Solution NMR Structure of the Iron–Sulfur Cluster Assembly Protein U (IscU) with Zinc Bound at the Active Site

doi:10.1016/j.jmb.2004.08.038

J. Mol. Biol. (2004) 344, 567–583

Solution NMR Structure of the Iron–Sulfur Cluster Assembly Protein U (IscU) with Zinc Bound at the Active Site Theresa A. Ramelot1,2, John R. Cort1,2, Sharon Goldsmith-Fischman2,3 Gregory J. Kornhaber2,4,5, Rong Xiao2,4, Ritu Shastry2,4 Thomas B. Acton2,4, Barry Honig2,3, Gaetano T. Montelione2,4,5 and Michael A. Kennedy1,2* 1

Biological Sciences Division Pacific Northwest National Laboratory, Richland, WA 99352, USA 2

Northeast Structural Genomics Consortium 3

Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, Columbia University New York, NY 10032, USA 4

Center for Advanced Biotechnology and Medicine Department of Molecular Biology and Biochemistry Rutgers University, Piscataway NJ 08854, USA

IscU is a highly conserved protein that serves as the scaffold for IscSmediated assembly of iron–sulfur ([Fe–S]) clusters. We report the NMR solution structure of monomeric Haemophilus influenzae IscU with zinc bound at the [Fe–S] cluster assembly site. The compact core of the globular structure has an a–b sandwich architecture with a three-stranded antiparallel b-sheet and four a-helices. A nascent helix is located N-terminal to the core structure. The zinc is ligated by three cysteine residues and one histidine residue that are located in and near conformationally dynamic loops at one end of the IscU structure. Removal of the zinc metal by chelation results in widespread loss of structure in the apo form. The zinc-bound IscU may be a good model for iron-loaded IscU and may demonstrate structural features found in the [Fe–S] cluster bound form. Structural and functional similarities, genomic context in operons containing other homologous genes, and distributions of conserved surface residues support the hypothesis that IscU protein domains are homologous (i.e. derived from a common ancestor) with the SufE/YgdK family of [Fe–S] cluster assembly proteins. q 2004 Elsevier Ltd. All rights reserved.

5 Department of Biochemistry Robert Wood Johnson Medical School, UMDNJ, Piscataway NJ 08854, USA

*Corresponding author

Keywords: IscU; NMR structure; Zn-binding; iron–sulfur clusters; zinc

Introduction Proteins containing iron–sulfur ([Fe–S]) clusters are involved in a variety of cellular processes, including regulation of gene expression, carbon metabolism, respiration, nitrogen fixation, and Abbreviations used: 2D, two-dimensional; 3D, threedimensional; DTT, 1,4-dithiothreitol; Ecol, Escherichia coli; Hinf, Haemophilus influenzae; Mes, 2-(Nmorpholino)ethanesulfonic acid; MWw, weight-average molecular mass; NMR, nuclear magnetic resonance; Tm, Thermatoga maritima; NOE, nuclear Overhauser effect; HSQC, heteronuclear single quantum coherence. E-mail address of the corresponding author: [email protected]

DNA repair. Multiple proteins are involved in the assembly and transfer of [Fe–S] clusters to their targets. The iron–sulfur cluster proteins IscS and IscU are involved in the first steps of [Fe–S] cluster biosynthesis in many prokaryotic organisms. They appear to work in concert with several other proteins, including the chaperone proteins HscA and HscB, whose corresponding genes are located in a gene cluster together with the iscS and iscU genes.1,2 The proteins IscS and IscU are homologous to the well-studied nitrogen fixation pathway proteins NifS and NifU, respectively, of Azotobacter vinelandii.1 Dean, Johnson, and co-workers 3,4 demonstrated that NifS and NifU are involved in the initial steps of the assembly of [Fe–S] clusters

0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

568 required by nitrogenase for activity. NifS is a homodimeric, pyridoxal phosphate-dependent L-cysteine desulfurase, catalyzing the removal of sulfur from L-cysteine to form L-alanine and a NifS persulfide intermediate.5,6 NifU is thought to function as a scaffold protein, providing the initial site for NifS-mediated [Fe–S] cluster assembly.4,7 The iscS and iscU genes, also found in A. vinelandii, were first identified by sequence similarity and apparent homology to nifS and nifU.1,8 IscU is homologous to the N-terminal domain of NifU, which contains one labile [2Fe–2S]2C binding site. For this reason, IscU proteins are often annotated as “NifU-like N-terminal domain” or NifU_N in SWISS-PROT/TrEMBL.9 IscS and IscU are found in the iscSUA gene cluster in a variety of prokaryotes, including Escherichia coli, Haemophilus influenzae, and nitrogen-fixing bacteria.1 Homologs of these proteins are highly conserved in all three superkingdoms, with IscU having a high level of sequence identity between species as divergent as humans and H. influenzae.10 IscS and IscU from E. coli and A. vinelandii have been subjects of extensive biochemical characterization.1,8,11–14 IscS, like NifS, is a homodimeric L-cysteine desulfurase that forms an IscS persulfide intermediate on a conserved cysteine residue.1,8 IscS then passes this sulfur to IscU, in a reaction that involves two IscU and two IscS proteins in an a2b2 heterotetrameric complex.11–13 Initially, IscS mediates assembly of one [2Fe–2S]2C center per IscU dimer.11 Kinetic studies of cluster assembly indicate that this product converts to a form containing two [2Fe–2S]2C clusters per IscU dimer, and then forms a [4Fe–4S]2C cluster bound to the IscU dimer.14 IscU thus acts as a scaffold for assembly and subsequent transfer to target proteins, as was demonstrated for the in vitro transfer of a [2Fe–2S]C2 cluster from Thermotoga maritima (Tm IscU) or the corresponding Schizosaccharomyces pombe IscU homolog to ferredoxin.15,16 As was shown for Tm IscU, an iron-loaded form of IscU will rapidly accept sulfur from the IscS persulfide intermediate to yield the [2Fe–2S]2C bound IscU.17 A mechanism for [Fe–S] cluster assembly, involving initial binding of Fe2C or Fe3C to IscU as a necessary prerequisite, has been proposed.17 The iron donor may be iron-loaded holo frataxin or the bacterial homolog CyaY. A recent study demonstrates that frataxin can supply iron to the Homo sapiens IscU homolog, and that subsequent cluster assembly can be NifS-mediated or induced by addition of inorganic sulfide.18 Three cysteine residues (Cys37, Cys63, and Cys106, numbered for E. coli and H. influenzae IscU) that have been proposed to be necessary for binding to iron in the NifU N-terminal domain are conserved across the IscU protein domain family (Figure 1). H. influenzae IscU (Hinf IscU) has greater than 80% sequence identity with the well-characterized IscUs from both E. coli and A. vinelandii (86% and 81%, respectively). In addition, Hinf IscU is similar to eukaryotic homologs from H. sapiens

