Integrating Hydrogen Deuterium Exchange-Mass Spectrometry with Molecular Simulations Enables Quantification of the Conformational Populations of the Sugar Transporter XylE.

A yet unresolved challenge in structural biology is to quantify the conformational states of proteins underpinning function. This challenge is particularly acute for membrane proteins owing to the difficulties in stabilizing them for in vitro studies. To address this challenge, we present an integrative strategy that combines hydrogen deuterium exchange-mass spectrometry (HDX-MS) with ensemble modeling. We benchmark our strategy on wild-type and mutant conformers of XylE, a prototypical member of the ubiquitous Major Facilitator Superfamily (MFS) of transporters. Next, we apply our strategy to quantify conformational ensembles of XylE embedded in different lipid environments. Further application of our integrative strategy to substrate-bound and inhibitor-bound ensembles allowed us to unravel protein-ligand interactions contributing to the alternating access mechanism of secondary transport in atomistic detail. Overall, our study highlights the potential of integrative HDX-MS modeling to capture, accurately quantify, and subsequently visualize co-populated states of membrane proteins in association with mutations and diverse substrates and inhibitors.

Structural dynamics in the evolution of SARS-CoV-2 spike glycoprotein.

SARS-CoV-2 spike glycoprotein mediates receptor binding and subsequent membrane fusion. It exists in a range of conformations, including a closed state unable to bind the ACE2 receptor, and an open state that does so but displays more exposed antigenic surface. Spikes of variants of concern (VOCs) acquired amino acid changes linked to increased virulence and immune evasion. Here, using HDX-MS, we identified changes in spike dynamics that we associate with the transition from closed to open conformations, to ACE2 binding, and to specific mutations in VOCs. We show that the RBD-associated subdomain plays a role in spike opening, whereas the NTD acts as a hotspot of conformational divergence of VOC spikes driving immune evasion. Alpha, beta and delta spikes assume predominantly open conformations and ACE2 binding increases the dynamics of their core helices, priming spikes for fusion. Conversely, substitutions in omicron spike lead to predominantly closed conformations, presumably enabling it to escape antibodies. At the same time, its core helices show characteristics of being pre-primed for fusion even in the absence of ACE2. These data inform on SARS-CoV-2 evolution and omicron variant emergence.

Insights into autoregulation of a membrane protein complex by its cytoplasmic domains.

Membrane transporters mediate the passage of molecules across membranes and are essential for cellular function. While the transmembrane region of these proteins is responsible for substrate transport, often the cytoplasmic regions are required for modulating their activity. However, it can be difficult to obtain atomic-resolution descriptions of these autoregulatory domains by classical structural biology techniques, especially if they lack a single, defined structure. The betaine permease, BetP, a homotrimer, is a prominent and well-studied example of a membrane protein whose autoregulation depends on cytoplasmic N- and C-terminal segments. These domains sense and transduce changes in K+ concentration and in lipid bilayer properties caused by osmotic stress. However, structural data for these terminal domains is incomplete, which hinders a clear description of the molecular mechanism of autoregulation. Here we used microsecond-scale molecular simulations of the BetP trimer to compare reported conformations of the 45-amino-acid long C-terminal tails. The simulations provide support for the idea that the conformation derived from electron microscopy (EM) data represents a more stable global orientation of the C-terminal segment under downregulating conditions while also providing a detailed molecular description of its dynamics and highlighting specific interactions with lipids, ions, and neighboring transporter subunits. A missing piece of the molecular puzzle is the N-terminal segment, whose dynamic nature has prevented structural characterization. Using Rosetta to generate ensembles of de novo conformations in the context of the EM-derived structure robustly identifies two features of the N-terminal tail, namely 1) short helical elements and 2) an orientation that would confine potential interactions to the protomer in the counterclockwise direction (viewed from the cytoplasm). Since each C-terminal tail only contacts the protomer in the clockwise direction, these results indicate an intricate interplay between the three protomers of BetP in the downregulated protein and a multidirectionality that may facilitate autoregulation of transport.

