Coupled equilibria of dimerization and lipid binding modulate SARS Cov 2 Orf9b interactions and interferon response

San Felipe, C.; Batra, J.; Muralidharan, M.; Malpotra, S.; Anand, D.; Bauer, R.; Verba, K. A.; Swaney, D. L.; Krogan, N. J.; Grabe, M.; Fraser, J. S.

doi:10.1101/2025.02.16.638509

bioRxiv · 10.1101/2025.02.16.638509

Coupled equilibria of dimerization and lipid binding modulate SARS Cov 2 Orf9b interactions and interferon response

San Felipe, C.·Batra, J.·Muralidharan, M.·Malpotra, S.·Anand, D.·Bauer, R.·Verba, K. A.·Swaney, D. L.·Krogan, N. J.·Grabe, M.·Fraser, J. S.

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Abstract

Open Reading Frame 9b (Orf9b), an accessory protein of SARS-CoV and -2, is involved in innate immune suppression through its binding to the mitochondrial receptor Translocase of Outer Membrane 70 (Tom70). Previous structural studies of Orf9b in isolation revealed a {beta}-sheet-rich homodimer, however, structures of Orf9b in complex with Tom70 revealed a monomeric helical fold. Here, we developed a biophysical model that quantifies how Orf9b switches between these conformations and binds to Tom70, a requirement for suppressing the type 1 interferon response. We used this model to characterize the effect of lipid binding and mutations in variants of concern to the Orf9b:Tom70 equilibrium. We found that the binding of a lipid to the Orf9b homodimer biases the Orf9b monomer:dimer equilibrium towards the dimer by reducing the dimer dissociation rate [~]100-fold. We also found that mutations in variants of concern can alter different microscopic rate constants without significantly affecting binding to Tom70. Together our results highlight how perturbations to different steps in these coupled equilibria can affect the apparent affinity of Orf9b to Tom70, with potential downstream implications for interferon signaling in coronavirus infection.

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San Felipe, C., Batra, J., Muralidharan, M., Malpotra, S., Anand, D., Bauer, R., Verba, K. A., Swaney, D. L., Krogan, N. J., Grabe, M., Fraser, J. S.. 2025-02-17. Coupled equilibria of dimerization and lipid binding modulate SARS Cov 2 Orf9b interactions and interferon response. https://doi.org/10.1101/2025.02.16.638509

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