Structural Insights into Plasticity and Discovery of Remdesivir Metabolite GS-441524 Binding in SARS-CoV-2 Macrodomain.

The nsP3 macrodomain is a conserved protein interaction module that plays essential regulatory roles in the host immune response by recognizing and removing posttranslational ADP-ribosylation sites during SARS-CoV-2 infection. Thus targeting this protein domain may offer a therapeutic strategy to combat current and future virus pandemics. To assist inhibitor development efforts, we report here a comprehensive set of macrodomain crystal structures complexed with diverse naturally occurring nucleotides, small molecules, and nucleotide analogues including GS-441524 and its phosphorylated analogue, active metabolites of remdesivir. The presented data strengthen our understanding of the SARS-CoV-2 macrodomain structural plasticity and provide chemical starting points for future inhibitor development.

Crystal structure of SARS-CoV-2 Orf9b in complex with human TOM70 suggests unusual virus-host interactions.

Although the accessory proteins are considered non-essential for coronavirus replication, accumulating evidences demonstrate they are critical to virus-host interaction and pathogenesis. Orf9b is a unique accessory protein of SARS-CoV-2 and SARS-CoV. It is implicated in immune evasion by targeting mitochondria, where it associates with the versatile adapter TOM70. Here, we determined the crystal structure of SARS-CoV-2 orf9b in complex with the cytosolic segment of human TOM70 to 2.2 Å. A central portion of orf9b occupies the deep pocket in the TOM70 C-terminal domain (CTD) and adopts a helical conformation strikingly different from the ß-sheet-rich structure of the orf9b homodimer. Interactions between orf9b and TOM70 CTD are primarily hydrophobic and distinct from the electrostatic interaction between the heat shock protein 90 (Hsp90) EEVD motif and the TOM70 N-terminal domain (NTD). Using isothermal titration calorimetry (ITC), we demonstrated that the orf9b dimer does not bind TOM70, but a synthetic peptide harboring a segment of orf9b (denoted C-peptide) binds TOM70 with nanomolar KD. While the interaction between C-peptide and TOM70 CTD is an endothermic process, the interaction between Hsp90 EEVD and TOM70 NTD is exothermic, which underscores the distinct binding mechanisms at NTD and CTD pockets. Strikingly, the binding affinity of Hsp90 EEVD motif to TOM70 NTD is reduced by ~29-fold when orf9b occupies the pocket of TOM70 CTD, supporting the hypothesis that orf9b allosterically inhibits the Hsp90/TOM70 interaction. Our findings shed light on the mechanism underlying SARS-CoV-2 orf9b mediated suppression of interferon responses.