Three-Dimensional Structure and Determinants of Stability of the Iron–Sulfur Cluster Scaffold Protein IscU from <i>Escherichia coli</i>

The highly conserved protein, IscU, serves as the scaffold for iron–sulfur cluster (ISC) assembly in the ISC system common to bacteria and eukaryotic mitochondria. The apo-form of IscU from <i>Escherichia coli</i> has been shown to populate two slowly interconverting conformational states: one structured (S) and one dynamically disordered (D). Furthermore, single-site amino acid substitutions have been shown to shift the equilibrium between the metamorphic states. Here, we report three-dimensional structural models derived from NMR spectroscopy for the S-state of wild-type (WT) apo-IscU, determined under conditions where the protein was 80% in the S-state and 20% in the D-state, and for the S-state of apo-IscU­(D39A), determined under conditions where the protein was ∼95% in the S-state. We have used these structures in interpreting the effects of single site amino acid substitutions that alter %S = (100 × [S])/([S] + [D]). These include different residues at the same site, %S: D39V > D39L > D39A > D39G ≈ WT, and alanine substitutions at different sites, %S: N90A > S107A ≈ E111A > WT. Hydrophobic residues at residue 39 appear to stabilize the S-state by decreasing the flexibility of the loops that contain the conserved cysteine residues. The alanine substitutions at positions 90, 107, and 111, on the other hand, stabilize the protein without affecting the loop dynamics. In general, the stability of the S-state correlates with the compactness and thermal stability of the variant

Nucleotide-Dependent Interactions within a Specialized Hsp70/Hsp40 Complex Involved in Fe–S Cluster Biogenesis

The structural mechanism by which Hsp70-type chaperones interact with Hsp40-type co-chaperones has been of great interest, yet still remains a matter of debate. Here, we used solution NMR spectroscopy to investigate the ATP-/ADP-dependent interactions between <i>Escherichia coli</i> HscA and HscB, the specialized Hsp70/Hsp40 molecular chaperones that mediate iron–sulfur cluster transfer. We observed that NMR signals assigned to amino acid residues in the J-domain and its “HPD” motif of HscB broadened severely upon the addition of ATP-bound HscA, but these signals were not similarly broadened by ADP-bound HscA or the isolated nucleotide binding domain of HscA complexed with either ATP or ADP. An HscB variant with an altered HPD motif, HscB­(H32A,P33A,D34A), failed to manifest WT-like NMR signal perturbations and also abolished WT-like stimulation of ATP hydrolysis by HscA. In addition, residues 153–171 in the C-terminal region of HscB exhibited NMR signal perturbations upon interaction with HscA, alone or complexed with ADP or ATP. These results demonstrate that the HPD motif in the J-domain of HscB directly interacts with ATP-bound HscA and suggest that a second, less nucleotide-dependent binding site for HscA resides in the C-terminal region of HscB

[2Fe-2S]-Ferredoxin Binds Directly to Cysteine Desulfurase and Supplies an Electron for Iron–Sulfur Cluster Assembly but Is Displaced by the Scaffold Protein or Bacterial Frataxin

Escherichia coli [2Fe-2S]-ferredoxin (Fdx) is encoded by the <i>isc</i> operon along with other proteins involved in the ‘house-keeping’ mechanism of iron–sulfur cluster biogenesis. Although it has been proposed that Fdx supplies electrons to reduce sulfane sulfur (S⁰) produced by the cysteine desulfurase (IscS) to sulfide (S^2–) as required for the assembly of Fe–S clusters on the scaffold protein (IscU), direct experimental evidence for the role of Fdx has been lacking. Here, we show that Fdx (in either oxidation state) interacts directly with IscS. The interaction face on Fdx was found to include residues close to its Fe–S cluster. In addition, C328 of IscS, the residue known to pick up sulfur from the active site of IscS and deliver it to the Cys residues of IscU, formed a disulfide bridge with Fdx in the presence of an oxidizing agent. Electrons from reduced Fdx were transferred to IscS only in the presence of l-cysteine, but not to the C328S variant. We found that Fdx, IscU, and CyaY (the bacterial frataxin) compete for overlapping binding sites on IscS. This mutual exclusion explains the mechanism by which CyaY inhibits Fe–S cluster biogenesis. These results (1) show that reduced Fdx supplies one electron to the IscS complex as S⁰ is produced by the enzymatic conversion of Cys to Ala and (2) explain the role of Fdx as a member of the <i>isc</i> operon

