Spike (S) protein is the primary antigenic target for neutralization and vaccine development for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It decorates the virus surface and ...undergoes large motions of its receptor binding domains (RBDs) to enter the host cell. Here, we observe Down, one-Up, one-Open, and two-Up-like structures in enhanced molecular dynamics simulations, and characterize the transition pathways via inter-domain interactions. Transient salt-bridges between RBD
and RBD
and the interaction with glycan at N343
support RBD
motions from Down to one-Up. Reduced interactions between RBD
and RBD
in one-Up induce RBD
motions toward two-Up. The simulations overall agree with cryo-electron microscopy structure distributions and FRET experiments and provide hidden functional structures, namely, intermediates along Down-to-one-Up transition with druggable cryptic pockets as well as one-Open with a maximum exposed RBD. The inherent flexibility of S-protein thus provides essential information for antiviral drug rational design or vaccine development.
Modern drug discovery increasingly focuses on the drug-target binding kinetics which depend on drug (un)binding pathways. The conventional molecular dynamics simulation can observe only a few binding ...events even using the fastest supercomputer. Here, we develop 2D gREST/REUS simulation with enhanced flexibility of the ligand and the protein binding site. Simulation (43 μs in total) applied to an inhibitor binding to c-Src kinase covers 100 binding and unbinding events. On the statistically converged free-energy landscapes, we succeed in predicting the X-ray binding structure, including water positions. Furthermore, we characterize hidden semibound poses and transient encounter complexes on the free-energy landscapes. Regulatory residues distant from the catalytic core are responsible for the initial inhibitor uptake and regulation of subsequent bindings, which was unresolved by experiments. Stabilizing/blocking of either the semibound poses or the encounter complexes can be an effective strategy to optimize drug-target residence time.
Recent advances in atomistic molecular dynamics (MD) simulations of biomolecules allow us to explore their conformational spaces widely, observing large-scale conformational fluctuations or ...transitions between distinct structures. To reproduce or refine experimental data using MD simulations, structure ensembles, which are characterized by multiple structures and their statistical weights on the rugged free-energy landscapes, are often used. Here, we summarize weight average approaches for various experimental measurements. Weight average approaches are now applied to hybrid quantum mechanics/molecular mechanics MD simulations to predict fast vibrational motions in a protein with a high accuracy for better understanding of molecular functions from atomic structures.
•Conformational dynamics of biomolecules involves fast vibrational motions and slow transitions between distinct structures.•The conformational dynamics is theoretically described via multidimensional free-energy landscapes with many basins.•structural ensemble from molecular dynamics simulations is used to reproduce dynamical properties observed experimentally.•Weight average approaches are useful to analyze not only slow conformational changes but also fast vibrational motions.
Abstract
The inside of a cell is highly crowded with proteins and other biomolecules. How proteins express their specific functions together with many off-target proteins in crowded cellular ...environments is largely unknown. Here, we investigate an inhibitor binding with c-Src kinase using atomistic molecular dynamics (MD) simulations in dilute as well as crowded protein solution. The populations of the inhibitor, 4-amino-5-(4-methylphenyl)−7-(
t
-butyl)pyrazolo3,4-dpyrimidine (PP1), in bulk solution and on the surface of c-Src kinase are reduced as the concentration of crowder bovine serum albumins (BSAs) increases. This observation is consistent with the reduced PP1 inhibitor efficacy in experimental c-Src kinase assays in addition with BSAs. The crowded environment changes the major binding pathway of PP1 toward c-Src kinase compared to that in dilute solution. This change is explained based on the population shift mechanism of local conformations near the inhibitor binding site in c-Src kinase.
