Explicit Solvent Molecular Dynamics Simulations Research Articles

Slower CDK4 and faster CDK2 activation in the cell cycle.

Dysregulation of cyclin-dependent kinases (CDKs) impacts cell proliferation, driving cancer. Here, we ask why the cyclin-D/CDK4 complex governs cell cycle progression through the longer G1 phase, whereas cyclin-E/CDK2 regulates the shorter G1/S phase transition. We consider available experimental cellular and structural data including cyclin-E's high-level burst, sustained duration of elevated cyclin-D expression, and explicit solvent molecular dynamics simulations of the inactive monomeric and complexed states, to establish the conformational tendencies along the landscape of the distinct activation scenarios of cyclin-D/CDK4 and cyclin-E/CDK2 in the G1 phase and G1/S transition of the cell cycle, respectively. These lead us to propose slower activation of cyclin-D/CDK4 and rapid activation of cyclin-E/CDK2. We provide the mechanisms through which this occurs, offering innovative CDK4 drug design considerations. Our insightful mechanistic work addresses a compelling cell cycle regulation question and illuminates the distinct activation speeds between the G1 and the G1/S phases, which are crucial for function.

A slow but steady nanoLuc: R162A mutation results in a decreased, but stable, nanoLuc activity

NanoLuc (NLuc) luciferase has found extensive application in designing a range of biological assays, including gene expression analysis, protein-protein interaction, and protein conformational changes due to its enhanced brightness and small size. However, questions related to its mechanism of interaction with the substrate, furimazine, as well as bioluminescence activity remain elusive. Here, we combined molecular dynamics (MD) simulation and mutational analysis to show that the R162A mutation results in a decreased but stable bioluminescence activity of NLuc in living cells and in vitro. Specifically, we performed multiple, all-atom, explicit solvent MD simulations of the apo and furimazine-docked (holo) NLuc structures revealing differential dynamics of the protein in the absence and presence of the ligand. Further, analysis of trajectories for hydrogen bonds (H-bonds) formed between NLuc and furimazine revealed substantial H-bond interaction between R162 and Q32 residues. Mutation of the two residues in NLuc revealed a decreased but stable activity of the R162A, but not Q32A, mutant NLuc in live cell and in vitro assays performed using lysates prepared from cells expressing the proteins and with the furimazine substrate. In addition to highlighting the role of the R162 residue in NLuc activity, we believe that the mutant NLuc will find wide application in designing in vitro assays requiring extended monitoring of NLuc bioluminescence activity. SignificanceBioluminescence has been extensively utilized in developing a variety of biological and biomedical assays. In this regard, engineering of brighter bioluminescent proteins, i.e. luciferases, has played a significant role. This is acutely exemplified by the engineering of the NLuc luciferase, which is small in size and displays much enhanced bioluminescence and thermal stability compared to previously available luciferases. While enhanced bioluminescent activity is desirable in a multitude of biological and biomedical assays, it would also be useful to develop variants of the protein that display a prolonged bioluminescence activity. This is specifically relevant in designing assays that require bioluminescence for extended periods, such as in the case of biosensors designed for monitoring slow enzymatic or cellular signaling reactions, without necessitating multiple rounds of luciferase substrate addition or any specialized reagents that result in increased assay costs. In the current manuscript, we report a mutant NLuc that possesses a stable and prolonged bioluminescence activity, albeit lower than the wild-type NLuc, and envisage a wider application of the mutant NLuc in designing biosensors for monitoring slower biological and biomedical events.

Situating the phosphonated calixarene-cytochrome C association by molecular dynamics simulations.

Protein-calixarenes binding plays an increasingly central role in many applications, spanning from molecular recognition to drug delivery strategies and protein inhibition. These ligands obey a specific bio-supramolecular chemistry, which can be revealed by computational approaches, such as molecular dynamics simulations. In this paper, we rely on all-atom, explicit-solvent molecular dynamics simulations to capture the electrostatically driven association of a phosphonated calix-[4]-arene with cytochome-C, which critically relies on surface-exposed paired lysines. Beyond two binding sites identified in direct agreement with the x-ray structure, the association has a larger structural impact on the protein dynamics. Then, our simulations allow a direct comparison to analogous calixarenes, namely, sulfonato, similarly reported as "molecular glue." Our work can contribute to a robust in silico predictive tool to assess binding sites for any given protein of interest for crystallization, with the specificity of a macromolecular cage whose endo/exo orientation plays a role in the binding.

