed.

Ligand-induced structural changes analysis of ribose-binding protein as studied by molecular dynamics simulations.

BACKGROUND: The ribose-binding protein (RBP) from Escherichia coli is one of the representative structures of periplasmic binding proteins. Binding of ribose at the cleft between two domains causes a conformational change corresponding to a closure of two domains around the ligand. The RBP has been crystallized in the open and closed conformations.OBJECTIVE: With the complex trajectory as a control, our goal was to study the conformation changes induced by the detachment of the ligand, and the results have been revealed from two computational tools, MD simulations and elastic network models.METHODS: Molecular dynamics (MD) simulations were performed to study the conformation changes of RBP starting from the open-apo, closed-holo and closed-apo conformations.RESULTS: The evolution of the domain opening angle clearly indicates large structural changes. The simulations indicate that the closed states in the absence of ribose are inclined to transition to the open states and that ribose-free RBP exists in a wide range of conformations. The first three dominant principal motions derived from the closed-apo trajectories, consisting of rotating, bending and twisting motions, account for the major rearrangement of the domains from the closed to the open conformation.CONCLUSIONS: The motions showed a strong one-to-one correspondence with the slowest modes from our previous study of RBP with the anisotropic network model (ANM). The results obtained for RBP contribute to the generalization of robustness for protein domain motion studies using either the ANM or PCA for trajectories obtained from MD.

Exploring Large Domain Motions in Proteins Using Atomistic Molecular Dynamics with Enhanced Conformational Sampling.

Conformational transitions in multidomain proteins are essential for biological functions. The Apo conformations are typically open and flexible, while the Holo states form more compact conformations stabilized by protein-ligand interactions. Unfortunately, the atomically detailed mechanisms for such open-closed conformational changes are difficult to be accessed experimentally as well as computationally. To simulate the transitions using atomistic molecular dynamics (MD) simulations, efficient conformational sampling algorithms are required. In this work, we propose a new approach based on generalized replica-exchange with solute tempering (gREST) for exploring the open-closed conformational changes in multidomain proteins. Wherein, selected surface charged residues in a target protein are defined as the solute region in gREST simulation and the solute temperatures are different in replicas and exchanged between them to enhance the domain motions. This approach is called gREST selected surface charged residues (gREST_SSCR) and is applied to the Apo and Holo states of ribose binding protein (RBP) in solution. The conformational spaces sampled with gREST_SSCR are much wider than those with the conventional MD, sampling open-closed conformational changes while maintaining RBP domains’ stability. The free-energy landscapes of RBP in the Apo and Holo states are drawn along with twist and hinge angles of the two moving domains. The inter-domain salt-bridges that are not observed in the experimental structures are also important in the intermediate states during the conformational changes.

Investigation of sequence features of hinge-bending regions in proteins with domain movements using kernel logistic regression

BackgroundHinge-bending movements in proteins comprising two or more domains form a large class of functional movements. Hinge-bending regions demarcate protein domains and collectively control the domain movement. Consequently, the ability to recognise sequence features of hinge-bending regions and to be able to predict them from sequence alone would benefit various areas of protein research. For example, an understanding of how the sequence features of these regions relate to dynamic properties in multi-domain proteins would aid in the rational design of linkers in therapeutic fusion proteins.ResultsThe DynDom database of protein domain movements comprises sequences annotated to indicate whether the amino acid residue is located within a hinge-bending region or within an intradomain region. Using statistical methods and Kernel Logistic Regression (KLR) models, this data was used to determine sequence features that favour or disfavour hinge-bending regions. This is a difficult classification problem as the number of negative cases (intradomain residues) is much larger than the number of positive cases (hinge residues). The statistical methods and the KLR models both show that cysteine has the lowest propensity for hinge-bending regions and proline has the highest, even though it is the most rigid amino acid. As hinge-bending regions have been previously shown to occur frequently at the terminal regions of the secondary structures, the propensity for proline at these regions is likely due to its tendency to break secondary structures. The KLR models also indicate that isoleucine may act as a domain-capping residue. We have found that a quadratic KLR model outperforms a linear KLR model and that improvement in performance occurs up to very long window lengths (eighty residues) indicating long-range correlations.ConclusionIn contrast to the only other approach that focused solely on interdomain hinge-bending regions, the method provides a modest and statistically significant improvement over a random classifier. An explanation of the KLR results is that in the prediction of hinge-bending regions a long-range correlation is at play between a small number amino acids that either favour or disfavour hinge-bending regions. The resulting sequence-based prediction tool, HingeSeek, is available to run through a webserver at hingeseek.cmp.uea.ac.uk.