Role of IscX in Iron–Sulfur Cluster Biogenesis in Escherichia coli

The Escherichia coli <i>isc</i> operon encodes key proteins involved in the biosynthesis of iron–sulfur (Fe–S) clusters. Whereas extensive studies of most ISC proteins have revealed their functional properties, the role of IscX (also dubbed YfhJ), a small acidic protein encoded by the last gene in the operon, has remained in question. Previous studies showed that IscX binds iron ions and interacts with the cysteine desulfurase (IscS) and the scaffold protein for cluster assembly (IscU), and it has been proposed that IscX functions either as an iron supplier or a regulator of Fe–S cluster biogenesis. We have used a combination of NMR spectroscopy, small-angle X-ray scattering (SAXS), chemical cross-linking, and enzymatic assays to enlarge our understanding of the interactions of IscX with iron ions, IscU, and IscS. We used chemical shift perturbation to identify the binding interfaces of IscX and IscU in their complex. NMR studies showed that Fe²⁺ from added ferrous ammonium sulfate binds IscX much more avidly than does Fe³⁺ from added ferric ammonium citrate and that Fe²⁺ strengthens the interaction between IscX and IscU. We found that the addition of IscX to the IscU–IscS binary complex led to the formation of a ternary complex with reduced cysteine desulfurase activity, and we determined a low-resolution model for that complex from a combination of NMR and SAXS data. We postulate that the inhibition of cysteine desulfurase activity by IscX serves to reduce unproductive conversion of cysteine to alanine. By incorporating these new findings with results from prior studies, we propose a detailed mechanism for Fe–S cluster assembly in which IscX serves both as a donor of Fe²⁺ and as a regulator of cysteine desulfurase activity

The Specialized Hsp70 (HscA) Interdomain Linker Binds to Its Nucleotide-Binding Domain and Stimulates ATP Hydrolysis in Both <i>cis</i> and <i>trans</i> Configurations

Proteins from the <i>isc</i> operon of <i>Escherichia coli</i> constitute the machinery used to synthesize iron–sulfur (Fe–S) clusters for delivery to recipient apoproteins. Efficient and rapid [2Fe-2S] cluster transfer from the holo-scaffold protein IscU depends on ATP hydrolysis in the nucleotide-binding domain (NBD) of HscA, a specialized Hsp70-type molecular chaperone with low intrinsic ATPase activity (0.02 min⁻¹ at 25 °C, henceforth reported in units of min^–1). HscB, an Hsp40-type cochaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer; however, while the interactions between HscA and HscB have been investigated, the role of HscA’s interdomain linker in modulating ATPase activity has not been explored. To address this issue, we created three variants of the 40 kDa NBD of HscA: NBD alone (HscA₃₈₆), NBD with a partial linker (HscA₃₈₉), and NBD with the full linker (HscA₃₉₅). We found that the rate of ATP hydrolysis of HscA₃₉₅ (0.45 min^–1) is nearly 15-fold higher than that of HscA₃₈₆ (0.035 min^–1), although their apparent affinities for ATP are equivalent. HscA₃₉₅, which contains the full covalently linked linker peptide, exhibited intrinsic tryptophan fluorescence and basal thermostability that were higher than those of HscA₃₈₆. Furthermore, HscA₃₉₅ displayed narrower ¹H^N line widths in its two-dimensional ¹H–¹⁵N TROSY-HSQC spectrum in comparison to HscA₃₈₆, indicating that the peptide in the <i>cis</i> configuration binds to and stabilizes the NBD. The addition to HscA₃₈₆ of a synthetic peptide with a sequence identical to that of the interdomain linker (L³⁸⁷LL­D­V­I­P­LS³⁹⁵) stimulated its ATPase activity and induced widespread NMR chemical shift perturbations indicative of a binding interaction in the <i>trans</i> configuration

Structure and Dynamics of the Iron−Sulfur Cluster Assembly Scaffold Protein IscU and Its Interaction with the Cochaperone HscB

IscU is a scaffold protein that functions in iron−sulfur cluster assembly and transfer. Its critical importance has been recently underscored by the finding that a single intronic mutation in the human <i>iscu</i> gene is associated with a myopathy resulting from deficient succinate dehydrogenase and aconitase [Mochel, F., Knight, M. A., Tong, W. H., Hernandez, D., Ayyad, K., Taivassalo, T., Andersen, P. M., Singleton, A., Rouault, T. A., Fischbeck, K. H., and Haller, R. G. (2008) <i>Am. J. Hum. Genet. 82</i>, 652−660]. IscU functions through interactions with a chaperone protein HscA and a cochaperone protein HscB. To probe the molecular basis for these interactions, we have used NMR spectroscopy to investigate the solution structure of IscU from <i>Escherichia coli</i> and its interaction with HscB from the same organism. We found that wild-type apo-IscU in solution exists as two distinct conformations: one largely disordered and one largely ordered except for the metal binding residues. The two states interconvert on the millisecond time scale. The ordered conformation is stabilized by the addition of zinc or by the single-site IscU mutation, D39A. We used apo-IscU(D39A) as a surrogate for the folded state of wild-type IscU and assigned its NMR spectrum. These assignments made it possible to identify the region of IscU with the largest structural differences in the two conformational states. Subsequently, by following the NMR signals of apo-IscU(D39A) upon addition of HscB, we identified the most perturbed regions as the two N-terminal β-strands and the C-terminal α-helix. On the basis of these results and analysis of IscU sequences from multiple species, we have identified the surface region of IscU that interacts with HscB. We conclude that the IscU−HscB complex exists as two (or more) distinct states that interconvert at a rate much faster than the rate of dissociation of the complex and that HscB binds to and stabilizes the ordered state of apo-IscU