Water Exchange from the Buried Binding Sites of Cytochrome P450 Enzymes 1A2, 2D6, and 3A4 Correlates with Conformational Fluctuations.

Human cytochrome P450 enzymes (CYPs) are critical for the metabolism of small-molecule pharmaceuticals (drugs). As such, the prediction of drug metabolism by and drug inhibition of CYP activity is an important component of the drug discovery and design process. Relative to the availability of a wide range of experimental atomic-resolution CYP structures, the development of structure-based CYP activity models has been limited. To better characterize the role of CYP conformational fluctuations in CYP activity, we perform multiple microsecond-scale all-atom explicit-solvent molecular dynamics (MD) simulations on three CYP isoforms, 1A2, 2D6, and 3A4, which together account for the majority of CYP-mediated drug metabolism. The MD simulations employ a variety of positional restraints, ranging from keeping all CYP atoms close to their experimentally determined coordinates to allowing full flexibility. We find that, with full flexibility, large fluctuations in the CYP binding sites correlate with efficient water exchange from these buried binding sites. This is especially true for 1A2, which, when restrained to its crystallographic conformation, is unable to exchange water between the binding site and bulk solvent. These findings imply that, in addition to crystal structures, a representative ensemble of conformational states ought to be included when developing structure-based CYP activity models.

Allosteric regulation in SARS-CoV-2 spike protein.

Allosteric regulation is common in protein-protein interactions and is thus promising in drug design. Previous experimental and simulation work supported the presence of allosteric regulation in the SARS-CoV-2 spike protein. Here the route of allosteric regulation in SARS-CoV-2 spike protein is examined by all-atom explicit solvent molecular dynamics simulations, contrastive machine learning, and the Ohm approach. It was found that peptide binding to the polybasic cleavage sites, especially the one at the first subunit of the trimeric spike protein, activates the fluctuation of the spike protein's backbone, which eventually propagates to the receptor-binding domain on the third subunit that binds to ACE2. Remarkably, the allosteric regulation routes starting from the polybasic cleavage sites share a high fraction (39-67%) of the critical amino acids with the routes starting from the nitrogen-terminal domains, suggesting the presence of an allosteric regulation network in the spike protein. Our study paves the way for the rational design of allosteric antibody inhibitors.

Probing the Nucleic Acid Flexibility to Disarm the Viral Counter-Defense Machinery: Design and Characterization of Potent p19 Inhibitors.

Plant viruses are highly destructive and significant contributors to several global pandemics and epidemics in plants. A viral disease outbreak in plants can cause a scarcity of food supply and is a severe concern to humanity. The siRNA (small interfering RNA)-mediated RNA-induced silencing complex (RISC) formation is a primary defense mechanism in plants against viruses, where the RISC binds and degrades viral mRNAs. As a counter-defense, many viruses encode RNA-silencing suppressor proteins (e.g., the p19 protein from the Tombusviridae family) for viral proliferation in plants. The functional form of p19 (homodimer) binds to plant siRNA with high affinities, thereby interrupting the RISC formation and thus preventing the viral mRNA silencing in plants. By altering the RISC formation, the p19 protein helps the virus invasion in the plant and ultimately stunts host growth. In this study, we designed several modified siRNA-based molecules for p19 inhibition. The viral p19 protein is known to interact predominantly through H-bonds with 2'-OH and phosphates of the plant siRNA. We utilized this information and in silico-designed flexible substituents of siRNA, where we removed the C2'-C3' bond in each nucleotide unit. We performed all-atom explicit-solvent molecular dynamics simulations (400 ns, 3 replicates each) for control/modified siRNA─p19 complexes (8 in total) followed by energetic estimations. Strikingly, in a few modified complexes, the siRNA not only retained the double-helical structural integrity but also displayed remarkably enhanced p19 binding compared to the control siRNA; hence, we consider it important to perform biological and chemical in vitro and in vivo studies on proposed flexible nucleic acids as p19 inhibitors for crop protection.

Coarse-grained explicit-solvent molecular dynamics simulations of semidilute unentangled polyelectrolyte solutions.

