Machine Learning Reveals the Critical Interactions for SARS-CoV-2 Spike Protein Binding to ACE2.

SARS-CoV and SARS-CoV-2 bind to the human ACE2 receptor in practically identical conformations, although several residues of the receptor-binding domain (RBD) differ between them. Herein, we have used molecular dynamics (MD) simulations, machine learning (ML), and free-energy perturbation (FEP) calculations to elucidate the differences in binding by the two viruses. Although only subtle differences were observed from the initial MD simulations of the two RBD-ACE2 complexes, ML identified the individual residues with the most distinctive ACE2 interactions, many of which have been highlighted in previous experimental studies. FEP calculations quantified the corresponding differences in binding free energies to ACE2, and examination of MD trajectories provided structural explanations for these differences. Lastly, the energetics of emerging SARS-CoV-2 mutations were studied, showing that the affinity of the RBD for ACE2 is increased by N501Y and E484K mutations but is slightly decreased by K417N.

Tuning Proton Transfer Thermodynamics in SARS-CoV-2 Main Protease: Implications for Catalysis and Inhibitor Design.

The catalytic reaction in SARS-CoV-2 main protease is activated by a proton transfer (PT) from Cys145 to His41. The same PT is likely also required for the covalent binding of some inhibitors. Here we use a multiscale computational approach to investigate the PT thermodynamics in the apo enzyme and in complex with two potent inhibitors, N3 and the α-ketoamide 13b. We show that with the inhibitors the free energy cost to reach the charge-separated state of the active-site dyad is lower, with N3 inducing the most significant reduction. We also show that a few key sites (including specific water molecules) significantly enhance or reduce the thermodynamic feasibility of the PT reaction, with selective desolvation of the active site playing a crucial role. The approach presented is a cost-effective procedure to identify the enzyme regions that control the activation of the catalytic reaction and is thus also useful to guide the design of inhibitors.

Inhibitor binding influences the protonation states of histidines in SARS-CoV-2 main protease.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of Mpro, a cysteine protease, have been determined, facilitating structure-based drug design. Mpro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41-Cys145, Mpro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nucleophile Cys145 have been debated in previous studies of SARS-CoV Mpro, but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 Mpro as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of Mpro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an α-ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored N δ (HD) and N ϵ (HE) protonation of His41 and His164, respectively, the α-ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 Mpro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts.

Tuning Proton Transfer Thermodynamics in SARS-Cov-2 Main Protease: Implications for Catalysis and Inhibitor Design.

In this comutational work a hybrid quantum mechanics/molecular mechanics approach, the MD-PMM approach, is used to investigate the proton transfer reaction the activates the catalytic activity of SARS-CoV-2 main protease. The proton transfer thermodynamics is investigated for the apo ensyme (i.e., without any bound substrate or inhibitor) and in the presence of a inhibitor, N3, which was previously shown to covalently bind SARS-CoV-2 main protease.

Inhibitor binding influences the protonation states of histidines in SARS-CoV-2 main protease.

The main protease (M pro ) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of M pro , a cysteine protease, have been determined, facilitating structure-based drug design. M pro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41-Cys145, M pro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nu-cleophile Cys145 have been debated in previous studies of SARS-CoV M pro , but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 M pro as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of M pro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an α -ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored N δ (HD) and N ϵ (HE) protonation of His41 and His164, respectively, the α -ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 M pro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts.

String Method for Protein-Protein Binding Free-Energy Calculations.

A powerful computational strategy to determine the equilibrium association constant of two macromolecules with explicit-solvent molecular dynamics (MD) simulations is the "geometric route", which considers the reversible physical separation of the bound complex in solution. Nonetheless, multiple challenges remain to render this type of methodology reliable and computationally efficient in practice. In particular, in one, formulation of the geometric route relies on the potential of mean force (PMF) for physically separating the two binding partners restrained along a straight axis, which must be selected prior to the calculation. However, practical applications indicate that the calculation of the separation PMF along the predefined rectilinear pathway may be suboptimal and slowly convergent. Recognizing that a rectilinear straight separation pathway is generally not representative of how the protein complex physically separates in solution, we put forth a novel theoretical framework for binding free-energy calculations, leaning on the optimal curvilinear minimum free-energy path (MFEP) determined from the string method. The proposed formalism is validated by comparing the results obtained using both rectilinear and curvilinear pathways for a prototypical host-guest complex formed by cucurbit[7]uril (CB[7]) binding benzene, and for the barnase-barstar protein complex. On the basis of multi-microsecond MD calculations, we find that the calculations following the traditional rectilinear pathway and the string-based curvilinear pathway agree quantitatively, but convergence is faster with the latter.

