Structural differences in 3C-like protease (Mpro) from SARS-CoV and SARS-CoV-2: molecular insights revealed by Molecular Dynamics Simulations

Abstract

Novel coronavirus SARS-CoV-2 has infected millions of people with thousands of mortalities globally. The main protease (Mpro) is vital in processing replicase polyproteins. Both the CoV's Mpro shares 97% identity, with 12 mutations, but none are present in the active site. Although many therapeutics and vaccines are available to combat SARS-CoV-2, these treatments may not be practical due to their high mutational rate. On the other hand, Mpro has a high degree of conservation throughout variants, making Mpro a stout drug target. Here, we report a detailed comparison of both the monomeric Mpro and the biologically active dimeric Mpro using MD simulation to understand the impact of the 12 divergent residues (T35V, A46S, S65N, L86V, R88K, S94A, H134F, K180N, L202V, A267S, T285A and I286L) on the molecular microenvironment and the interaction between crucial residues. The present study concluded that the change in the microenvironment of residues at the entrance (T25, T26, M49 and Q189), near the catalytic site (F140, H163, H164, M165 and H172) and in the substrate-binding site (V35, N65, K88 and N180) is due to 12 mutations in the SARS-CoV-2 Mpro. Furthermore, the involvement of F140, E166 and H172 residues in dimerization stabilizes the Mpro dimer, which should be considered. We anticipate that networks and microenvironment changes identified here might guide repurposing attempts and optimization of new Mpro inhibitors.

Supplementary information: The online version contains supplementary material available at 10.1007/s11224-022-02089-6.

Keywords: 3C-like proteases; COVID-19; Drug repurposing; Molecular dynamics; Mpro; SARS-CoV-2.

Fig. 1

SARS-CoV-2 Mpro in monomer and dimer state: A monomer structure of SARS-CoV-2 Mpro with domain assignment marked with residue ranges. B Dimer structure of SARS-CoV-2 Mpro coloured by domain assignment. C Dimer structure of SARS-CoV-2 Mpro in surface representation coloured by domain assignment at two different angles. In all panels, domain I is marked in red, domain II in green, domain III in blue and the loop region with magenta

Fig. 2

Monomer structure alignment of SARS-CoV Mpro (red) and SARS-CoV-2 Mpro (blue) aligned with the Cα backbone. A The overall structure of SARS-CoV and SARS-CoV-2 Mpro with 12 differing amino acids is marked and shown in the sticks as red (SARS-CoV Mpro) and blue (SARS-CoV-2 Mpro). The active site residues are coloured green. Close-up of active site residues represented in the sticks (side chains) as cyan (SARS-CoV-2 Mpro) and dark green (SARS-CoV Mpro). B The structure of SARS-CoV-2 Mpro in surface display in two angles (180° rotation) with 12 different residues from SARS-CoV Mpro marked and shown in blue sticks. The catalytic HIS41 and CYS145 residues are shown in green sticks, and other active site residues are shown on the green surface

Fig. 3

Structural superposition of 12 CoV Mpro from different strains. A Superposition of 12 Mpro structures (Cα backbone) from different CoV onto SARS-CoV-2 Mpro as a query. B Representation of 12 Mpro superposed structures using variable tube depiction, where the radius is proportional to the RMSD differences in Cα between SARS-CoV-2 Mpro and 12 other homologous Mpro structures. In both panels, blue-to-red colour ramping is used to visualize and correlate conservation from strong to weak conservation areas. Domain assignments are marked with residue ranges in both panels

Fig. 4

Molecular Dynamics Simulations of Mpro from SARS-CoV and SARS-CoV-2 in monomer and dimer structures, computing the deviation (nm) vs function of time (50 ns): RMSD of the protein Cα backbone atoms of SARS-CoV and SARS-CoV-2 Mpro in monomer (A) and dimer (B) forms. ROG of the protein Cα backbone atoms of SARS-CoV Mpro and SARS-CoV-2 Mpro in monomer (C) and dimer (D) forms. The inset graph represents average values with standard deviations, and SARS-CoV and SARS-CoV-2 Mpro are plotted in black and red, respectively

Fig. 5

The plot of SASA values for individual residues in all four MD systems: A the plot of 12 divergent residues in SARS-CoV-2 Mpro. The X-axis represents the residue present in SARS-CoV-2, followed by the residue number and ending with the residue found in SARS-CoV. B The plot of residues in the active site conserved in SARS-CoV and SARS-CoV-2 Mpro

Fig. 6

Residue-wise RMSF deviations (nm) of Mpro from SARS-CoV and SARS-CoV-2 in monomer and dimer form: A RMSF plot of both CoV Mpro in monomer form. The 12 divergent residues in SARS-CoV-2 are marked with a green line in the lower plot. The active site residues are marked with a red line in the upper plot. B RMSF plot of both CoV Mpro in dimer form. In both panels, the domain I, II and III residues are marked in red, green and blue, respectively

Fig. 7

RMSF plots showing deviations (nm) of selected residues from SARS-CoV and SARS-CoV-2 Mpro in monomer and dimer form: RMSF plots of 12 divergent residues from both the CoV Mpro in monomer (A) and dimer (B). The X-axis represents the residue present in SARS-CoV-2, followed by the residue number and ending with the residue found in SARS-CoV. RMSF plots of active site residues from the CoV Mpro in monomer (C) and dimer (D)

Fig. 8

PCA 2D projection scatters plot of SARS-CoV and SARS-CoV-2 Mpro: A Overlay of 2D scatter plot projection of the motion of the proteins in phase space for the two principal components, PC1 and PC3, derived from four MD simulation setups. Panel B, C, D and E represent individual 2D plots of SARS-CoV_monomer, SARS-CoV-2_monomer, SARS-CoV_dimer and SARS-CoV-2_dimer, respectively. F Plot representing Eigenvalues calculated from the covariance matrix of Cα backbone fluctuations vs the respective eigenvector indices for the first 50 eigenvectors from 1000 eigenvectors. For all the panels, the colour representation is SARS-CoV_monomer (black), SARS-CoV-2_monomer (red), SARS-CoV_dimer (green) and SARS-CoV-2_dimer (blue)

Fig. 9

The porcupine plot for conformational variability is computed from the crystal structure and average MD simulations ensemble. Porcupine plots of A SARS-CoV_monomer, B SARS-CoV-2_monomer, C SARS-CoV_dimer and D SARS-CoV-2_dimer. The length of the cone is proportional to the conformational variability, while the colour of the cone is represented by deviation in RMSD as indicated in the respective colour scale in each plot

Fig. 10

Free energy surface of Mpro computed over entire simulations (50ns): FES (in kcal/mol) for Mpro from A SARS-CoV_monomer, B SARS-CoV-2_monomer, C SARS-CoV_dimer and D SARS-CoV-2_dimer considering the conformational variability in terms of ROG and RMSD took together and represented by Gibbs free energy