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.

Iron-sulfur (Fe-S) clusters are essential cofactors required for diverse cellular processes, yet how the Fe-S cluster biogenesis machinery selectively recognizes apo-client proteins remain poorly understood. In eukaryotes, many cytosolic and nuclear Fe-S proteins are recruited to the cytosolic iron-sulfur cluster assembly (CIA) system through a short C-terminal targeting complex recognition (TCR) motif having a [ILM]-[DES]-FW] consensus. Currently, the physicochemical properties underlying this molecular recognition event are undefined. By combining quantitative binding measurements, bioinformatic analysis, and structural modeling, we define the molecular basis for TCR peptide recognition by the CIA targeting complex (CTC). This systematic energetic dissection reveals a hierarchy of binding determinants, in which the side chain and C-terminal carboxylate of the aromatic residue provide the dominant energetic contributor, whereas the upstream residues modulate affinity in a sequence context-dependent manner. Computational docking and molecular dynamics simulations identify an interfacial binding site at the Cia1-Cia2 interface that can accommodate these TCR moieties complementary interaction surfaces. Mutational analysis the identified interaction site is consistent with an aromatic pocket and an adjacent hydrophobic groove on Cia2 accommodating the TCR's terminal aromatic and antepenultimate aliphatic residues. Together, these results reveal the physicochemical decoding grammar by which the CTC recognizes targeting peptides with divergent sequences, illustrating how short targeting motifs can achieve both the specificity and adaptability required for Fe-S protein maturation.

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.

The cytosolic iron-sulfur cluster assembly (CIA) pathway maturates essential nuclear and cytosolic Fe-S proteins required for genome maintenance and cellular metabolism. Nar1 (also called CIAO3 or IOP1) is a conserved Fe-S protein that connects the early and late steps of the CIA pathway, yet the molecular basis for its proposed function as a metallocluster carrier remains poorly defined. In particular, the interactions responsible for Nar1 recruitment to the CIA targeting complex (CTC) during cluster delivery remain unknown. Here, we define the molecular basis for Nar1 recruitment to the CTC using biochemical reconstitution, quantitative protein-protein interaction assays, and AlphaFold modeling. Our data reveal that Nar1 binds the CTC through two distinct interfaces. A primary interface comprises an electrostatic interaction that anchors Nar1 to a conserved acidic surface on the Cia1 subunit of the CTC and a secondary interface involves binding of Nar1's divergent targeting complex recognition peptide at the Cia1-Cia2 interface. Thus, Nar1 engages a conserved CTC surface that serves as a recruitment platform for multiple binding partners, including CIA clients. Computational structural models position the putative Fe-S cluster donor site of Nar1 adjacent to a proposed acceptor site on Cia2, suggesting that this bipartite binding mechanism positions Nar1 for potential transfer of an Fe-S cluster to the targeting complex. Together, these findings resolve conflicting models for Nar1 recruitment and establish a mechanistic framework for understanding how the CTC engages multiple binding partners during cytosolic iron-sulfur protein maturation.

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.

The cytosolic iron-sulfur cluster assembly (CIA) targeting complex is responsible for maturation of cytosolic and nuclear iron-sulfur enzymes, numbering >30 proteins critical for fundamental processes such as DNA replication and repair. Up to 25% of these client proteins terminate in a targeting complex recognition (TCR) motif. This carboxy-terminal tripeptide motif recruits the CIA targeting complex (CTC) to the client so that the metallocluster can be inserted. Herein, we use a combination of computational, biochemical and biophysical approaches to determine that the clients bearing a TCR motif docks at the interface of the Cia1 and Cia2 subunits of the CTC. Thus, mutations destabilizing the Cia1-Cia2 complex also disrupt TCR-based client identification by the CTC. Our study also reveals that the understudied human Cia2 paralog CIAO2A, which is proposed to be a specific targeting factor for iron regulatory protein 1, can recruit clients terminating in the TCR peptide. These data signal that CIAO2A plays a more general role in iron-sulfur protein maturation than previously appreciated. Taken together, our findings deepen our understanding of the molecular basis for client recognition by the CTC that is critical to understand the impact of CIA function in human health and disease.

