ABSTRACT
The main protease (Mpro) of SARS-CoV-2 is critical for viral function and a key drug target. Mpro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized Mpro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. Mpro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein's crystallization behavior is linked to its redox state. Oxidized Mpro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of Mpro and the crystallization conditions necessary to study this structurally.
Subject(s)
Catalytic Domain , Coronavirus 3C Proteases , Cysteine , Disulfides , Oxidation-Reduction , SARS-CoV-2 , Disulfides/chemistry , Disulfides/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/chemistry , Coronavirus 3C Proteases/metabolism , Coronavirus 3C Proteases/chemistry , Cysteine/chemistry , Cysteine/metabolism , Crystallography, X-Ray , Humans , Models, Molecular , Protein Multimerization , COVID-19/virologyABSTRACT
Liquid drops on deformable soft substrates exhibit quite complicated wetting behavior as compared to those on rigid solid substrates. We report on a soft wetting behavior of Co nanoparticles (NPs) on a sapphire substrate during pulsed laser-induced dewetting (PLID). Co NPs produced by PLID wetted the sapphire substrate with a contact angle near 70°, which is in contrast to typical dewetting behavior of metal thin films exhibiting contact angles greater than 90°. In addition, a nanoscale γ-Al2O3 wetting ridge about 15 nm in size and a thin amorphous Al2O3 interlayer were observed around and beneath the Co NP, respectively. The observed soft wetting behavior strongly indicates that the sapphire substrate became soft and deformable during PLID. Moreover, the soft wetting was augmented under PLID in air due to the formation of a CoO shell, resulting in a smaller contact angle near 30°.
