Predicting molecular properties of α-synuclein using force fields for intrinsically disordered proteins.

Independent force field validation is an essential practice to keep track of developments and for performing meaningful Molecular Dynamics simulations. In this work, atomistic force fields for intrinsically disordered proteins (IDP) are tested by simulating the archetypical IDP α-synuclein in solution for 2.5 µs. Four combinations of protein and water force fields were tested: ff19SB/OPC, ff19SB/TIP4P-D, ff03CMAP/TIP4P-D, and a99SB-disp/TIP4P-disp, with four independent repeat simulations for each combination. We compare our simulations to the results of a 73 µs simulation using the a99SB-disp/TIP4P-disp combination, provided by D. E. Shaw Research. From the trajectories, we predict a range of experimental observations of α-synuclein and compare them to literature data. This includes protein radius of gyration and hydration, intramolecular distances, NMR chemical shifts, and 3 J-couplings. Both ff19SB/TIP4P-D and a99SB-disp/TIP4P-disp produce extended conformational ensembles of α-synuclein that agree well with experimental radius of gyration and intramolecular distances while a99SB-disp/TIP4P-disp reproduces a balanced α-synuclein secondary structure content. It was found that ff19SB/OPC and ff03CMAP/TIP4P-D produce overly compact conformational ensembles and show discrepancies in the secondary structure content compared to the experimental data.

Molecular mechanism of sugar transport in plants unveiled by structures of glucose/H+ symporter STP10.

Sugars are essential sources of energy and carbon and also function as key signalling molecules in plants. Sugar transport proteins (STP) are proton-coupled symporters responsible for uptake of glucose from the apoplast into plant cells. They are integral to organ development in symplastically isolated tissues such as seed, pollen and fruit. Additionally, STPs play a vital role in plant responses to stressors such as dehydration and prevalent fungal infections like rust and mildew. Here we present a structure of Arabidopsis thaliana STP10 in the inward-open conformation at 2.6 Å resolution and a structure of the outward-occluded conformation at improved 1.8 Å resolution, both with glucose and protons bound. The two structures describe key states in the STP transport cycle. Together with molecular dynamics simulations that establish protonation states and biochemical analysis, they pinpoint structural elements, conserved in all STPs, that clarify the basis of proton-to-glucose coupling. These results advance our understanding of monosaccharide uptake, which is essential for plant organ development, and set the stage for bioengineering strategies in crops.

Baseline Comparisons of Complementary Sampling Methods for Assembly Driven by Short-Ranged Pair Potentials toward Fast and Flexible Hybridization.

This work measures baseline sampling characteristics that highlight fundamental differences between sampling methods for assembly driven by short-ranged pair potentials. Such granular comparison is essential for fast, flexible, and accurate hybridization of complementary methods. Besides sampling speed, efficiency, and accuracy of uniform grid coverage, other sampling characteristics measured are (i) accuracy of covering narrow low energy regions that have low effective dimension (ii) ability to localize sampling to specific basins, and (iii) flexibility in sampling distributions. As a proof of concept, we compare a recently developed geometric methodology EASAL (Efficient Atlasing and Search of Assembly Landscapes) and the traditional Monte Carlo (MC) method for sampling the energy landscape of two assembling trans-membrane helices, driven by short-range pair potentials. By measuring the above-mentioned sampling characteristics, we demonstrate that EASAL provides localized and accurate coverage of crucial regions of the energy landscape of low effective dimension, under flexible sampling distributions, with much fewer samples and computational resources than MC sampling. EASAL's empirically validated theoretical guarantees permit credible extrapolation of these measurements and comparisons to arbitrary number and size of assembling units. Promising avenues for hybridizing the complementary advantages of the two methods are discussed.

Simulating Multiple Substrate-Binding Events by γ-Glutamyltransferase Using Accelerated Molecular Dynamics.

γ-Glutamyltransferase (GGT) is an enzyme that uses γ-glutamyl compounds as substrates and catalyzes their transfer to a water molecule or an acceptor substrate with varied physiological function in bacteria, plants, and animals. Crystal structures of GGT are known for different species and in different states of the chemical reaction; however, the structural dynamics of the substrate binding to the catalytic site of GGT are unknown. Here, we modeled Escherichia coli GGT's glutamine binding by using a swarm of accelerated molecular dynamics (aMD) simulations. Characterization of multiple binding events identified three structural binding motifs composed of polar residues in the binding pocket that govern glutamine binding into the active site. Simulated open and closed conformations of a lid-loop protecting the binding cavity suggest its role as a gating element by allowing or blocking substrates entry into the binding pocket. Partially open states of the lid-loop are accessible within thermal fluctuations, while the estimated free energy cost of a complete open state is 2.4 kcal/mol. Our results suggest that both specific electrostatic interactions and GGT conformational dynamics dictate the molecular recognition of substrate-GGT complexes.

A Hierarchical Approach to Predict Conformation-Dependent Histidine Protonation States in Stable and Flexible Proteins.