An ensemble of flexible conformations underlies mechanotransduction by the cadherin–catenin adhesion complex

The cadherin-catenin adhesion complex is the central component of the cell-cell adhesion adherens junctions that transmit mechanical stress from cell to cell. We have determined the nanoscale structure of the adherens junction complex formed by the α-catenin•β-catenin•epithelial cadherin cytoplasmic domain (ABE) using negative stain electron microscopy, small-angle X-ray scattering, and selective deuteration/small-angle neutron scattering. The ABE complex is highly pliable and displays a wide spectrum of flexible structures that are facilitated by protein-domain motions in α- and β-catenin. Moreover, the 107-residue intrinsically disordered N-terminal segment of β-catenin forms a flexible "tongue" that is inserted into α-catenin and participates in the assembly of the ABE complex. The unanticipated ensemble of flexible conformations of the ABE complex suggests a dynamic mechanism for sensitivity and reversibility when transducing mechanical signals, in addition to the catch/slip bond behavior displayed by the ABE complex under mechanical tension. Our results provide mechanistic insight into the structural dynamics for the cadherin-catenin adhesion complex in mechanotransduction.

Simulating and analysing configurational landscapes of protein-protein contact formation.

Interacting proteins can form aggregates and protein-protein interfaces with multiple patterns and different stabilities. Using molecular simulation one would like to understand the formation of these aggregates and which of the observed states are relevant for protein function and recognition. To characterize the complex configurational ensemble of protein aggregates, one needs a quantitative measure for the similarity of structures. We present well-suited descriptors that capture the essential features of non-covalent protein contact formation and domain motion. This set of collective variables is used with a nonlinear multi-dimensional scaling-based dimensionality reduction technique to obtain a low-dimensional representation of the configurational landscape of two ubiquitin proteins from coarse-grained simulations. We show that this two-dimensional representation is a powerful basis to identify meaningful states in the ensemble of aggregated structures and to calculate distributions and free energy landscapes for different sets of simulations. By using a measure to quantitatively compare free energy landscapes we can show how the introduction of a covalent bond between two ubiquitin proteins at different positions alters the configurational states of these dimers.

Functional Water Dynamics in Rhodopsin Using Solid‐State Deuterium NMR Spectroscopy

G‐protein‐coupled receptors (GPCRs) are the largest family of cell membrane‐bound proteins that detect extracellular stimuli and transduce signals to activate intracellular functions. Drugs that target GPCRs also account for about 27% of the global market share of therapeutic drugs, with aggregated sales for 2011–2015 of ca. US $890 billion [1]. Hydration is essential for protein tertiary folding and activity, and therefore knowledge of hydration water dynamics is vital for understanding the GPCR activation mechanism [2]. We studied rhodopsin hydration as a prototype for GPCRs, where the role of water in the rhodopsin activation mechanism has been recently explored using quasi‐elastic neutron scattering (QENS) methods [3]. We developed methods for generating powdered samples, notable for exceptionally high protein content while retaining photochemical functionality. The powdered GPCR samples are suitable for biophysical experiments to study the dynamic process of rhodopsin activation [4], where the protein is hydrated using vapor phase absorption in a controlled manner. We propose that the internal protein dynamics (β‐fluctuations) are coupled to the solvent shell, while large‐scale protein domain motions (α‐fluctuations) are coupled to the bulk solvent. To test our hypothesis of functional water interactions, we studied rhodopsin hydration shell dynamics using solid‐state 2H NMR spectroscopy at various hydration levels and temperatures [5]. From the spectral features at 200 K, we identified two dynamically distinguishable protein hydration regions. The broad quadrupolar splitting at approximately 180 kHz is due to bulk water; however, the anisotropic narrow component is a result of water molecules dynamically perturbed by rhodopsin. Additional solid‐state 2H NMR relaxation investigations of molecular dynamics of the rhodopsin‐bound water shell are in progress. Our future focus is on a comparative study of water dynamics in the light activated state and the dark state of the visual photoreceptor.Support or Funding InformationThis work was supported by the NIH (EY026041 and EY012049) and the NSF (MCB 1817862).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Localised contacts lead to nanosecond hinge motions in dimeric bovine serum albumin.

Domain motions in proteins are crucial for biological function. In the present manuscript, we present a neutron spin-echo spectroscopy (NSE) study of native bovine serum albumin (BSA) in solution. NSE allows to probe both global and internal dynamics of the BSA monomer and dimer equilibrium that is formed in solution. Using a model independent approach, we were able to identify an internal dynamic process in BSA that is visible in addition to global rigid-body diffusion of the BSA monomer and dimer mixture. The observed internal protein motion is characterised by a relaxation time of 43 ns. The overdamped Brownian oscillator was considered as an alternative analytical theory that was able to describe the internal process as first-order approximation. More detailed information on the physical nature of the internal protein motion was extracted from the q-dependent internal diffusion coefficients ΔDeff(q) that were detected by NSE in addition to global rigid-body translational and rotational diffusion. The ΔDeff(q) were interpreted using normal mode analysis based on the available crystal structures of the BSA monomer and dimer as structural test models. Normal mode analysis demonstrates that the observed internal dynamic process can be attributed to bending motion of the BSA dimer. The native BSA monomer does not show any internal dynamics on the time- and length-scales probed by NSE. An intermolecular disulphide bridge or a direct structural contact between the BSA monomers forms a localised link acting as a molecular hinge in the BSA dimer. The effect of that hinge on the observed motion of BSA in the used dimeric structural model is discussed in terms of normal modes in a molecular picture.

