8E4J

Room-temperature X-ray structure of SARS-CoV-2 main protease H41A miniprecursor mutant

Summary for 8E4J

• Entry DOI: 10.2210/pdb8e4j/pdb
• Descriptor: Replicase polyprotein 1ab (2 entities in total)
• Functional Keywords: homodimer, cysteine protease, precursor enzyme, h41a mutation, hydrolase
• Biological source: Severe acute respiratory syndrome coronavirus 2
• Total number of polymer chains: 2
• Total formula weight: 69006.52

Authors

Coates, L.,Kneller, D.W.,Kovalevsky, A. (deposition date: 2022-08-18, release date: 2022-11-23, Last modification date: 2023-10-25)

Primary citation

Kovalevsky, A.,Coates, L.,Kneller, D.W.,Ghirlando, R.,Aniana, A.,Nashed, N.T.,Louis, J.M.
Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor.
J.Mol.Biol., 434:167876-167876, 2022

PubMed Abstract: We recently demonstrated that inhibitor binding reorganizes the oxyanion loop of a monomeric catalytic domain of SARS CoV-2 main protease (MPro) from an unwound (E) to a wound (active, E*) conformation, independent of dimerization. Here we assess the effect of the flanking N-terminal residues, to imitate the MPro precursor prior to its autoprocessing, on conformational equilibria rendering stability and inhibitor binding. Thermal denaturation (T) of C145A mutant, unlike H41A, increases by 6.8 °C, relative to wild-type mature dimer. An inactivating H41A mutation to maintain a miniprecursor containing TSAVL[Q or E] of the flanking nsp4 sequence in an intact form [MPro and MPro, respectively], and its corresponding mature MPro were systematically examined. While the H41A mutation exerts negligible effect on T and dimer dissociation constant (K) of MPro, relative to the wild type MPro, both miniprecursors show a 4-5 °C decrease in T and > 85-fold increase in K as compared to MPro. The K for the binding of the covalent inhibitor GC373 to MPro increases ∼12-fold, relative to MPro, concomitant with its dimerization. While the inhibitor-free dimer exhibits a state in transit from E to E* with a conformational asymmetry of the protomers' oxyanion loops and helical domains, inhibitor binding restores the asymmetry to mature-like oxyanion loop conformations (E*) but not of the helical domains. Disorder of the terminal residues 1-2 and 302-306 observed in both structures suggest that N-terminal autoprocessing is tightly coupled to the E-E* equilibrium and stable dimer formation.

PubMed: 36334779
DOI: 10.1016/j.jmb.2022.167876

Experimental method

X-RAY DIFFRACTION (1.9 Å)