Maria-Eirini Pandelia

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.

The last step in the biosynthesis of the lipoyl cofactor (LipCo) is the addition of two sulfur atoms at C6 and C8 of an n-octanoyl chain attached by an amide linkage to a target lysyl residue of a lipoyl carrier protein. This reaction is catalyzed by lipoyl synthase, a member of the radical S-adenosylmethionine (SAM) superfamily. Lipoyl synthase requires two [4Fe-4S] clusters. One cluster is used to cleave SAM reductively to generate two 5′-deoxyadenosyl 5′-radicals (5′-dA•), which abstract the C6 and C8 hydrogen atoms (H•) of the substrate in two sequential steps. The second cluster, termed the auxiliary cluster, is consumed during turnover to provide the attached sulfur atoms. The auxiliary cluster is ligated by three cysteines in a CX4CX5C motif and one serine residue (Ser308 in Escherichia coli) in a highly conserved R306SS308Y motif in the C-terminal region of the protein. Here, we show that Arg306 and Ser308 are absolutely required for LipCo formation. Substitution of Arg306 with Lys results in an essentially inactive protein due to poor substrate binding and positioning in the active site. Multiple substitutions of Ser308 were engineered. Most notably, the S308C and S308A variants greatly diminished LipCo formation. However, the S308C variant resulted in greater production of the 6-mercaptooctanoyl intermediate and the formation of a desaturated product, identified as a 6-octenoyl group. Furthermore, the 3Fe cluster formed during degradation of the auxiliary cluster in the course of C6 sulfur substitution in the wild-type reaction is not observed with the S308C variant. Instead, the auxiliary cluster remains tetranuclear and forms a monothiolated cross-linked species with a high-spin, S = 7/2 configuration that decays to the 6-octenoyl-containing product. Other amino acids in the RSSY motif were not essential for catalysis.

1. Iron-sulfur (Fe-S) cluster-containing proteins are the most ubiquitous and functionally versatile class of Fe-containing metalloproteins across all domains of life, including viruses [1–7]. Fe-S...

SAMHD1 is the lone human dNTP triphosphohydrolase and is linked to antiviral defense, nucleotide pool homeostasis, chemotherapy resistance, and the autoinflammatory Aicardi-Goutières syndrome. Although its substrate specificity and nucleotide-dependent oligomerization have been extensively studied, the identity and mechanistic roles of its metal cofactors remain poorly understood. Here, we integrate selective metal enrichment, spectroscopy, biochemical reconstitution, and enzyme kinetics to define the metal requirements underlying SAMHD1 activation and catalysis. We show that robust SAMHD1 activity is preferentially supported by transition metals and that the enzyme readily assembles multiple iron-containing dinuclear active sites in solution. Iron preferentially binds to one position of the bimetallic core and promotes recruitment of a second divalent metal required for catalysis. Although manganese can substitute for iron, it alters metal-binding equilibria and less efficiently supports dinuclear cofactor assembly, highlighting a specialized organizational role for iron. In contrast, the second site remains comparatively permissive and accommodates various divalent metal ions with distinct functional consequences. Mixed-metal active sites further retain catalytic activity across redox conditions that otherwise suppress activity in homodinuclear diiron configurations, suggesting that metal plasticity buffers SAMHD1 against oxidative inhibition. Transition metals additionally act as higher-affinity allosteric activators than Mg2+, revealing that metal identity contributes to both catalytic and regulatory layers of SAMHD1 function. Cumulatively, these findings redefine the metal requirements of SAMHD1 and establish a framework in which iron-dependent active site organization and mixed-metal flexibility cooperate to sustain dNTP hydrolysis under changing cellular environments and metal flux conditions.

Porcine reproductive and respiratory syndrome virus (PRRSV; Betaarterivirus suid ) is a major global threat to swine production, yet effective antiviral therapies are lacking. The leader protease Nsp1α is essential for viral replication and innate immune suppression, and its N-terminal zinc-finger (ZF) domain is critical for function, although its molecular role remains unclear. Here, we show that the ZF domain plays only a minor role in protease activity and that Nsp1α is largely inactive following release from the polyprotein. Using Mössbauer and UV/visible spectroscopy combined with chemoproteomics, we demonstrate that the ZF site binds not only Zn but also a [4Fe-4S] cluster. Notably, the Fe-S cluster, but not Zn, allosterically modulates residual protease activity. Nsp1α directly engages the cytosolic iron-sulfur cluster assembly machinery via CIAO1 and competes with the Fe-S carrier CIAO3, establishing the [4Fe-4S] cluster as a bona fide cofactor. These findings redefine Nsp1α as an Fe-S–dependent viral protein and reveal previously unidentified opportunities for metal-targeted antiviral strategies.