Mapping Calcium-Sensitive Regions in GCAPs by Site-Specific Fluorescence Labelling.

Signal transduction processes that are under control of changes in cytoplasmic Ca2+-concentration involve Ca2+-sensor proteins, which often undergo pronounced conformational transitions triggered by Ca2+. Consequences of conformational changes can be the structural rearrangement of single amino acids, exposition of small patches of several amino acids, or the movement of whole protein regions or domains. Furthermore, these conformational changes can lead to the exposure or movement of posttranslationally attached acyl groups. These processes could then control the function of target proteins, for example, by Ca2+-dependent protein-protein interaction. Fluorescence spectroscopy allows for mapping these Ca2+-sensitive regions but needs site-specific fluorescence labelling. We describe the application of a new group of diaminoterephthalate-derived fluorescence probes targeting either cysteines in guanylate cyclase-activating proteins, named GCAPs, or azide moieties in covalently attached acyl groups. By monitoring Ca2+-dependent changes in fluorescence emission, we identify Ca2+-sensitive protein regions in GCAPs and correlate conformational changes to protein function.

Methyl-Based NMR Spectroscopy Methods for Uncovering Structural Dynamics in Large Proteins and Protein Complexes

NMR spectroscopy is particularly adept at site-specifically monitoring dynamic processes in proteins, such as protein folding, domain movements, ligand binding, and side-chain rotations. By coupling the favorable spectroscopic properties of highly dynamic side-chain methyl groups with transverse-relaxation-optimized spectroscopy (TROSY), it is now possible to routinely study such dynamic processes in high-molecular-weight proteins and complexes approaching 1 MDa. In this Perspective, we describe many elegant methyl-based NMR experiments that probe slow (second) to fast (picosecond) dynamics in large systems. To demonstrate the power of these methods, we also provide interesting examples of studies that utilized each methyl-based NMR technique to uncover functionally important dynamics. In many cases, the NMR experiments are paired with site-directed mutagenesis and/or other biochemical assays to put the dynamics and function into context. Our vision of the future of structural biology involves pairing methyl-based NMR spectroscopy with biochemical studies to advance our knowledge of the motions large proteins and macromolecular complexes use to choreograph complex functions. Such studies will be essential in elucidating the critical structural dynamics that underlie function and characterizing alterations in these processes that can lead to human disease.

Enhanced Sampling of Interdomain Motion Using Map-Restrained Langevin Dynamics and NMR: Application to Pin1

Many signaling proteins consist of globular domains connected by flexible linkers that allow for substantial domain motion. Because these domains often serve as complementary functional modules, the possibility of functionally important domain motions arises. To explore this possibility, we require knowledge of the ensemble of protein conformations sampled by interdomain motion.Measurements of NMR residual dipolar couplings (RDCs) of backbone HN bonds offer a per-residue characterization of interdomain dynamics, as the couplings are sensitive to domain orientation. A challenge in reaching this potential is the need to interpret the RDCs as averages over dynamic ensembles of domain conformations.Here, we address this challenge by introducing an efficient protocol for generating conformational ensembles appropriate for flexible, multi-domain proteins. The protocol uses map-restrained self-guided Langevin dynamics simulations to promote collective, interdomain motion while restraining the internal domain motion to near rigidity. Critically, the simulations retain an all-atom description for facile inclusion of site-specific NMR RDC restraints. The result is the rapid generation of conformational ensembles consistent with the RDC data.We illustrate this protocol on human Pin1, a two-domain peptidyl-prolyl isomerase relevant for cancer and Alzheimer's disease. The results include the ensemble of domain orientations sampled by Pin1, as well as those of a dysfunctional variant, I28A-Pin1. The differences between the ensembles corroborate our previous spin relaxation results that showed weakened interdomain contact in the I28A variant relative to wild type. Our protocol extends our abilities to explore the functional significance of protein domain motions.

Patterns of coevolving amino acids unveil structural and dynamical domains

Patterns of interacting amino acids are so preserved within protein families that the sole analysis of evolutionary comutations can identify pairs of contacting residues. It is also known that evolution conserves functional dynamics, i.e., the concerted motion or displacement of large protein regions or domains. Is it, therefore, possible to use a pure sequence-based analysis to identify these dynamical domains? To address this question, we introduce here a general coevolutionary coupling analysis strategy and apply it to a curated sequence database of hundreds of protein families. For most families, the sequence-based method partitions amino acids into a few clusters. When viewed in the context of the native structure, these clusters have the signature characteristics of viable protein domains: They are spatially separated but individually compact. They have a direct functional bearing too, as shown for various reference cases. We conclude that even large-scale structural and functionally related properties can be recovered from inference methods applied to evolutionary-related sequences. The method introduced here is available as a software package and web server (spectrus.sissa.it/spectrus-evo_webserver).