SARS-CoV-2 Nsp16 activation mechanism and a cryptic pocket with pan-coronavirus antiviral potential

Abstract

Coronaviruses have caused multiple epidemics in the past two decades, in addition to the current COVID-19 pandemic that is severely damaging global health and the economy. Coronaviruses employ between 20 and 30 proteins to carry out their viral replication cycle, including infection, immune evasion, and replication. Among these, nonstructural protein 16 (Nsp16), a 2'-O-methyltransferase, plays an essential role in immune evasion. Nsp16 achieves this by mimicking its human homolog, CMTr1, which methylates mRNA to enhance translation efficiency and distinguish self from other. Unlike human CMTr1, Nsp16 requires a binding partner, Nsp10, to activate its enzymatic activity. The requirement of this binding partner presents two questions that we investigate in this manuscript. First, how does Nsp10 activate Nsp16? Although experimentally derived structures of the active Nsp16/Nsp10 complex exist, structures of inactive, monomeric Nsp16 have yet to be solved. Therefore, it is unclear how Nsp10 activates Nsp16. Using over 1 ms of molecular dynamics simulations of both Nsp16 and its complex with Nsp10, we investigate how the presence of Nsp10 shifts Nsp16's conformational ensemble to activate it. Second, guided by this activation mechanism and Markov state models, we investigate whether Nsp16 adopts inactive structures with cryptic pockets that, if targeted with a small molecule, could inhibit Nsp16 by stabilizing its inactive state. After identifying such a pocket in SARS-CoV2 Nsp16, we show that this cryptic pocket also opens in SARS-CoV1 and MERS but not in human CMTr1. Therefore, it may be possible to develop pan-coronavirus antivirals that target this cryptic pocket.

Figure 1

Substrate-binding pockets and Nsp10 binding interface of Nsp16 observed in the crystal structure of the Nsp16/Nsp10 complex (PDB: 6wks). (A) Surface representation of Nsp16 showing the SAM-binding pocket (cyan), RNA-binding pocket (yellow), and Nsp10-binding interface (green). (B) Overlay of Nsp16 structures from structures of the Nsp16/Nsp10 complex with RNA (PDB: 6wks, shown in gray) and without RNA (PDB: 6w4h, shown in cyan), showing structural heterogeneity in the RNA binding site. Gate loop 1 and gate loop 2 of the RNA-binding pocket, and SAM-binding loop 1 (SAMBL1) and SAM-binding loop 2 (SAMBL2) lining the SAM-binding pocket are highlighted. To see this figure in color, go online.

Figure 2

Nsp10 binding shifts Nsp16’s conformational ensemble, increasing its propensity to adopt structural states that are ligand binding compatible. (A) 10 structures of Nsp16 that represent the DiffNet prediction changing from inactive to active (white to purple). The DiffNet output label scales from 0 to 1 (white to purple) reflecting the extent the DiffNet predicts a structure to be associated with Nsp16 activation. (B) Comparison of the DiffNet-predicted active and inactive states (purple plus white, respectively) to the starting simulation state (yellow), a known SAM- and RNA-bound structural state (orange), and a known SAM- (but not RNA-) bound state (teal). All structures are aligned to 6wks (orange). (C) Probability-weighted distance distribution between RNA-binding gate loops 1 and 2, comparing monomeric Nsp16 (black) to the Nsp10-Nsp16 complex (gray). (D) Probability-weighted distance distribution between SAM-binding loop 2 and gate loop 2, comparing monomeric Nsp16 (black) to the Nsp10-Nsp16 complex (gray). For (C) and (D), the distance for a SAM- and RNA-bound crystal structure is also plotted (red dotted line). To see this figure in color, go online.

Figure 3

Cryptic pocket opening in SARS-CoV-2 Nsp16. (A) Structural states with the cryptic pocket closed and open. The insets show surface views of the closed and open pocket. Residues exposed upon pocket opening are shown in cyan, and the regions undergoing the opening motion are shown in blue. Collapse of the SAM-binding pocket is measured as the distance between SAMBL2 and gate loop 2, shown in yellow. (B) Equilibrium probability-weighted two-dimensional histograms of solvent-accessible surface area (SASA) of pocket residues (shown in cyan in (A)) and the distance between SAMBL2 and gate loop 2 in Nsp16 for monomeric Nsp16 (upper panel) and the Nsp16/Nsp10 complex (lower panel). The black dotted line separates the pocket closed and open states in Nsp16. The Equilibrium probability-weighted distribution scales from 0 to 0.11 (blue to red) for Nsp16 monomer, and from 0 to 0.26 (blue to red) for Nsp16/Nsp10 complex. To see this figure in color, go online.

Figure 4

Comparison of cryptic pocket opening in Nsp16 homologs and human CMTr1. (A) Equilibrium probability-weighted distribution of the solvent exposure of pocket-forming residues for SARS-CoV-2 (black), SARS-CoV-1 (blue), MERS (red), and CMTr1 (cyan). Structures representing the open pocket are shown for each homolog, with β3 colored in cyan and other pocket-forming residues from α3 colored in green. Black dotted line depicts SASA of pocket residues in the crystal structure of Nsp16/Nsp10 complex (PDB: 6wks). (B) Structure-based sequence alignment of Nsp16 homologs (SARS-CoV-2, SARS-CoV-1, and MERS) and human CMTr1 is shown for the cryptic pocket-forming regions. Residues of β3 are marked inside the black-colored box, and other pocket-forming residues from α3 are marked by green-colored stars. To see this figure in color, go online.