Facilitated transfer of IscU-[2Fe2S] clusters by chaperone-mediated ligand exchange.

The scaffold protein IscU and molecular chaperones HscA and HscB play central roles in biological assembly of iron-sulfur clusters and maturation of iron-sulfur proteins. However, the structure of IscU-FeS complexes and the molecular mechanism whereby the chaperones facilitate cluster transfer to acceptor proteins are not well understood. We have prepared amino acid substitution mutants of Escherichia coli IscU in which potential ligands to the FeS cluster (Cys-37, Cys-63, His-105, and Cys-106) were individually replaced with alanine. The properties of the IscU-FeS complexes formed were investigated by measuring both their ability to transfer preformed FeS clusters to apo-ferredoxin and the activity of the IscU proteins in catalyzing cluster assembly on apo-ferredoxin using inorganic iron with inorganic sulfide or with IscS and cysteine as a sulfur source. The ability of the HscA/HscB chaperone system to accelerate ATP-dependent cluster transfer from each IscU substitution mutant to apo-ferredoxin was also determined. All of the mutants formed FeS complexes with a stoichiometry similar to the wild-type holo-protein, i.e., IscU(2)[2Fe2S], raising the possibility that different cluster ligation states may occur during iron-sulfur protein maturation. Spectroscopic properties of the mutants and the kinetics of transfer of performed IscU-FeS clusters to apo-ferredoxin indicate that the most stable form of holo-IscU involves iron coordination by Cys-63 and Cys-106. Results of studies on the ability of mutants to catalyze formation of holo-ferredoxin using iron and different sulfur sources were consistent with proposed roles for Cys-63 and Cys-106 in FeS cluster binding and also indicated an essential role for Cys-106 in sulfide transfer to IscU from IscS. Measurements of the ability of the chaperones HscA and HscB to facilitate cluster transfer from holo-IscU to apo-ferredoxin showed that only IscU(H105A) behaved similarly to wild-type IscU in exhibiting ATP-dependent stimulation of cluster transfer. IscU(C63A) and IscU(C106A) displayed elevated rates of cluster transfer in the ±ATP whereas IscU(C37A) exhibited low rates of cluster transfer ±ATP. In interpreting these findings, we propose that IscU(2)[2Fe2S] is able undergo structural isomerization to yield conformers having different cysteine residues bound to the cluster. On the basis of the crystal structure of HscA complexed with an IscU-derived peptide, we propose that the chaperone binds and stabilizes an isomer of IscU(2)[2Fe2S] in which the cluster is bound by cysteine residues 37 and 63 and that the [2Fe2S] cluster, being held less tightly than that coordinated by Cys-63 and Cys-106 in free IscU(2)[2Fe2S], is more readily transferred to acceptor proteins such as apo-ferredoxin.

Three hydrophobic amino acids in Escherichia coli HscB make the greatest contribution to the stability of the HscB-IscU complex.

BACKGROUND: General iron-sulfur cluster biosynthesis proceeds through assembly of a transient cluster on IscU followed by its transfer to a recipient apo-protein. The efficiency of the second step is increased by the presence of HscA and HscB, but the reason behind this is poorly understood. To shed light on the function of HscB, we began a study on the nature of its interaction with IscU. Our work suggested that the binding site of IscU is in the C-terminal domain of HscB, and two different triple alanine substitutions ([L92A, M93A, F153A] and [E97A, E100A, E104A]) involving predicted binding site residues had detrimental effects on this interaction. However, the individual contribution of each substitution to the observed effect remains to be determined as well as the possible involvement of other residues in the proposed binding site. RESULTS: In the work reported here, we used isothermal titration calorimetry to characterize the affinity of single alanine HscB mutants for IscU, and subsequently confirmed our results with nuclear magnetic resonance spectroscopy. Alanine substitutions of L92, L96, and F153 severely impaired the ability of HscB to form a complex with IscU; substitutions of R87, R99, and E100 had more modest effects; and substitutions of T89, M93, E97, D103, E104, R152, K156, and S160 had only minor or no detectable effects. CONCLUSIONS: Our results show that the residues of HscB most important for strong interaction with IscU include three hydrophobic residues (L92, L96, and F153); in addition, we identified a number of other residues whose side chains contribute to a lesser extent to the interaction. Our results suggest that the triple alanine substitution at HscB positions 92, 96, and 153 will destabilize the HscB-IscU complex by ΔΔGb≅ 5.7 kcal/mol, equivalent to a ≅ 15000-fold reduction in the affinity of HscB for IscU. We propose that this triple mutant could provide a more definitive test of the functional importance of the HscB-IscU interaction in vivo than those used previously that yielded inconclusive results.