Integrin α5β1 is a major cellular receptor for the extracellular matrix protein fibronectin and plays a fundamental role during mammalian development. A crystal structure of the α5β1 integrin ...headpiece fragment bound by an allosteric inhibitory antibody was determined at a 2.9-Å resolution both in the absence and presence of a ligand peptide containing the Arg-Gly-Asp (RGD) sequence. The antibody-bound β1 chain accommodated the RGD ligand with very limited structural changes, which may represent the initial step of cell adhesion mediated by nonactivated integrins. Furthermore, a molecular dynamics simulation pointed to an important role for Ca(2+) in the conformational coupling between the ligand-binding site and the rest of the molecule. The RGD-binding pocket is situated at the center of a trenchlike exposed surface on the top face of α5β1 devoid of glycosylation sites. The structure also enabled the precise prediction of the acceptor residue for the auxiliary synergy site of fibronectin on the α5 subunit, which was experimentally confirmed by mutagenesis and kinetic binding assays.
Alchemical free energy simulations have long been utilized to predict free energy changes for binding affinity and solubility of small molecules. However, while the theoretical foundation of these ...methods is well established, seamlessly handling many of the practical aspects regarding the preparation of the different thermodynamic end states of complex molecular systems and the numerous processing scripts often remains a burden for successful applications. In this work, we present CHARMM-GUI
(http://www.charmm-gui.org/input/fec) that provides various alchemical free energy perturbation molecular dynamics (FEP/MD) systems with input and post-processing scripts for NAMD and GENESIS. Four submodules are available:
(for absolute ligand binding FEP/MD),
(for relative ligand binding FEP/MD),
(for absolute ligand solvation FEP/MD), and
(for relative ligand solvation FEP/MD). Each module is designed to build multiple systems of a set of selected ligands at once for high-throughput FEP/MD simulations. The capability of
is illustrated by absolute and relative solvation FEP/MD of a set of ligands and absolute and relative binding FEP/MD of a set of ligands for T4-lysozyme in solution and the adenosine A
receptor in a membrane. The calculated free energy values are overall consistent with the experimental and published free energy results (within ∼1 kcal/mol). We hope that
is useful to carry out high-throughput FEP/MD simulations in the field of biomolecular sciences and drug discovery.
The coronavirus disease 2019 (COVID-19) pandemic has necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The main protease (M
) is a ...promising target for COVID-19 treatment. Here, we report an irreversible SARS-CoV-2 M
inhibitor possessing chlorofluoroacetamide (CFA) as a warhead for the covalent modification of M
. Ugi multicomponent reaction using chlorofluoroacetic acid enabled the rapid synthesis of dipeptidic CFA derivatives that identified 18 as a potent inhibitor of SARS-CoV-2 M
. Among the four stereoisomers, (
,
)-18 exhibited a markedly higher inhibitory activity against M
than the other isomers. Reaction kinetics and computational docking studies suggest that the
configuration of the CFA warhead is crucial for the rapid covalent inhibition of M
. Our findings highlight the prominent influence of the CFA chirality on the covalent modification of proteinous cysteines and provide the basis for improving the potency and selectivity of CFA-based covalent inhibitors.
The introduction of bisecting GlcNAc and core fucosylation in N-glycans is essential for fine functional regulation of glycoproteins. In this paper, the effect of these modifications on the ...conformational properties of N-glycans is examined at the atomic level by performing replica-exchange molecular dynamics (REMD) simulations. We simulate four biantennary complex-type N-glycans, namely, unmodified, two single-substituted with either bisecting GlcNAc or core fucose, and disubstituted forms. By using REMD as an enhanced sampling technique, five distinct conformers in solution, each of which is characterized by its local orientation of the Manα1-6Man glycosidic linkage, are observed for all four N-glycans. The chemical modifications significantly change their conformational equilibria. The number of major conformers is reduced from five to two and from five to four upon the introduction of bisecting GlcNAc and core fucosylation, respectively. The population change is attributed to specific inter-residue hydrogen bonds, including water-mediated ones. The experimental NMR data, including nuclear Overhauser enhancement and scalar J-coupling constants, are well reproduced taking the multiple conformers into account. Our structural model supports the concept of "conformer selection", which emphasizes the conformational flexibility of N-glycans in protein-glycan interactions.
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