Solution NMR Structure of IscU

(75%), Drosophila melanogaster (78%), Caenorhabditis elegans (75%), Arabidopsis thaliana (72%), and Saccharomyces cerevisiae (72%). Genetic studies suggest that the isc genes are ubiquitous and have a crucial function in [Fe–S] cluster assembly in many organisms.1,19 The sequence similarity between nif genes and isc genes suggests that they are homologs (i.e. derived from a common ancestor) and share the common function of [Fe–S] cluster synthesis. However, it was found for A. vinelandii that even in an organism expressing nif gene products, isc genes are essential for growth.1 Also, in nifU deletion strains, IscU cannot compensate for NifU under conditions of diazotrophic growth.20 These results demonstrate that NifU and IscU, although homologous, are divergent in their functions and probably also their interactions with other proteins. In E. coli, deletion of isc genes can be compensated for by genes in the suf operon, although growth is impaired.19 The suf operon is found in various bacteria and Archaea, and includes a gene encoding a cysteine desulfurase, SufS and an interacting protein SufE.19,21–23 SufE accepts a sulfur atom from SufS via a persulfide linkage to an invarient cysteine residue.23,24 In contrast to IscU, SufE does not serve as a template for [Fe–S] cluster assembly, but rather accepts sulfur from SufS and may transfer it to an alternative [Fe–S] cluster scaffold protein such as SufA. E. coli also contains a third cysteine desulfurase, CsdA homologous to SufS,21,25 and a gene ygdK homologous to sufE from the suf operon.22 The biological function of YgdK has not yet been identified, although it shares 35% identity with SufE as well as the invarient cyteine residue. As they have only limited sequence identity (w10%), sequencebased methods do not indicate homology between IscU and SufE; however, they are both involved in [Fe–S] cluster synthesis and each interacts with its corresponding cysteine desulfurase.11–13,22–24 Efforts to determine high-quality structures of IscU by NMR have been challenged by flexibility and internal dynamics in some members of the IscU family that have been studied. In the case of Tm IscU, both the apo dimer (with oxidized cysteine residues) and the [Fe–S]-coordinated dimer preparations exhibit molten globule-like characteristics; using heteronuclear NMR methods, several pieces of secondary structure, which appear to be dynamic with respect to one another, have been characterized, but not the tertiary structure.26,27 A report of structural characterization of human and yeast homologs of IscU found that they lack secondary or tertiary structure under the conditions studied.16 Here, we report the first high-resolution three-dimensional structure of IscU. The structure was determined under reducing conditions with zinc bound at the iron–sulfur cluster assembly site. Under these conditions (10 mM DTT, w0.6 mM protein concentration, pH 6.5, bound to Zn2C), Hinf IscU is primarily monomeric, and does not exhibit the dynamic behavior seen for Tm IscU, although key structural features are preserved across the IscU

Solution NMR Structure of IscU

569

Figure 1. Multiple sequence alignment of H. influenzae IscU (GenBank accession number NP_438538) with 11 other homologs from E. coli (NP_289086), A. vinelandii (ZP_00091677), D. melanogaster (NP_649840), H. sapiens (NP_055116), C. elegans (NP_502658), A. thaliana (NP_192317), S. cerevisiae (Isu1p, NP_015190), Aquifex aeolicus (NP_213607), B. subtilis (NP_391147), Streptococcus pyogenes (NP_268637), and T. maritima (NP_229173). Alignment was constructed with the aid of CLUSTAL_X.77 Secondary structure of Hinf IscU are shown above: B, b strand; H, a helix; h, possible a helix. Stars indicate zinc ligands, Hinf IscU residues Cy37, C63, K103, H105, and C106. Conserved residues are highlighted in black and gray. Buried residues with average exposed surface of less than 10% are indicated by an I (for internal). Secondary structure for apo Asp40Ala Tm IscU dimer comes from Bertini et al.26

family. Comparison of the structure of IscU with the structures of E. coli SufE and YgdK proteins (PDB codes 1MGZ and 1NI7), reveals structural similarities and distributions of conserved residues that indicate that these three proteins are probably homologous, despite the limited (w10%) sequence identity between IscU and the SufE/YgdK protein domain families.

Results Oligomerization state of Zn-bound IscU IscU was overexpressed and purified from E. coli cell extracts grown in MJ9 minimal medium which contains 8.3 mM ZnSO4 and 59.9 mM FeCl3 along with other metal ions.28 Preparative gel-filtration chromatography under reducing conditions (10 mM Tris buffer, 5 mM DTT, pH 7.5) revealed monomers, dimers, and higher-order oligomers of IscU during purification from cell extracts of E. coli cells overproducing IscU. Fractions corresponding to monomeric IscU, accounting for about 40% of the total IscU, were combined and exchanged into NMR buffer (20 mM Mes (pH 6.5), 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02% NaN3) for further

biophysical characterization. Analytical gel-filtration followed by static light-scattering measurements on this sample (Figure 2) demonstrated that Hinf IscU is primarily monomeric (MWWZ17.5 kDa) under the reducing conditions used, with a small amount (!5%) of higher-order oligomers (possibly tetramer). Under these conditions, the sample remains monomeric even following several weeks of storage at 4 8C. Inductively-coupled plasma mass spectroscopy (ICP-MS) results, coupled with protein concentration determination by measurement of UV absorbance at 280 nm, revealed approximately stoichiometric zinc in IscU NMR samples (w0.5 mM Zn and 0.65(G0.05) mM IscU). The aggregation state of IscU was also determined to be primarily monomeric (translational diffusion coefficient of 10.3!10K7 cm 2/s, compared to 13.2!10K7 cm2/s and 8.6!10K7 cm2/s for 9.0 kDa and 23.6 kDa control proteins at 20 8C, respectively) by translational diffusion measurements using pulsed field gradient NMR methods.29 Further addition of Zn2C to this sample resulted in no significant changes in the 1H–15N-heteronuclear single quantum coherence (HSQC) spectrum of IscU, suggesting that the metal-binding site in this preparation of IscU is saturated. Taken together, these data demonstrate the Hinf IscU sample used

570

Solution NMR Structure of IscU

Figure 2. Analytical gel-filtration followed by static light-scattering, demonstrate that IscU is largely monomeric with only a small amount of oligomer (*) under the NMR measurement conditions. An aliquot of the IscU NMR sample (w0.6 mM IscU in 20 mM Mes, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, pH 6.5) was applied to an analytical gelfiltration column pre-equilibrated with gel filtration buffer (20 mM Mes (pH 6.5), 100 mM NaCl, 10 mM DTT). Static light-scattering at three different angles (blue trace is for the 908 detector) and the index of refraction (red trace) were observed. A Debye plot (upper left inset) was utilized in estimating the mass at different elution points. The estimated molar mass throughout most of the peak region (right inset) remains constant at w17.5 kDa. Static light-scattering measurements at multiple angles, together with protein concentration estimates based on refractive index measurements, provide a shape-independent estimate of the weight-average molecular mass MWw.60,61