Machine learning (ML) is rapidly gaining traction in many areas of experimental molecular science for elucidating relationships and patterns in large or complex data sets. Historically, ML was largely the preserve of those with specialized training in fields such as statistics or cheminformatics. Increasingly, however, ML methodologies are becoming part of the standard toolkit for experimental scientists across a range of disciplines. For scientists without a significant background in computer science or statistics, lowering the barrier of entry to these ML techniques is important to broadening access to these powerful methods. Here we provide detailed, step‐by‐step protocols for performing four ML methods that are particularly useful for applications in biochemistry, cell biology, and drug discovery: hierarchical clustering, principal component analysis (PCA), partial least squares discriminant analysis (PLSDA), and partial least squares regression (PLSR). The protocols are written for the widely used software MATLAB, but no prior experience with MATLAB is required to use them. We include an explanation of each step, pitched at a level to be understood by investigators without any prior experience with ML, MATLAB, or any kind of coding. We also highlight the scientific issues pertaining to selecting and scaling the data to be analyzed. Throughout, we emphasize the relationship between the scientific question and how to choose data and methods that will allow it to be addressed in a meaningful way. Our aim is to provide a basic introduction that will equip experimental chemical biologists, chemists, and other biomedical scientists with the knowledge required to use ML to aid in the design of experiments, the formulation and data‐driven testing of hypotheses, and the analysis of experimental data. © 2025 Wiley Periodicals LLC. Basic Protocol 1 : Clustering Basic Protocol 2 : Principal component analysis Basic Protocol 3 : Partial least squares‐discriminant analysis Basic Protocol 4 : Partial least squares regression

The cytosolic iron-sulfur cluster assembly (CIA) targeting complex maturates over 30 cytosolic and nuclear Fe-S proteins, raising the question of how a single complex recognizes such a diverse set of clients. The discovery of a C-terminal targeting complex recognition (TCR) peptide in up to 25% of CIA clients provided a clue to substrate specificity, yet the molecular and energetic basis for this interaction remained unresolved. By integrating computational and biochemical approaches, we show that the TCR peptide binds a conserved interface between the Cia1 and Cia2 subunits of the targeting complex, even in the absence of the Fe-S cluster. Since this same site also mediates binding of predominantly apo-Nar1, the proposed Fe-S cluster carrier, we provide in vitro evidence for Nar1's role as a cluster trafficking protein in the CIA pathway. We further show that Cia1-Cia2 complex formation is essential for CIA function as substitutions disrupting this interface, including the disease-linked R65W Cia1 variant, impair TCR peptide-dependent client recruitment. Our findings also clarify the role of the poorly characterized human paralog Cia2a, proposed to act solely in iron regulatory protein 1 (IRP1) maturation. We find that a Cia1-Cia2a complex can bind the TCR peptide, suggesting a broader role for Cia2a in Fe-S protein biogenesis, as IRP1 lacks a TCR motif. Together, these findings define a well-conserved molecular mechanism for client recognition in the CIA pathway and uncover how CIA targeting complex assembly and client identification are mechanistically linked to human disease.

Iron-sulfur clusters are essential for life and defects in their biosynthesis lead to human diseases. The mechanism of cluster assembly and delivery to cytosolic and nuclear client proteins via the cytosolic iron-sulfur cluster assembly (CIA) pathway is not well understood. Here we report cryo-EM structures of the HEAT-repeat protein Met18 from Saccharomyces cerevisiae , a key component of the CIA targeting complex (CTC) that identifies cytosolic and nuclear client proteins and delivers a mature iron-sulfur cluster. We find that in the absence of other CTC proteins, Met18 adopts tetrameric and hexameric states. Using mass photometry and negative stain EM, we show that upon the addition of Cia2, these higher order oligomeric states of Met18 disassemble. We also use pulldown assays to identify residues of critical importance for Cia2 binding and recognition of the Leu1 client, many of which are buried when Met18 oligomerizes. Our structures show conformations of Met18 that have not been previously observed in any Met18 homolog, lending support to the idea that a highly flexible Met18 may be key to how the CTC is able to deliver iron-sulfur clusters to client proteins of various sizes and shapes, i.e. Met18 conforms to the dimensions needed.

