Abstract
Iron-sulfur (Fe-S) clusters and Zn are ubiquitous metal cofactors across all domains of life, including viruses. While Fe-S clusters can serve a structural role that stabilizes protein folding and are uniquely competent for redox chemistry, supporting electron transfer, catalytic, and redox-sensing, they are highly oxygen-sensitive and prone to degradation. As a result, Fe-S clusters are frequently replaced by the redox-inert Zn due to their shared coordination geometry with cysteine and histidine residues. Recently, a growing number of proteins involved in viral and bacterial infection have been found to harbor an Fe-S cluster rather than Zn as their bona fide cofactor, despite being initially misannotated as Zn-binding proteins. Therefore, defining the authentic metallocofactor and its metal-binding site in these pathogen proteins is a critical step toward understanding infection mechanisms and identifying new therapeutic vulnerabilities. This dissertation addresses the metallobiology of infection by characterizing Fe-S clusters and Zn coordination in three representative pathogen proteins. In Chapter 2, we examine the hepatitis B virus (HBV) HBx protein, which is essential for viral replication and hepatocarcinogenesis. By integrating chemoproteomics with spectroscopic and mutagenesis studies, we reveal that Fe-S and Zn bind competitively at a common tetracysteine site, with these coordinating cysteines associated with HBx transactivation, indicating a possible functional role for the Fe-S cluster in viral replication. We further establish that HBx is an authentic Fe-S cluster-containing protein by demonstrating its interaction with human cytosolic Fe-S assembly factors, with its sensitivity to Fe-S-targeting reagents points to a potential antiviral vulnerability in HBV infection. In Chapter 3, we extend this characterization to the papain-like cysteine protease (PCP) domain of the Hepatitis E virus ORF1 polyprotein, which is also required for viral replication. Mössbauer and UV-Vis spectroscopy combined with mutagenesis show that PCP binds an Fe-S cluster. The Fe-S cluster is ligated by four conserved cysteine residues, each of which is important for viral replication, challenging the prevailing hexacysteine metal-binding motif in PCP and aligning with the emerging role of Fe-S cofactors in viral replication. Finally, in Chapter 4, we studied the Mycobacterium tuberculosis sulfide transfer protein SufU, uncovering a regulatory mechanism in which Fe-S cluster binding competes with Zn at the same site to drive cluster-dependent tetramerization of SufU, inhibiting activation of the cysteine desulfurase SufS and thereby limiting sulfide supply for Fe-S cluster biogenesis in bacteria.
Together, these studies highlight an increasingly recognized theme in which Fe-S clusters play a vital role in viral and bacterial pathogen proteins. This work reveals an underexplored connection between metallobiology and infectious disease, providing new insight into metallocofactor coordination and infection mechanisms and identifying the Fe-S cluster as a potentially druggable vulnerability.