in these studies to be largely (O95%) monomeric, stable, and predominantly Zn-bound. Hinf IscU resonance assignments The Hinf IscU protein includes 126 amino acid residues plus an eight-residue C-terminal hexahistidine (HexaHis) affinity purification tag (L127– H134). Samples were prepared with uniform 13C and 15N-enrichment at w0.6 mM protein concentration in H2O and 2H2O solvent at pH 6.5 and 20 8C. Standard triple-resonance NMR methods were used to determine an extensive set of sequence-specific 1H, 15N, and 13C resonance assignments. No 1H-15N-HSQC cross-peaks were observed for residues M1–N16, V40, T60, G64, A68, S70, H105, and H130–H134 (in the HexaHis tag). Side-chain assignments were also missing for residues M1–N16, F58, C63–G64, A68, S70, E98– P100, K103, K122, K124, and H129–H134 (in the HexaHis tag). Aside from these residues, all Asn and Gln side-chain NH2 resonances, and aromatic resonances were assigned. Stereospecific assignments were obtained for the isopropyl groups of six Val and five Leu residues (11/17 residues). Overall, 94% of backbone, and 89% of side-chain 1H, 15N, and 13C resonances routinely observed in NMR spectra were assigned for the protein segment residues V17–E128. Identification of zinc ligands The location of the single Zn-binding sites in Hinf IscU was inferred by combined analysis of cysteine

chemical shift information and structural information. Cys residues that are coordinated to Zn2C have 13 Cb chemical shifts that are characteristic of the sulfhydryl “free” cysteine state (w26–32 ppm versus 37–48 ppm for oxidized).30 In Hinf IscU, residues Cys37 and Cys106 exhibit 13 Cb chemical shifts (30.0 ppm and 28.1 ppm, respectively) that are characteristic of free-reduced or metal-bound states, whereas the 13 Cb chemical shift for the third conserved cysteine residue, Cys63, could not be assigned from the available data. However, residue Cys63 cannot be disulfide-bound, as Hinf IscU is monomeric under these conditions (as demonstrated above) and the other two cysteine residues in the protein are not oxidized. Efforts were made to identify a fourth Zn2C ligand by examination of initial structures of Hinf IscU generated without bound metal constraints. These structures reveal that the three conserved cysteine residues cluster together with His105 to form a potential Zn-binding site. These residues are located in or near loops with few nuclear Overhauser effect (NOE) restraints and therefore have a large variability in their locations in the calculated structures. It is not possible to determine the sidechain orientation for His105 from preliminary structures due to this variability. However, chemical shifts of the resonances in the histidine ring allow for identification of the tautomeric state and the coordinating nitrogen resonance in the ring. Residue His105 has an unusually downfield 13 Cd2 resonance shift of 129.7 ppm (e.g. histidine 13 Cd2 shifts of the unstructured HexaHis tag resonances are near 120 ppm) that is characteristic of a histidine

571

Solution NMR Structure of IscU

coordinated to Zn2C.31 In addition, the 15 N32 (225.5 ppm) and 15 Nd1 (171.4 ppm) chemical shifts of His105 are shifted downfield and upfield relative to values of residues in the HexaHis tag (177 ppm and 205 ppm, respectively), resulting in a pattern in a histidine-optimized 1H-15N-HMQC characteristic of a histidine tautomer with Nd1 protonated and the zinc coordinated through the N32 position.32 These results indicate that the single Zn atom in Hinf IscU identified by ICP-MS analysis is coordinated by side-chains of residues Cys37, Cys63, His105 (via N32 ), and Cys106. Efforts to better characterize the zinc-binding region of Hinf IscU by NOE data analysis were complicated by broad lines observed for many of the resonances in this region of the protein structure. This apparent exchange broadening suggests conformational flexibility in and/or around the Zn-binding site. Adding these Cys-Zn and His-Zn constraints results in a small decrease in

the backbone and side-chain r.m.s.d. values for the ensemble and no additional NOE violations (data not shown), demonstrating that this coordination is completely consistent with the available NOE data. The observation of exchange broadening in and around the metal binding site is also consistent with transient weak binding of Zn2C,33 and/or dynamic flexibility in this region of Zn-bound IscU. However, as mentioned above, further addition of Zn2C did not result in any change in the 1H-15N-HSQC cross-peaks. In particular, the weak Cys63 and Cys106 cross-peaks did not shift or become more intense upon addition of Zn2C. Zn2C bound structure of Hinf IscU The solution structure of Hinf IscU was determined from 829 conformationally restricting experimental constraints (Table 1) over 108 residues, including ten distance constraints for

Table 1. Statistics for Hinf IscU NMR structure determination Completeness of resonance assignments for residues 17–128 Backbone (%) Side-chainsa (%) Conformationally restricting NOE constraintsb Intra-residue [iZj] Sequential [jiKjjZ1] Medium range [1!jiKjj!5] Long range [jiKjjR5] Total NOE constraints per residuec Zinc-binding constraints Dihedral angle constraints f j Hydrogen bond constraints Total Long range [jiKjjR5] Total number of conformationally restricting constraints Number of constraints per residuec Number of long-range constraints per residuec Number of structures calculated Number of structures used Distance violations/structure ˚ 0.1–0.2 A ˚ 0.2–0.5 A ˚ O0.5 A ˚) r.m.s.d. of distance violation/constraint (A ˚) Maximum distance violation (A Dihedral angle violations/structure 0–108 O108 r.m.s.d. of dihedral angle violation/constraint (8) Maximum dihedral angle violation (8) ˚) Average r.m.s.d from the average structure (A Backbone atoms (N, Ca , C 0 ) Heavy atoms Ramachandran plot summary from PROCHECK34 Most favored regions (%) Additionally allowed regions (%) Generously allowed regions (%) Disallowed regions (%)

94 89 6 148 184 255 608 5.4 10 133 66 67 78 22 829 7.7 2.6 25 20 0.1 0 0 0.004 0.12 0.9 0 0.01 0.66 Residues 26–123 0.72 1.19

Ordered residuesd 0.59 1.06

78 18 3 0

84.3 14.5 0 0

a C Lys NHC 3 , Arg NH2, Cys SH, Ser/Thr/Tyr OH, Pro N, N-terminal NH3 , C-terminal carbonyl, side-chain carbonyl and aromatic non-protonated carbon atoms were not considered to be routinely NMR assignable resonances. b An additional 106 non-conformationally restricting constraints were used in the calculation. c For 108 residues with conformationally restricting NOEs, 20–99, 101–128. d Ordered residues ranges: 27–34, 40–47, 51–63, 66–87, 90–101, 105–124, with the sum of f and j order parameters O1.8.78

572 Zn ligation, and corresponding to w7.7 constraints (w2.6 long-range constraints) per residue. The resulting ensemble of 20 NMR structures (Figure 3(a)) satisfy essentially all of these distance and dihedral constraints (Table 1), and exhibit r.m.s.d values for well-defined regions ˚ for backbone atoms and 1.06 A ˚ for all of 0.59 A heavy atoms. Secondary structural elements (with the exception of helix a1) are well defined, while other regions are disordered in the superimposed ensemble of structures. PROCHECK34 analysis indicates that 100% of well-defined residues have backbone f,j dihedral angles in most-favored or additionally allowed regions of the Ramachandran map. Hinf IscU has an aCb tertiary fold, with three antiparallel b-strands and four a-helices in a compact globular structure (Figure 3(a)–(d)). In addition, the protein exhibits NOEs characteristic of a nascent helix (residues D21–N26) located N-terminal to the globular fold, referred to as helix a1. Secondary structural elements are ordered a1-b1-b2-b3-a2-a3-a4-a5 along the sequence. The core structure has an a–b sandwich architecture with helices packed against a b-sheet (Figure 3). Helices a2 (residues I67–V77) and a5 (residues H105–K124) are packed against the three-stranded antiparallel b-sheet (b1, residues V27–A34; b2, residues D39–V47; b3, residues I53–T60). Helix a2 is antiparallel to strand b3, and the longer helix a5 is