Solution acidity measured by pH is an important environmental factor that affects protein structure. It influences the protonation state of protein residues, which in turn may be coupled to protein conformational changes, unfolding, and ligand binding. It remains difficult to compute and measure the p Ka of individual residues, as well as to relate them to pH-dependent protein transitions. This paper presents a hierarchical approach to compute the p Ka of individual protonatable residues, specifically histidines, coupled with underlying structural changes of a protein. A fast and efficient free energy perturbation (FEP) algorithm has also been developed utilizing a fast implementation of standard molecular dynamics (MD) algorithms. Specifically, a CUDA version of the AMBER MD engine is used in this paper. Eight histidine p Ka's are computed in a diverse set of pH stable proteins to demonstrate the proposed approach's utility and assess the predictive quality of the AMBER FF99SB force field. A reference molecule is carefully selected and tested for convergence. A hierarchical approach is used to model p Ka's of the six histidine residues of the diphtheria toxin translocation domain (DTT), which exhibits a diverse ensemble of individual conformations and pH-dependent unfolding. The hierarchical approach consists of first sampling equilibrium conformational ensembles of a protein with protonated and neutral histidine residues via long equilibrium MD simulations (Flores-Canales, J. C.; et al. bioRxiv, 2019, 572040). A clustering method is then used to identify sampled protein conformations, and p Ka's of histidines in each protein conformation are computed. Finally, an ensemble averaging formalism is developed to compute weighted average histidine p Ka's. These can be compared with an apparent experimentally measured p Ka of the DTT protein and thus allows us to propose a mechanism of pH-dependent unfolding of the DTT protein.

Protein structure modeling and refinement by global optimization in CASP12.

For protein structure modeling in the CASP12 experiment, we have developed a new protocol based on our previous CASP11 approach. The global optimization method of conformational space annealing (CSA) was applied to 3 stages of modeling: multiple sequence-structure alignment, three-dimensional (3D) chain building, and side-chain re-modeling. For better template selection and model selection, we updated our model quality assessment (QA) method with the newly developed SVMQA (support vector machine for quality assessment). For 3D chain building, we updated our energy function by including restraints generated from predicted residue-residue contacts. New energy terms for the predicted secondary structure and predicted solvent accessible surface area were also introduced. For difficult targets, we proposed a new method, LEEab, where the template term played a less significant role than it did in LEE, complemented by increased contributions from other terms such as the predicted contact term. For TBM (template-based modeling) targets, LEE performed better than LEEab, but for FM targets, LEEab was better. For model refinement, we modified our CASP11 molecular dynamics (MD) based protocol by using explicit solvents and tuning down restraint weights. Refinement results from MD simulations that used a new augmented statistical energy term in the force field were quite promising. Finally, when using inaccurate information (such as the predicted contacts), it was important to use the Lorentzian function for which the maximal penalty arising from wrong information is always bounded.

Configurational Preference of the Glutamate Receptor Ligand Binding Domain Dimers.

Ionotropic glutamate receptors are a family of tetrameric ion channels with functional states consisting of nonconducting, conducting, and desensitized states that are starting to become well characterized by electrophysiological and biophysical studies. However, the structure and relative energetics of these states beyond the general structure of the receptor are still not well understood. It is known that the interface between monomeric subunits of the tetramer plays a major role in distinguishing these functional states. We have used umbrella sampling and multimicrosecond molecular dynamics simulations of the GluA2 AMPA subtype glutamate receptor ligand-binding domain (LBD) dimers to characterize a natural propensity of the LBD dimers for various configurational states. Our results show a proposed desensitized conformation of the LBD dimer is a highly preferable conformation of the LBD dimer without the influence of other receptor domains or crystallographic conditions. This has been demonstrated by both free protein simulations of 5 µs duration, as well as by computed free energy difference between the active and desensitized states. At the same time, the simulations performed using the same protocols revealed that for the LBD mutant L483Y, known to lack desensitization, the postulated active state of the LBD dimer is indeed the preferred configurational state, which remained stable in the simulations. Our findings pave the path for developing more detailed hypotheses of the full receptor activation mechanism. Combined with the energetics of glutamate binding to the LBD and the energy required to open the transmembrane pore helices, our results strongly support a hypothesis that the low absolute free-energy state is the desensitized state of the intact AMPA receptor.

Targeting electrostatic interactions in accelerated molecular dynamics with application to protein partial unfolding.

Accelerated molecular dynamics (aMD) is a promising sampling method to generate an ensemble of conformations and to explore the free energy landscape of proteins in explicit solvent. Its success resides in its ability to reduce barriers in the dihedral and the total potential energy space. However, aMD simulations of large proteins can generate large fluctuations of the dihedral and total potential energy with little conformational changes in the protein structure. To facilitate wider conformational sampling of large proteins in explicit solvent, we developed a direct intrasolute electrostatic interactions accelerated MD (DISEI-aMD) approach. This method aims to reduce energy barriers within rapidly changing electrostatic interactions between solute atoms at short-range distances. It also results in improved reconstruction quality of the original statistical ensemble of the system. Recently, we characterized a pH-dependent partial unfolding of diphtheria toxin translocation domain (T-domain) using microsecond long MD simulations. In this work, we focus on the study of conformational changes of a low-pH T-domain model in explicit solvent using DISEI-aMD. On the basis of the simulations of the low-pH T-domain model, we show that the proposed sampling method accelerates conformational rearrangement significantly faster than multiple standard aMD simulations and microsecond long conventional MD simulations.