The last step in the biosynthesis of the lipoyl cofactor (LipCo) is the addition of two sulfur atoms at C6 and C8 of an n-octanoyl chain attached in an amide linkage to a target lysyl residue of a lipoyl carrier protein. The enzyme that catalyzes this reaction, lipoyl synthase (LipA in bacteria, and LIAS in humans), is a member of the radical S-adenosylmethionine (SAM) superfamily. As such, it requires a [4Fe-4S] cluster cofactor to cleave SAM reductively to generate two 5′-deoxyadenosyl 5′-radicals (5′-dA•) that abstract the C6 and C8 hydrogen atoms (H•) of the substrate in two distinct steps. LipAs also contain a second [4Fe-4S] cluster, termed the auxiliary cluster, degraded during turnover as the source of the attached sulfur atoms. The auxiliary cluster is ligated by three cysteines in a CX₄CX₅C motif and one serine residue (Ser308 in Escherichia coli) in a highly conserved R³⁰⁶SS³⁰⁸Y motif in the C-terminal region of the protein. Here, we show that Arg306 and Ser308 are absolutely required for LipCo formation. Substitution of Arg306 with Lys results in a protein that is essentially inactive due to poor substrate binding and positioning in the active site. Multiple different substitutions of Ser308 were engineered. Most notable were the S308C and S308A variants, which gave greatly diminished LipCo formation. However, the S308C variant resulted in greater production of the 6-mercaptooctanoyl peptide, an intermediate in the reaction, and the formation of a desaturated product, determined to be a 6-octenoyl group attached to the peptide substrate. Furthermore, the 3Fe cluster formed during cannibalization of the auxiliary cluster during C6 sulfur substitution in the wild-type reaction is not observed with the S308C variant. Instead, the auxiliary cluster remains tetranuclear and forms a monothiolated cross-linked species with a high-spin, S = 7/2, configuration that decays to the 6-octenoyl-containing product. Other amino acids in the RSSY motif were not essential for catalysis.

SAMHD1 is the lone human dNTP triphosphohydrolase and is intimately linked to HIV viral restriction, dNTP pool maintenance, resistance to chemotherapy, and the autoinflammatory Aicardi-Goutières syndrome. While its substrate promiscuity and nucleotide basis of activity have been extensively studied, the identity and mechanistic roles of its metal cofactors remain poorly defined. Here, we integrate elemental analysis, spectroscopy, protein cross-linking, and enzyme kinetics to elucidate the molecular mechanisms underlying metal-dependent activation and catalysis in SAMHD1. Our findings establish that transition metals are essential components of SAMHD1 function, highlight their overlooked role in allosteric regulation, and reveal a central role for iron in organizing the dinuclear active site. We show that iron is preferentially incorporated in one position of the bimetallic core, where it promotes recruitment of a second divalent metal ion required for activity. While manganese can substitute for iron, it alters the metal binding equilibria, highlighting the unique functional properties of iron. Notably, SAMHD1 exhibits metal cofactor promiscuity at the second metal site, accommodating diverse divalent metals with distinct effects on activity. Cumulatively, our findings establish iron as a core structural and functional determinant of SAMHD1 catalysis and reveal how transition metal selectivity and flexibility enable enzymatic activity across diverse cellular environments and metal flux conditions.

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 Statement: Fe-S clusters are emerging as key cofactors in viral replication but are often mistaken for Zn due to O ₂ -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.

Porcine reproductive and respiratory syndrome virus (PRRSV; Betaarterivirus suid ) is a major global threat to swine production, yet effective antiviral therapies are lacking. The leader protease Nsp1α is essential for viral replication and innate immune suppression, and its N-terminal zinc-finger (ZF) domain is critical for function, although its molecular role remains unclear. Here, we show that the ZF domain plays only a minor role in protease activity and that Nsp1α is largely inactive following release from the polyprotein. Using Mössbauer and UV/visible spectroscopy combined with chemoproteomics, we demonstrate that the ZF site binds not only Zn but also a [4Fe-4S] cluster. Notably, the Fe-S cluster, but not Zn, allosterically modulates residual protease activity. Nsp1α directly engages the cytosolic iron-sulfur cluster assembly machinery via CIAO1 and competes with the Fe-S carrier CIAO3, establishing the [4Fe-4S] cluster as a bona fide cofactor. These findings redefine Nsp1α as an Fe-S-dependent viral protein and reveal new opportunities for metal-targeted antiviral strategies.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in biological systems. Given their central role in bacterial metabolism and pathogenesis, the biogenesis of Fe-S clusters is tightly controlled. We reveal a feedback regulatory mechanism involving the sulfide producing SufS/SufU complex within the sulfur utilization (SUF) system of Mycobacterium tuberculosis , the bacterium that causes tuberculosis. In this mechanism, [2Fe-2S] clusters compete with zinc ions for binding to the sulfide transfer protein SufU. Cluster binding induces SufU tetramerization, which prevents its interaction with the cysteine desulfurase SufS, thereby inhibiting SufS activation and limiting sulfide supply for Fe-S cluster biogenesis. These findings uncover an unrecognized regulatory mechanism in M. tuberculosis , ensuring strict control of Fe-S cluster production.