Large Protein Conformational Changes Research Articles

DynamicBind: predicting ligand-specific protein-ligand complex structure with a deep equivariant generative model

While significant advances have been made in predicting static protein structures, the inherent dynamics of proteins, modulated by ligands, are crucial for understanding protein function and facilitating drug discovery. Traditional docking methods, frequently used in studying protein-ligand interactions, typically treat proteins as rigid. While molecular dynamics simulations can propose appropriate protein conformations, they’re computationally demanding due to rare transitions between biologically relevant equilibrium states. In this study, we present DynamicBind, a deep learning method that employs equivariant geometric diffusion networks to construct a smooth energy landscape, promoting efficient transitions between different equilibrium states. DynamicBind accurately recovers ligand-specific conformations from unbound protein structures without the need for holo-structures or extensive sampling. Remarkably, it demonstrates state-of-the-art performance in docking and virtual screening benchmarks. Our experiments reveal that DynamicBind can accommodate a wide range of large protein conformational changes and identify cryptic pockets in unseen protein targets. As a result, DynamicBind shows potential in accelerating the development of small molecules for previously undruggable targets and expanding the horizons of computational drug discovery.

Characterization of Methyl- and Acetyl-Ni Intermediates in Acetyl CoA Synthase Formed during Anaerobic CO2 and CO Fixation.

The Wood-Ljungdahl Pathway is a unique biological mechanism of carbon dioxide and carbon monoxide fixation proposed to operate through nickel-based organometallic intermediates. The most unusual steps in this metabolic cycle involve a complex of two distinct nickel-iron-sulfur proteins: CO dehydrogenase and acetyl-CoA synthase (CODH/ACS). Here, we describe the nickel-methyl and nickel-acetyl intermediates in ACS completing the characterization of all its proposed organometallic intermediates. A single nickel site (Nip) within the A cluster of ACS undergoes major geometric and redox changes as it transits the planar Nip, tetrahedral Nip-CO and planar Nip-Me and Nip-Ac intermediates. We propose that the Nip intermediates equilibrate among different redox states, driven by an electrochemical-chemical (EC) coupling process, and that geometric changes in the A-cluster linked to large protein conformational changes control entry of CO and the methyl group.

Multiplexed Isobaric Quantitative Cross-Linking Reveals Drug-Induced Interactome Changes in Breast Cancer Cells.

The study of protein structures and interactions is critical to understand their function. Chemical cross-linking of proteins with mass spectrometry (XL-MS) is a rapidly developing structural biology technique able to provide valuable insight into protein conformations and interactions, even as they exist within their native cellular environment. Quantitative analysis of cross-links can reveal protein conformational and interaction changes that occur as a result of altered biological states, environmental conditions, or pharmacological perturbations. Our laboratory recently developed an isobaric quantitative protein interaction reporter (iqPIR) cross-linking strategy for comparative interactome studies. This strategy relies on isotope encoded chemical cross-linkers that have the same molecular mass yet produce unique and specific isotope signatures upon fragmentation in the mass spectrometer which can be used for quantitative analysis of cross-linked peptides. The initial set of iqPIR molecules allowed for binary comparisons. Here, we describe the in vivo application of an extended set of six iqPIR reagents (6-plex iqPIR), allowing multiplexed quantitative interactome analysis of up to six biological samples in a single LC-MS acquisition. Multiplexed iqPIR is demonstrated on MCF-7 breast cancer cells treated with five different Hsp90 inhibitors revealing large scale protein conformational and interaction changes specific to the molecular class of the inhibitors.

Rapid-Scan Time-Resolved ATR-FTIR Study on the Photoassembly of the Water-Oxidizing Mn4CaO5 Cluster in Photosystem II.

The catalytic center of photosynthetic water oxidation, the Mn4CaO5 cluster, is assembled in photosystem II (PSII) through a light-driven process called photoactivation, whose mechanism remains elusive. Here, we used rapid-scan time-resolved Fourier transform infrared (FTIR) spectroscopy combined with the attenuated total reflection (ATR) technique to monitor the photoactivation process. Rapid-scan ATR-FTIR spectra of apo-PSII with Mn2+ upon flash illumination showed spectral features typical of carboxylate stretching vibrations, which were attributed to two carboxylate ligands, D1-D170 and D1-E189, by quantum chemical calculations. The FTIR signal decayed with a time constant of ∼0.7 s, showing that the subsequent "dark rearrangement" step occurred with a low quantum yield and Mn3+ ions were mostly released during this decay. Simulation of the kinetic process provided a slow intrinsic rate of the dark rearrangement, which was attributed to a large protein conformational change. The photoassembly mechanism of the Mn4CaO5 cluster is proposed based on these findings.

