12vu
The structure of the C145D variant of the COVID-2 main protease
Structural highlights
FunctionR1AB_SARS2 Multifunctional protein involved in the transcription and replication of viral RNAs. Contains the proteinases responsible for the cleavages of the polyprotein.[UniProtKB:P0C6X7] Inhibits host translation by interacting with the 40S ribosomal subunit. The nsp1-40S ribosome complex further induces an endonucleolytic cleavage near the 5'UTR of host mRNAs, targeting them for degradation. Viral mRNAs are not susceptible to nsp1-mediated endonucleolytic RNA cleavage thanks to the presence of a 5'-end leader sequence and are therefore protected from degradation. By suppressing host gene expression, nsp1 facilitates efficient viral gene expression in infected cells and evasion from host immune response.[UniProtKB:P0C6X7] May play a role in the modulation of host cell survival signaling pathway by interacting with host PHB and PHB2. Indeed, these two proteins play a role in maintaining the functional integrity of the mitochondria and protecting cells from various stresses.[UniProtKB:P0C6X7] Responsible for the cleavages located at the N-terminus of the replicase polyprotein. In addition, PL-PRO possesses a deubiquitinating/deISGylating activity and processes both 'Lys-48'- and 'Lys-63'-linked polyubiquitin chains from cellular substrates. Participates together with nsp4 in the assembly of virally-induced cytoplasmic double-membrane vesicles necessary for viral replication. Antagonizes innate immune induction of type I interferon by blocking the phosphorylation, dimerization and subsequent nuclear translocation of host IRF3. Prevents also host NF-kappa-B signaling.[UniProtKB:P0C6X7] Participates in the assembly of virally-induced cytoplasmic double-membrane vesicles necessary for viral replication.[UniProtKB:P0C6X7] Cleaves the C-terminus of replicase polyprotein at 11 sites. Recognizes substrates containing the core sequence [ILMVF]-Q-|-[SGACN] (PubMed:32198291). Also able to bind an ADP-ribose-1-phosphate (ADRP).[UniProtKB:P0C6X7][1] Plays a role in the initial induction of autophagosomes from host reticulum endoplasmic. Later, limits the expansion of these phagosomes that are no longer able to deliver viral components to lysosomes.[UniProtKB:P0C6X7] Forms a hexadecamer with nsp8 (8 subunits of each) that may participate in viral replication by acting as a primase. Alternatively, may synthesize substantially longer products than oligonucleotide primers.[UniProtKB:P0C6X7] Forms a hexadecamer with nsp7 (8 subunits of each) that may participate in viral replication by acting as a primase. Alternatively, may synthesize substantially longer products than oligonucleotide primers.[UniProtKB:P0C6X7] May participate in viral replication by acting as a ssRNA-binding protein.[UniProtKB:P0C6X7] Plays a pivotal role in viral transcription by stimulating both nsp14 3'-5' exoribonuclease and nsp16 2'-O-methyltransferase activities. Therefore plays an essential role in viral mRNAs cap methylation.[UniProtKB:P0C6X7] Responsible for replication and transcription of the viral RNA genome.[UniProtKB:P0C6X7] Multi-functional protein with a zinc-binding domain in N-terminus displaying RNA and DNA duplex-unwinding activities with 5' to 3' polarity. Activity of helicase is dependent on magnesium.[UniProtKB:P0C6X7] Enzyme possessing two different activities: an exoribonuclease activity acting on both ssRNA and dsRNA in a 3' to 5' direction and a N7-guanine methyltransferase activity. Acts as a proofreading exoribonuclease for RNA replication, thereby lowering The sensitivity of the virus to RNA mutagens.[UniProtKB:P0C6X7] Mn(2+)-dependent, uridylate-specific enzyme, which leaves 2'-3'-cyclic phosphates 5' to the cleaved bond.[UniProtKB:P0C6X7] Methyltransferase that mediates mRNA cap 2'-O-ribose methylation to the 5'-cap structure of viral mRNAs. N7-methyl guanosine cap is a prerequisite for binding of nsp16. Therefore plays an essential role in viral mRNAs cap methylation which is essential to evade immune system.[UniProtKB:P0C6X7] Publication Abstract from PubMedThe SARS-CoV-2 main protease (M(pro)) is essential for viral replication and functions as a homodimer, with dimerization being critical for catalytic activity. M(pro) contains an unusually high number of cysteine residues. Among these, C117 and the catalytic nucleophile C145 can form a reversible disulfide bond under oxidative conditions. To investigate the structural and functional consequences of irreversible oxidation of these residues, we generated oxidation mimics by substituting these residues with aspartate (C117D and C145D), to mimic the sulfinic acid oxidation state. Kinetic assays revealed that both variants are catalytically inactive, with C117D exhibiting at least 100-fold lower activity than wild-type (WT) M(pro). Small-angle X-ray scattering (SAXS) and differential scanning fluorimetry (DSF) demonstrated that C117D adopts a monomeric, destabilized state in solution, whereas C145D retains a dimeric conformation similar to WT. Crystallographic analysis of C117D revealed a dramatic rearrangement of domain III, involving a ~40 degrees rotation relative to domains I and II, and disorder in the N- and C-terminal regions, disrupting the canonical dimerization interface. Local structural changes propagated from the C117D site to the active site, including an unwound oxyanion loop that provides structural evidence for the observed inactivity. Rescue of the stable, dimeric state for C117D was achieved through formation of the covalent C117D-GC376 complex. These findings establish a high-resolution structure of monomeric full-length SARS-CoV-2 M(pro) and underscore a critical role of C117 in maintaining dimerization and enzymatic function. Furthermore, the unique monomeric domain II-III interface present in the monomeric form may offer opportunities for allosteric inhibitor design targeting M(pro) dimerization. The C117D oxidation mimic reveals the monomeric structure of SARS-CoV-2 main protease.,Andress S, McLeod MJ, Holyoak T Protein Sci. 2026 Sep;35(9):e70753. doi: 10.1002/pro.70753. PMID:42572186[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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