Journal:Acta Cryst D:S2059798322000948

A new inactive conformation of SARS-CoV-2 main protease

Emanuele Fornasier, Maria Ludovica Macchia, Gabriele Giachin, Alice Sosic, Matteo Pavan, Mattia Sturlese, Cristiano Salata, Stefano Moro, Barbara Gatto, Massimo Bellanda and Roberto Battistutta ^[1]

Molecular Tour
The SARS-CoV-2 main protease, M^pro or 3CL^pro, is a cysteine peptidase involved in the processing of the two overlapping polyproteins pp1a and pp1ab, with the formation of individual mature non-structural proteins (nsp). Playing a pivotal role in genome replication and transcription, it is a validated antiviral drug target against Covid-19 pandemic. M^pro forms homo-dimers, the only enzymatically active form of the enzyme. Dimeric assembly of the protease with the main structural features discussed in the text highlighted (PDB entry 6y2e). Protomer A in blue-based colors, protomer B in yellow/red-based colors. In green the two oxyanion loops and the two catalytic cysteines 145. Each M^pro protomer is composed of three structural domains; the chymotrypsin- and 3C protease-like domains I and II directly control the catalytic event. The substrate-binding site is between domains I and II, and, at variance with the classical catalytic triad of chymotrypsin-like proteases, SARS-CoV-2 M^pro has a catalytic dyad, consisting of His41 and Cys145. A structural element essential for the catalysis is the so-called “oxyanion loop” (in green), comprising residues 138-145.

In this paper, we describe a new inactive structure of the main protease of SARS-CoV-2 (7nij). Movements in the substrate-binding region and near the catalytic site result in a significant reshaping of the reaction center. Comparison between different oxyanion loop conformations of M^pro: “active” in SARS-CoV-2 M^pro 6y2e in pink; “collapsed-inactive” in SARS-CoV M^pro 1uj1 chain B in magenta; “new-inactive” in SARS-CoV-2 M^pro 7nij in green. Consequently, the conformation adopted by residues 139-144 of the oxyanion loop is catalytically-incompetent. New conformation of the oxyanion loop is stabilized by several hydrogen bonds (white dashed lines). The side-chain of catalytic Cys145 has a double conformation. Phe140, Leu141 and Asn142 play a major role in the shift between the new inactive and active conformations. Comparison between new-inactive and active oxyanion loop. The new-inactive oxyanion loop (7nij) is in green and the active loop (6y2e) is in pink. There are large movements (white dashed lines) for the side-chains of Asn142 and Phe140. In the new-inactive conformation, Asn142 moves from an exposed position, ASA 153.74 Ų, to a buried one, ASA 49.00 Ų and Phe140 moves from a buried position, ASA 14.79 Ų, to an exposed one, ASA 143.29 Ų. Gly143-NH (G-NH) of the oxyanion hole, involved in the stabilization of the tetrahedral intermediate, moves 8.8 Å away.

The movements of the oxyanion loop and of the N- and C-termini result in the weakening of the dimeric architecture, as evidenced by the decreases in the interaction surface area and in the number of inter-protomer interactions. This novel conformation is relevant both for the comprehension of the mechanism of action of M^pro within the catalytic cycle and for the success of the structure-based drug design of anti-viral drugs.

Reshaping of the S1 and S2' subsites. Molecular dynamics modeling of the hypothetical interaction of new-inactive M^pro with substrates is shown. Putative interaction with the 11_mer pseudo-substrate peptide from structure 2q6g:

• New-inactive Mpro from 7nij.
• SARS-CoV Mpro from 2q6g.

As a result of the rearrangement of the oxyanion loop, a new cavity near the S2´ 9 site, indicated as “NEW”, is formed.

Putative interaction with the acyl-intermediate of M^pro C-terminal autoprocessing site:

• New-inactive Mpro from 7nij.
• Mpro from 7khp.

PDB reference: SARS-CoV-2 main protease in a novel conformational state, 7nij

References

1. ↑ Fornasier E, Macchia ML, Giachin G, Sosic A, Pavan M, Sturlese M, Salata C, Moro S, Gatto B, Bellanda M, Battistutta R. A new inactive conformation of SARS-CoV-2 main protease. Acta Crystallogr D Struct Biol. 2022 Mar 1;78(Pt 3):363-378. doi:, 10.1107/S2059798322000948. Epub 2022 Feb 21. PMID:35234150 doi:http://dx.doi.org/10.1107/S2059798322000948