Modeling the native ensemble of PhuS using enhanced sampling MD and HDX-ensemble reweighting.

The cytoplasmic heme binding protein from Pseudomonas aeruginosa, PhuS, plays two essential roles in regulating heme uptake and iron homeostasis. First, PhuS shuttles exogenous heme to heme oxygenase (HemO) for degradation and iron release. Second, PhuS binds DNA and modulates the transcription of the prrF/H small RNAs (sRNAs) involved in the iron-sparing response. Heme binding to PhuS regulates this dual function, as the unliganded form binds DNA, whereas the heme-bound form binds HemO. Crystallographic studies revealed nearly identical structures for apo- and holo-PhuS, and yet numerous solution-based measurements indicate that heme binding is accompanied by large conformational rearrangements. In particular, hydrogen-deuterium exchange mass spectrometry (HDX-MS) of apo- versus holo-PhuS revealed large differences in deuterium uptake, notably in α-helices 6, 7, and 8 (α6,7,8), which contribute to the heme binding pocket. These helices were mostly labile in apo-PhuS but largely protected in holo-PhuS. In contrast, in silico-predicted deuterium uptake levels of α6,7,8 from molecular dynamics (MD) simulations of the apo- and holo-PhuS structures are highly similar, consistent only with the holo-PhuS HDX-MS data. To rationalize this discrepancy between crystal structures, simulations, and observed HDX-MS, we exploit a recently developed computational approach (HDXer) that fits the relative weights of conformational populations within an ensemble of structures to conform to a target set of HDX-MS data. Here, a combination of enhanced sampling MD, HDXer, and dimensionality reduction analysis reveals an apo-PhuS conformational landscape in which α6, 7, and 8 are significantly rearranged compared to the crystal structure, including a loss of secondary structure in α6 and the displacement of α7 toward the HemO binding interface. Circular dichroism analysis confirms the loss of secondary structure, and the extracted ensembles of apo-PhuS and of heme-transfer-impaired H212R mutant, are consistent with known heme binding and transfer properties. The proposed conformational landscape provides structural insights into the modulation by heme of the dual function of PhuS.

Testing the Limitations of MD-Based Local Electric Fields Using the Vibrational Stark Effect in Solution: Penicillin G as a Test Case.

Noncovalent interactions underlie nearly all molecular processes in the condensed phase from solvation to catalysis. Their quantification within a physically consistent framework remains challenging. Experimental vibrational Stark effect (VSE)-based solvatochromism can be combined with molecular dynamics (MD) simulations to quantify the electrostatic forces in solute-solvent interactions for small rigid molecules and, by extension, when these solutes bind in enzyme active sites. While generalizing this approach toward more complex (bio)molecules, such as the conformationally flexible and charged penicillin G (PenG), we were surprised to observe inconsistencies in MD-based electric fields. Combining synthesis, VSE spectroscopy, and computational methods, we provide an intimate view on the origins of these discrepancies. We observe that the electric fields are correlated to conformation-dependent effects of the flexible PenG side chain, including both the local solvation structure and solute conformational sampling in MD. Additionally, we identified that MD-based electric fields are consistently overestimated in three-point water models in the vicinity of charged groups; this cannot be entirely ameliorated using polarizable force fields (AMOEBA) or advanced water models. This work demonstrates the value of the VSE as a direct method for experiment-guided refinements of MD force fields and establishes a general reductionist approach to calibrating vibrational probes for complex (bio)molecules.

Chloride-dependent conformational changes in the GlyT1 glycine transporter.