The eukaryotic cytosolic Fe-S protein assembly (CIA) machinery inserts iron-sulfur (Fe-S) clusters into cytosolic and nuclear proteins. In the final maturation step, the Fe-S cluster is transferred to the apo-proteins by the CIA-targeting complex (CTC). However, the molecular recognition determinants of client proteins are unknown. We show that a conserved [LIM]-[DES]-[WF]-COO - tripeptide present at the C-terminus of clients is necessary and sufficient for binding to the CTC in vitro and directing Fe-S cluster delivery in vivo . Remarkably, fusion of this TCR (target complex recognition) signal enables engineering of cluster maturation on a non-native protein via recruitment of the CIA machinery. Our study significantly advances our understanding of Fe-S protein maturation and paves the way for bioengineering applications. One-Sentence Summary A C-terminal tripeptide guides eukaryotic iron-sulfur cluster insertion into cytosolic and nuclear proteins.

Iron-sulfur ([Fe-S]) clusters are essential cofactors for sustaining life. Because [Fe-S] clusters play a role in a wide range of cellular processes, defects in their biosynthesis, trafficking, and incorporation into target proteins lead to human diseases. The molecular details of how iron-sulfur clusters are assembled and transferred to their target proteins are not well understood. Therefore, the goal of this work is to structurally and biophysically characterize proteins involved in the Cytosolic Iron-Sulfur Cluster Assembly (CIA) pathway. One of these proteins, Met18, is in the CIA targeting complex, and this complex is believed to receive a mature [4Fe-4S] cluster and transfer it to target proteins downstream. To better understand its function, we determined a 3.3 Å resolution cryo-EM structure of Met18. In the absence of other CIA targeting complex proteins and target proteins, we observed that Met18 forms a hexamer with its N-terminus exposed and its C-terminus buried. Conserved sequences within Met18 assist in forming the hexamer. Upon the addition of Cia2, a CIA targeting complex protein, the hexamer appears to be broken up. We hypothesize that the hexamer is a storage form of Met18 that protects itself from ubiquitination and proteosomal degradation. Here we present the cryo-EM structure determination of the Met18 hexamer.

Complex biosynthetic pathways are required for the assembly and insertion of iron-sulfur (Fe-S) cluster cofactors. Each of the four cluster biogenesis systems that have been discovered requires at least one ATPase. Generally, the function of nucleotide hydrolysis in Fe-S cluster biogenesis is understudied. For example, the cytosolic Fe-S cluster assembly (CIA) pathway is proposed to begin with a scaffold, which assembles nascent Fe-S clusters destined for cytosolic and nuclear enzymes. This scaffold, comprised of Nbp35 and Cfd1 in yeast, possesses an ATPase site that is necessary for CIA function, but the role of nucleotide hydrolysis is poorly understood. Herein, we describe the in vitro methods that have been developed to uncover how the ATPase site of the scaffold regulates interaction with one of its partner proteins, Dre2. We describe a qualitative affinity copurification assay and a quantitative assay for evaluating the dissociation constant for the scaffold-partner protein complex. Finally, we describe kinetic methods to measure the kcat and KM values for ATP hydrolysis by the scaffold-partner protein complex and the execution of the ATPase assays in an anaerobic environment. These methods could be applied to study other ATPases to advance our mechanistic understanding of nucleotide hydrolases involved in metallocluster biogenesis.