A pH-Induced Reversible Conformational Switch Able to Control the Photocurrent Efficiency in a Peptide Supramolecular System.

External stimuli are potent tools that Nature uses to control protein function and activity. For instance, during viral entry and exit, pH variations are known to trigger large protein conformational changes. In Nature, also the electron transfer (ET) properties of ET proteins are influenced by pH-induced conformational changes. In this work, a pH-controlled, reversible 310 -helix to α-helix conversion (from acidic to highly basic pH values and vice versa) of a peptide supramolecular system built on a gold surface is described. The effect of pH on the ability of the peptide SAM to generate a photocurrent was investigated, with particular focus on the effect of the pH-induced conformational change on photocurrent efficiency. The films were characterized by electrochemical and spectroscopic techniques, and were found to be very stable over time, also in contact with a solution. They were also able to generate current under illumination, with an efficiency that is the highest recorded so far with biomolecular systems.

Photoreceptors Take Charge: Emerging Principles for Light Sensing.

The first stage in biological signaling is based on changes in the functional state of a receptor protein triggered by interaction of the receptor with its ligand(s). The light-triggered nature of photoreceptors allows studies on the mechanism of such changes in receptor proteins using a wide range of biophysical methods and with superb time resolution. Here, we critically evaluate current understanding of proton and electron transfer in photosensory proteins and their involvement both in primary photochemistry and subsequent processes that lead to the formation of the signaling state. An insight emerging from multiple families of photoreceptors is that ultrafast primary photochemistry is followed by slower proton transfer steps that contribute to triggering large protein conformational changes during signaling state formation. We discuss themes and principles for light sensing shared by the six photoreceptor families: rhodopsins, phytochromes, photoactive yellow proteins, light-oxygen-voltage proteins, blue-light sensors using flavin, and cryptochromes.

Multiscale Kinetic Modeling Reveals an Ensemble of Cl-/H+ Exchange Pathways in ClC-ec1 Antiporter.

Despite several years of research, the ion exchange mechanisms in chloride/proton antiporters and many other coupled transporters are not yet understood at the molecular level. Here, we present a novel approach to kinetic modeling and apply it to ion exchange in ClC-ec1. Our multiscale kinetic model is developed by (1) calculating the state-to-state rate coefficients with reactive and polarizable molecular dynamics simulations, (2) optimizing these rates in a global kinetic network, and (3) predicting new electrophysiological results. The model shows that the robust Cl:H exchange ratio (2.2:1) can indeed arise from kinetic coupling without large protein conformational changes, indicating a possible facile evolutionary connection to chloride channels. The E148 amino acid residue is shown to couple chloride and proton transport through protonation-dependent blockage of the central anion binding site and an anion-dependent pKa value, which influences proton transport. The results demonstrate how an ensemble of different exchange pathways, as opposed to a single series of transitions, culminates in the macroscopic observables of the antiporter, such as transport rates, chloride/proton stoichiometry, and pH dependence.

Nanosecond pump-probe device for time-resolved serial femtosecond crystallography developed at SACLA.

X-ray free-electron lasers (XFELs) have opened new opportunities for time-resolved X-ray crystallography. Here a nanosecond optical-pump XFEL-probe device developed for time-resolved serial femtosecond crystallography (TR-SFX) studies of photo-induced reactions in proteins at the SPring-8 Angstrom Compact free-electron LAser (SACLA) is reported. The optical-fiber-based system is a good choice for a quick setup in a limited beam time and allows pump illumination from two directions to achieve high excitation efficiency of protein microcrystals. Two types of injectors are used: one for extruding highly viscous samples such as lipidic cubic phase (LCP) and the other for pulsed liquid droplets. Under standard sample flow conditions from the viscous-sample injector, delay times from nanoseconds to tens of milliseconds are accessible, typical time scales required to study large protein conformational changes. A first demonstration of a TR-SFX experiment on bacteriorhodopsin in bicelle using a setup with a droplet-type injector is also presented.

Mechanism of DNA loading by the DNA repair helicase XPD.

The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5′ to 3′ helicase with an essential iron–sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase from Thermoplasma acidophilum has been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD from Sulfolobus acidocaldiarius that lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD.