Solution NMR Structure of IscU

antiparallel to strand b1. Two short helices a3 (residues L82–A85) and a4 (residues N90–L97) are packed against helices a2 and a5. The end of helix a2, nearest the Zn2C-binding site, may be longer than it appears in the calculated structures, as line broadening of resonances in residues G64–S70 prevents an accurate structure determination in this region of the protein structure. However, residues Ser65 and Ala66 have 13 Ca chemical shifts consistent with helical conformation, and helical backbone torsion angles in the calculated structures, suggesting that they are also part of helix a2. Resonances of Gly64 could not be assigned. Residues V40–Q42, T60–L72, and L99–L109, which are localized in or near the Zn2C-binding site, had very weak or missing 1H–15N-HSQC cross-peaks and very few, weak amide NOEs, suggesting exchange broadening due to motions on an intermediate time scale. These regions include the b3-a2 and a4-a5 loops, the N-terminal portions of helices a2 and a5, and a few residues in strand b2 that are near the mobile helical regions. The nascent helix, a1, is located on the opposite side of the b-sheet from the other helices (Figure 3). The N-terminal 20 residues of Hinf IscU are unstructured under the conditions of this study, and the C-terminal segment includes the unstructured HexaHis tag. The protein has a compact hydrophobic core that is composed of side-chains from all of the secondary structural elements, excluding a1.

Figure 3. (a) Backbone atoms of 20 NMR structures of Hinf IscU optimally superimposed with respect to the average backbone coordinates (N, Ca , C 0 , and O). (b) Schematic diagram with Zn2C coordinating residues indicated, C37, C63, H105, and C106. (c) Ribbon diagram representation of Hinf IscU structure residues 21–128. Active site residues believed to play a role in the coordination of Zn2C are shown in green. (d) Ribbon diagram of zinc-binding region end view. Figures were generated by the program MOLMOL.35 The b-sheet is colored blue, helices a2 and a5 are red, and helices a3 and a4 are yellow. Nascent helix a1 is cyan.

Solution NMR Structure of IscU

All four core-structure helices of IscU are amphipathic, with hydrophobic side-chains on the inside of the protein. Residues that have less than 10% average solvent-exposed surface area for the ensemble as predicted by MOLMOL35 are indicated by the symbol I in Figure 1. The N and C termini are located at one side of the IscU structure, opposite the side with the three conserved cysteine residues and three solvent-exposed loops that connect secondary structural elements (residues P35–G38, Y61–S65, and L99–V104). Conserved cysteine residue Cys37 is located in the b-hairpin turn between sheets b1 and b2, residue Cys63 is in the loop between b3 and a2, and residue Cys106 is near the N-terminal end of helix a5. The side-chain of residue Cys37 is largely solvent-accessible and on the surface of IscU, while the side-chain of residue Cys106 is mostly buried inside the structure. In a view of IscU facing the Zn-binding site, the protein appears to be barrel-shaped with a tunnel leading to the hydrophobic center of the protein (Figure 3(d)). Although we have identified the residues coordinating Zn2C in Hinf IscU, other residues in this region may also (or alternatively) be involved in coordinating Fe2C, Fe3C, or different forms of [Fe–S] clusters. The side-chains of residues Asp39, Ser65, and Lys103 are close to the Zn-binding site, and are therefore potential metal-binding ligands. In particular, the side-chain amino group of Lys103 is near the zinc-binding site in all the calculated structures. Positively charged lysine amino groups can be part of the outer-sphere coordination of zinc. The lysine amino group can also form a hydrogen bond to the cysteine thiolate of one of the Cys ligands to help balance the negative charge.36,37 Our results suggest that residue Lys103 is close enough to perform this function and maintain the overall charge balance. In

573 addition, Lys103 is conserved as Lys or Arg across the IscU family (Figure 1), further supporting a possible functional role for this residue. Apo Hinf IscU is less structured than Zn-bound IscU Addition of 10 mM EDTA to the Hinf IscU NMR sample, which removes the bound Zn2C, results in the disappearance or reduced intensity of many 1 H–15N HSQC cross-peaks (Figure 4). The resulting spectrum has less chemical shift dispersion in the HN dimension, indicating that the protein is destabilized by the removal of the zinc ion. Addition of EDTA to 20 mM resulted in no further change of the 1 H–15N HSQC spectrum, and remained unchanged several weeks later with the apo form remaining soluble. Figure 5 shows that the regions of Hinf IscU affected by addition of EDTA cover a large portion of the protein. Notably, 1 H–15N HSQC peaks from the backbone amides of residues Cys37, Cys63, and Cys106 disappear altogether, as do many other nearby residues. Helices a1, a3 and a4, which are furthest from the Zn-binding site, were the least affected. Missing or weakened cross-peaks were found for most residues in the b-sheet and in helices a2 and a5. These results indicate that IscU becomes less structured upon conversion from the Zn-bound form to the apo form. Peak linewidths in the 1H-15N HSQC spectrum after addition of EDTA remain unchanged, suggesting that the Hinf IscU remains primarily monomeric in the apo form. Interestingly, zinc-bound IscU could be reconstituted from the apo IscU, after dialysis to remove EDTA and addition of zinc to the buffer.

Figure 4. 1H–15N HSQC spectra of (a) Zn-bound [U-15N; U-13C]IscU (0.6 mM) in 20 mM Mes (pH 6.5), 100 mM NaCl, 10 mM DTT, and 0.02% NaN3, and (b) after addition of EDTA to 10 mM. Both spectra were recorded at 20 8C and 600 MHz. Cys37, Cys63, and C106, are labeled in (a) and are missing in (b).

574

Solution NMR Structure of IscU

terminus of a2 (A66–S71), and the N terminus of a5 (H105–S107) shown in Figure 6(b). The three active-site cysteine residues and H105 are conserved in all cases. All four potential non-cysteinyl ligands for binding to iron and [Fe–S] clusters are highly conserved (D39, S65, K103, and H105). The electrostatic surface potential has a conserved basic (K103) and acidic (D39) area in proximity to the active-site cysteine residues (Figure 6(d) and (f)). Many of the Gly residues were conserved, which is likely due to their role in structured turns at the ends of secondary structural elements. The least conserved residues are found in the b2-b3 loop and in helices a3 and a4. Some residues in the unstructured N terminus are also conserved across the domain family (Figure 6(c)). Secondary structure prediction algorithms (PSIPRED,40 PHD41) predict residues 4–11 to be helical. Taken together, these observations suggest that the disordered N-terminal segment of IscU (particularly residues 4–11) may have functional significance, perhaps becoming structurally ordered and clustered only in the presence of a binding partner like IscS.

Structural similarity analyses

Figure 5. IscU ribbon of residues 21–128 color-coded with information regarding 1H–15N-HSQC cross-peaks that disappear or shift (orange) or weaken (yellow) upon addition of EDTA. The ribbon is colored blue for 1H–15NHSQC cross-peaks that show no change in structural environment upon metal chelation, and gray for those for which data are not available even in the absence of EDTA (including proline residues).

