Molecular dynamics and in silico mutagenesis on the reversible inhibitor-bound SARS-CoV-2 main protease complexes reveal the role of lateral pocket in enhancing the ligand affinity

Abstract

The 2019 novel coronavirus pandemic caused by SARS-CoV-2 remains a serious health threat to humans and there is an urgent need to develop therapeutics against this deadly virus. Recent scientific evidences have suggested that the main protease (M^pro) enzyme in SARS-CoV-2 can be an ideal drug target due to its crucial role in the viral replication and transcription processes. Therefore, there are ongoing research efforts to identify drug candidates against SARS-CoV-2 M^pro that resulted in hundreds of X-ray crystal structures of ligand-bound M^pro complexes in the Protein Data Bank (PDB) describing the interactions of different fragment chemotypes within different sites of the M^pro. In this work, we performed rigorous molecular dynamics (MD) simulation of 62 reversible ligand-M^pro complexes in the PDB to gain mechanistic insights about their interactions at the atomic level. Using a total of over 3 µs long MD trajectories, we characterized different pockets in the apo M^pro structure, and analyzed the dynamic interactions and binding affinity of ligands within those pockets. Our results identified the key residues that stabilize the ligands in the catalytic sites and other pockets of M^pro. Our analyses unraveled the role of a lateral pocket in the catalytic site in M^pro that is critical for enhancing the ligand binding to the enzyme. We also highlighted the important contribution from HIS163 in the lateral pocket towards ligand binding and affinity against M^pro through computational mutation analyses. Further, we revealed the effects of explicit water molecules and M^pro dimerization in the ligand association with the target. Thus, comprehensive molecular-level insights gained from this work can be useful to identify or design potent small molecule inhibitors against SARS-CoV-2 M^pro.

Figure 1

Three dimensional (3D) structure (a), and topology (b) of SARS CoV-2 M^pro, along with the variable residues with reference to SARS-CoV-1 M^pro (c) and the different ligand binding sites described by the known PDB complexes of M^pro. (a) A 3D structure of SARS-CoV-2 M^pro is shown with Domains I, II and III colored in blue, red and yellow respectively. Two important loops close to the catalytic dyad (HIS41 and CYS145) are shown in green: CYS44–PRO52 loop flanks the catalytic dyad and PHE185–THR201 connects Domain II with Domain III. (b) A topology diagram showing the secondary structural elements in SARS-CoV-2 M^pro with β-sheets marked as A-M. (c) Structural alignment of SARS-CoV-2 M^pro and Sars-CoV-1 M^pro shows only 12 mutations of amino acids that are shown as a stick representation in purple. (d) The binding of 62 reversible ligands bound at different sites in the SARS-CoV-2 M^pro that is shown as a surface representation in blue. The ligands bound within the catalytic site are highlighted with a circle. The molecular graphics in this figure were generated using VMD 1.9.3 (a,c) and UCSF Chimera 1.14 (d) while the secondary structure topology diagram in (b) was created using information from the EMBL-EBI online server PDBsum^,.

Figure 2

The backbone RMSD (a) and RMSF (b), of apo M^pro (PDB: 6M2Q) and the different pockets on the M^pro (c,d) identified through MD simulation. RMSD of the apo structure changed between 1–3 Å during MD. The per-residue fluctuation during MD is shown as RMSF with the different domains in the enzyme labelled to reflect this. Using the “MDpocket” tool, various transient pockets (named as Pocket 1–8) were identified and characterized within the apo structure during the MD simulation. The graphs in this figure (a,b) were generated using GNUplot (v5.2 patchlevel 8; http://www.gnuplot.info/) while the molecular graphics were generated using VMD 1.9.3 (c,d).

Figure 3

The binding free energies and the binding poses of the ligand–M^pro complexes in which the ligands were bound in pocket 3 (a) and in pocket 4 (b). Both in pocket 3 and pocket 4, a few ligands remained stable due to their ability to make key electrostatic interactions with the residues in the pocket, while the other ligands failed to make such interactions that resulted in their weak affinity against M^pro. The molecular graphics in this figure were generated using UCSF Chimera 1.14 (a,b) while the plots were generated using Microsoft Excel 365 (https://www.office.com/).

Figure 4

The scatter plot comparing the binding free energies of the ligand–M^pro complexes in which the ligands were bound in catalytic site (a) and the identification of four key components that are critical for ligand-binding in this site (b). Two loops flank the active site pocket: the L1 loop shown in yellow, and the L2 loop (or the domain II-III linker loop) shown in green. In addition, the ‘L’ β-sheet is shown in blue, and the residues such as HIS41, LEU27, and MET49 located deep inside the pocket are shown as stick representation. The molecular graphic in this figure was generated using VMD 1.9.3 (b) while the scatter plot was generated using Microsoft Excel 365 (a) (https://www.office.com/).

Figure 5

The pair-wise decomposition analyses (a) for the binding mode (b) of ligand in 6W63 complex and its hydrogen bond interactions with the key residues in the pocket (c), along with the 2D interaction diagram for the ligand–M^pro binding mode are shown. The decomposition plot (a) identified the key residues that helped in a favourable binding mode of the ligand (b) that was supported by its interactions with the L1 loop, L2 loop, L β-sheet and the catalytic diad residues. The ligand in this complex made stable H-bond interactions with a few key resiudes including HIS41, GLY143, HIS163, and GLY166 (c). In the presented 2D interaction diagram for the ligand–M^pro binding pose (d), the ligand is shown as red lines, the hydrophobic residues are shown in green; the polar residues in torquise; the negatively charged residues in orange; and the positively charged residues are shown in blue. The hydrogen bonds are displayed as purple arrows. The three dimensional molecular graphic used in this figure was generated using VMD 1.9.3 (b) while the two-dimensional interaction diagram was developed using Maestro Schrodinger (d). The plots were generated using GNUplot (v5.2 patchlevel 8; http://www.gnuplot.info/) (c) and Microsoft Excel 365 (a) (https://www.office.com/).