Probing Conformational Changes of Secondary Active Transport Proteins

Secondary active transporters (SATs) require large protein conformational changes, which some believe follow the alternating access model. The structure of each characteristic conformational state is important for understanding the complete substrate transport process. However, the structure of certain state(s) can be very difficult to obtain by experiment, while certain enhanced simulation techniques may provide an approach to predict unknown structures. Self-guided Langevin dynamics (SGLD) (Chem. Phys. Lett., 381: 512-518) simulations allows proteins to overcome energy barriers and thus it can be used to search the structures of all the conformational states efficiently by enhancing the conformational sampling of low frequency motion. SGLD accelerates the conformational change while preserves the conformational distribution well. Therefore, this method is applied to study a SAT - lactose permease of E.coli. (LacY), which belongs to major facilitator superfamily (MFS). LacY structures of the cytoplasmic-open and occluded-like structure have been determined. To search the periplasmic-open LacY, SGLD simulations are performed with inward-facing LacY as initial structure, with different residues protonated, with and without ββ-(Galp)2, in the implicit membrane and in an explicit membrane in the NVT ensemble (available recently). The SGLD parameters adjusted are guiding factor and friction factor to control guiding force, and also local average time to set the boundary of low frequency motion to enhance. The preliminary broad SGLD parameters sweep are applied to find reasonable parameter set range for sampling protein structures. Then, a fine parameter sweep is applied to obtain the optimized structure. The effect of each parameter set, the protonation states, and presence of sugar on LacY residue distances and pore radii will be presented. These optimal SGLD parameter values may be applied to study conformational changes of other similar SAT proteins.

Atomic view of the histidine environment stabilizing higher-pH conformations of pH-dependent proteins.

External stimuli are powerful tools that naturally control protein assemblies and functions. For example, during viral entry and exit changes in pH are known to trigger large protein conformational changes. However, the molecular features stabilizing the higher pH structures remain unclear. Here we elucidate the conformational change of a self-assembling peptide that forms either small or large nanotubes dependent on the pH. The sub-angstrom high-pH peptide structure reveals a globular conformation stabilized through a strong histidine-serine H-bond and a tight histidine-aromatic packing. Lowering the pH induces histidine protonation, disrupts these interactions and triggers a large change to an extended β-sheet-based conformation. Re-visiting available structures of proteins with pH-dependent conformations reveals both histidine-containing aromatic pockets and histidine-serine proximity as key motifs in higher pH structures. The mechanism discovered in this study may thus be generally used by pH-dependent proteins and opens new prospects in the field of nanomaterials.

GneimoSim: a modular internal coordinates molecular dynamics simulation package.

The generalized Newton-Euler inverse mass operator (GNEIMO) method is an advanced method for internal coordinates molecular dynamics (ICMD). GNEIMO includes several theoretical and algorithmic advancements that address longstanding challenges with ICMD simulations. In this article, we describe the GneimoSim ICMD software package that implements the GNEIMO method. We believe that GneimoSim is the first software package to include advanced features such as the equipartition principle derived for internal coordinates, and a method for including the Fixman potential to eliminate systematic statistical biases introduced by the use of hard constraints. Moreover, by design, GneimoSim is extensible and can be easily interfaced with third party force field packages for ICMD simulations. Currently, GneimoSim includes interfaces to LAMMPS, OpenMM, and Rosetta force field calculation packages. The availability of a comprehensive Python interface to the underlying C++ classes and their methods provides a powerful and versatile mechanism for users to develop simulation scripts to configure the simulation and control the simulation flow. GneimoSim has been used extensively for studying the dynamics of protein structures, refinement of protein homology models, and for simulating large scale protein conformational changes with enhanced sampling methods. GneimoSim is not limited to proteins and can also be used for the simulation of polymeric materials.

Structural diversity of neuronal calcium sensor proteins and insights for activation of retinal guanylyl cyclase by GCAP1

Neuronal calcium sensor (NCS) proteins, a sub-branch of the calmodulin superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite different. Retinal recoverin controls Ca2+-dependent inactivation of light-excited rhodopsin during phototransduction, guanylyl cyclase activating proteins 1 and 2 (GCAP1 and GCAP2) promote Ca2+-dependent activation of retinal guanylyl cyclases, and neuronal frequenin (NCS-1) modulates synaptic activity and neuronal secretion. Here we review the molecular structures of myristoylated forms of NCS-1, recoverin, and GCAP1 that all look very different, suggesting that the attached myristoyl group helps to refold these highly homologous proteins into different three-dimensional folds. Ca2+-binding to both recoverin and NCS-1 cause large protein conformational changes that ejects the covalently attached myristoyl group into the solvent exterior and promotes membrane targeting (Ca2+-myristoyl switch). The GCAP proteins undergo much smaller Ca2+-induced conformational changes and do not possess a Ca2+-myristoyl switch. Recent structures of GCAP1 in both its activator and Ca2+-bound inhibitory states will be discussed to understand structural determinants that control their Ca2+-dependent activation of retinal guanylyl cyclases.

