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• W2163871231 abstract "The fhuD2 gene encodes a lipoprotein that has previously been shown to be important for the utilization of iron(III)-hydroxamates by Staphylococcus aureus. We have studied the function of the FhuD2 protein in greater detail, and demonstrate here that the protein binds several iron(III)-hydroxamates. Mutagenesis of FhuD2 identified several residues that were important for the ability of the protein to function in iron(III)-hydroxamate transport. Several residues, notably Tyr-191, Trp-197, and Glu-202, were found to be critical for ligand binding. Moreover, mutation of two highly conserved glutamate residues, Glu-97 and Glu-231, had no affect on ligand binding, but did impair iron(III)-hydroxamate transport. Interestingly, the transport defect was not equivalent for all iron(III)-hydroxamates. We modeled FhuD2 against the high resolution structures of Escherichia coli FhuD and BtuF, two structurally related proteins, and showed that the three proteins share a similar overall structure. FhuD2 Glu-97 and Glu-231 were positioned on the surface of the N and C domains, respectively. Characterization of E97A, E231A, or E97A/E231A mutants suggests that these residues, along with the ligand itself, play a cumulative role in recognition by the ABC transporter FhuBGC2. In addition, small angle x-ray scattering was used to demonstrate that, in solution, FhuD2 does not undergo a detectable change in conformation upon binding iron(III)-hydroxamates. Therefore, the mechanism of binding and transport of ligands for binding proteins within this family is significantly different from that of other well studied binding protein families, such as that represented by maltose-binding protein. The fhuD2 gene encodes a lipoprotein that has previously been shown to be important for the utilization of iron(III)-hydroxamates by Staphylococcus aureus. We have studied the function of the FhuD2 protein in greater detail, and demonstrate here that the protein binds several iron(III)-hydroxamates. Mutagenesis of FhuD2 identified several residues that were important for the ability of the protein to function in iron(III)-hydroxamate transport. Several residues, notably Tyr-191, Trp-197, and Glu-202, were found to be critical for ligand binding. Moreover, mutation of two highly conserved glutamate residues, Glu-97 and Glu-231, had no affect on ligand binding, but did impair iron(III)-hydroxamate transport. Interestingly, the transport defect was not equivalent for all iron(III)-hydroxamates. We modeled FhuD2 against the high resolution structures of Escherichia coli FhuD and BtuF, two structurally related proteins, and showed that the three proteins share a similar overall structure. FhuD2 Glu-97 and Glu-231 were positioned on the surface of the N and C domains, respectively. Characterization of E97A, E231A, or E97A/E231A mutants suggests that these residues, along with the ligand itself, play a cumulative role in recognition by the ABC transporter FhuBGC2. In addition, small angle x-ray scattering was used to demonstrate that, in solution, FhuD2 does not undergo a detectable change in conformation upon binding iron(III)-hydroxamates. Therefore, the mechanism of binding and transport of ligands for binding proteins within this family is significantly different from that of other well studied binding protein families, such as that represented by maltose-binding protein. Iron, the fourth most abundant element on the earth's crust, is one of the most important micronutrients for almost all forms of life. The majority of that iron, however, exists in a biologically unavailable form as a result of the fact that ferric iron is highly insoluble at a physiological pH. To overcome this iron shortage, bacteria have developed a variety of mechanisms to sequester what little available