Abstract 2062 Biophysical basis of interaction of ORF9b of SARS-CoV-2 with human Tom70

SARS-CoV-2 caused the Covid-19 pandemic and continues to pose a major global health challenge. Despite many studies, the mechanistic basis of how SARS-CoV-2 evades the host immune response remains elusive. Open reading frame 9b (ORF9b), an accessory protein of SARS-CoV-2, is a key antagonist of the host innate immune system. ORF9b binds to the translocase of mitochondrial outer membrane 70 (Tom70), a vital adapter protein mediating the activation of host interferon expression upon viral infection. ORF9b is a metamorphic protein that exists as a β-sheet rich dimer but transitions to a α-helical conformation upon binding to Tom70. Using a combination of biophysical studies such as bio-layer interferometry (BLI) and size exclusion chromatography-multi angle light scattering (SEC-MALS), we revealed that only the ORF9b monomer, not the dimer, can bind to Tom70. We also found that the high affinity of ORF9b to Tom70 is due to the slow dissociation rate constant of the complex. We characterized the dynamic structural transition of ORF9b using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. We observed that ORF9b undergoes a conformational switch from a random coil to a α-helical conformation in the free state, prior to its binding to Tom70. Consequently, we elucidated the mechanistic basis of the dynamic metamorphism of ORF9b and its role in the hijacking of Tom70. The findings of this study provide key insights into the immune evasion strategy employed by SARS-CoV-2. This work is supported by the Welch Foundation (Grant No. A-2028-20230405).