Mitogen-activated protein/extracellular signal-regulated kinase kinase 1act/tubulin interaction is an important determinant of mitotic stability in cultured HT1080 human fibrosarcoma cells.

Activation of the mitogen-activated protein kinase (MAPK) pathway plays a major role in neoplastic cell transformation. Using a proteomics approach, we identified alpha tubulin and beta tubulin as proteins that interact with activated MAP/extracellular signal-regulated kinase kinase 1 (MEK1), a central MAPK regulatory kinase. Confocal analysis revealed spatiotemporal control of MEK1-tubulin colocalization that was most prominent in the mitotic spindle apparatus in variant HT1080 human fibrosarcoma cells. Peptide arrays identified the critical role of positively charged amino acids R108, R113, R160, and K157 on the surface of MEK1 for tubulin interaction. Overexpression of activated MEK1 caused defects in spindle arrangement, chromosome segregation, and ploidy. In contrast, chromosome polyploidy was reduced in the presence of an activated MEK1 mutant (R108A, R113A) that disrupted interactions with tubulin. Our findings indicate the importance of signaling by activated MEK1-tubulin in spindle organization and chromosomal instability.

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 iscu 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) Am. J. Hum. Genet. 82, 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 Escherichia coli 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 beta-strands and the C-terminal alpha-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.

Studies on the mechanism of catalysis of iron-sulfur cluster transfer from IscU[2Fe2S] by HscA/HscB chaperones.

The HscA/HscB chaperone/cochaperone system accelerates transfer of iron-sulfur clusters from the FeS-scaffold protein IscU (IscU(2)[2Fe2S], holo-IscU) to acceptor proteins in an ATP-dependent manner. We have employed visible region circular dichroism (CD) measurements to monitor chaperone-catalyzed cluster transfer from holo-IscU to apoferredoxin and to investigate chaperone-induced changes in properties of the IscU(2)[2Fe2S] cluster. HscA-mediated acceleration of [2Fe2S] cluster transfer exhibited an absolute requirement for both HscB and ATP. A mutant form of HscA lacking ATPase activity, HscA(T212V), was unable to accelerate cluster transfer, suggesting that ATP hydrolysis and conformational changes accompanying the ATP (T-state) to ADP (R-state) transition in the HscA chaperone are required for catalysis. Addition of HscA and HscB to IscU(2)[2Fe2S] did not affect the properties of the [2Fe2S] cluster, but subsequent addition of ATP was found to cause a transient change of the visible region CD spectrum, indicating distortion of the IscU-bound cluster. The dependence of the rate of decay of the observed CD change on ATP concentration and the lack of an effect of the HscA(T212V) mutant were consistent with conformational changes in the cluster coupled to ATP hydrolysis by HscA. Experiments carried out under conditions with limiting concentrations of HscA, HscB, and ATP further showed that formation of a 1:1:1 HscA-HscB-IscU(2)[2Fe2S] complex and a single ATP hydrolysis step are sufficient to elicit the full effect of the chaperones on the [2Fe2S] cluster. These results suggest that acceleration of iron-sulfur cluster transfer involves a structural change in the IscU(2)[2Fe2S] complex during the T --> R transition of HscA accompanying ATP hydrolysis.

Solution structure of the iron-sulfur cluster cochaperone HscB and its binding surface for the iron-sulfur assembly scaffold protein IscU.