We present results from explicit-solvent coarse-grained molecular dynamics (MD) simulations of fully charged, salt-free, and unentangled polyelectrolytes in semidilute solutions. The inclusion of a polar solvent in the model allows for a more physical representation of these solutions at concentrations, where the assumptions of a continuum dielectric medium and screened hydrodynamics break down. The collective dynamic structure factor of polyelectrolytes, S(q,t), showed that at [Formula: see text], where [Formula: see text] is the polyelectrolyte peak in the structure factor S(q) and [Formula: see text] is the correlation length, the relaxation time obtained from fits to stretched exponential was [Formula: see text], which describes unscreened Zimm-like dynamics. This is in contrast to implicit-solvent simulations using a Langevin thermostat where [Formula: see text]. At [Formula: see text], a crossover region was observed that eventually transitions to another inflection point [Formula: see text] at length scales larger than [Formula: see text] for both implicit- and explicit-solvent simulations. The simulation results were also compared to scaling predictions for correlation length, [Formula: see text], specific viscosity, [Formula: see text], and diffusion coefficient, [Formula: see text], where [Formula: see text] is the polyelectrolyte concentration. The scaling prediction for [Formula: see text] holds; however, deviations from the predictions for [Formula: see text] and D were observed for systems at higher [Formula: see text], which are in qualitative agreements with recent experimental results. This study highlights the importance of explicit-solvent effects in molecular dynamics simulations, particularly in semidilute solutions, for a better understanding of polyelectrolyte solution behavior.

Insights into the Differential Composition of Stem-Loop Structures of Nanoviruses and Their Impacts.

Multipartite viruses package their genomic segments independently and mainly infect plants; few of them target animals. Nanoviridae is a family of multipartite single-stranded DNA (ssDNA) plant viruses that individually encapsidate ssDNAs of ~1 kb and transmit them through aphids without replication in aphid vectors, thereby causing important diseases in host plants, mainly leguminous crops. All of these components constitute an open reading frame to perform a specific role in nanovirus infection. All segments contain conserved inverted repeat sequences, potentially forming a stem-loop structure and a conserved nonanucleotide, TAGTATTAC, within a common region. This study investigated the variations in the stem-loop structure of nanovirus segments and their impact using molecular dynamics (MD) simulations and wet lab approaches. Although the accuracy of MD simulations is limited by force field approximations and simulation time scale, explicit solvent MD simulations were successfully used to analyze the important aspects of the stem-loop structure. This study involves the mutants' design, based on the variations in the stem-loop region and construction of infectious clones, followed by their inoculation and expression analysis, based on nanosecond dynamics of the stem-loop structure. The original stem-loop structures showed more conformational stability than mutant stem-loop structures. The mutant structures were expected to alter the neck region of the stem-loop by adding and switching nucleotides. Changes in conformational stability are suggested expression variations of the stem-loop structures found in host plants with nanovirus infection. However, our results can be a starting point for further structural and functional analysis of nanovirus infection. IMPORTANCE Nanoviruses comprise multiple segments, each with a single open reading frame to perform a specific function and an intergenic region with a conserved stem-loop region. The genome expression of a nanovirus has been an intriguing area but is still poorly understood. We attempted to investigate the variations in the stem-loop structure of nanovirus segments and their impact on viral expression. Our results show that the stem-loop composition is essential in controlling the virus segments' expression level.

Molecular Insights into the Self-Assembly of a Full-Length hIAPP Trimer: β-Protofibril Formed by β-Hairpin Lateral or Longitudinal Association.

The fibrillar protein deposits of the human islet amyloid polypeptide (hIAPP) in the pancreatic islet of Langerhans are pathological hallmark of type II diabetes. Extensive experimental studies have revealed that the oligomeric formations of the hIAPP are more toxic than the mature fibrils. Exploring the oligomeric conformations in the early aggregation state is valuable for effective therapeutics. In this work, using the all-atom explicit-solvent replica exchange molecular dynamic (REMD) simulations, we investigated the structural features and the assembly mechanisms of the full-length hIAPP trimer in solution. The hIAPP trimer adopted more β-sheets than a-helix conformations, and three types of ordered conformations including open β-barrel, single-layer, and double-layer U-shaped β-sheet structures with five β-strands were captured in our simulations. A representative single-layer β-sheet conformation with a CCS value of 1400 Å2 in our simulations matches exactly the experimentally ESI-IMS-MS-derived hIAPP trimer sample. These five β-strand conformations formed via the β-hairpin lateral and longitudinal association, respectively, showing two β-protofibril formation models. To the best of our knowledge, it is the first time to reveal two routes to β-sheet formation in the hIAPP trimers on the atomic level. The contact probabilities between pairs of the β-stranded residue show that the hydrophobic interactions between the residues F15 ∼ V17 and A25 ∼ L27 are responsible for the inter- and intra-peptide β-hairpin formations. All of these results indicate that the β-sheet formation is the first step in the conformational changes toward pathological aggregation and provides evidence of the β-sheet assembly mechanism into hIAPP aggregation.