ConSurf analysis of IscU protein domain family The family of IscU homologs in TIGRFAM† includes 42 homologs from a variety of different organisms from all three superkingdoms of life. A phylogenetic analysis of these homologs was performed with the program ConSurf,38,39 which evaluates the degree of conservation of each amino acid and maps this to the tertiary structure to identify surface clusters of conserved residues. These results mapped onto the structure of Hinf IscU are shown in Figure 6. Conserved residues (dark pink, Figure 6(c) and (e)) form a cluster near the Zn-binding site. Regions contributing to the conserved surface patch in the IscU domain family include the loop connecting b1 and b2 (P35–G38), the b3-a2 turn (Y61–S65), the a4-a5 loop (L99– K103), the C terminus of b3 (F58–T60), the N † www.tigr.org

A 3D structure search using the programs Dali,42 SKAN,43 and CE44 found structural similarity of Hinf IscU (average structure, residues 21–126) to domains in several large multi-domain proteins, although none of the matches have high sequence identity to Hinf IscU. Most of the Dali hits with Z-score O3.0 belong to the fold designated the “CO dehydrogenase flavoprotein C-domain-like” fold in the SCOP database.45,46 This fold has an a–b sandwich topology (three or four b-strands and three a-helices) with the first and last helix packed against the three or four-stranded b-sheet. No evidence for metal binding or conserved cysteine or histidine residues is observed in any of the Dali hits. In addition to this CO-dehydrogenase domain, present in several flavoproteins, IscU is structurally similar to the E. coli SufE and YgdK proteins (pairwise Dali Z-scores 5.4 and 6.4), whose 3D structures were also determined from the Northeast Structural Genomics Consortium (PDB codes 1MGZ and 1NI7, respectively). The structure of SufE (NESG target ER30) is presented in the accompanying paper,79 and YgdK (NESG target ER75) was determined by Szyperski and colleagues (unpublished results). While there is minimal sequence identity (!10%) between SufE/YgdK proteins and IscU, these three proteins exhibit substantial structural similarity (the Ca r.m.s.d. ˚ for 88 between Ecol SufE and Hinf IscU is 3.5 A ˚ structurally aligned residues and 3.0 A for 90 structurally aligned residues in YgdK, from Dali alignment), suggesting that there is a distant evolutionary relationship between the protein domain families. The structural alignment includes residues in all structural elements of IscU, SufE and YgdK, with the exception of the nascent helix in

Solution NMR Structure of IscU

575

Figure 6. (a) Ribbon representation of the backbone structure of IscU residues 1–126. (b) Surface of IscU structure with Zn2C in purple, Zn-coordinating side-chains in green, residues from the b1-b2, b3-a2, a3-a4 loops are shown in blue, red, and yellow, respectively. (c) ConSurf image, in the same orientation as above, for the IscU domain family. (d) Electrostatic surface potential prepared with MOLMOL.35 (e) ConSurf and (f) electrostatic surfaces rotated 1808 about the y-axis relative to the images shown in (c) and (d).

IscU and the two N-terminal helices of SufE and YgdK (Figure 7). This will be referred to as the core structure of IscU and SufE.

Discussion Zn2C binding may be similar to Fe2C binding Although the zinc-bound form of Hinf IscU was purified from E. coli cell extracts containing both zinc and iron ions, it remains to be determined whether this form is physiologically relevant. Zinc binding to IscU is favored over iron binding under the conditions of our study. Iron binding to Tm IscU was characterized under anaerobic conditions in

the absence of zinc, suggesting that these conditions may be necessary for iron binding to IscU, in general.17 It has previously been observed for at least a few iron-binding proteins that a stable Zn2C derivative can be obtained that is more tightly bound than the iron-bound form.47,48 These zincsubstituted proteins have essentially the same structure as native iron-bound proteins. It has also been demonstrated for the [Fe–S] cluster binding protein, ferredoxin, that Zn2C can be incorporated into these clusters.49 Therefore, the possibility that zinc-ligation of IscU plays a physiological role cannot be ruled out. Based on the structural similarity with SufE and YgdK, the zinc-bound IscU structure appears to be a good structural model for a stabilized form of IscU. The Hinf IscU

576

Solution NMR Structure of IscU

Figure 7. Ribbon representations with conserved surfaces facing out (08 orientation) of (a) Hinf IscU structure (residues 26–126 shown, transient helix a1 not shown), (d) E. coli SufE and (g) E. coli YgdK. ConSurf images in same orientation as above for (b) IscU, (e) SufE, and (h) YgdK. ConSurf images of (c) IscU (f) SufE and (i) YgdK rotated 1808 about the y-axis relative to the images shown in (b), (e) and (h), respectively. The sequences used in the ConSurf analysis of SufE and YgdK were obtained using PsiBlast59 for three iterations to achieve convergence and aligned with ClustalX.78 The SufE and YgdK sequence families contain 31 and 32 sequences, respectively, and only three sequences that are not found in both families.

structure characterized here may be the same as an iron-bound form, perhaps as it is stabilized by chaperones, and may demonstrate structural features of the [Fe–S] cluster bound form. Preliminary experiments to incorporate iron into Hinf IscU by titration of Fe2C to the zinc-bound form or to the apo form after dialysis to remove the EDTA were unsuccessful. These reactions were performed under aerobic conditions and at the specific pH, and buffer conditions used for the NMR study. Incorporation of iron ions may be possible under different buffer conditions or under anaerobic conditions. Additionally, it may require donation of iron from a holo donor, such as frataxin, for incorporation, or stabilization via protein– protein interactions, such as from the chaperone proteins HscA and HscB. Comparison with the molten-globule structure of T. maritima IscU Efforts to characterize the solution structure of Tm IscU are challenged by extensive internal dynamics and molten-globule behavior.16,26,27 However, some secondary-structure elements in the molten globule-like apo Tm IscU dimer (Asp40Ala mutant) were identified.26 These local structures are similar to the corresponding secondary structural elements of the Zinc-bound Hinf IscU monomer structure reported here. However, in the case of both the apo and holo dimeric Tm IscU samples, the packing of secondary structural elements and the overall fold of the protein could not be determined. Peak doubling and missing resonances for many protons in aliphatic residues indicated conformational exchange and motion on an intermediate time-scale.26 Further characterization suggests that Tm IscU structure samples multiple discrete conformations on a slow time-

scale.27 The main differences between the secondary structures of Tm and Hinf IscU include an N-terminal helix in Tm IscU that does not align with the N-terminal nascent helix a1 observed in Hinf IscU, and an additional sixth helix in the region of the Tm IscU sequence where sequence alignment indicates an w18 residue insertion (Figure 1). Although some regions of the Hinf IscU protein are disordered, in contrast to Tm IscU, most of the Hinf IscU protein is sufficiently structured under the conditions of our study to allow 3D structure characterization by NMR. Comparison of 15N dynamics between the two homologs is complicated by either the small amount of oligomerization present in the Hinf IscU sample or the presence of non-specific aggregation. We measured a mean value of 17(G3) sK1 for the relaxation parameter R2 fit to a singleexponential decay for each HN cross-peak (data not shown), which is much larger than those expected for monomeric IscU.26 Adinolfi et al. have reported mean R1 and R2 values of 1.6 and 12.3 sK1, respectively for monomeric E. coli IscU.50 Dilution of a Hinf IscU sample, as well as addition of 5% glycerol, result in decreasing linewidths of HN cross-peaks in a 1H–15 N HSQC spectrum, suggesting that an equilibrium can be shifted towards monomer by these perturbations (data not shown). These results suggest that the 15N dynamics measured for Hinf IscU under our NMR conditions are influenced by a fast equilibrium with a specific higher-order oligomer and/or nonspecific aggregation and do not provide representative dynamics for the monomeric form of IscU. Binding to [Fe–S] clusters and future studies It is likely that Hinf IscU will bind [Fe–S] clusters as a dimer, as has been shown for other prokaryotic