The interaction between IscU and HscB is critical for successful assembly of iron-sulfur clusters. NMR experiments were performed on HscB to investigate which of its residues might be part of the IscU binding surface. Residual dipolar couplings ( (1) D HN and (1) D CalphaHalpha) indicated that the crystal structure of HscB [Cupp-Vickery, J. R., and Vickery, L. E. (2000) Crystal structure of Hsc20, a J-type cochaperone from Escherichia coli, J. Mol. Biol. 304, 835-845] faithfully represents its solution state. NMR relaxation rates ( (15)N R 1, R 2) and (1)H- (15)N heteronuclear NOE values indicated that HscB is rigid along its entire backbone except for three short regions which exhibit flexibility on a fast time scale. Changes in the NMR spectrum of HscB upon addition of IscU mapped to the J-domain/C-domain interface, the interdomain linker, and the C-domain. Sequence conservation is low in the interface and in the linker, and NMR changes observed for these residues likely result from indirect effects of IscU binding. NMR changes observed in the conserved patch of residues in the C-domain (L92, M93, L96, E97, E100, E104, and F153) were suggestive of a direct interaction with IscU. To test this, we replaced several of these residues with alanine and assayed for the ability of HscB to interact with IscU and to stimulate HscA ATPase activity. HscB(L92A,M93A,F153A) and HscB(E97A,E100A,E104A) both showed decreased binding affinity for IscU; the (L92A,M93A,F153A) substitution also strongly perturbed the allosteric interaction within the HscA.IscU.HscB ternary complex. We propose that the conserved patch in the C-domain of HscB is the principal binding site for IscU.

Molecular chaperones HscA/Ssq1 and HscB/Jac1 and their roles in iron-sulfur protein maturation.

Genetic and biochemical studies have led to the identification of several cellular pathways for the biosynthesis of iron-sulfur proteins in different organisms. The most broadly distributed and highly conserved system involves an Hsp70 chaperone and J-protein co-chaperone system that interacts with a scaffold-like protein involved in [FeS]-cluster preassembly. Specialized forms of Hsp70 and their co-chaperones have evolved in bacteria (HscA, HscB) and in certain fungi (Ssq1, Jac1), whereas most eukaryotes employ a multifunctional mitochondrial Hsp70 (mtHsp70) together with a specialized co-chaperone homologous to HscB/Jac1. HscA and Ssq1 have been shown to specifically bind to a conserved sequence present in the [FeS]-scaffold protein designated IscU in bacteria and Isu in fungi, and the crystal structure of a complex of a peptide containing the IscU recognition region bound to the HscA substrate binding domain has been determined. The interaction of IscU/Isu with HscA/Ssq1 is regulated by HscB/Jac1 which bind the scaffold protein to assist delivery to the chaperone and stabilize the chaperone-scaffold complex by enhancing chaperone ATPase activity. The crystal structure of HscB reveals that the N-terminal J-domain involved in regulation of HscA ATPase activity is similar to other J-proteins, whereas the C-terminal domain is unique and appears to mediate specific interactions with IscU. At the present time the exact function(s) of chaperone-[FeS]-scaffold interactions in iron-sulfur protein biosynthesis remain(s) to be established. In vivo and in vitro studies of yeast Ssq1 and Jac1 indicate that the chaperones are not required for [FeS]-cluster assembly on Isu. Recent in vitro studies using bacterial HscA, HscB and IscU have shown that the chaperones destabilize the IscU[FeS] complex and facilitate cluster delivery to an acceptor apo-protein consistent with a role in regulating cluster release and transfer. Additional genetic and biochemical studies are needed to extend these findings to mtHsp70 activities in higher eukaryotes.

Structural determinants of HscA peptide-binding specificity.

The Hsp70-class molecular chaperone HscA interacts specifically with a conserved (99)LPPVK(103) motif of the iron-sulfur cluster scaffold protein IscU. We used a cellulose-bound peptide array to perform single-site saturation substitution of peptide residues corresponding to Glu(98)-Ile(104) of IscU to determine positional amino acid requirements for recognition by HscA. Two mutant chaperone forms, HscA(F426A) with a DnaK-like arch structure and HscA(M433V) with a DnaK-like substrate-binding pocket, were also studied. Wild-type HscA and HscA(F426A) exhibited a strict preference for proline in the central peptide position (ELPPVKI), whereas HscA(M433V) bound a peptide containing a Pro-->Leu substitution at this location (ELPLVKI). Contributions of Phe(426) and Met(433) to HscA peptide specificity were further tested in solution using a fluorescence-based peptide-binding assay. Bimane-labeled HscA and HscA(F426A) bound ELPPVKI peptides with higher affinity than leucine-substituted peptides, whereas HscA(M433V) favored binding of ELPLVKI peptides. Fluorescence-binding studies were also carried out with derivatives of the peptide NRLLLTG, a model substrate for DnaK. HscA and HscA(F426A) bound NRLLLTG peptides weakly, whereas HscA(M433V) bound NRLLLTG peptides with higher affinity than IscU-derived peptides ELPPVKI and ELPLVKI. These results suggest that the specificity of HscA for the LPPVK recognition sequence is determined in part by steric obstruction of the hydrophobic binding pocket by Met(433) and that substitution with the Val(433) sidechain imparts a broader, more DnaK-like, substrate recognition pattern.