The human GlyT1 glycine transporter requires chloride for its function. However, the mechanism by which Cl- exerts its influence is unknown. To examine the role that Cl- plays in the transport cycle, we measured the effect of Cl- on both glycine binding and conformational changes. The ability of glycine to displace the high-affinity radioligand [3H]CHIBA-3007 required Na+ and was potentiated over 1,000-fold by Cl- We generated GlyT1b mutants containing reactive cysteine residues in either the extracellular or cytoplasmic permeation pathways and measured changes in the reactivity of those cysteine residues as indicators of conformational changes in response to ions and substrate. Na+ increased accessibility in the extracellular pathway and decreased it in the cytoplasmic pathway, consistent with stabilizing an outward-open conformation as observed in other members of this transporter family. In the presence of Na+, both glycine and Cl- independently shifted the conformation of GlyT1b toward an outward-closed conformation. Together, Na+, glycine, and Cl- stabilized an inward-open conformation of GlyT1b. We then examined whether Cl- acts by interacting with a conserved glutamine to allow formation of an ion pair that stabilizes the closed state of the extracellular pathway. Molecular dynamics simulations of a GlyT1 homolog indicated that this ion pair is formed more frequently as that pathway closes. Mutation of the glutamine blocked the effect of Cl-, and substituting it with glutamate or lysine resulted in outward- or inward-facing transporter conformations, respectively. These results provide an unexpected insight into the role of Cl- in this family of transporters.

Interpreting Hydrogen-Deuterium Exchange Experiments with Molecular Simulations: Tutorials and Applications of the HDXer Ensemble Reweighting Software [Article v1.0].

Hydrogen-deuterium exchange (HDX) is a comprehensive yet detailed probe of protein structure and dynamics and, coupled to mass spectrometry, has become a powerful tool for investigating an increasingly large array of systems. Computer simulations are often used to help rationalize experimental observations of exchange, but interpretations have frequently been limited to simple, subjective correlations between microscopic dynamical fluctuations and the observed macroscopic exchange behavior. With this in mind, we previously developed the HDX ensemble reweighting approach and associated software, HDXer, to aid the objective interpretation of HDX data using molecular simulations. HDXer has two main functions; first, to compute H-D exchange rates that describe each structure in a candidate ensemble of protein structures, for example from molecular simulations, and second, to objectively reweight the conformational populations present in a candidate ensemble to conform to experimental exchange data. In this article, we first describe the HDXer approach, theory, and implementation. We then guide users through a suite of tutorials that demonstrate the practical aspects of preparing experimental data, computing HDX levels from molecular simulations, and performing ensemble reweighting analyses. Finally we provide a practical discussion of the capabilities and limitations of the HDXer methods including recommendations for a user's own analyses. Overall, this article is intended to provide an up-to-date, pedagogical counterpart to the software, which is freely available at https://github.com/Lucy-Forrest-Lab/HDXer.

Structural predictions of the functions of membrane proteins from HDX-MS.

HDX-MS has emerged as a powerful tool to interrogate the structure and dynamics of proteins and their complexes. Recent advances in the methodology and instrumentation have enabled the application of HDX-MS to membrane proteins. Such targets are challenging to investigate with conventional strategies. Developing new tools are therefore pertinent for improving our fundamental knowledge of how membrane proteins function in the cell. Importantly, investigating this central class of biomolecules within their native lipid environment remains a challenge but also a key goal ahead. In this short review, we outline recent progresses in dissecting the conformational mechanisms of membrane proteins using HDX-MS. We further describe how the use of computational strategies can aid the interpretation of experimental data and enable visualisation of otherwise intractable membrane protein states. This unique integration of experiments with computations holds significant potential for future applications.

The Role of Electrostatics in Enzymes: Do Biomolecular Force Fields Reflect Protein Electric Fields?

Preorganization of large, directionally oriented, electric fields inside protein active sites has been proposed as a crucial contributor to catalytic mechanism in many enzymes, and it may be efficiently investigated at the atomistic level with molecular dynamics simulations. Here, we evaluate the ability of the AMOEBA polarizable force field, as well as the additive Amber ff14SB and Charmm C36m models, to describe the electric fields present inside the active site of the peptidyl-prolyl isomerase cyclophilin A. We compare the molecular mechanical electric fields to those calculated with a fully first-principles quantum mechanical (QM) representation of the protein, solvent, and ions, and find that AMOEBA consistently shows far greater correlation with the QM electric fields than either of the additive force fields tested. Catalytically relevant fields calculated with AMOEBA were typically smaller than those observed with additive potentials, but were generally consistent with an electrostatically driven mechanism for catalysis. Our results highlight the accuracy and the potential advantages of using polarizable force fields in systems where accurate electrostatics may be crucial for providing mechanistic insights.