Microsecond Simulations of the Diphtheria Toxin Translocation Domain in Association with Anionic Lipid Bilayers.

Diphtheria toxin translocation (T) domain undergoes conformational changes in acidic solution and associates with the lipid membranes, followed by refolding and transmembrane insertion of two nonpolar helices. This process is an essential step in delivery of the toxic catalytic domain of the diphtheria toxin to the infected cell, yet its molecular determinants are poorly characterized and understood. Therefore, an atomistic model of the T-domain-membrane interaction is needed to help characterize factors responsible for such association. In this work, we present atomistic model structures of T-domain membrane-bound conformations and investigate structural factors responsible for T-domain affinity with the lipid bilayer in acidic solution using all-atom molecular dynamics (MD) simulations. The initial models of the protein conformations and protein-membrane association that serve as starting points in the present work were developed using atomistic simulations of partial unfolding of the T-domain in acidic solution (Kurnikov, I. V.; et al. J. Mol. Biol. 2013, 425, 2752-2764), and coarse-grained simulations of the T-domain association with the membranes of various compositions (Flores-Canales, J. C.; et al. J. Membr. Biol. 2015, 248, 529-543). In this work we present atomistic level modeling of two distinct configurations of the T-domain in association with the anionic lipid bilayer. In microsecond-long MD simulations both conformations retain their compact structure and gradually penetrate deeper into the bilayer interface. One membrane-bound conformation is stabilized by the protein contacts with the lipid hydrophobic core. The second modeled conformation is initially inserted less deeply and forms multiple contacts with the lipid at the interface (headgroup) region. Such contacts are formed by the charged and hydrophilic groups of partially unfolded terminal helixes and loops. Neutralization of the acidic residues at the membrane interface allows for deeper insertion of the protein and reorientation of the protein at the membrane interface, which corroborates that acidic residue protonation as well as presence of the anionic lipids may play a role in the membrane association and further membrane insertion of the T-domain as implicated in experiments. All simulations reported in this work were performed using AMBER force-field on Anton supercomputer. To perform these reported simulations, we developed and carefully tested a force-field for the anionic 1-palmitoyl-2-oleoyl-phosphatidyl-glycerol (POPG) lipid, compatible with the Amber 99SB force-field and stable in microsecond-long MD simulations in isothermal-isobaric ensemble.

Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain.

The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8-TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8-TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

Membrane Association of the Diphtheria Toxin Translocation Domain Studied by Coarse-Grained Simulations and Experiment.

Diphtheria toxin translocation (T) domain inserts in lipid bilayers upon acidification of the environment. Computational and experimental studies have suggested that low pH triggers a conformational change of the T-domain in solution preceding membrane binding. The refolded membrane-competent state was modeled to be compact and mostly retain globular structure. In the present work, we investigate how this refolded state interacts with membrane interfaces in the early steps of T-domain's membrane association. Coarse-grained molecular dynamics simulations suggest two distinct membrane-bound conformations of the T-domain in the presence of bilayers composed of a mixture of zwitteronic and anionic phospholipids (POPC:POPG with a 1:3 molar ratio). Both membrane-bound conformations show a common near parallel orientation of hydrophobic helices TH8-TH9 relative to the membrane plane. The most frequently observed membrane-bound conformation is stabilized by electrostatic interactions between the N-terminal segment of the protein and the membrane interface. The second membrane-bound conformation is stabilized by hydrophobic interactions between protein residues and lipid acyl chains, which facilitate deeper protein insertion in the membrane interface. A theoretical estimate of a free energy of binding of a membrane-competent T-domain to the membrane is provided.

pH-triggered conformational switching of the diphtheria toxin T-domain: the roles of N-terminal histidines.

pH-induced conformational switching is essential for functioning of diphtheria toxin, which undergoes a membrane insertion/translocation transition triggered by endosomal acidification as a key step of cellular entry. In order to establish the sequence of molecular rearrangements and side-chain protonation accompanying the formation of the membrane-competent state of the toxin's translocation (T) domain, we have developed and applied an integrated approach that combines multiple techniques of computational chemistry [e.g., long-microsecond-range, all-atom molecular dynamics (MD) simulations; continuum electrostatics calculations; and thermodynamic integration (TI)] with several experimental techniques of fluorescence spectroscopy. TI calculations indicate that protonation of H257 causes the greatest destabilization of the native structure (6.9 kcal/mol), which is consistent with our early mutagenesis results. Extensive equilibrium MD simulations with a combined length of over 8 µs demonstrate that histidine protonation, while not accompanied by the loss of structural compactness of the T-domain, nevertheless results in substantial molecular rearrangements characterized by the partial loss of secondary structure due to unfolding of helices TH1 and TH2 and the loss of close contact between the C- and N-terminal segments. The structural changes accompanying the formation of the membrane-competent state ensure an easier exposure of the internal hydrophobic hairpin formed by helices TH8 and TH9, in preparation for its subsequent transmembrane insertion.