The cytosolic iron-sulfur cluster assembly (CIA) scaffold, comprising Nbp35 and Cfd1 in yeast, assembles iron-sulfur (FeS) clusters destined for cytosolic and nuclear enzymes. ATP hydrolysis by the CIA scaffold plays an essential but poorly understood role in cluster biogenesis. Here we find that mutation of conserved residues in the four motifs comprising the ATPase site of Nbp35 diminished the scaffold's ability to both assemble and transfer its FeS cluster in vivo. The mutants fall into four phenotypic classes that can be understood by how each set of mutations affects ATP binding and hydrolysis. In vitro studies additionally revealed that occupancy of the bridging FeS cluster binding site decreases the scaffold's affinity for the nucleotide. On the basis of our findings, we propose that nucleotide binding and hydrolysis by the CIA scaffold drive a series of protein conformational changes that regulate association with other proteins in the pathway and with its newly formed FeS cluster. Our results provide insight into how the ATPase and cluster scaffolding activities are allosterically integrated.

Immune checkpoint inhibitors, such as pembrolizumab, are transforming clinical oncology. Yet, insufficient overall response rate, and accelerated tumor growth rate in some patients, highlight the need for identifying potential responders. To construct a computational model, identifying response predictors, and enabling immunotherapy personalization. The combined dynamics of cellular immunity, pembrolizumab, and the melanoma cancer were modeled by a set of ordinary differential equations. The model relies on a scheme of T memory stem cells, progressively differentiating into effector CD8+ T cells, and additionally includes T cell exhaustion, reinvigoration and senescence. Clinical data of a pembrolizumab-treated patient with advanced melanoma (Patient O') were used for model calibration and simulations. Virtual patient populations, varying in one parameter or more, were generated for retrieving clinical studies. Simulations captured the major features of Patient O's disease, displaying a good fit to her clinical data. A temporary increase in tumor burden, as implied by the clinical data, was obtained only when assuming aberrant self-renewal rates. Variation in effector T cell cytotoxicity was sufficient for simulating dynamics that vary from rapid progression to complete cure, while variation in tumor immunogenicity has a delayed and limited effect on response. Simulations of a-specific clinical trial were in good agreement with the clinical results, demonstrating positive correlations between response to pembrolizumab and the ratio of reinvigoration to baseline tumor load. These results were obtained by assuming inter-patient variation in the toxicity of effector CD8+ T cells, and in their intrinsic division rate, as well as by assuming that the intrinsic division rate of cancer cells is correlated with the baseline tumor burden. In conclusion, hyperprogression can result from lower patient-specific effector cytotoxicity, a temporary increase in tumor load is unlikely to result from real tumor growth, and the ratio of reinvigoration to tumor load can predict personal response to pembrolizumab. Upon further validation, the model can serve for immunotherapy personalization.

The cytosolic iron sulfur cluster assembly (CIA) scaffold biosynthesizes iron sulfur cluster cofactors for enzymes residing in the cytosol and the nucleus. In fungi and animals, it comprises two homologous ATPases, called Nbp35 and Cfd1 in yeast, which can form homodimeric and heterodimeric complexes. Both proteins are required for CIA function, but their individual roles are not well understood. Here we investigate the nucleotide affinity of each form of the scaffold for ATP and ADP to reveal any differences that could shed light on the functions of the different oligomeric forms of the protein or any distinct roles of the individual subunits. All forms of the CIA scaffold are specific for adenosine nucleotides and not guanosine nucleotides. Although the Cfd1 homodimer has no detectable ATPase activity, it binds ATP with an affinity comparable to that of the hydrolysis competent forms, Nbp352 and Nbp35-Cfd1. Titrations to determine the number of nucleotide binding sites combined with site-directed mutagenesis demonstrate that the nucleotide must bind to the Cfd1 subunit of the heterodimer before it can bind to Nbp35 and that the Cfd1 subunit is hydrolysis competent when bound to Nbp35 in the heterodimer. Altogether, our work reveals the distinct roles of the Nbp35 and Cfd1 subunits in their heterodimeric complex. Cfd1 controls nucleotide binding, and the Nbp35 subunit is required to activate nucleotide hydrolysis.