Sequence-dependent peptide binding orientation by the molecular chaperone DnaK.

Hsp70-class molecular chaperones interact with diverse polypeptide substrates, but there is limited information on the structures of different Hsp70-peptide complexes. We have used a site-directed fluorescence labeling and quenching strategy to investigate the orientation of different peptides bound to DnaK from Escherichia coli. DnaK was selectively labeled on opposite sides of the substrate-binding domain (SBD) with the fluorescent probe bimane, and the ability of peptides containing N- or C-terminal tryptophan residues to quench bimane fluorescence was measured. Tryptophan-labeled derivatives of the model peptide NRLLLTG bound with the same forward orientation previously observed in the crystal structure of the DnaK(SBD)-NRLLLTG complex. Derivatives of this peptide containing arginine in the C-terminal rather than N-terminal region, NTLLLRG, also bound in the forward direction indicating that charged residues in the flanking regions of the peptide are not the major determinant of peptide binding orientation. We also tested peptides having proline in one (ELPLVKI) or two (ELPPVKI) central positions. Tryptophan derivatives of each of these peptides bound with a strong preference for the reverse direction relative to that observed for the NRLLLTG and NTLLLRG peptides. Computer modeling the peptides NRLLLTG and ELPPVKI in both the forward and reverse orientations into the DnaK(SBD) indicated that differential hydrogen-bonding patterns and steric constraints of the central peptide residues are likely causes for differences in their binding orientations. These findings establish that DnaK is able to bind substrates in both forward and reverse orientations and suggest that the central residues of the peptide are the major determinants of directional preference.

Multiple turnover transfer of [2Fe2S] clusters by the iron-sulfur cluster assembly scaffold proteins IscU and IscA.

IscU/Isu and IscA/Isa (and related NifU and SufA proteins) have been proposed to serve as molecular scaffolds for preassembly of [FeS] clusters to be used in the biogenesis of iron-sulfur proteins. In vitro studies demonstrating transfer of preformed scaffold-[FeS] complexes to apoprotein acceptors have provided experimental support for this hypothesis, but investigations to date have yielded only single-cluster transfer events. We describe an in vitro assay system that allows for real-time monitoring of [FeS] cluster formation using circular dichroism spectroscopy and use this to investigate de novo [FeS] cluster formation and transfer from Escherichia coli IscU and IscA to apo-ferredoxin. Both IscU and IscA were found to be capable of multiple cycles of [2Fe2S] cluster formation and transfer suggesting that these scaffold proteins are capable of acting "catalytically." Kinetic studies further showed that cluster transfer exhibits Michaelis-Menten behavior indicative of complex formation of holo-IscU and holo-IscA with apoferredoxin and consistent with a direct [FeS] cluster transfer mechanism. Analysis of the dependence of the rate of cluster transfer, however, revealed enhanced efficiency at low ratios of scaffold to acceptor protein suggesting participation of a transient, labile scaffold-[FeS] species in the transfer process.

X-ray diffraction analysis of a crystal of HscA from Escherichia coli.

HscA is a constitutively expressed Hsp70 that interacts with the iron-sulfur cluster assembly protein IscU. Crystals of a truncated form of HscA (52 kDa; residues 17-505) grown in the presence of an IscU-recognition peptide, WELPPVKI, have been obtained by hanging-drop vapor diffusion using ammonium sulfate as the precipitant. A complete native X-ray diffraction data set was collected from a single crystal at 100 K to a resolution of 2.9 A. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 158.35, b = 166.15, c = 168.26 A, and contains six molecules per asymmetric unit. Phases were determined by molecular replacement using the nucleotide-binding domain from DnaK and the substrate-binding domain from HscA as models. This is the first reported crystallization of an Hsp70 containing both nucleotide- and substrate-binding domains.