iron may exist within their environment. In particular, one important iron acquisition strategy is through the production of small molecules, termed siderophores, which have an extremely high affinity for Fe(III) and which can interact with cognate transporters that effectively move Fe(III)-siderophore complexes across the bacterial envelope.ABC transporters are protein conduits that use energy derived from the hydrolysis of ATP to move solutes across the cell membrane. Typically, the basic unit of an ABC transporter is (i) two transmembrane domains that span the membrane usually 6 times each for a total of 12 membrane-spanning domains, (ii) two cytoplasmic ATP-binding proteins that serve to energize the system, and (iii) a binding protein that interacts with the transmembrane domains (1.Higgins C.F. Linton K.J. Science. 2001; 293: 1782-1784Crossref PubMed Scopus (70) Google Scholar). Binding proteins in Gram-negative bacteria are present within the periplasm, whereas those in Gram-positive bacteria are tethered to the cell membrane via the acylation of a cysteine residue that is an integral component of a lipoprotein signal sequence.Solute binding proteins typically exhibit a common structural design in that they contain a cleft or groove region that serves as the ligand binding site, and this ligand binding site is surrounded by two globular domains typically consisting of a central β-sheet surrounded by α-helixes (2.Quiocho F.A. Ledvina P.S. Mol. Microbiol. 1996; 20: 17-25Crossref PubMed Scopus (450) Google Scholar). The canonical binding protein, maltose-binding protein (MBP), 1The abbreviations used are: MBPmaltose-binding proteinAPSAdvanced Photon SourceDESYDeutsches Elektronen-SynchrotronSAXSsmall angle x-ray scattering.1The abbreviations used are: MBPmaltose-binding proteinAPSAdvanced Photon SourceDESYDeutsches Elektronen-SynchrotronSAXSsmall angle x-ray scattering. undergoes a significant structural rearrangement upon ligand binding. Indeed, the closure of the two domains around the ligand has been likened to the active motion of a Venus flytrap. Recent x-ray crystallographic studies have defined a new class of binding proteins that include Escherichia coli FhuD (3.Clarke T.E. Braun V. Winkelmann G. Tari L.W. Vogel H.J. J. Biol. Chem. 2002; 277: 13966-13972Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 4.Clarke T.E. Ku S.Y. Dougan D.R. Vogel H.J. Tari L.W. Nat. Struct. Biol. 2000; 7: 287-291Crossref PubMed Scopus (104) Google Scholar), which binds iron(III)-hydroxamates; E. coli BtuF (5.Karpowich N.K. Huang H.H. Smith P.C. Hunt J.F. J. Biol. Chem. 2003; 278: 8429-8434Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 6.Borths E.L. Locher K.P. Lee A.T. Rees D.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16642-16647Crossref PubMed Scopus (177) Google Scholar), which binds vitamin B12; and Treponema pallidum TroA (7.Lee Y.H. Dorwart M.R. Hazlett K.R. Deka R.K. Norgard M.V. Radolf J.D. Hasemann C.A. J. Bacteriol. 2002; 184: 2300-2304Crossref PubMed Scopus (78) Google Scholar), a zinc-binding protein. These proteins possess an architecture that differs from the sugar- and amino acid-binding proteins (represented by MBP) in that they have adopted a single, more inflexible backbone α-helix that connects two globular domains. This topology is not found within the interdomain connections of group I or group II binding proteins (2.Quiocho F.A. Ledvina P.S. Mol. Microbiol. 