A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process

Abstract

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral M^pro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. The lack of structural information for intermediary forms of M^pro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 M^pro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the M^pro bound to its endogenous N and C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.

Keywords: COVID; M(pro); SARS-CoV-2; drug discovery; maturation.

Graphical abstract

Figure 1

(a) Schematic showing different constructs of M^pro. (b) Time-course reactions of M^pro constructs against fluorogenic peptide substrate. (c) Differential scanning fluorimetry of M^pro constructs. M^pro is shown as blue squares, IMT M^pro is shown as red spheres and C145S M^pro is shown as black triangles (d) SEC elution profiles with overlaid calculated molar mass from elution peaks. M^pro (blue) elutes as a single peak with a calculated molecular mass consistent with a dimer. IMT M^pro (red) exhibits a single peak with a mass compatible with a monomer in solution. The monomeric SEC peak of C145S M^pro (grey) elutes as an equilibrium between dimers and monomers in solution. The tetrameric SEC peak of C145S M^pro (black) contains peaks of monomers, dimer, trimers and tetramers. (e) SDS-PAGE of N-terminal cleavage over time from C145S M^pro. At top, reaction containing 10 µM C145S M^pro, and at the bottom the same reaction supplemented with 5 nM M^pro. Red arrows are pointing to the band of cleaved M^pro. The bar graph shows the relative band intensity of cleaved M^pro overtime for both reactions in blue and salmon, respectively. (f) SEC elution profiles of monomeric peak of C145S M^pro over time. (g) SEC elution profiles of monomeric peak of C145S M^pro over time supplemented with 10 nM M^pro. In SEC-MAL graphs, curves correspond to the change in the normalized scattered light intensity at 90° (lines) and calculated molar mass of the corresponding peak (dots) are given for each peak.

Figure 2

(a) Overview of DIII region from IMT M^pro (chain A yellow and B cyan) superposed with M^pro (grey ghost). N-terminal residues are depicted as spheres. (b) Rotated view showing IMT M^pro DIII from a distinct angle. (c) Active site residues of IMT M^pro chain B (cyan cartoon) superposed with M^pro. Catalytic residues are depicted as yellow sticks. N-terminal chain A residues are depicted as spheres. M^pro structure and residues are shown as a grey ghost.

Figure 3

(a) Location of IMT M^pro probing fragments identified during screening. Chain A is colored as yellow surface, chain B as cyan surface. Fragments are depicted as red spheres. For comparison, fragments of previous manuscript using monoclinic M^pro were aligned to the structure of IMT M^pro and are depicted as grey spheres. (b) Contact details of identified fragments bound to IMT M^pro. Chain A is colored as yellow cartoon and chain B as cyan cartoon. Fragments are depicted as yellow sticks. Residues forming polar contacts are depicted as green lines. Contacts are depicted as black dashes.

Figure 4

(a) C145S M^pro chain A active site (cyan surface) in complex with processed N-terminal residues (yellow sticks). Main interacting residues are depicted as blue lines. (b) C-terminal peptide (yellow) main interactions with C145S M^pro chain A active site residues (blue). (c) C145S M^pro chain B active site (blue surface) in complex with processed C-terminal residues (yellow sticks). Main interacting residues are depicted as blue lines. (d) C-terminal peptide (yellow) main interactions with C145S M^pro chain B active site residues (blue). For (b) and (d), the 2mFo-DFc electron density contoured at 0.8σ. Ser1 from respective dimerization partners are depicted with green letters. *Ser145 is the site-direct mutant of Cys145. Simulated annealing omit map is available in Figure S7.

Figure 5

Overview of the dimer-dimer association intermediary formed by C145S M^pro tetramer during self-processing. Chain A is colored as yellow surface, chain B as cyan surface. Trans-cleavage M^pro partner is show as green cartoon. N-terminal residues are depicted as blue spheres, and C-terminal residues are depicted as red spheres.

Figure 6

Time-curse reactions of C145S M^pro monomeric construct determined after different incubation periods. Activity of the construct was monitored after 0 h (black), 24 h (red) and 48 h (blue) incubation.

Figure 7

Scheme containing steps of SARS-CoV-2 M^pro self-maturation process. (a) At first, two protomers assembly as an immature dimer during N-terminal cis and trans-cleavage. After processing, the M^pro with the matured active site permit the correct positioning of DIII, which allows the stabilization of the dimeric form. The dimer C-terminal is them trans-cleaved by another full or at least half mature dimer, forming a transient dimer-dimer association and producing the full mature form of M^pro. (b) Surface view of chain B active site from immature form. (c) Surface view of chain A active site during N-terminal residues recognition. (d) Surface view of chain B active site during C-terminal residues recognition. (e) Surface view of full mature M^pro active site.