Molecular Mechanism of Ligand Binding to the Minor Groove of DNA

Although DNA–ligand binding is pervasive in biology, little is known about molecular-level binding mechanisms. Using all-atom, explicit-solvent molecular dynamics simulations in conjunction with weighted ensemble (WE)-enhanced sampling, an ensemble of 2562 binding trajectories of Hoechst 33258 (H33258) to d(CGC AAA TTT GCG) was generated from which the binding mechanism was extracted. In particular, the electrostatic interaction between the positively charged H33258 and the negatively charged DNA backbone drives the formation of initial H33258–DNA contacts. After this initial contact, a hinge-like intermediate state is formed in which one end of H33258 inserts into the minor groove of DNA. Following hinge state formation is a concerted motion whereby the second end of H33258 swings into the minor groove and the spine of hydration along the minor groove causing dehydration. This study illustrates how WE-enhanced simulations of biomolecular ligation processes can offer novel mechanistic insights by generating ensembles of binding events.

Dissecting the Molecular Mechanisms of the Co-Aggregation of Aβ40 and Aβ42 Peptides: A REMD Simulation Study.

The aggregation of amyloid-β protein (Aβ) into oligomers and amyloid fibrils is closely related to Alzheimer's disease (AD). Aβ40 and Aβ42, as two most prominent isoforms of Aβ peptides, can cross-interact with each other and form co-aggregates, which affect the progression of the disease. However, the molecular determinants underlying Aβ40 and Aβ42 cross-interaction and the structural details of their co-oligomers remain elusive. Herein, we performed all-atom explicit-solvent replica exchange molecular dynamics simulations on Aβ40-Aβ42 heterogeneous and Aβ40/Aβ42 homogeneous dimer systems to dissect the co-aggregation mechanisms of the two isoforms. Our results show that the interpeptide main-chain interaction of Aβ40-Aβ42 is stronger than that of Aβ40-Aβ40 and Aβ42-Aβ42. The positions of hotspot residues in heterodimers and homodimers display high similarity, implying similar molecular recognition sites for both cross-interaction and self-interaction. Contact maps of Aβ40-Aβ42 heterodimers reveal that residue pairs crucial for cross-interaction are mostly located in the C-terminal hydrophobic regions of Aβ40 and Aβ42 peptides. Conformational analysis shows that Aβ40 and Aβ42 monomers can co-assemble into β-sheet-rich heterodimers with shorter β-sheets than those in homodimers, which is decremental to monomer addition. Similar molecular recognition sites and β-sheet distribution of Aβ40 and Aβ42 peptides are observed in heterodimers and homodimers, which may provide the molecular basis for the two isoforms' co-aggregation and cross-seeding. Our work dissects the co-aggregation mechanisms of Aβ40 and Aβ42 peptides at the atomic level, which will help for in-depth understanding of the cross-talk between the two Aβ isoforms and the pathogenesis of AD.

Gamma-Hemolysin Components: Computational Strategies for LukF-Hlg2 Dimer Reconstruction on a Model Membrane.

The gamma-hemolysin protein is one of the most common pore-forming toxins expressed by the pathogenic bacterium Staphylococcus aureus. The toxin is used by the pathogen to escape the immune system of the host organism, by assembling into octameric transmembrane pores on the surface of the target immune cell and leading to its death by leakage or apoptosis. Despite the high potential risks associated with Staphylococcus aureus infections and the urgent need for new treatments, several aspects of the pore-formation process from gamma-hemolysin are still unclear. These include the identification of the interactions between the individual monomers that lead to the formation of a dimer on the cell membrane, which represents the unit for further oligomerization. Here, we employed a combination of all-atom explicit solvent molecular dynamics simulations and protein-protein docking to determine the stabilizing contacts that guide the formation of a functional dimer. The simulations and the molecular modeling reveal the importance of the flexibility of specific protein domains, in particular the N-terminus, to drive the formation of the correct dimerization interface through functional contacts between the monomers. The results obtained are compared with the experimental data available in the literature.