577

Solution NMR Structure of IscU

IscU homologs.11,13 The structure of IscU in its dimeric apo or holo forms remains to be determined. It also remains to be determined which IscU side-chains ligate the iron in either a ferrous or ferric ion-bound form, and which ligate the different [Fe–S] cluster forms. Raman studies of the A. vinelandii IscU homodimer coordinating one [2Fe–2S]2C cluster indicate that at least one noncysteinyl residue is involved in [Fe–S] cluster ligation.14 Considering that there are only three cysteine residues in the IscU sequence, coordination of two [2Fe–2S]2C clusters per dimer must include non-cysteinyl ligation. However, ligation of the different cluster forms may involve ligand swapping as the [Fe–S] cluster changes during assembly. Our structural studies find several potential ironcoordinating ligands near the zinc-binding site, but do not provide certain identification of iron ligands for these [Fe–S] cluster/IscU complexes. Whereas Hinf IscU ligates zinc with three Cys and one His, other prokaryotic IscU homologs, such as Tm IscU and IscU from Bacillus subtilis, do not have a conserved histidine residue. Therefore, in order to form a four coordinated zinc or iron-bound form, ligation needs to involve another nearby side-chain or even a water molecule as the fourth ligand. Studies of prokaryotic IscU and other Isc proteins are relevant to higher organisms because of the high level of conservation and importance of this system for [Fe–S] cluster assembly. Higher organisms have homologs of IscU, as well as homologs of IscS, HscA, and HscB. Homologs of IscU in yeast are found in the mitochondria and are essential for cellular function.53,54 However, unlike prokaryotic IscU, human D37A-IscU (Asp37Ala mutant) can bind to one [2Fe–2S] cluster as a monomer.55 Several eukaryotic homologs, including IscU from S. pombe, H. sapiens, Mus musculus, and C. elegans have an additional cysteine residue that could coordinate to the cluster; all four of these cysteine residues are necessary for cluster formation in human D37A-IscU.55 However, residue His105 (Hinf IscU numbering) is also conserved in these sequences, suggesting a potential role for histidine ligation in [Fe–S] cluster synthesis even in systems with additional cysteine ligands. Implications of IscU structure in understanding IscU–IscS interactions Although several binding protein partners for IscU are known, aside from the specific cysteine residues of IscU that are involved in binding little is known about the surface that mediates its interactions with these other proteins. Residue Cys63 is essential for formation of a covalent IscU homodimer.13 It is also involved in forming an intermolecular disulfide-bond in the a2b2 heterotetrameric complex with IscS,13 although it is not required for complex formation as the Cys35Ser mutant of IscU (C63S-IscU) also forms a noncovalent a2b2 heterotetrameric complex with IscS.13 In an analysis of a recently determined crystal

structure of T. maritima IscS, it was noted that Cys328, the residue that forms a Cys-persulfide intermediate, is located in a mobile surface loop that is proposed to undergo significant structural rearrangement in order to move between the pyroxidal phosphate cofactor and the solventaccessible location.54 In our IscU structure, residue Cys63, which receives the persulfide from IscS,13 is located in a solvent-exposed loop that is easily accessible for the sulfur transfer reaction or disulfide bond formation. However, in our structure Cys63 is coordinated to a zinc ion. If an iron-loaded form of IscU is the precursor to [Fe–S] cluster assembly as was suggested by Nuth et al.,17 then the previously mentioned results involving Cys63 covalent bonds to IscS or in the IscU dimer may be artifactual. It is unknown whether covalent disulfide bonds are involved in the in vivo [Fe–S] cluster assembly reactions. In vitro IscS-mediated cluster formation can proceed under reducing conditions (typically O4 mM DTT or 2-mercaptoethanol) in an anaerobic atmosphere, further suggesting that covalent disulfide bonds are not obligatory.11,14,17,18,50 IscU interactions with other proteins IscU also interacts with two chaperone proteins HscA and HscB (also known as Hsc66 and Hsc20, respectively), encoded by the genes hscA and hscB, which are located immediately downstream from the iscSUA gene cluster.1,2 Both HscA and HscB interact with IscU, resulting in stimulation of the ATPase activity of these chaperones.55,56 HscA interacts with the LPPVK motif (residues 99–103) of IscU,56,57 whereas HscB requires Cys37 and Cys63.2 The interaction and stimulation of HscA by IscU is greatly enhanced by the co-chaperone HscB, which directly binds to both HscA and IscU.55,56 The sequence motif LPPVK, which is almost invariant among IscU homologs, is located in a surface-exposed loop between helices a4 and a5, near the [Fe–S] cluster-binding site in our structure of the Zn-bound Hinf IscU. This loop had broad lines and few NOEs, indicative of structural mobility, and consistent with the observation that HscA interacts with peptides containing this sequence motif.56 IscU interacts with and stimulates HscA (with or without HscB) in both the apo and the [2Fe–2S]2C-bound state.57 For this reason, it has been suggested that these chaperones help to stabilize the IscU scaffold, and may not be involved directly in [Fe–S] cluster assembly for the transfer of the cluster to the apoprotein targets.58 These chaperones may stabilize the dimeric form of IscU containing the [Fe–S] cluster. Relationships between IscU/NifU and SufE/YgdK domain families A protein–protein BLAST60 analysis seeded with Hinf IscU identified 144 sequences with an E-value less than 10K03 (O25% sequence identity). These sequences, which include the IscU domains from