1996; 20: 17-25Crossref PubMed Scopus (450) Google Scholar), and, as such, this class of proteins can be placed into a new group. Structural constraints imposed by the backbone α-helix have led to the suggestion that this new class of binding protein does not undergo a significant change in conformation upon ligand binding. Indeed, crystal structures of apo and holo forms of BtuF indicate a relatively minor conformational change upon ligand binding (5.Karpowich N.K. Huang H.H. Smith P.C. Hunt J.F. J. Biol. Chem. 2003; 278: 8429-8434Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). High resolution structures of E. coli FhuD bound to different hydroxamate siderophores have been determined (3.Clarke T.E. Braun V. Winkelmann G. Tari L.W. Vogel H.J. J. Biol. Chem. 2002; 277: 13966-13972Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 4.Clarke T.E. Ku S.Y. Dougan D.R. Vogel H.J. Tari L.W. Nat. Struct. Biol. 2000; 7: 287-291Crossref PubMed Scopus (104) Google Scholar). These structures identify a shallow binding pocket that exists between the two protein domains; siderophores are recognized by the side chains of a few key residues lining the binding pocket. The structures of different hydroxamate siderophores are accommodated in the binding pocket by subtle rearrangements in the positions of the side chains. In a recent study of the high resolution structure of BtuF, Borths et al. (6.Borths E.L. Locher K.P. Lee A.T. Rees D.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16642-16647Crossref PubMed Scopus (177) Google Scholar) highlight two highly conserved glutamate residues that are located on the surface of each of the two lobes of the protein. When they aligned the BtuF structure above the structure of BtuCD, the ABC transporter, it was found that the BtuF glutamates align with the position of positively charged pockets of arginines present on the periplasmic surface of BtuCD. This has led to the suggestion that interprotein Glu-Arg salt bridges might form important docking contacts between the binding protein and the ABC transporter. Notably, both the glutamates and the arginines are conserved in a number of iron(III)-siderophore transport systems in different bacteria (6.Borths E.L. Locher K.P. Lee A.T. Rees D.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16642-16647Crossref PubMed Scopus (177) Google Scholar).Staphylococcus aureus is a Gram-positive human pathogen that possesses an ABC transporter (FhuCBG, FhuD1, FhuD2) for the import of iron(III)-hydroxamates (8.Sebulsky M.T. Heinrichs D.E. J. Bacteriol. 2001; 183: 4994-5000Crossref PubMed Scopus (86) Google Scholar, 9.Sebulsky M.T. Hohnstein D. Hunter M.D. Heinrichs D.E. J. Bacteriol. 2000; 182: 4394-4400Crossref PubMed Scopus (108) Google Scholar). FhuB and FhuG constitute the membrane-spanning components, whereas FhuC is the ATP-binding protein and two acylated proteins, FhuD1 and FhuD2, are thought to serve as the binding proteins for the system. FhuD2 participates in the transport of a wider range of substrates than FhuD1 (8.Sebulsky M.T. Heinrichs D.E. J. Bacteriol. 2001; 183: 4994-5000Crossref PubMed Scopus (86) Google Scholar), and is the subject of the investigations presented herein. FhuD2, representative of a large family of putative iron-binding proteins in Gram-positive bacteria (10.Clarke T.E. Rohrbach M.R. Tari L.W. Vogel H.J. Koster W. Biometals. 2002; 15: 121-131Crossref PubMed Scopus (13) Google Scholar), was used as a model protein to characterize the role of several conserved amino acid residues in iron(III)-hydroxamate transport. We have shown that FhuD2 binds a variety of iron(III)-hydroxamates with differing affinities, possessing significantly higher affinity for iron(III)-ferrichrome and iron(III)-desferrioxamine (used in this study as Desferal™) than the E. coli FhuD protein. Moreover, solution x-ray scattering experiments, which are sensitive to protein conformation, indicated that, in solution, little or no conformational change is associated with ligand binding, the first such demonstration for this class of ligand-binding proteins. This result, in combination with mutations affecting ligand binding and transport, suggests a novel mode of recognition between the binding protein-ligand complex and the ABC transporter (FhuBGC2).EXPERIMENTAL PROCEDURESBacterial Strains and Plasmids—Bacterial strains and plasmids used in this study are listed in Table I.Table IBacterial strains and plasmidsStrain or plasmidDescriptionSource or referenceS. aureus RN2564(80α) ω25[Tn551] pig-131J. Iandolo RN4220rk-mk+30.Kreiswirth B.N. Lofdahl S. Bentley M.J. O'Reilly M. Schlievert P.M. Bergdoll M.S. Novick R.P. Nature. 