Regulation of the HscA ATPase reaction cycle by the co-chaperone HscB and the iron-sulfur cluster assembly protein IscU.

The ATPase activity of HscA, a specialized hsp70 molecular chaperone from Escherichia coli, is regulated by the iron-sulfur cluster assembly protein IscU and the J-type co-chaperone HscB. IscU behaves as a substrate for HscA, and HscB enhances the binding of IscU to HscA. To better understand the mechanism by which HscB and IscU regulate HscA, we examined binding of HscB to the different conformational states of HscA and the effects of HscB and IscU on the kinetics of the individual steps of the HscA ATPase reaction cycle. Affinity sensor studies revealed that whereas IscU binds both ADP (R-state) and ATP (T-state) HscA complexes, HscB interacts only with an ATP-bound state. Studies of ATPase activity under single-turnover and rapid mixing conditions showed that both IscU and HscB interact with the low peptide affinity T-state of HscA (HscA++.ATP) and that both modestly accelerate (3-10-fold) the rate-determining steps in the HscA reaction cycle, k(hyd) and k(T-->R). When present together, IscU and HscB synergistically stimulate both k(hyd) (approximately = 500-fold) and k(T-->R) (approximately = 60-fold), leading to enhanced formation of the HscA.ADP-IscU complex (substrate capture). Following ADP/ATP exchange, IscU also stimulates k(R-->T) (approximately = 50-fold) and thereby accelerates the rate at which the low peptide affinity HscA++.ATP T-state is regenerated. Because HscA nucleotide exchange is fast, the overall rate of the chaperone cycle in vivo will be determined by the availability of the IscU-HscB substrate-co-chaperone complex.

Crystal structure of the molecular chaperone HscA substrate binding domain complexed with the IscU recognition peptide ELPPVKIHC.

HscA, a specialized bacterial Hsp70-class molecular chaperone, interacts with the iron-sulfur cluster assembly protein IscU by recognizing a conserved LPPVK sequence motif. We report the crystal structure of the substrate-binding domain of HscA (SBD, residues 389-616) from Escherichia coli bound to an IscU-derived peptide, ELPPVKIHC. The crystals belong to the space group I222 and contain a single molecule in the asymmetric unit. Molecular replacement with the E.coli DnaK(SBD) model was used for phasing, and the HscA(SBD)-peptide model was refined to Rfactor=17.4% (Rfree=21.0%) at 1.95 A resolution. The overall structure of HscA(SBD) is similar to that of DnaK(SBD), although the alpha-helical subdomain (residues 506-613) is shifted up to 10 A relative to the beta-sandwich subdomain (residues 389-498) when compared to DnaK(SBD). The ELPPVKIHC peptide is bound in an extended conformation in a hydrophobic cleft in the beta-subdomain, which appears to be solvent-accessible via a narrow passageway between the alpha and beta-subdomains. The bound peptide is positioned in the reverse orientation of that observed in the DnaK(SBD)-NRLLLTG peptide complex placing the N and C termini of the peptide on opposite sides of the HscA(SBD) relative to the DnaK(SBD) complex. Modeling of the peptide in the DnaK-like forward orientation suggests that differences in hydrogen bonding interactions in the binding cleft and electrostatic interactions involving surface residues near the cleft contribute to the observed directional preference.

Preferential substrate binding orientation by the molecular chaperone HscA.

HscA, a specialized bacterial hsp70-class chaperone, interacts with the iron-sulfur cluster assembly protein IscU by recognizing a conserved LPPVK sequence motif at positions 99-103. We have used a site-directed fluorescence labeling and quenching strategy to determine whether HscA binds to IscU in a preferred orientation. HscA was selectively labeled on opposite sides of the substrate binding domain with the fluorescent probe bimane, and the ability of LPPVK-containing peptides having tryptophan at the N or C terminus to quench bimane fluorescence was measured. Quenching was highly dependent on the position of tryptophan in the peptide and the location of bimane on HscA implying a strong directional preference for peptide binding. Similar experiments showed that full-length IscU binds in the same orientation as IscU-derived peptides and that binding orientation is unaffected by the co-chaperone HscB. The preferred orientation of the HscA-IscU complex is the reverse of that previously described for peptide complexes of Escherichia coli DnaK and rat Hsc70 substrate binding domain fragments establishing that hsp70 isoforms can bind peptide/polypeptide substrates in different orientations.