NMR Detection of pH-Dependent Histidine–Water Proton Exchange Reveals the Conduction Mechanism of a Transmembrane Proton Channel

The acid-activated proton channel formed by the influenza M2 protein is important for the life cycle of the virus. A single histidine, His37, in the M2 transmembrane domain (M2TM) is responsible for pH activation and proton selectivity of the channel. Recent studies suggested three models for how His37 mediates proton transport: a shuttle mechanism involving His37 protonation and deprotonation, a H-bonded imidazole-imidazolium dimer model, and a transporter model involving large protein conformational changes in synchrony with proton conduction. Using magic-angle-spinning (MAS) solid-state NMR spectroscopy, we examined the proton exchange and backbone conformational dynamics of M2TM in a virus-envelope-mimetic membrane. At physiological temperature and pH, (15)N NMR spectra show fast exchange of the imidazole (15)N between protonated and unprotonated states. To quantify the proton exchange rates, we measured the (15)N T(2) relaxation times and simulated them for chemical-shift exchange and fluctuating N-H dipolar fields under (1)H decoupling and MAS. The exchange rate is 4.5 × 10(5) s(-1) for Nδ1 and 1.0 × 10(5) s(-1) for Nε2, which are approximately synchronized with the recently reported imidazole reorientation. Binding of the antiviral drug amantadine suppressed both proton exchange and ring motion, thus interfering with the proton transfer mechanism. By measuring the relative concentrations of neutral and cationic His as a function of pH, we determined the four pK(a) values of the His37 tetrad in the viral membrane. Fitting the proton current curve using the charge-state populations from these pK(a)'s, we obtained the relative conductance of the five charge states, which showed that the +3 channel has the highest time-averaged unitary conductance. At physiologically relevant pH, 2D correlation spectra indicated that the neutral and cationic histidines do not have close contacts, ruling out the H-bonded dimer model. Moreover, a narrowly distributed nonideal helical structure coexists with a broadly distributed ideal helical conformation without interchange on the sub-10 ms time scale, thus excluding the transporter model in the viral membrane. These data support the shuttle mechanism of proton conduction, whose essential steps involve His-water proton exchange facilitated by imidazole ring reorientations.

Abstract 2327: Structural and kinetic characterization of crizotinib with wild-type and mutant anaplastic lymphoma kinase

Abstract Dysregulation of Anaplastic Lymphoma Kinase (ALK), primarily through gene translocations, has been shown to be involved in a variety of cancers. Crizotinib, an orally available small molecule inhibitor of the ALK tyrosine kinase has demonstrated marked efficacy in clinical trials of NSCLC patients harboring the EML4-ALK oncogenic gene rearrangement. Mutation of some residues within the ALK kinase domain have been reported to confer acquired or de novo resistance to crizotinib. To understand the binding of crizotinib to ALK and the mechanism of resistance of specific mutations we generated and kinetically characterized wild-type (WT) and mutant ALK kinase domains (KD). Additionally, ALK KD crystal structures were determined of the WT nonphosphorylated apoenzyme and complexes with crizotinib bound to WT and a L1196M gatekeeper mutation. No large protein conformational changes are necessary for crizotinib to bind to unliganded ALK. The interactions which crizotinib makes with ALK are similar to its binding to c-Met with the exception of notable differences in the position of the activation loop between ALK and c-Met. Mutation of the L1196 gatekeeper residue to methionine results in a ∼8-fold increase in catalytic efficiency of phosphorylation of an activation loop peptide and also more rapid enzyme auto-phosphorylation. In addition, inhibition of L1196M ALK by crizotinib was reduced ∼9-fold, compared to wild-type enzyme, from Ki determinations. The crystal structures show that L1196 or M1196 make direct contact with crizotinib. The diminished activity of crizotinib against L1196M ALK is therefore likely due to both higher intrinsic kinase activity and a subtle change in the ALK-crizotinib binding interactions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2327. doi:10.1158/1538-7445.AM2011-2327