1983; 305: 680-685Crossref Scopus (1001) Google Scholar RN6390Prophage-cured wild type strain31.Peng H.L. Novick R.P. Kreiswirth B. Kornblum J. Schlievert P. J. Bacteriol. 1988; 170: 4365-4372Crossref PubMed Scopus (409) Google Scholar H431RN6390 fhuD1::Km fhuD2::Tet; KmrTcr8.Sebulsky M.T. Heinrichs D.E. J. Bacteriol. 2001; 183: 4994-5000Crossref PubMed Scopus (86) Google ScholarE. coli DH5αφ80dlacZΔM15 recA1 endA1 gyrA96 thi-1 hsdR17(rk-mk+) supE44 relA1 deoR Δ(lacZYA-argF)U169Promega BL21 (DE3)F-ompT [lon] hsdSB (rB-m-; an E. coli B strain) with a DE3 λ prophage carrying the T7 RNA polymerase gene H584DH5α containing pMTS57; AprThis studyPlasmids pET-28A(+)5.3-kb E. coli expression vector; KmrNovagen pGEX-2T-TEV4.9-kb E. coli expression vector, modified to contain a tobacco etch virus (TEV) protease cleavage sequence; AprF. Sicheri pL1505.2-kb E. coli-S. aureus shuttle vector; AprCmr32.Lee C.Y. Mol. Microbiol. 1992; 6: 1515-1522Crossref PubMed Scopus (31) Google Scholar pMTS37pLI50, containing the S. aureus fhuD2 gene; Apr Cmr8.Sebulsky M.T. Heinrichs D.E. J. Bacteriol. 2001; 183: 4994-5000Crossref PubMed Scopus (86) Google Scholar pMTS57pGEX-2T-TEV, digested with BamHI/EcoRI containing the S. aureus fhuD2 gene minus the first 24 codons; AprThis study pMTS90pET28a(+) carrying the E. coli fhuD gene inserted into the BamHI/EcoRI sites. Expresses FhuD with an N-terminal His6 tag; KmrThis study Open table in a new tab Growth Media and Conditions—Unless otherwise specified, E. coli and S. aureus were routinely cultivated in Luria-Bertani broth (Difco) and tryptic soy broth (Difco), respectively. Solid media were obtained by the addition of 1.5% (w/v) Bacto-agar (Difco). Tris-minimal succinate was the iron-deficient medium used throughout (9.Sebulsky M.T. Hohnstein D. Hunter M.D. Heinrichs D.E. J. Bacteriol. 2000; 182: 4394-4400Crossref PubMed Scopus (108) Google Scholar). FeCl3 (50 μm) was added to Tris-minimal succinate media as required. Where appropriate, tetracycline (2 μg/ml), chloramphenicol (5 μg/ml), and kanamycin (50 μg/ml) were incorporated into media for the cultivation of S. aureus, whereas tetracycline (10 μg/ml), chloramphenicol (30 μg/ml), kanamycin (30 μg/ml), and ampicillin (100 μg/ml) were incorporated into media for the growth of E. coli. Growth of bacterial cultures was performed at 37 °C. Iron-free water was used for all experiments and was obtained by passage through a Milli-Q water filtration system (Millipore Corp.).Siderophores—Ferrichrome and rhodotorulic acid were purchased from Sigma (Mississauga, Ontario, Canada), aerobactin and coprogen were purchased from EMC Microcollections GmbH (Tübingen, Germany), and desferrioxamine, used as Desferal™ (Novartis), was obtained from the London Health Sciences Centre. Enterobactin and pyoverdine were prepared by methods previously described (11.Payne S.M. Methods Enzymol. 1994; 235: 329-344Crossref PubMed Scopus (361) Google Scholar, 12.Meyer J.M. Stintzi A. De Vos D. Cornelis P. Tappe R. Taraz K. Budzikiewicz H. Microbiology. 