Dynamically-Driven Inactivation of the Catalytic Machinery of the SARS 3C-Like Protease by the N214A Mutation on the Extra Domain

Despite utilizing the same chymotrypsin fold to host the catalytic machinery, coronavirus 3C-like proteases (3CLpro) noticeably differ from picornavirus 3C proteases in acquiring an extra helical domain in evolution. Previously, the extra domain was demonstrated to regulate the catalysis of the SARS-CoV 3CLpro by controlling its dimerization. Here, we studied N214A, another mutant with only a doubled dissociation constant but significantly abolished activity. Unexpectedly, N214A still adopts the dimeric structure almost identical to that of the wild-type (WT) enzyme. Thus, we conducted 30-ns molecular dynamics (MD) simulations for N214A, WT, and R298A which we previously characterized to be a monomer with the collapsed catalytic machinery. Remarkably, three proteases display distinctive dynamical behaviors. While in WT, the catalytic machinery stably retains in the activated state; in R298A it remains largely collapsed in the inactivated state, thus implying that two states are not only structurally very distinguishable but also dynamically well separated. Surprisingly, in N214A the catalytic dyad becomes dynamically unstable and many residues constituting the catalytic machinery jump to sample the conformations highly resembling those of R298A. Therefore, the N214A mutation appears to trigger the dramatic change of the enzyme dynamics in the context of the dimeric form which ultimately inactivates the catalytic machinery. The present MD simulations represent the longest reported so far for the SARS-CoV 3CLpro, unveiling that its catalysis is critically dependent on the dynamics, which can be amazingly modulated by the extra domain. Consequently, mediating the dynamics may offer a potential avenue to inhibit the SARS-CoV 3CLpro

Resistant machanisms, dynamic properties and inhibitor development of neuraminidase in influenza virus : molecular dynamics simulations studies

Since the beginning of the last century, several pandemics with high morbidity and mortality caused by influenza viruses have occurred, and posed great threat to human life. Although vaccines and antiviral drugs have been developed to treat the upcoming influenza viruses, the accumulating mutant viral strains weaken the power of antiviral strategies. In order to efficiently treat this infectious disease, it is critical to understand the molecular basis of the drug resistance mechanisms of influenza viruses. The aim of this work is to investigate drug resistant mechanisms at the molecular level, to explore structural plasticity of the target protein and to provide promising ligands that can effectively bind the influenza target protein through computational methodologies. Recently, H1N1 strains of influenza A carrying a mutation of Q136K in neuraminidase (NA) were reported. This new strain showed a strong zanamivir (ZMR, an antiviral NA inhibitor) neutralization effect. In the first study, normal molecular dynamics (MD) simulations and metadynamics simulations were employed to explore the mechanism of ZMR resistance. Hydrogen-bond network analysis showed weakened interaction between the ZMR drug and E276/D151 on account of the electrostatic interaction between K136 and D151. Metadynamics simulations showed that the free energy landscape in the mutant is different from that in the wild type (WT) NA, suggesting weaker binding. This study indicates that the deformation of the 150-loop combined with the induced altered hydrogen-bond network is the reason for development of ZMR resistance. In addition, Hamiltonian replica exchange molecular dynamics (HREMD) simulations were performed to explore the plasticity of the 150-loop, which had been found to be crucial in maintaining the stability of the ZMR. The free energy landscape of the 150-loop was extensively sampled, and the most dynamical motif was identified. This enhanced sampling simulation together with the discovery of a drug resistance mechanism provides invaluable information in structural-based drug discovery against influenza viruses. Based on these two findings, the 150-loop seems to be a hotspot for influenza drug development. Thus, a combined virtual screening and MD simulations method was applied to design ligands that specifically target the 150-cavity based on the structure template of ZMR. Finally, one ligand was found to stably interact with NA and lock the 150-loop in an open configuration. After comparing to both positive and negative controls, this newly designed ligand was shown to possess the highest binding affinity. More generally, the key interactions between NA and inhibitors were also identified which provides references for novel inhibitors design. Our study provides a possible route in designing new NA inhibitors for combating the spread of influenza virus.​Doctor of Philosophy (SBS

Locking the 150-cavity open : in silico design and verification of influenza neuraminidase inhibitors

Neuraminidase (NA) of influenza is a key target for virus infection control and the recently discovered open 150-cavity in group-1 NA provides new opportunity for novel inhibitors design. In this study, we used a combination of theoretical methods including fragment docking, molecular linking and molecular dynamics simulations to design ligands that specifically target at the 150-cavity. Through in silico screening of a fragment compound library on the open 150-cavity of NA, a few best scored fragment compounds were selected to link with Zanamivir, one NA-targeting drug. The resultant new ligands may bind both the active site and the 150-cavity of NA simultaneously. Extensive molecular dynamics simulations in explicit solvent were applied to validate the binding between NA and the designed ligands. Moreover, two control systems, a positive control using Zanamivir and a negative control using a low-affinity ligand 3-(p-tolyl) allyl-Neu5Ac2en (ETT, abbreviation reported in the PDB) found in a recent experimental work, were employed to calibrate the simulation method. During the simulations, ETT was observed to detach from NA, on the contrary, both Zanamivir and our designed ligand bind NA firmly. Our study provides a prospective way to design novel inhibitors for controlling the spread of influenza virus.Published versio