578 many organisms as well as NifU N-terminal domain sequences from nitrogen-fixing bacteria, were analyzed using the program ConSurf38,39 and mapped onto the structure of Hinf IscU (Figure 7(b) and (c)). The larger NifU/IscU family used in this analysis has a smaller conserved surface cluster than the one previously shown for the IscU TIGRFAM homologs in Figure 6(c), but is located in the same region of the protein structure (Figure 7(b)). The conserved residues still include the three active-site cysteine residues and residues in the three loops at the [Fe–S] cluster-binding site; however, less total residues are conserved in this surface patch. The larger conserved surface patch seen for the IscU family may include residues important for protein–protein interactions such as with IscS or a second IscU protein. The much smaller conserved surface seen for the combined IscU/NifU family suggests that although the activesite residues are conserved, the set of protein interactions are different. The NifU N-terminal domain interactions with NifS, NifU in the homodimer, or other proteins probably involves different residues than the corresponding IscU interaction and therefore, residues involved in these interactions are not conserved. Even though IscU and NifU N-terminal domains have different functional interactions and are found in different gene clusters, the sequence similarity and the conserved active site strongly support the view that these proteins are homologous (derived from a common ancestor). The 3D core structure of IscU is similar to the core structures of both SufE and YgdK (Figure 7). Analysis of the conserved residues within the IscU, SufE, and YgdK families, in the context of the 3D structure, reveals that all three proteins contain a similar cluster of conserved surface residues in structurally equivalent locations. Figures generated by ConSurf38,39 are shown in Figure 7. This conserved patch includes the active-site Cys51 of SufE and the corresponding Cys61 in YgdK, as well as a conserved acidic and basic residue in close proximity. Nearby residues that make up the conserved surfaces in the SufE/YgdK families may confer specificity for binding with their corresponding cysteine desulfurase or other ligands. The conserved surface patch is larger than the patch seen for the IscU/NifU family, and may reflect the binding interface between SufE and YgdK and their corresponding cysteine desulfurase. It is interesting to note that in SufE and YgdK, the conserved surface is expanded due to the presence of two N-terminal helices not present in IscU, and that conserved residues in the a3 helix of SufE and YgdK (which corresponds structurally to helix a2 in IscU) form hydrophobic contacts with one of these N-terminal helices. The presence of similarly conserved and surface-accessible residues in the a2 helix of IscU may indicate a region involved in mediating protein–protein interactions. This analysis supports the hypothesis that conserved residues in the flexible N-terminal segment of IscU (which is predicted to be helical) may become ordered upon ligand binding

Solution NMR Structure of IscU

and pack against conserved residues of helix a2, in a manner structurally similar to that observed in SufE and YgdK. There is a high level of structural similarity yet minimal sequence identity (!10% based on a structure-based sequence alignment), between the core structures of IscU and SufE or YgdK. The sequence conservation within each family reveals that both the IscU/NifU and SufE/YgdK families maintain a common conserved surface in structurally and functionally similar regions. These proteins have a common role in accepting sulfur from their corresponding cysteine desulfurase and are all implicated in [Fe–S] cluster assembly. Furthermore, the active sites are located in structurally equivalent positions. Taken together, these data support the view that the IscU and SufE/YgdK protein families are indeed homologous, with common evolutionary origins that can be detected by similarities in genomic context, 3D structure, conserved surface features, and roles in [Fe–S] cluster-assembly processes, but which cannot be recognized from their very limited sequence similarity.

Materials and Methods Sample preparation IscU from H. influenzae was produced for structure analysis as target IR24 of the Northeast Structural Genomics Consortium†. The iscU gene from H. influenzae (NIFU_HAEIN) was PCR amplified from genomic DNA and cloned into a pET21d (Novagen) derivative, generating plasmid pIR24-21. The resulting construct codes for the full length protein (126 residues), with an additional eight non-native residues (residues LEHHHHHH). This construct sequence was verified by standard DNA sequence analysis in both directions. E. coli BL21 (DE3) pMGK cells, a rare codon enhanced strain, were transformed with pIR24-21. A single isolate was cultured in MJ9 minimal medium (25 mg/l of kanamycin)28 containing 100% 15 N-enriched ammonium sulfate and unenriched glucose or either 5% or 100% uniformly 13 C-enriched glucose, as the sole nitrogen and carbon sources, respectively. MJ9 medium is fortified with metal ions and vitamins compared to conventional M9 medium, and includes 8.3 mM ZnSO4 and 59.9 mM FeCl3. Initial growth was carried out at 37 8C until the A600 nm of the culture reached 0.6–0.9 unit. The incubation temperature was then decreased to 17 8C and protein expression was induced by the addition of IPTG (isopropyl-b-D-thiogalactopyranoside) at a final concentration of 1 mM. Following overnight incubation at 17 8C, the cells were harvested by centrifugation. IscU protein samples were purified using standard protocols. Cell pellets were resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 5 mM b-mercaptoethanol (pH 8.0)) and disrupted by sonication. The resulting lysate was clarified by centrifugation at 26,000g for 45 minutes at 4 8C. The supernatant was loaded onto a Ni-NTA column (Qiagen), washed with lysis buffer containing 20 mM imidazole, and eluted in lysis buffer containing 250 mM imidazole. Fractions † www.nesg.org

Solution NMR Structure of IscU

containing partially purified IscU were pooled and loaded onto a gel-filtration column (HiLoad 26/60 Superdex 75 PG, Amersham Biosciences), and eluted in 10 mM Tris, 5 mM DTT at pH 7.5. Chromatograms revealed approximately 40% monomer, 30% dimer, along with 30% uncharacterized higher-order aggregates, although these numbers are rough due to the lack of baseline resolution in the chromatogram. Fractions corresponding to the monomeric form of [U-15 N], [U-15N; U-13C], or [U-15N; 5%-13C]IscU were collected and concentrated by ultracentrifugation (Amicon) to 0.6–0.8 mM IscU in 20 mM Mes, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02% (w/v) NaN3 at pH 6.5, containing 5% (v/v) 2H2O, and placed in 5 mm Shigemi susceptibility-matched NMR tubes. The standard buffer used in screening proteins with NMR and prioritizing them for structural analysis in the Northeast Structural Genomics Consortium includes 5 mM CaCl2. For some proteins, this buffer provides better spectra than buffer lacking calcium. The IscU spectra, however, are not sensitive to calcium. Removal of CaCl2 by dialysis into NMR buffer without CaCl2 had no effect on the 2D 1H-15N HSQC spectrum, indicating that the CaCl2 was unnecessary. The buffer was not optimized further to determine the importance of other components. Sample purity (O97%) and molecular mass were verified by SDS-PAGE (w15 kDa) and MALDI-TOF (15.1 kDa) mass spectrometry (Applied Biosystems DE-PRO), respectively. The yield of purified monomeric IscU was approximately 20 mg/l. Metal analysis After concentration in the NMR buffer, 50 ml of the [U-15N]IscU NMR sample (at 0.65(G0.05) mM protein concentration) was analyzed by ICP-MS (Agilent Technologies 4500 ICP-MS). Addition of 10 mM EDTA and filtration of the buffer by ultracentrifugation (Amicon Microcon 3) disassociated zinc from the protein. ICP-MS analysis of the filtrate measured w0.5 mM of zinc that was not present in the buffer prior to EDTA addition and filtration (!0.01 mM zinc). Dialysis of a [U-15N]IscU sample into 20 mM Mes (pH 6.5), 100 mM NaCl, 0.1 mM ZnOAc, 10 mM DTT, 0.02% NaN3 for five days reconstituted the zinc-bound Hinf IscU. Oligomer distribution analysis Oligomerization distributions were analyzed by analytical gel-filtration followed by static light-scattering detection.60,61 In this system, analytical gel-filtration was carried out using an Agilent 1100 liquid chromatography system with a Shodex Protein KW-802.5 size-exclusion column. The effluent was then detected using (i) static light-scattering at three angles (458, 908, and 1358) using a using a miniDawn static light-scattering system (Wyatt Technology), (ii) absorbance at 280 nm, and (iii) refractive index using an Optilab Interferometric Refractometer (Wyatt Technology). Analysis of these data provides estimates of shape-independent weight-average molecular mass (MW w) and characteristics of the biopolymer mass distributions.60,61 Hinf IscU NMR spectroscopy, processing and resonance assignments NMR spectra were recorded at 20 8C on 600 MHz, 750 MHz, and 800 MHz Varian Inova spectrometers. Both 2D 1H-15N HSQC and 1H-13C HSQC spectra recorded on [U-15N; U-13C]IscU exhibit good chemical shift dispersion

