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bioRxiv · 10.64898/2026.01.20.700672

Promiscuous Metal Site in Hepatitis B Virus X Protein Binds anFe-S Cluster

Chen, J.·Langton, M.·Cao, P.·Aaron, A.·Ho, J.·Weerapana, E.·Perlstein, D. L.·Silakov, A.·Bak, D. W.·Pandelia, M.-E.
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Abstract

The Hepatitis B virus (HBV) regulatory protein HBx is essential for viral replication and pathogenesis, yet its cofactor specificity and ligand environment remain poorly defined. Although HBx binds either an Fe-S cluster or Zn, its intrinsic disorder and mutational tolerance have hindered its precise characterization. Here, we integrate chemoproteomics with HYSCORE spectroscopy to identify the metal-coordinating ligands in HBx. Histidine coordination is excluded, while C61, C69, C143, and C148 emerge as primary cysteine ligands for the Fe-S cluster, with C137 acting as a conditional ligand. These residues also bind Zn and are associated with HBx transactivation and clinically relevant variants. HBx engages the host cytosolic Fe-S machinery and displays sensitivity to Fe-S-targeting reagents, behavior consistent with Fe-S cluster acquisition and lability. Together, these findings suggest that HBx functionally behaves as an Fe-S cluster-associated protein, highlighting a potentially druggable vulnerability in HBV replication. Significance StatementFe-S clusters are emerging as key cofactors in viral replication but are often mistaken for Zn due to O2-sensitivity and shared cysteine coordination. The Hepatitis B virus HBx protein, essential for viral replication and hepatocarcinogenesis, has long been mechanistically intractable, with debate over its metallocofactor. Here, we provide evidence that HBx coordinates an Fe-S cluster, placing it within the growing family of viral Fe-S-cluster-containing proteins. Using chemoproteomics, we identify its cysteine ligands, overcoming limitations of mutational analysis in disordered proteins. Although HBx binds both an Fe-S cluster and Zn, its interaction with human Fe-S assembly factors suggests a functional link to Fe-S cluster biology, while its sensitivity to TEMPOL and NO reveals a potentially druggable vulnerabilityin HBx.

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Chen, J., Langton, M., Cao, P., Aaron, A., Ho, J., Weerapana, E., Perlstein, D. L., Silakov, A., Bak, D. W., Pandelia, M.-E.. 2026-01-21. Promiscuous Metal Site in Hepatitis B Virus X Protein Binds anFe-S Cluster. https://doi.org/10.64898/2026.01.20.700672

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