Promiscuous Metal Site in Hepatitis B Virus X Protein Binds an Fe–S Cluster

Abstract Fe–S clusters are emerging as important cofactors in viral replication but are frequently misassigned as Zn due to their O2-sensitivity and overlapping cysteine-based coordination chemistry. The Hepatitis B virus regulatory protein HBx, which is essential for viral replication and hepatocarcinogenesis, has long remained mechanistically intractable because of uncertainty surrounding its physiologically relevant metallocofactor. Although HBx can bind either an Fe–S cluster or Zn, its intrinsic disorder and extensive mutational tolerance have hindered precise characterization of its metal-binding environment. Here, we combine chemoproteomics with HYSCORE spectroscopy to define the metal-coordinating ligands in HBx and overcome limitations associated with conventional mutational analysis of disordered proteins. We exclude histidine coordination and identify C61, C69, C143, and C148 as the primary cysteine ligands for Fe–S cluster binding, with C137 functioning as a conditional auxiliary ligand. These residues also support Zn binding and overlap with regions implicated in HBx transactivation and clinically relevant variants. In addition, HBx engages the host cytosolic Fe–S cluster assembly machinery and displays sensitivity to Fe–S-targeting reagents, behavior consistent with Fe–S cluster acquisition and cofactor lability. Together, these findings propose HBx as an Fe–S cluster-associated viral protein and expand the growing class of viral Fe–S proteins that are critical for infection.