The Cytosolic Iron Sulfur Cluster Assembly (CIA) pathway is a highly conserved pathway that assembles and inserts iron sulfur (FeS) cluster cofactors into a variety of target proteins. These targets are involved in many cellular processes including DNA repair, iron homeostasis, and nucleotide metabolism. For CIA targets to receive the FeS cluster, they must be recruited for FeS cluster insertion by the CIA targeting complex. Although it is known that the targeting complex recognizes and binds to targets, the mechanisms of target recognition are not fully understood. In this work, mutagenesis and affinity co‐purification were utilized to elucidate the binding interface of Met18, a protein in the CIA targeting complex, and Rad3, a DNA helicase that receives an FeS cluster from the CIA pathway. Although this interaction has been investigated by other groups, there has been contradiction as to which portion of Rad3 binds to the targeting complex. Vashist et al. (J. Biol. Chem., 2012, 287, 43351) reported that the N‐terminus of Rad3 is responsible for binding to the targeting complex, while Ito et al. (Mol. Cell., 2010, 39, 632) reported that the C‐terminus of Rad3 is responsible for binding. To help resolve this discrepancy and gain insight into the targeting mechanism of the CIA targeting complex, truncated versions of Rad3 were expressed and isolated, and it was found that the N‐terminal region of the protein is responsible for binding to the targeting complex. Our current work involves investigating a 10 amino acid N‐terminal region of Rad3 identified by Vashist et al. Their group identified this region as participating in binding to Met18. In this work, we split the region into multiple alanine scans to further narrow down the Rad3‐Met18 binding interface. The identification of a region involved in Rad3‐Met18 binding could help define a target recognition motif for the CIA targeting complex.Support or Funding InformationBU Undergraduate Research Opportunities Program; NIH R01 GM121673This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Iron‐sulfur (FeS) clusters are essential protein cofactors required for numerous biological functions including iron regulation, DNA synthesis and DNA repair. The Cytosolic Iron‐Sulfur Cluster Assembly (CIA) pathway is responsible for the metallocofactor assembly of extra mitochondrial FeS proteins. The CIA pathway culminates with the targeting complex, which contains the proteins Met18, Cia1 and Cia2. This complex is crucial for the last step in the CIA pathway, which is the recognition of apo‐FeS targets and insertion of their cofactors. To begin understanding the structure of this complex, we wanted to identify the residues responsible for the complex formation. We began by identifying the clusters of conserved residues required for the formation of the Cia1‐Cia2 sub‐complex since both of these proteins are essential for yeast viability, whereas the Met18 subunit is not. We alanine scanned these residues, then determined how the mutation of these residues affect Cia1‐Cia2 complex formation and the ability to support viability via genetic complementation assays. If the interactions are important in vitro, then there could be phenotypic changes in yeast cells that can alter CIA function. Our lab has previously determined that Glutamate208 within one of Cia2's conserved motifs was vital for Cia2's ability to bind to Cia1. Therefore, we scanned the surrounding conserved residues to identify additional residues important for Cia1‐Cia2 interaction. Several residues close to E208 also disrupted the Cia1‐Cia2 interaction in vitro, confirming that this conserved motif in Cia2 is the docking site for Cia1. Previously, E208A supported yeast viability, whereas, an adjacent residue, Aspartate 206 (D206), appears to be particularly important as it was the only one that failed to support viability in the complementation assay. This shows that this residue does not allow for the targeting complex to form and identify targets, negatively impacting CIA function. Now that we understand which residues are required for Cia1‐Cia2 binding, this allows for further investigation of which specific targets the complex binds to for proper CIA function.Support or Funding InformationBoston University Undergraduate Research Opportunities Program (UROP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nucleotide hydrolases play integral yet poorly understood roles in several metallocluster biosynthetic pathways. For example, the cytosolic iron-sulfur cluster assembly (CIA) is initiated by the CIA scaffold, an ATPase which builds new iron-sulfur clusters for proteins localized to the cytosol and the nucleus in eukaryotic organisms. While in vivo studies have demonstrated the scaffold's nucleotide hydrolase domain is vital for its function, in vitro approaches have not revealed tight allosteric coupling between the cluster scaffolding site and the ATPase site. Thus, the role of ATP hydrolysis has been hard to pinpoint. Herein, we describe methods to probe the nucleotide affinity and hydrolysis activity of the CIA scaffold from yeast, which is comprised of two homologous polypeptides called Nbp35 and Cfd1. In particular, we report two different equilibrium binding assays that make use of commercially available fluorescent nucleotide analogs. Importantly, these assays can be applied to probe nucleotide affinity of both the apo-and holo-forms of the CIA scaffold. Generally, these fluorescent nucleotide analogs have been underutilized to probe metal trafficking NTPase because one of the most commonly used probes, mantATP, which is labeled with the methylanthraniloyl probe via the 20 or 30 sugar hydroxyls, has an absorption which overlaps with the UV-Vis features of many metal-binding proteins. However, by exploiting analogs like BODIPY-FL and trinitrophenyl-labeled nucleotides which have better photophysical properties for metalloprotein applications, these approaches have the potential to reveal the mechanistic underpinnings of NTPases required for metallocluster biosynthesis.