Interpretation of HDX Data by Maximum-Entropy Reweighting of Simulated Structural Ensembles.

Hydrogen-deuterium exchange combined with mass spectrometry (HDX-MS) is a widely applied biophysical technique that probes the structure and dynamics of biomolecules without the need for site-directed modifications or bio-orthogonal labels. The mechanistic interpretation of HDX data, however, is often qualitative and subjective, owing to a lack of quantitative methods to rigorously translate observed deuteration levels into atomistic structural information. To help address this problem, we have developed a methodology to generate structural ensembles that faithfully reproduce HDX-MS measurements. In this approach, an ensemble of protein conformations is first generated, typically using molecular dynamics simulations. A maximum-entropy bias is then applied post hoc to the resulting ensemble such that averaged peptide-deuteration levels, as predicted by an empirical model, agree with target values within a given level of uncertainty. We evaluate this approach, referred to as HDX ensemble reweighting (HDXer), for artificial target data reflecting the two major conformational states of a binding protein. We demonstrate that the information provided by HDX-MS experiments and by the model of exchange are sufficient to recover correctly weighted structural ensembles from simulations, even when the relevant conformations are rarely observed. Degrading the information content of the target data-e.g., by reducing sequence coverage, by averaging exchange levels over longer peptide segments, or by incorporating different sources of uncertainty-reduces the structural accuracy of the reweighted ensemble but still allows for useful insights into the distinctive structural features reflected by the target data. Finally, we describe a quantitative metric to rank candidate structural ensembles according to their correspondence with target data and illustrate the use of HDXer to describe changes in the conformational ensemble of the membrane protein LeuT. In summary, HDXer is designed to facilitate objective structural interpretations of HDX-MS data and to inform experimental approaches and further developments of theoretical exchange models.

Evaluating Anti-CD32b F(ab) Conformation Using Molecular Dynamics and Small-Angle X-Ray Scattering.

Complementary strategies of small-angle x-ray scattering (SAXS) and crystallographic analysis are often used to determine atomistic three-dimensional models of macromolecules and their variability in solution. This combination of techniques is particularly valuable when applied to macromolecular complexes to detect changes within the individual binding partners. Here, we determine the x-ray crystallographic structure of a F(ab) fragment in complex with CD32b, the only inhibitory Fc-γ receptor in humans, and compare the structure of the F(ab) from the crystal complex to SAXS data for the F(ab) alone in solution. We investigate changes in F(ab) structure by predicting theoretical scattering profiles for atomistic structures extracted from molecular dynamics (MD) simulations of the F(ab) and assessing the agreement of these structures to our experimental SAXS data. Through principal component analysis, we are able to extract principal motions observed during the MD trajectory and evaluate the influence of these motions on the agreement of structures to the F(ab) SAXS data. Changes in the F(ab) elbow angle were found to be important to reach agreement with the experimental data; however, further discrepancies were apparent between our F(ab) structure from the crystal complex and SAXS data. By analyzing multiple MD structures observed in similar regions of the principal component analysis, we were able to pinpoint these discrepancies to a specific loop region in the F(ab) heavy chain. This method, therefore, not only allows determination of global changes but also allows identification of localized motions important for determining the agreement between atomistic structures and SAXS data. In this particular case, the findings allowed us to discount the hypothesis that structural changes were induced upon complex formation, a significant find informing the drug development process. The methodology described here is generally applicable to deconvolute global and local changes of macromolecular structures and is well suited to other systems.

Evaluation of solvation free energies for small molecules with the AMOEBA polarizable force field.