Figure 6

The binding mode of ligand in the 5RG1 complex within the catalytic site of M^pro (a), the decomposition of key residues contributing to this ligand–receptor complex (b), along with the time evolution of H-bond interactions of the bound ligand with residues such as GLN189, HIS163 and GLU166 (c). In the ligand bind pose in this complex (a), the ligand is seen interacting with all the key segments in the catalytic site of M^pro, which include the L1 loop, L2 loop, L β-sheet and the catalytic diad residues. Evidently, the ligand occupied the lateral pocket in the catalytic site of M^pro, where it formed stable H-bonds with HIS163 residue. This binding mode was supported by other key residues such as GLN189 and GLU166, as seen in the pairwise decomposition plot (b). The molecular graphic used in this figure was generated using VMD 1.9.3 (a) while the plots were generated using GNUplot (v5.2 patchlevel 8 http://www.gnuplot.info/) (c) and Microsoft Excel 365 (b) (https://www.office.com/).

Figure 7

(a) EDA variance of the atomic fluctuation in the combined ensemble. Fractional variance (blue bars) and cumulative fractional variance (blue curve) of the atomic fluctuations for the first 100 PC modes from an EDA over the Grand ensemble of MD conformations. (b–e) Mobility profile per mode in the combined ensemble. Dissection of the MSqF (in Ų) for the first 4 PC modes. Highlighted with colors residues belonging to active-site loops according to segment [24–28] (red), L3: [39–54] (blue), segment [118–119] (green), L1: [140–146] (cyan), “L” strand: [163–172] (purple), L2: [181–192] (yellow). (f) MD Grand ensemble deformations into the essential space. Tube representation of the Cα-trace of M^pro (residues 5..300) depicting vectors that characterize the direction and relative magnitude of the deformations determined by the indicated PC modes: PC1 (blue), PC2 (red), and PC3 (green). Each set of vectors scaled to 1 Å of $〈\text{RMSF}〉$ for visualization. The molecular graphic used in this figure was generated using ProDY and rendered using VMD 1.9.3 (f) while the plots were created using Microsoft Excel 365 (a–e) (https://www.office.com/).

Figure 8

The identification of steric water sites in the apo and ligand-bound M^pro complexes (a) along with the comparison of the binding free energy of the wildtype (WT) and HIS163ALA mutant complexes of the select systems (b). (a) The presence of steric water molecules (shown as yellow spheres) are found within the lateral binding pocket in the apo protein structure. When a ligand occupied the lateral site in the 5RG1 complex, the steric water molecules were displaced to the edge of the pocket, allowing for more stable ligand-binding. Nevertheless, when the ligand did not occupy the lateral pocket (as in the case of 5RGH structure), the steric water molecules are again observed within this lateral site. (b) Mutation of HIS163 to an alanine (ALA163) clearly led to a weak binding affinity between the selected ligand–M^pro complexes. The molecular graphic in this figure was generated using VMD 1.9.3 (a) while the comparison plot in (b) was generated using Microsoft Excel 365 (https://www.office.com/).

Figure 9

A polar plot describing the change in binding affinity scores of the select ligand–M^pro complexes in response to the presence of varying numbers of explicit water molecules (NWAT = 0–6) (a), and the 3D snapshots describing the different water interactions with the ligand (b–d) are also shown. In the 5RHD complex, a water molecule bridged the protein and the ligand acting as a proton donor to M^pro and proton acceptor for the ligand (b). In PDBs 5RGZ and 5RG1, a key water molecule interacted with the carboxamide and hydroxyl side chains of the ligand that helped to maintain the association of the ligands with the lateral pocket (c,d) in M^pro. The molecular graphics in the figure were generated using VMD 1.9.3 (b–d) while the radial plot in (a) was generated using Microsoft Excel 365 (https://www.office.com/).

Figure 10

The binding of ligand from 5REH within a dimer model of M^pro (shown as surface representation) (a), along with the RMSD of the ligand when bound with the monomer and dimer (b) and the binding pose of the ligand within the dimer. The RMSD plots described that the ligand was unstable when bound to a monomer of M^pro; however, it was more stable within a dimer condition. This is likely due to its favourable binding in the active site containing pocket where the ligand made stable H-bonds with HIS163 and GLU166 of monomer B thus occupying the lateral pocket that was extensively explored by other ligands part of our set (c). The N terminal of the second monomer, specifically SER 1, interacted in a hydrophobic manner, which indeed helped in the stable ligand interactions with M^pro. In the 2D interaction diagram, the hydrophobic residues are shown in green; the polar residues in torquise; the negatively charged residues in orange; and the positively charged residues are shown in blue. The hydrogen bonds are displayed as purple arrows; and the solvent exposed sites in the bound ligand are noted with grey circles over the chemical structure of the ligand shown in red lines. The dimer molecular graphic shown in (a) was generated using VMD 1.9.3 (a) while the two-dimensional interaction diagram was developed using Maestro Schrodinger (c). The plot in (c) was generated using GNUplot (v5.2 patchlevel 8 http://www.gnuplot.info/).