Exploration of potent antiviral phytomedicines from Lauraceae family plants against SARS-CoV-2 RNA-dependent RNA polymerase

RNA-dependent RNA polymerase, also known as RdRp, is a possible therapeutic target that could be used to suppress the proliferation of RNA viruses such as SARS-CoV-2. This protein has two major functional sites (a) catalytic and (b) substrate entry, which regulate the natural substrate entry and its corresponding interaction with the protein. In this study, a computational drug design pipeline was applied to investigate potential inhibitors against SARS-CoV-2 RdRp from Lauraceae plants, and five top hits were selected based on the docked score (< −7 kcal/mol). The docking study suggested that the Glochidioboside had a minimum binding score of −7.8 kcal/mol. This compound showed total five hydrogen bonds while two of them were with catalytic residues Asp618 and Asp760. However, another compound, Sitogluside showed a binding score of −7.3 kcal/mol with four hydrogen bonds targeting three functional residues (Arg555, Ser759, and Asp760). Later, 100 ns explicit solvent molecular dynamics (MD) simulation was performed to evaluate the stability of the protein-ligand docked system. These compounds translocated their positions from the catalytic site to the substrate entry site, as observed in the MD simulation trajectory. However, translocation did not affect the binding strength of these compounds, and they retained the strong binding affinity (ΔG < −11.5 kcal/mol), estimated using the MM/GBSA method. In general, the findings of this study indicated the potential therapeutic compounds that may be used targeting SARS-CoV-2 RdRp. However, these compounds still need to be validated by experimentation in order to determine their inhibitory function. Communicated by Ramaswamy H. Sarma

Influence of ALS-linked M337V mutation on the conformational ensembles of TDP-43321-340 peptide monomer and dimer.

The transactive response (TAR) DNA/RNA-binding protein 43 (TDP-43) can self-assemble into both functional stress granules via liquid-liquid phase separation (LLPS) and pathogenic amyloid fibrillary aggregates that are closely linked to amyotrophic lateral sclerosis. Previous experimental studies reported that the low complexity domain (LCD) of TDP-43 plays an essential role in the LLPS and aggregation of the full-length protein, and it alone can also undergo LLPS to form liquid droplets mainly via intermolecular interactions in the 321-340 region. And the ALS-associated M337V mutation impairs LCD's LLPS and facilitates liquid-solid phase transition. However, the underlying atomistic mechanism is not well understood. Herein, as a first step to understand the M337V-caused LLPS disruption of TDP-43 LCD mediated by the 321-340 region and the fibrillization enhancement, we investigated the conformational properties of monomer/dimer of TDP-43321-340 peptide and its M337V mutant by performing extensive all-atom explicit-solvent replica exchange molecular dynamic simulations. Our simulations demonstrate that M337V mutation alters the residue regions with high helix/β-structure propensities and thus affects the conformational ensembles of both monomer and dimer. M337V mutation inhibits helix formation in the N-terminal Ala-rich region and the C-terminal mutation site region, while facilitating their long β-sheet formation, albeit with a minor impact on the average probability of both helix structure and β-structure. Further analysis of dimer system shows that M337V mutation disrupts inter-molecular helix-helix interactions and W334-W334 π-π stacking interactions which were reported to be important for the LLPS of TDP-43 LCD, whereas enhances the overall peptide residue-residue interactions and weakens peptide-water interactions, which is conducive to peptide fibrillization. This study provides mechanistic insights into the M337V-mutation-induced impairment of phase separation and facilitation of fibril formation of TDP-43 LCD.

A comparative study on inclusion complex formation between formononetin and β-cyclodextrin derivatives through multiscale classical and umbrella sampling simulations