The effects of electronic polarization in biomolecular interactions will differ depending on the local dielectric constant of the environment, such as in solvent, DNA, proteins, and membranes. Here the performance of the AMOEBA polarizable force field is evaluated under nonaqueous conditions by calculating the solvation free energies of small molecules in four common organic solvents. Results are compared with experimental data and equivalent simulations performed with the GAFF pairwise-additive force field. Although AMOEBA results give mean errors close to "chemical accuracy," GAFF performs surprisingly well, with statistically significantly more accurate results than AMOEBA in some solvents. However, for both models, free energies calculated in chloroform show worst agreement to experiment and individual solutes are consistently poor performers, suggesting non-potential-specific errors also contribute to inaccuracy. Scope for the improvement of both potentials remains limited by the lack of high quality experimental data across multiple solvents, particularly those of high dielectric constant. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

Advanced Potential Energy Surfaces for Molecular Simulation.

Advanced potential energy surfaces are defined as theoretical models that explicitly include many-body effects that transcend the standard fixed-charge, pairwise-additive paradigm typically used in molecular simulation. However, several factors relating to their software implementation have precluded their widespread use in condensed-phase simulations: the computational cost of the theoretical models, a paucity of approximate models and algorithmic improvements that can ameliorate their cost, underdeveloped interfaces and limited dissemination in computational code bases that are widely used in the computational chemistry community, and software implementations that have not kept pace with modern high-performance computing (HPC) architectures, such as multicore CPUs and modern graphics processing units (GPUs). In this Feature Article we review recent progress made in these areas, including well-defined polarization approximations and new multipole electrostatic formulations, novel methods for solving the mutual polarization equations and increasing the MD time step, combining linear-scaling electronic structure methods with new QM/MM methods that account for mutual polarization between the two regions, and the greatly improved software deployment of these models and methods onto GPU and CPU hardware platforms. We have now approached an era where multipole-based polarizable force fields can be routinely used to obtain computational results comparable to state-of-the-art density functional theory while reaching sampling statistics that are acceptable when compared to that obtained from simpler fixed partial charge force fields.

Evaluating Parametrization Protocols for Hydration Free Energy Calculations with the AMOEBA Polarizable Force Field.

Hydration free energy (HFE) calculations are often used to assess the performance of biomolecular force fields and the quality of assigned parameters. The AMOEBA polarizable force field moves beyond traditional pairwise additive models of electrostatics and may be expected to improve upon predictions of thermodynamic quantities such as HFEs over and above fixed-point-charge models. The recent SAMPL4 challenge evaluated the AMOEBA polarizable force field in this regard but showed substantially worse results than those using the fixed-point-charge GAFF model. Starting with a set of automatically generated AMOEBA parameters for the SAMPL4 data set, we evaluate the cumulative effects of a series of incremental improvements in parametrization protocol, including both solute and solvent model changes. Ultimately, the optimized AMOEBA parameters give a set of results that are not statistically significantly different from those of GAFF in terms of signed and unsigned error metrics. This allows us to propose a number of guidelines for new molecule parameter derivation with AMOEBA, which we expect to have benefits for a range of biomolecular simulation applications such as protein-ligand binding studies.

Comparing experimental and computational alanine scanning techniques for probing a prototypical protein-protein interaction.

The central role of protein-protein interactions in a wide range of cellular processes makes them a target for research and drug discovery. A variety of methods, both experimental and theoretical, exist for probing protein interfaces for residues that affect activity and binding affinity. Using as an example a protein-protein complex between trypsin and a nine-residue synthetic peptide, we experimentally assay-binding affinities for a variety of mutants and determine their relative free energy of binding, ΔΔG, to rank the importance of interface residues to binding. We then compare how accurately, precisely and reliably computational methods for calculating ΔΔG can replicate these results. We find that a 'post-process alanine scanning' protocol of a single native complex trajectory gives results with better accuracy than running separate molecular dynamics (MD) trajectories for individual mutants. Compared across 10 independent simulations, we find that results from the post-process alanine scanning are also more precise and are obtained over five times faster than their equivalent with the 'full MD' protocol. These results suggest that, although not suitable in every case, post-process alanine scanning is a useful and reliable tool in predicting important residues at protein interfaces with potential for modulation.