Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD, 20892-0520, USA.
Neutron Scattering Division, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN, 37831, USA.
Commun Biol. 2022 Sep 16;5(1):976. doi: 10.1038/s42003-022-03910-y.
The monomeric catalytic domain (residues 1-199) of SARS-CoV-2 main protease (MPro) fused to 25 amino acids of its flanking nsp4 region mediates its autoprocessing at the nsp4-MPro junction. We report the catalytic activity and the dissociation constants of MPro and its analogs with the covalent inhibitors GC373 and nirmatrelvir (NMV), and the estimated monomer-dimer equilibrium constants of these complexes. Mass spectrometry indicates the presence of the accumulated adduct of NMV bound to MPro and MPro and not of GC373. A room temperature crystal structure reveals a native-like fold of the catalytic domain with an unwound oxyanion loop (E state). In contrast, the structure of a covalent complex of the catalytic domain-GC373 or NMV shows an oxyanion loop conformation (E* state) resembling the full-length mature dimer. These results suggest that the E-E* equilibrium modulates autoprocessing of the main protease when converting from a monomeric polyprotein precursor to the mature dimer.
SARS-CoV-2 主蛋白酶（MPro）的单体催化结构域（残基 1-199）与侧翼 nsp4 区域的 25 个氨基酸融合，介导 nsp4-MPro 连接处的自身加工。我们报告了 MPro 及其类似物与共价抑制剂 GC373 和 nirmatrelvir（NMV）的催化活性和离解常数，以及这些复合物的估计单体-二聚体平衡常数。质谱分析表明存在与 MPro 和 MPro 结合的 NMV 积累加合物，而不存在 GC373。室温晶体结构揭示了催化结构域的天然样折叠，带有展开的氧阴离子环（E 态）。相比之下，催化结构域-GC373 或 NMV 的共价复合物结构显示出氧阴离子环构象（E态）类似于全长成熟二聚体。这些结果表明，E-E平衡在从单体多蛋白前体转化为成熟二聚体时调节主蛋白酶的自身加工。
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