Reevaluating Herpes Simplex Virus Hemifusion

Membrane fusion induced by enveloped viruses proceeds through the actions of viral fusion proteins. Once activated, viral fusion proteins undergo large protein conformational changes to execute membrane fusion. Fusion is thought to proceed through a "hemifusion" intermediate in which the outer membrane leaflets of target and viral membranes mix (lipid mixing) prior to fusion pore formation, enlargement, and completion of fusion. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins-glycoprotein D (gD), glycoprotein B (gB), and a heterodimer of glycoprotein H and L (gH/gL)-to accomplish fusion. gD is primarily thought of as a receptor-binding protein and gB as a fusion protein. The role of gH/gL in fusion has remained enigmatic. Despite experimental evidence that gH/gL may be a fusion protein capable of inducing hemifusion in the absence of gB, the recently solved crystal structure of HSV-2 gH/gL has no structural homology to any known viral fusion protein. We found that in our hands, all HSV entry proteins-gD, gB, and gH/gL-were required to observe lipid mixing in both cell-cell- and virus-cell-based hemifusion assays. To verify that our hemifusion assay was capable of detecting hemifusion, we used glycosylphosphatidylinositol (GPI)-linked hemagglutinin (HA), a variant of the influenza virus fusion protein, HA, known to stall the fusion process before productive fusion pores are formed. Additionally, we found that a mutant carrying an insertion within the short gH cytoplasmic tail, 824L gH, is incapable of executing hemifusion despite normal cell surface expression. Collectively, our findings suggest that HSV gH/gL may not function as a fusion protein and that all HSV entry glycoproteins are required for both hemifusion and fusion. The previously described gH 824L mutation blocks gH/gL function prior to HSV-induced lipid mixing.

Free Energy Profiles of Large Scale Protein Conformational Changes

The function of many enzymes requires a transformation between widely different conformational states. Often simulations of all-atom models are not capable of determining the transformation between experimentally known end states because of the large system size and wide range of these conformational changes. However, the characterization of such transition pathways using coarse-grained models can identify significant features such as free energy barriers. In this study we employed a Monte Carlo simulation framework where bond lengths and bond angles are preserved in order to generate an initial pathway along the change in RMSD connecting these end point conformations. Within this framework, rotatable dihedral bonds along the main chain and side chain serve as the effective degrees of freedom. We then sampled conformations for each of the intermediates along this pathway without bias. The resulting conformations were combined in order to calculate the free energy profiles for these conformational changes in different proteins. These profiles yield realistic free energy barriers and indicate the degree to which conformational change is coupled to ligand binding in these enzymes.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Protein Conformational Changes in the Bacteriorhodopsin Photocycle: Comparison of Findings from Electron and X-Ray Crystallographic Analyses

Light-driven conformational changes in the membrane protein bacteriorhodopsin have been studied extensively using X-ray and electron crystallography, resulting in the deposition of >30 sets of coordinates describing structural changes at various stages of proton transport. Using projection difference Fourier maps, we show that coordinates reported by different groups for the same photocycle intermediates vary considerably in the extent and nature of conformational changes. The different structures reported for the same intermediate cannot be reconciled in terms of differing extents of change on a single conformational trajectory. New measurements of image phases obtained by cryo-electron microscopy of the D96G/F171C/F219L triple mutant provide independent validation for the description of the large protein conformational change derived at 3.2 Å resolution by electron crystallography of 2D crystals, but do not support atomic models for light-driven conformational changes derived using X-ray crystallography of 3D crystals. Our findings suggest that independent determination of phase information from 2D crystals can be an important tool for testing the accuracy of atomic models for membrane protein conformational changes.

Structural basis for gate-DNA recognition and bending by type IIA topoisomerases

Type II topoisomerases disentangle DNA to facilitate chromosome segregation, and represent a major class of therapeutic targets. Although these enzymes have been studied extensively, a molecular understanding of DNA binding has been lacking. Here we present the structure of a complex between the DNA-binding and cleavage core of Saccharomyces cerevisiae Topo II (also known as Top2) and a gate-DNA segment. The structure reveals that the enzyme enforces a 150 degrees DNA bend through a mechanism similar to that of remodelling proteins such as integration host factor. Large protein conformational changes accompany DNA deformation, creating a bipartite catalytic site that positions the DNA backbone near a reactive tyrosine and a coordinated magnesium ion. This configuration closely resembles the catalytic site of type IA topoisomerases, reinforcing an evolutionary link between these structurally and functionally distinct enzymes. Binding of DNA facilitates opening of an enzyme dimerization interface, providing visual evidence for a key step in DNA transport.

Mathematical and biological models of blood coagulation

Mathematical and biological models of blood coagulation