1997; 143: 35-43Crossref PubMed Scopus (206) Google Scholar).Generation and Selection of fhuD2 Mutants—Plasmid DNA was isolated using QIAprep plasmid spin columns (Qiagen Inc., Santa Clarita, CA) as described by the manufacturer. Site-directed mutations were introduced into the fhuD2 gene, carried on plasmid pMTS37, using the QuikChange™ site-directed mutagenesis kit and procedure from Stratagene. Primers used for generation of site-directed mutants are listed in Table II. Multiple mutations were made progressively by performing mutagenesis using templates and primers with the desired combination of mutations. Once automated DNA sequencing confirmed the mutations, the mutated plasmids were introduced into S. aureus RN4220 by electroporation before being mobilized into S. aureus H431 via transduction. Random mutations were introduced into the fhuD2 gene by treating plasmid pMTS37 (5 μg) with 400 mm hydroxylamine at 72 °C for 2 h, and introducing plasmid thus treated into H431. Plasmids not allowing growth in the presence of 2.5 mm Desferal™ (H431 will not grow on media containing 2.5 mm Desferal™ whereas H431 carrying pMTS37 will grow well under these conditions) were selected for further study.Table IIPrimers used for site-directed mutagenesis of fhuD2Mutation constructedMutagenic primeraSense primer is shown, antisense primer (not shown) is exactly complementary. Nucleotides shown in bold are those that are changed relative to wild type sequence.K48A/R49A5′-AAGACCCTGCAGCCATTGCAGTAG-3′Y57G5′-GTTGCGCCAACAGGAGCTGGTGGACTT-3′E97A5′-GGCGATGTAGCAAAAGTTGCTAAAG-3′E97Q5′-GGCGATGTACAAAAAGTTGCTAAAG-3′D105A5′-AGAAAAGCCAGCTTTAATTATTGTA-3′T125A5′-GTAGCACCAGCAGTAGTTGTTGAC-3′W155A5′-GTAAAAGCTGCGAAGAAAGATTGG-3′Y191A5′-GATAAAAAATTAGCGACTTACGGCGAT-3′W197A5′-TACGGCGATAACGCAGGTCGTGGTGGA-3′E231A5′-GAAGTGAAACAAGCAGAAATTGAAA-3′E231Q5′-GAAGTGAAACAACAAGAAATTGAAA-3′D239A5′-TATGCTGGTGCTTACATTGTGAGT-3′W259A5′-ACAAACATGGCGAAGAATTTGAAA-3′a Sense primer is shown, antisense primer (not shown) is exactly complementary. Nucleotides shown in bold are those that are changed relative to wild type sequence. Open table in a new tab Siderophore Plate Bioassays—Siderophore plate bioassays were performed essentially as previously described (9.Sebulsky M.T. Hohnstein D. Hunter M.D. Heinrichs D.E. J. Bacteriol. 2000; 182: 4394-4400Crossref PubMed Scopus (108) Google Scholar). Siderophores were used at concentrations of 50 μm except for aerobactin (100 μm) and rhodotorulic acid (14 mm). For assessment of mutations, the wild type fhuD2 gene (including its own iron-regulated promoter) and mutated derivatives were expressed from the low copy E. coli/S. aureus shuttle vector pLI50. The recombinant vectors were then introduced into S. aureus H431 (RN6390 fhuD1 fhuD2). Under the conditions of the bioassay, H431 alone does not grow in the presence of the iron(III)-hydroxamates.Expression and Purification of FhuD2 and Derivatives—The fhuD2 gene was PCR-amplified as a 1.1-kb fragment that was cloned into the BamHI/EcoRI sites of pGEX-2T-TEV. The resulting vector, pMTS57, which encodes a glutathione S-transferase-FhuD2 fusion protein (the first 24 amino acid residues of the FhuD2 are not included in the construct), was introduced into E. coli strain DH5α for protein overexpression. Similarly, the fhuD2 gene containing the E97A, E231A, or E97A/E231A mutations was PCR-amplified and cloned into the BamHI/EcoRI sites of pGEX-2T-TEV. Strains expressing fusion proteins were grown to an approximate A600 of 0.8. Following the addition of isopropyl-1-thio-β-d-galactopyranoside (0.4 mm), growth was allowed to continue another 3 h before the cells were lysed. The resulting supernatants were centrifuged at 40,000 × g to remove insolubles and then passed across a 5-ml GSTrap column (Amersham Biosciences) for purification. Fusion proteins were eluted from GSTrap columns and treated with TEV protease (4 h at 23 °C in 50 mm Tris-Cl, pH 8.0, and 10 mm reduced glutathione). FhuD2 and derivatives were purified by ion-exchange chromatography across an 8-ml Mono-S column (Amersham Biosciences), using a linear gradient of NaCl (0–1 m). Protein purity was assessed by SDS-PAGE.Expression and Purification of E. coli fhuD—The E. coli fhuD gene was PCR-amplified on a 850-bp fragment that was cloned into BamHI/ SmaI-digested pGEX-2T-TEV. E. coli fhuD was then