579 and resolution. Backbone and side-chain assignments were made using the following triple resonance experiments recorded on [U-15N; U-13C]IscU: 3D HNCO, HNCACB, CBCA(CO)NH, HNHA, HCCH-TOCSY, H(CC)(CO)NH-TOCSY, and (H)CC(CO)NH-TOCSY.62–64 NOE constraints were derived from 3D 15N-edited NOESY-HSQC (tmZ120 ms), 13C-edited NOESY-HSQC centered on the aliphatic or aromatic regions (tmZ100 ms), and 4D CC-NOESY (2H2O, tmZ80 ms) experiments.62,65,66 Coupling constants 3 JðHN –Ha Þ were obtained from the HNHA experiment.64 A 2D 1H–13C HSQC and a 1H–15N HMQC spectrum centered on the aromatic regions were used to assign aromatic chemical shifts and to identify the histidine ligand for Zn2C.32 Stereospecific assignments of isopropyl methyl groups of Val and Leu residues were determined by stereospecific biosynthetic isotope incorporation67 using a 1H–13C HSQC spectrum of the [U-15N; 5% 13C]IscU sample. Slowly exchanging amide protons were identified from a time-course of 2D 1H–15N HSQC spectra recorded at 10, 20, 40, and 60 minutes, and two, three, and 18 hours after a lyophilized sample was redissolved in 2H2O. Translational diffusion coefficients (D) were measured using the water-sLED experiment29 An array of 31 spectra (32 scans each, 5 s recycle delay) at gradient strengths from 2 to 33 Gauss/cm with 118 ms between pulsed field gradient pulses. Calibration of the magnetic field gradient was determined using the Varian program “profile” (gzcalZ 2.085). The D value for the ubiquitin standard was first measured and found to be within 0.1!10K7 cm2/s of the literature value (14.9!10K7 cm2/s at 25 8C).29 The 15N transverse relaxation rates, R2, measurements were recorded on a [U-15N]IscU sample using a typical Carr–Purcell–Meiboom–Gill (CPMG) containing pulse sequence,68,69 with relaxation delays of 10, 30, 50, 70, 90, 110, 130, 150, and 170 ms. R2 rate constants were obtained from the cross-peak heights in the 1N–15N correlation spectra by fitting to a single-exponential decay. Spectra were processed with Felix (MSI) and analyzed with SPARKY (SGI65 version)†. SPARKY was used to peak pick the spectra and to generate NOE peak lists with intensities for each peak. Dihedral angle constraints for f and j (G408 and G508, respectively) dihedral angles were derived from chemical shift data using the program TALOS.70 NOE peak lists, chemical shift data, 3 JðHN –Ha Þ values, and a list of residues with slowly exchanging amide protons (i.e. those observed after one hour in 2H2O at pH* 6.5 and temperature of 20 8C) were used by the program AutoStructure 1.1.2 to generate the preliminary conformational restraints.71,72 AutoStructure uses either DYANA or XPLOR to generate protein NMR structures.73,74 The preliminary structures calculated for this work were generated with DYANA-1.5 and a typical AutoStructure calculation, providing an ensemble of 20 structures, required 150 minutes on a 14-node 450 MHz Linux Pentium III CPU cluster. AutoStructure generates restraints, including dihedral, NOE distance restraints and hydrogen bond restraints. Several additional cycles of calculations were completed after examining the NOE assignments generated by AutoStructure and updating the peak lists to remove noise peaks and add real peaks. This process was continued until the initial structures had consistent folds and the final structures could account for a large percentage (O70%) of the NOEs. NOE assignments were then carefully examined and manually evaluated. Those that were determined to be † Goddard, T. D. & Kneller, D. G. http://cgl.ucsf.edu/ home/sparky. University of California, San Francisco.

580 wrongly assigned, overlapped, or produced by spin diffusion were loosened or removed. Additional NOE cross-peaks were iteratively assigned. Hydrogen bond constraints were added for 39 slowly exchanging amide protons for which an acceptor could be identified from ˚ and dHNZ1.8– preliminary structures (dNOZ2.8–3.3 A ˚ ). When Zn was incorporated into the calculations, 2.3 A ten additional constraints were added to coordinate the Zn to Sg atoms of the three cysteine residues and the N3 of H105 ˚ or 2.0– (four Sg – or N3 –Zn distance constraints of 2.3–2.4 A ˚ , respectively, and six Sg K Sg or Sg –N3 constraints of 2.1 A ˚ to restrain tetrahedral geometry). 3.6–3.85 A

Solution NMR Structure of IscU

3.

4.

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3D structure calculations A total of 829 conformationally restricting constraints (819 for calculations without Zn) (Table 1) were used to generate 25 structures of IscU using the Xplor-NIH software with the Xplor-NIH (v. 2.0.6) routines mkpsf.inp and generate_template.inp, and the standard Xplor-3.84 routine sa.inp.74,75 No pseudoatom corrections were added to the upper bounds because sum averaging was used.76 Ramachandran backbone conformation statistics were analyzed with PROCHECK-NMR.34

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Data bank accession codes The ensemble of 20 lowest-energy structures for structures calculated with and without zinc bound were deposited in the RCSB Protein Data Bank (PDB codes 1R9P and1Q48, respectively) along with a list of NOE, hydrogen bond, ligand constraints, and dihedral constraints in Xplor format. Chemical shift data for 1H, 15 N, and 13C resonances and raw fids were deposited in BioMagResBank (BMRB) under accession code 5842.

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Acknowledgements We thank J. Liu and B. Rost for providing IscU as target IR24 of the Northeast Structural Genomics Consortium, T. W. Weitsma for ICP-MS analysis, and A. Bhattacharya for assistance in validation of the IscU structure. Acquisition and processing of NMR spectra and structure calculations were performed at the Environmental Molecular Sciences Laboratory (a national scientific user facility sponsored by the US DOE Office of Biological and Environmental Research) located at Pacific Northwest National Laboratory and operated by Battelle for the Department of Energy (contract KP130103). This work was supported by the NIH Protein Structure Initiative (grant P50-GM62413) and by NSF grant DBI-9904841 to B.H.

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References 1. Zheng, L., Cash, V. L., Flint, D. H. & Dean, D. R. (1998). Assembly of iron–sulfur clusters. Identification of an iscSUA-hscBA-fdx gene cluster from Azotobacter vinelandii. J. Biol. Chem. 273, 13264–13272. 2. Tokumoto, U., Nomura, S., Minami, Y., Mihara, H., Kato, S., Kurihara, T. et al. (2002). Network of protein–

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Edited by M. F. Summers (Received 5 May 2004; received in revised form 10 August 2004; accepted 11 August 2004)