Crystal structure of IscA, an iron-sulfur cluster assembly protein from Escherichia coli.

IscA, an 11 kDa member of the hesB family of proteins, binds iron and [2Fe-2S] clusters, and participates in the biosynthesis of iron-sulfur proteins. We report the crystal structure of the apo-protein form of IscA from Escherichia coli to a resolution of 2.3A. The crystals belong to the space group P3(2)21 and have unit cell dimensions a=b=66.104 A, c=150.167 A (alpha=beta=90 degrees, gamma=120 degrees ). The structure was solved using single-wavelength anomalous dispersion (SAD) phasing of a selenomethionyl derivative, and the IscA model was refined to R=21.4% (Rfree=25.4%). IscA exists as an (alpha1alpha2)2 homotetramer with the (alpha1alpha2) dimer comprising the asymmetric unit. Cys35, implicated in Fe-S cluster assembly, is located in a central cavity formed at the tetramer interface with the gamma-sulfur atoms of residues from the alpha1 and alpha2' monomers (and alpha1'alpha2) positioned close to one another (approximately equal 7 A). C-terminal residues 99-107 are disordered, and the exact positions of Cys99 and Cys101 could not be determined. However, computer modeling of C-terminal residues in the tetramer suggests that Cys99 and Cys101 in the alpha1 monomer and those of the alpha1' monomer (or alpha2 and alpha2') are positioned sufficiently close to coordinate [2Fe-2S] clusters between the two dimers, whereas this is not possible within the (alpha1alpha2) or (alpha1'alpha2') dimer. This symmetrical arrangement allows for binding of two [2Fe-2S] clusters on opposite sides of the tetramer. Modeling further reveals that Cys101 is positioned sufficiently close to Cys35 to allow Cys35 to participate in cluster assembly, formation, or transfer.

Substitutions in an active site loop of Escherichia coli IscS result in specific defects in Fe-S cluster and thionucleoside biosynthesis in vivo.

IscS catalyzes the fragmentation of l-cysteine to l-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. In Escherichia coli IscS, the active site cysteine Cys(328) resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys(328) identified additional residues important for IscS function in vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesis in vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s(2)C and ms(2)i(6)A) yet showed wild type levels of Fe-S-independent thionucleosides (s(4)U and mnm(5)s(2)U) that require persulfide formation and transfer. In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesis in vivo without detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.

Identification of a novel candidate gene in the iron-sulfur pathway implicated in ataxia-susceptibility: human gene encoding HscB, a J-type co-chaperone.

Iron-sulfur proteins participate in a wide range of biochemical processes, including many that are central to mitochondrial electron transfer and energy metabolism. Mutations in two such proteins, frataxin and ABCB7, cause Friedreich ataxia and X-linked sideroblastic anemia with ataxia, respectively, rendering other participants in this pathway functional candidates for hereditary ataxia syndromes. Recently frataxin was shown to have an identical phylogenetic distribution with two genes and was most likely specifically involved in the same sub-process in iron-sulfur cluster assembly as one gene, designated hscB, in bacteria. To set the stage for an analysis of the potential role of this candidate gene in human disease, we defined the human HscB cDNA, its genomic locus, and its pattern of expression in normal human tissues. The isolated human HscB cDNA spans 785 bp and encodes a conserved 235-amino-acid protein, including a putative mitochondrial import leader. The HscB gene is found at chromosome 22q11-12 and is composed of six exons and five introns. Northern blot analyses of RNA from adult and fetal tissues defined a pattern of expression in mitochondria-rich tissues similar to that of frataxin, an expression pattern compatible with its implied role in mitochondrial energetics and related disease phenotypes.

Crystal structure of IscS, a cysteine desulfurase from Escherichia coli.