Formononetin, a naturally occurring isoflavone exhibits a wide range of therapeutic applications including antioxidant, anti-tumor, antiviral, anti-diabetic and neuroprotective activities. However, the low hydro-solubility of formononetin has limited its prospective use in cosmetic, neutraceutical and pharmaceutical industries. Cyclodextrins (CDs), especially β-CD and its derivatives have emerged as promising agents to improve the water solubility of poorly hydrosoluble compounds by the formation of inclusion complexes. We employed multiscale (1000 ns) explicit solvent and umbrella sampling molecular dynamics (MD) simulations to study the interactions and thermodynamic parameters of inclusion complex formation between formononetin and five most commonly used β-CD derivatives. Classical MD simulations revealed two possible binding conformations of formononetin inside the central cavity of hydroxypropyl-β-CD (HP-β-CD), randomly methylated-β-CD (ME-β-CD), and sulfobutylether-β-CD (SBE-β-CD). The binding conformation with the benzopyrone ring of formononetin inside the central cavity of β-CD derivatives was more frequent than the phenyl group occupying the hydrophobic cavity. These interactions were supported by a variety of non-bonded contacts including hydrogen bonds, pi-lone pair, pi-sigma, and pi-alkyl interactions. Formononetin showed favorable end-state MD-driven thermodynamic binding free energies with all the selected β-CD derivatives, except succinyl-β-CD (S-β-CD). Furthermore, umbrella sampling simulations were used to investigate the interactions and thermodynamic parameters of the host-guest inclusion complexes. The SBE-β-CD/formononetin inclusion complex showed the lowest binding energy signifying the highest affinity among all the selected host-guest inclusion complexes. Our study could be used as a standard for analyzing and comparing the ability of different β-CD derivatives to enhance the hydro-solubility of poorly soluble molecules.

Atomistic insights into the role of Sar1 GTPase in COPII mediated protein secretary pathway.

In the protein secretory pathway of the cell, the Coat Protein Complex II (COPII) is a crucial molecular machine that transports protein payloads from the Endoplasmic Reticulum (ER) to the Golgi Body. Sar1, a COPII component protein, initiates the protein transport mechanism by budding vesicles on the subdomains of ER after being activated by GDP/GTP exchange. Based on crystallographic and cryo-EM study, the conformational change of Sar1's amino terminal amphipathic helix following GTP binding is considered to be a critical factor in the initiation of the vesicle budding step. However, such GTP triggered conformational switch mechanism remains incomplete without the explicit molecular details of the penetration of the amino terminus of GDP and GTP bound state into the membrane. We perform all-atom, explicit solvent Molecular Dynamics simulation to capture atomistic insights into the differential membrane binding and bending ability of GDP and GTP bound Sar1. We demonstrate that in the GDP-bound state, Sar1 partially inserts its amino terminus into the membrane (residues 1-12), whereas in the GTP-bound state, the whole amino terminus stays horizontally buried inside the membrane (residues 1-23). Thus, GTP bound Sar1 triggers strong inter-leaflet stress due to excess volume insertion in the membrane leading to ∼10-20 fold enhancement in positive curvature induction compared to that due to GDP bound form. We reveal that the magnitude of membrane curvature formation is further amplified by the dimerization of the GTP bound state. Although amphipathic helices are known to generate positive curvature on membranes our simulations reinforce the dominance of the amount of volume inclusion over its penetration depth inside the membrane in this process. Together, we propose a thorough molecular basis for the activity of Sar1-mediated membrane remodeling activity.

Effects of cryo-EM cooling on structural ensembles of biomolecules and their solvation shell.

The recent revolution in cryo electron microscopy (cryo-EM) allows the determination of structures of macromolecular complexes at atomic resolution. Cryo-EM also provides information on structural heterogeneity and ensembles of macromolecules. To obtain cryo-EM images of macromolecules, the samples are first rapidly cooled down to liquid nitrogen temperatures. The rapid cooling prevents ice-crystal formation and preserves some information of the room-temperature ensemble. However, to what extent the ensemble of the macromolecues and of the solvation shell is perturbed by the cooling is currently unknown. To quantify the cooling effects, we first estimated the temperature drop rate by solving the heat equation which suggested rapid cooling within microseconds. Then, we started all-atom explicit-solvent molecular dynamics (MD) simulations of the ribosome from 41 snapshots taken from a room-temperature ensemble with linearly decreasing temperatures at 11 different cooling rates (simulation lengths 0.1-128 ns). In the simulations, we observed that cooling markedly decreased the structural heterogeneity of the cooled ensemble. To investigate if and how this effect depends on the cooling rate, we used Bayesian statistics to test three thermodynamic and kinetic models of the cooling process. A kinetic two-state model improves the prediction of the decrease in heterogeneity compared to the cooling-rate independent thermodynamic model suggesting that kinetic effects contribute markedly. The combination of the estimated temperature drop rate with the kinetic model suggests that small barriers between states (<10 kJ/mol) are overcome during cooling and do not contribute to the heterogeneity of the structural ensemble obtained by cryo-EM. In contrast, conformational states separated by larger barriers are expected to be trapped during plunge-freezing. The obtained parameters for the kinetic model will allow one to quantify the heterogeneity of biologically relevant room-temperature ensembles from cryo-EM structures.