Plasticity of 150-Loop in Influenza Neuraminidase Explored by Hamiltonian Replica Exchange Molecular Dynamics Simulations

<div><p>Neuraminidase (NA) of influenza is a key target for antiviral inhibitors, and the 150-cavity in group-1 NA provides new insight in treating this disease. However, NA of 2009 pandemic influenza (09N1) was found lacking this cavity in a crystal structure. To address the issue of flexibility of the 150-loop, Hamiltonian replica exchange molecular dynamics simulations were performed on different groups of NAs. Free energy landscape calculated based on the volume of 150-cavity indicates that 09N1 prefers open forms of 150-loop. The turn A (residues 147–150) of the 150-loop is discovered as the most dynamical motif which induces the inter-conversion of this loop among different conformations. In the turn A, the backbone dynamic of residue 149 is highly related with the shape of 150-loop, thus can function as a marker for the conformation of 150-loop. As a contrast, the closed conformation of 150-loop is more energetically favorable in N2, one of group-2 NAs. The D147-H150 salt bridge is found having no correlation with the conformation of 150-loop. Instead the intimate salt bridge interaction between the 150 and 430 loops in N2 variant contributes the stabilizing factor for the closed form of 150-loop. The clustering analysis elaborates the structural plasticity of the loop. This enhanced sampling simulation provides more information in further structural-based drug discovery on influenza virus.</p> </div

Exploring the mechanism of zanamivir resistance in a neuraminidase mutant: a molecular dynamics study.

It is critical to understand the molecular basis of the drug resistance of influenza viruses to efficiently treat this infectious disease. Recently, H1N1 strains of influenza A carrying a mutation of Q136K in neuraminidase were found. The new strain showed a strong Zanamivir neutralization effect. In this study, normal molecular dynamics simulations and metadynamics simulations were employed to explore the mechanism of Zanamivir resistance. The wild-type neuraminidase contained a 3(10) helix before the 150 loop, and there was interaction between the 150 and 430 loops. However, the helix and the interaction between the two loops were disturbed in the mutant protein due to interaction between K136 and nearby residues. Hydrogen-bond network analysis showed weakened interaction between the Zanamivir drug and E276/D151 on account of the electrostatic interaction between K136 and D151. Metadynamics simulations showed that the free energy landscape was different in the mutant than in the wild-type neuraminidase. Conformation with the global minimum of free energy for the mutant protein was different from the wild-type conformation. While the drug fit completely into the active site of the wild-type neuraminidase, it did not match the active site of the mutant variant. This study indicates that the altered hydrogen-bond network and the deformation of the 150 loop are the key factors in development of Zanamivir resistance. Furthermore, the Q136K mutation has a variable effect on conformation of different N1 variants, with conformation of the 1918 N1 variant being more profoundly affected than that of the other N1 variants studied in this paper. This observation warrants further experimental investigation

Force distribution analysis between the active site of 09N1 and ETT as well as ZMR.

<p>The forces between the active site of 09N1 and ETT are shown in red color curve, and the forces between ZMR and the active site are shown in black curve as a positive control. The pair-wise forces of other protomers and systems can be found in supporting information (Figures S2-S5).</p

Potential of mean force (PMF) based on volume of 150-cavity.

<p>PMF calculated based on volume of 150-cavity. Structure representing two local minima together with 16 ų and 24 ų is shown as cartoon with green color. As a comparison, crystal structures with closed and open configuration of 150-loop are aligned together and colored cyan and green respectively.</p

Detailed information of all the MD simulation systems.

<p>Detailed information of all the MD simulation systems.</p

Root Mean Squared Deviation (RMSD) of ligands in different systems.

<p>RMSD values of the linked ligands as well as ZMR and ETT bound with 09N1 are shown in panel A-H. Lines colored in black, red, green and blue represented RMSD of protomer A-D in each complex system.</p

Dihedral PCA indicated turn A of 150-loop is dominant changing place.

<p>Free energy landscape obtained from dihedral PCA analysis of N1o and N1c system is shown in panel (A) and (B) respectively. Four local minima were highlight and their representative structures were shown on its right panel by clustering analysis. Structure of cluster 1, 2, 3, 4 is shown in forest green, green, lemon and yellow color. Crystal structure with closed and open 150-loop is shown in cyan and magenta color respectively.</p