IscS is a widely distributed cysteine desulfurase that catalyzes the pyridoxal phosphate-dependent desulfuration of L-cysteine and plays a central role in the delivery of sulfur to a variety of metabolic pathways. We report the crystal structure of Escherichia coli IscS to a resolution of 2.1A. The crystals belong to the space group P2(1)2(1)2(1) and have unit cell dimensions a=73.70A, b=101.97A, c=108.62A (alpha=beta=gamma=90 degrees ). Molecular replacement with the Thermotoga maritima NifS model was used to determine phasing, and the IscS model was refined to an R=20.6% (R(free)=23.6%) with two molecules per asymmetric unit. The structure of E.coli IscS is similar to that of T.maritima NifS with nearly identical secondary structure and an overall backbone r.m.s. difference of 1.4A. However, in contrast to NifS a peptide segment containing the catalytic cysteine residue (Cys328) is partially ordered in the IscS structure. This segment of IscS (residues 323-335) forms a surface loop directed away from the active site pocket. Cys328 is positioned greater than 17A from the pyridoxal phosphate cofactor, suggesting that a large conformational change must occur during catalysis in order for Cys328 to participate in nucleophilic attack of a pyridoxal phosphate-bound cysteine substrate. Modeling suggests that rotation of this loop may allow movement of Cys328 to within approximately 3A of the pyridoxal phosphate cofactor.

Contributions of the LPPVK motif of the iron-sulfur template protein IscU to interactions with the Hsc66-Hsc20 chaperone system.

Hsc66 (HscA) and Hsc20 (HscB) from Escherichia coli comprise a specialized chaperone system that selectively binds the iron-sulfur cluster template protein IscU. Hsc66 interacts with peptides corresponding to a discrete region of IscU including residues 99-103 (LPPVK), and a peptide containing residues 98-106 stimulates Hsc66 ATPase activity in a manner similar to IscU. To determine the relative contributions of individual residues in the LPPVK motif to Hsc66 binding and regulation, we have carried out an alanine mutagenesis scan of this motif in the Glu98-Cys106 peptide and the IscU protein. Alanine substitutions in the Glu98-Cys106 peptide resulted in decreased ATPase stimulation (2-10-fold) because of reduced binding affinity, with peptide(P101A) eliciting <10% of the parent peptide stimulation. Alanine substitutions in the IscU protein also revealed lower activities resulting from decreased apparent binding affinity, with the greatest changes in Km observed for the Pro101 (77-fold), Val102 (4-fold), and Lys103 (15-fold) mutants. Calorimetric studies of the binding of IscU mutants to the Hsc66.ADP complex showed that the P101A and K103A mutants also exhibit decreased binding affinity for the ADP-bound state. When ATPase stimulatory activity was assayed in the presence of the co-chaperone Hsc20, each of the mutants displayed enhanced binding affinity, but the P101A and V102A mutants exhibited decreased ability to maximally simulate Hsc66 ATPase. A charge mutant containing the motif sequence of NifU, IscU(V102E), did not bind the ATP or ADP states of Hsc66 but did bind Hsc20 and weakly stimulated Hsc66 ATPase in the presence of the co-chaperone. These results indicate that residues in the LPPVK motif are important for IscU interactions with Hsc66 but not for the ability of Hsc20 to target IscU to Hsc66. The results are discussed in the context of a structural model based on the crystallographic structure of the DnaK peptide-binding domain.

Preliminary crystallographic analysis of the cysteine desulfurase IscS from Escherichia coli.

IscS is a widely distributed cysteine desulfurase that catalyzes the pyridoxal phosphate dependent beta-elimination of sulfur from L-cysteine and plays a central role in the delivery of sulfur to a variety of metabolic pathways. Crystals of Escherichia coli IscS have been obtained by the hanging-drop vapor-diffusion method using polyethylene glycol (PEG) as a precipitant. Initial seed crystals were obtained using PEG 6000 and sodium acetate, and diffraction-quality crystals were grown using a mixture of PEG 2000 and PEG 10 000 in the presence of sodium citrate. A complete native X-ray diffraction data set was collected from a single crystal at 103 K to a resolution of 2.1 A. The crystals belong to space group P2(1)2(1)2(1) and have unit-cell parameters a = 73.7086, b = 101.9741, c = 108.617 A (alpha = beta = gamma = 90 degrees ). Analysis of the Matthews equation and self-rotation function suggest two molecules per asymmetric unit, consistent with the presence of a single dimeric molecule.