removed as a BamHI/EcoRI fragment and cloned into pET28A(+), to produce pMTS90. This construct encodes E. coli FhuD tagged at the N terminus with His6. The recombinant pET28A(+) vector was introduced into E. coli BL21(λ DE3) for protein overexpression. BL21(λ DE3) (pMTS90) cells were grown to an approximate A600 of 0.8. before isopropyl-1-thio-β-d-galactopyranoside (1.0 mm) was added and growth allowed to continue for another 3 h before the cells were lysed. The resulting supernatant was centrifuged at 40,000 × g to remove insoluble material and then passed across a 5-ml HiTrap™ chelating HP column (Amersham Biosciences) for purification. His6 FhuD was eluted from the column with a linear gradient of imidazole (10–500 mm). Protein purity was assessed by SDS-PAGE.Fluorescence Titrations—Fluorescence titration experiments were performed using protein concentrations of 8–16 nm, in 2 ml of 10 mm sodium phosphate buffer, pH 7.5, using a Fluorolog 3 spectrofluorimeter (ISA Instruments). All reactions were performed at 23 °C. The excitation and emission slits were set at 1 and 10 nm, respectively, with excitation and emission wavelengths set at 283 and 348 nm, respectively. Experiments involving the E. coli His6-tagged FhuD were performed as previously described (13.Rohrbach M.R. Braun V. Köster W. J. Bacteriol. 1995; 177: 7186-7193Crossref PubMed Google Scholar). The value for relative minimum fluorescence (Fmin) was obtained using Equation 1.F0-FF0=Fmin[ligand][ligand]+c(Eq. 1) F0 is the starting fluorescence, F is the fluorescence after addition(s) of Fe(III)-siderophores, and c is a constant. The KD was then calculated using the following equation (14.Miller 3rd, D.M. Olson J.S. Pflugrath J.W. Quiocho F.A. J. Biol. Chem. 1983; 258: 13665-13672Abstract Full Text PDF PubMed Google Scholar), with the KD as the single variable.F0-FF0-Fmin=KD+[ligand]+[FhuD2]-(KD+[ligand]+[FhuD2])2-4[ligand][FhuD2]2[FhuD2](Eq. 2) The solver function in Microsoft Excel was used to fit the data to the equations and obtain values for the relevant parameters. All titration experiments were done in triplicate for each ligand.Proteinase K Digests—Samples of FhuD2 (2 μg) were incubated with proteinase K (2 μg added to each reaction) in 30 mm sodium phosphate buffer, pH 7.5, for 30 min in the presence or absence of ferric siderophores at 55 °C. The resulting proteins were resolved on SDS-polyacrylamide as previously described (15.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar).Computer Analysis—DNA sequence analysis, oligonucleotide primer design, and sequence alignments were performed using the Vector NTI Suite software package (Informax, Inc., Bethesda, MD).Small Angle X-ray Scattering—Initial low angle measurements and some high angle measurements were made at BioCAT (beamline 18ID) of the Advanced Photon Source (APS). A second round of experiments was conducted at the European Molecular Biology Laboratory Outstation at the Deutsches Elektronen-Synchrotron (DESY, Hamburg, Germany), beamline X33 (16.Koch M.H.J. Bordas J. Nucl. Instr. Methods. 1983; 208: 461-469Crossref Scopus (287) Google Scholar). Note that Q = 2π sinθ/λ and S = sinθ/λ.To ensure that the protein preparations were free of aggregates, they were subjected to gel filtration chromatography several days prior to small angle x-ray scattering (SAXS) measurements. Protein samples (0.5 ml, 20–25 mg/ml) were applied to a Superdex 200 HR 10/30 gel filtration column (Amersham Biosciences), equilibrated and developed with 100 mm KCl, 20 mm Tris-HCl, 1 mm EDTA, pH 8.5. Samples were then dialyzed against 50 mm Tris-HCl, 100 mm KCl, 10 mm MgCl2, 10 mm β-mercaptoethanol, pH 8.0. The dialysis buffers were reserved for background measurements. For measurements at APS, FhuD2 in complex with iron-loaded siderophores was obtained by adding iron-loaded siderophores directly to the protein