The cytosolic iron-sulfur cluster assembly (CIA) system assembles iron-sulfur (FeS) cluster cofactors and inserts them into >20 apoprotein targets residing in the cytosol and nucleus. Three CIA proteins, called Cia1, Cia2, and Met18 in yeast, form the targeting complex responsible for apo-target recognition. There is little information about the structure of this complex or its mechanism of CIA substrate recognition. Herein, we exploit affinity co-purification and size exclusion chromatography to determine the subunit connectivity and stoichiometry of the CIA targeting complex. We conclude that Cia2 is the organizing center of the targeting complex, which contains one Met18, two Cia1, and four Cia2 polypeptides. To probe target recognition specificity, we utilize the CIA substrates Leu1 and Rad3 as well as the Escherichia coli FeS-binding transcription factor FNR (fumerate nitrate reductase). We demonstrate that both of the yeast CIA substrates are recognized, whereas the bacterial protein is not. Thus, while the targeting complex exhibits flexible target recognition in vitro, it cannot promiscuously recognize any FeS protein. Additionally, we demonstrate that the full CIA targeting complex is required to stably bind Leu1 in vitro, whereas the Met18-Cia2 subcomplex is sufficient to recognize Rad3. Together, these results allow us to propose a unifying model for the architecture of this highly conserved complex and demonstrate what component or subcomplexes are vital for target identification.

The cytosolic iron-sulfur cluster assembly (CIA) system biosynthesizes iron-sulfur (FeS) cluster cofactors for cytosolic and nuclear proteins. The yeast Cia2 protein is the central component of the targeting complex which identifies apo-protein targets in the final step of the pathway. Herein, we determine that Cia2 contains five conserved motifs distributed between an intrinsically disordered N-terminal domain and a C-terminal domain of unknown function 59 (DUF59). The disordered domain is dispensible for binding the other subunits of the targeting complex, Met18 and Cia1, and the apo-target Rad3 in vitro. While in vivo assays reveal that the C-terminal domain is sufficient to support viability, several phenotypic assays indicate that deletion of the N-terminal domain negatively impacts CIA function. We additionally establish that Glu208, located within a conserved motif found only in eukaryotic DUF59 proteins, is important for the Cia1-Cia2 interaction in vitro. In vivo, E208A-Cia2 results in a diminished activity of the cytosolic iron sulfur cluster protein, Leu1 but only modest effects on hydroxyurea or methylmethane sulfonate sensitivity. Finally, we demonstrate that neither of the two highly conserved motifs of the DUF59 domain are vital for any of Cia2's interactions in vitro yet mutation of the DPE motif in the DUF59 domain results in a nonfunctional allele in vivo. Our observation that four of the five highly conserved motifs of Cia2 are dispensable for targeting complex formation and apo-target binding suggests that Cia2 is not simply a protein-protein interaction mediator but it likely possesses an additional, currently cryptic, function during the final cluster insertion step of CIA.