Characterization of alternate encounter assemblies of SARS-CoV-2 main protease

The assembly of two monomeric constructs spanning segments 1-199 (MPro^1-199) and 10-306 (MPro^10-306) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by ∼7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro^1-199 and MPro^10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201-214 onto a second helical domain of MPro^10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro^1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro^10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at ∼6 μM MPro^1-199. Catalytic activity arises primarily from MPro^1-199 and is dependent on the interface interactions involving the N-finger residues 1-9 of MPro^1-199 and E290 of MPro^10-306. Importantly, our results confirm that a single N-finger region with its associated inter-subunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity.

SARS-CoV-2 main protease; alternate folding pathways; dimer interface; fold-switching; inhibitor binding; monomer-dimer equilibrium; precursor processing; protein folding; protein fragment assembly; structure comparison; swapped dimer.