Scrutinizing the protein hydration shell from MD simulations against SAXS/SANS data from a worldwide round robin benchmark.

The hydration shell of proteins plays key roles in protein stability, folding, recognition, and enzyme activity. However, it remained unclear whether the hydration shell predicted by explicit-solvent molecular dynamics (MD) simulations matches experimental conditions, while accurate experimental probes of the hydration shell structure remained limited. Small-angle scattering (SAS) in solution using X-rays (SAXS) or neutrons (SANS) in principle provides information on the hydration shell, since both the radius of gyration (Rg) and the zero-angle scattering (I0) depend on the hydration shell contrast relative to bulk solvent. Using MD simulations and explicit-solvent SAXS/SANS calculations, we computed Rg and I0 for five different proteins and a set of 18 different combinations of protein force fields and water models, and we validated the simulation results against consensus data from a recent worldwide round robin benchmark (Trewhella, Vachette et al., doi:10.1107/S2059798322009184). Overall, we find remarkable agreement between MD simulations and consensus SAS data; however the agreement is force field dependent. Certain force field/water model combinations underestimate the hydration layer contrast significantly, while water models with increased dispersion interactions may, for some proteins, overestimate the hydration layer contrast. Our study shows that explicit-solvent SAS calculation and consensus SAS data provide a novel route for scrutinizing the hydration layer of proteins.

Dynamics of the SARS-CoV-2 main protease binding site.

The COVID-19 pandemic has been controlled through quarantines, travel bans, mask mandates, and vaccines. However, due to viral mutations and the unlikelihood of reaching herd immunity, there remains a need for therapeutics. The SARS-CoV-2 main protease (MPro) is necessary for the virus’ replication, thus it is a promising drug target. Additionally, it can be crystallized consistently and its sequence has few changes across virus mutants. MPro crystal structures depict general residue-residue interactions and backbone conformations, but they do not account for the dynamics of flexible regions. Instead, molecular dynamics (MD) simulations are used to identify the flexibility of the binding site, data we hypothesize is essential for successfully designing drugs to bind to MPro using computational drug docking methods. 600 ns of all-atom, explicit solvent MD simulations of the MPro homodimer were conducted, resulting in 1.2 million frames of monomeric data. Principal component analysis (PCA) was conducted on pairwise Cα-Cα distances between selected binding residues within apo and holo PDBs and the MD frames. These distances provide an intuitive set of measurements to characterize the dynamics of the binding site. Contour mapping was used to visualize the presence of clusters in the space defined by a few of the most contributive PC's. Snapshots representative of the most common conformations within the distribution of data were identified through the use of clustering methods and will be used in future drug docking simulations.

Elucidating the dynamics of individual IDPs in a biomolecular condensate using single-molecule FRET and molecular simulation.

A wide range of biomolecules in solution can phase-separate and form membraneless organelles in the cell. These assemblies often have liquid-like properties, and the corresponding dynamics and exchange of molecules with the environment are important for biological function. The dynamics and materials properties of these systems are commonly assessed based on translational diffusion or rheological properties, typically covering timescales of milliseconds and longer. However, information on the structure and dynamics at the molecular level is lacking. We have studied coacervates of two intrinsically disordered highly oppositely charged nuclear proteins as a prototypical example of heterotypic liquid-liquid phase separation of components abundant in the cell nucleus. Using single-molecule fluorescence spectroscopy, we show that the proteins in the condensates remain disordered, and that their chain dynamics occur on timescale surprisingly close to the ∼100-nanosecond dynamics of the 1:1 complex in dilute solution, even though the condensate is almost three orders of magnitude more viscous than the dilute solution and translational diffusion of the same proteins is ∼50 times slower in the dense than in the dilute phase. The experimental results are in good agreement with large-scale all-atom explicit-solvent molecular dynamics simulations consisting of ∼200 proteins and ∼4 million atoms in total, which can thus provide detailed insights into the protein interactions and dynamics within the condensate. The simulations indicate that the rapid protein chain dynamics in the dense phase are linked to the similarity of the local environment experienced by the proteins in the dilute and dense phases and the short lifetime of intermolecular contacts in the dense phase.