Inhibitor induced conformational changes in SARS-COV-2 papain-like protease

Abstract

SARS-CoV-2's papain-like protease (PL^pro) interaction with ligands has recently been explored with a myriad of crystal structures. We used molecular dynamics (MD) simulations to study different PL^pro-ligand complexes, their ligand-induced conformational changes, and interactions. We focused on inhibitors reported with known IC₅₀ against PL^pro, namely GRL-0617, XR8-89, PLP_Snyder530, and Sander's recently published compound 7 (CPD7), and compared these trajectories against the apostructure (Apo), with a total of around 60 µs worth simulation data. We aimed to study the conformational changes using molecular dynamics simulations for the inhibitors in the PL^pro. PCA analyses and the MSM models revealed distinct conformations of PL^pro in the absence/presence of ligands and proposed that BL2-loop contributes to the accessibility of these inhibitors. Further, bulkier substituents closer to Tyr268 and Gln269 could improve inhibition of SARS-CoV-2 PL^pro by occupying the region between BL2-groove and BL2-loop, but we also expand on the relevance of exploring multiple PL^pro sub-pockets to improve inhibition.

Figure 1

SARS-CoV-2 PL^pro structure overview, ligands from the protein data bank (PDB). (A) The tertiary structure of the SARS-CoV-2 PL^pro with domains UBL (1–60, in dark grey), Thumb (61–176, in grey), (B) Ligand binding pocket and catalytic triad (Cys111, His272, and Asp286), Fingers (177–238), and Palm (239–315) and the BL2-loop (259–277, in magenta). (C) Two-dimensional representation of the ligands GRL-0617 (from PDB: 7jir), PLP_Snyder530 (from PDB: 7jiw), and XR8-89 (from PDB: 7lbr) and CPD7 (modelled as described in the methods section).

Figure 2

SARS-CoV-2 PL^pro principal component analysis. (A) Distributions over the two significant PCs (PC1 and PC2) separated for each simulated system apostructure (PDB: 7lbr) is highlighted in grey; GRL-0617 bound structures (GLR067-7JIR) in sky-blue; PLP_Synder530 bound structures (PLP_Synder530 - 7JIW) in magenta, XR8-89 bound structures (XR8-89 - 7LBR) in orange and CPD7 bound structures (CPD7) in dark brown. (B) Extreme motions from the PC1 displayed over the PL^pro tertiary structure represented by arrows. (C) Extreme motions from the PC2 displayed over the PL^pro tertiary structure represented by arrows. (D) Ubl-Catalytic connection path, with relevant residues highlighted as sticks (E) Distance values between C111 to H272 along with the simulations depicting the full extension of the inhibitor binding site opening. (F) S2 binding site opening representation, and (G) S2 vector angle analysis describing the opening/closing of this region. For E and G, ligands are coloured according to figure legend.

Figure 3

Metastable states of PL^pro in the presence and absence of the ligands as revealed by Markov state modelling. (A) Pseudo free energy map of distributions along with time-lagged independent components (ICs) 1 and 2. (B) Separation of five metastable states (S₁–S₅) by PCCA++ analysis. (C) Committor probability of the most representative metastable states. Each metastable state (S) is illustrated with ten representative structures (coloured ribbons), superimposed to a transparent cartoon with the original crystal structure. Equilibrium probability (πi) of distributions for each state is indicated below the conformations together with circles with an area representing the changes induced by the systems, values near to 1.0 more probable than values close to 0.

Figure 4

Dihedral angle distribution analysis for the residues BL2 loop (χ₁ (CA to CB) and χ₂ (CB to CD/CZ) angles conformation). (A) 3D structure representation of the distance between R166 (in grey), P299 (in sand) and Y268 (in magenta). (B) Distance between R166 to Y268 in absence and presence of the ligands and distance between Pro299 to Tyr268 in absence and presence of the ligands. (C) 2D and 3D representation of the χ angles in Y268 and Gln269 from apostructure and in presence of the ligands. For each graphic in (D,E) Dark coloured angles (black, dark blue, pink, dark orange and red) represents the angle χ₁, which is the movement of Tyr289 and Gln269, while the light coloured bars represents the χ₂, which is the angle of Tyr289 and Gln269. Graphics are coloured according to Apostructure by grey, GRL-0617 sky blue, PLP_Snyder530, magenta, XR8-89, orange and CPD7 covalently bound, red.

Figure 5

Overall interactions of the SARS-CoV-2 PL^pro with inhibitors. Snapshot frames of the simulations displaying relevant interactions between the PL^pro in the presence of the ligands GRL-0617 (A, depicted in sky blue); PLP_Snyder530 (B, magenta), XR8-89 (C, orange) and CPD7 (D, dark brown). 2D structures representations of XR8-89 (E) and CPD7 (F) and frequencies of interactions. PL^pro residues are colored according to the types of atoms in the interacting amino acid residues with the protein carbon, light grey; nitrogen, blue; oxygen, red.