Hepatitis C virus sequence divergence preserves p7 viroporin structural and dynamic features.

The hepatitis C virus (HCV) viroporin p7 oligomerizes to form ion channels, which are required for the assembly and secretion of infectious viruses. The 63-amino acid p7 monomer has two putative transmembrane domains connected by a cytosolic loop, and has both N- and C- termini exposed to the endoplasmic reticulum (ER) lumen. NMR studies have indicated differences between p7 structures of distantly related HCV genotypes. A critical question is whether these differences arise from the high sequence variation between the different isolates and if so, how the divergent structures can support similar biological functions. Here, we present a side-by-side characterization of p7 derived from genotype 1b (isolate J4) in the detergent 6-cyclohexyl-1-hexylphosphocholine (Cyclofos-6) and p7 derived from genotype 5a (isolate EUH1480) in n-dodecylphosphocholine (DPC). The 5a isolate p7 in conditions previously associated with a disputed oligomeric form exhibits secondary structure, dynamics, and solvent accessibility broadly like those of the monomeric 1b isolate p7. The largest differences occur at the start of the second transmembrane domain, which is destabilized in the 5a isolate. The results show a broad consensus among the p7 variants that have been studied under a range of different conditions and indicate that distantly related HCVs preserve key features of structure and dynamics.

Rotational Mechanism Model of the Bacterial V1 Motor Based on Structural and Computational Analyses.

V1-ATPase exemplifies the ubiquitous rotary motor, in which a central shaft DF complex rotates inside a hexagonally arranged catalytic A3B3 complex, powered by the energy from ATP hydrolysis. We have recently reported a number of crystal structures of the Enterococcus hirae A3B3DF (V1) complex corresponding to its nucleotide-bound intermediate states, namely the forms waiting for ATP hydrolysis (denoted as catalytic dwell), ATP binding (ATP-binding dwell), and ADP release (ADP-release dwell) along the rotatory catalytic cycle of ATPase. Furthermore, we have performed microsecond-scale molecular dynamics simulations and free-energy calculations to investigate the conformational transitions between these intermediate states and to probe the long-time dynamics of the molecular motor. In this article, the molecular structure and dynamics of the V1-ATPase are reviewed to bring forth a unified model of the motor's remarkable rotational mechanism.

The substrate specificity of the human ADP/ATP carrier AAC1.

The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [(32)P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [(14)C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificity. Next we tried to understand the molecular basis of this specificity by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity. The binding of the phosphate groups of guanine and adenine nucleotides is similar, yet there is a low probability for the base moiety to be bound, likely to be rooted in the greater polarity of guanine compared to adenine. AMP is unlikely to engage fully with all contact points of the substrate binding site, suggesting that it cannot trigger translocation.

Impaired transport of nucleotides in a mitochondrial carrier explains severe human genetic diseases.

The mitochondrial ADP/ATP carrier (AAC) is a prominent actor in the energetic regulation of the cell, importing ADP into the mitochondria and exporting ATP toward the cytoplasm. Severe genetic diseases have been ascribed to specific mutations in this membrane protein. How minute, well-localized modifications of the transporter impact the function of the mitochondria remains, however, largely unclear. Here, for the first time, the relationship between all documented pathological mutations of the AAC and its transport properties is established. Activity measurements combined synergistically with molecular-dynamics simulations demonstrate how all documented pathological mutations alter the binding affinity and the translocation kinetics of the nucleotides. Throwing a bridge between the pathologies and their molecular origins, these results reveal two distinct mechanisms responsible for AAC-related genetic disorders, wherein the mutations either modulate the association of the nucleotides to the carrier by modifying its electrostatic signature or reduce its conformational plasticity.