solutions, which resulted in a 1.5:1 ratio of ligand:protein. For experiments at DESY, the samples (0.5 ml) were dialyzed against a 50-ml solution containing 20 μm iron-loaded Desferal™.Measurements at BioCAT (beamline 18ID) at APS were made as follows. The sample temperature was 20 °C, and the protein solution was moved continuously through a 1-mm quartz capillary during the course of the measurement to minimize the effects of radiation damage. For each sample, five 10-s exposures were recorded, consisting of three measurements from the protein solution bracketed by two measurements of the buffer solution. Data were integrated using Fit2D (17.Hammersley A.P. Svensson S.O. Hanfland M. Fitch A.N. Häusermann D. High Pressure Res. 1996; 14: 235-248Crossref Google Scholar), and exported into a spreadsheet. The three protein solution curves and two background buffer curves were inspected and averaged, and the background buffer curve was subtracted, with no correction, from the protein solution curve to yield scattering from the hydrated protein. Data were collected at 2779 and 335 mm using a CCD detector, with x-rays at a wavelength of 1.03 Å, to cover the momentum transfer ranges 0.08 < Q < 1.87 nm–1 and 0.7 < Q < 10.5 nm–1, respectively. The overlapping region in the data sets (0.85 < Q < 1.85 nm–1) was used to scale the two curves to produce a composite curve covering the momentum transfer range 0.08 < Q < 10.5 nm–1.SAXS measurements at DESY beamline X33 were carried out as follows. The wavelength of the radiation was 1.5 Å, the path length was 1 mm, and the sample was maintained at a temperature of 14 °C. Low angle measurements were made using a protein concentration of ∼5 mg/ml, and a sample-detector distance of 2650 mm to cover the momentum transfer range 0.03 < S < 0.55 nm–1. Mid- and high angle x-ray scattering measurements were made, simultaneously, using a protein concentration of ∼20 mg/ml, with a detector at 2650 mm and a second detector (offset) at a distance of 1.2 m, to cover the momentum transfer range from 0.3 < S < 1.5 nm–1. Data were processed and analyzed using Sapoko and Otoko (18.Boulin C. Kempf R. Koch M.H.J. McLaughlin S. Nucl. Instr. Methods. 1986; A249: 399-407Crossref Scopus (296) Google Scholar) and Gnom (19.Semenyuk A.V. Svergun D.I. J. Appl. Crystallogr. 1991; 24: 537-540Crossref Scopus (567) Google Scholar) programs. The three scattering curves were scaled and merged using a spreadsheet.Molecular Modeling of S. aureus FhuD2—To model the structure of FhuD2, we began with a structural alignment between E. coli FhuD (4.Clarke T.E. Ku S.Y. Dougan D.R. Vogel H.J. Tari L.W. Nat. Struct. Biol. 2000; 7: 287-291Crossref PubMed Scopus (104) Google Scholar) and BtuF (5.Karpowich N.K. Huang H.H. Smith P.C. Hunt J.F. J. Biol. Chem. 2003; 278: 8429-8434Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 6.Borths E.L. Locher K.P. Lee A.T. Rees D.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16642-16647Crossref PubMed Scopus (177) Google Scholar). The structural alignment was carried out with the program O (20.Jones T.A. Bergdoll M. Kjeldgaard M. Bugg C.F. Ealick S.E. Crystallographic and Modeling Methods in Molecular Design. Springer-Verlag, New York1990: 189-195Google Scholar); an initial superposition of residues 130–166 of FhuD and 111–147 of BtuF (the two long α-helices on the underside of the proteins) was refined using the lsq_improve function in O, matching CA atoms with a cutoff of 3 Å. The final alignment yielded matches for 148 CA atoms throughout the FhuD and BtuF polypeptides, with a root mean square fit of 2.2 Å. The structures diverged mainly in loop regions between secondary structure elements. We aligned the sequences of six iron-siderophore-binding proteins from Gram-positive bacteria (FhuD1 and FhuD2 from S. aureus, and FhuD proteins from Clostridium acetobutylicum, Bacillus halodurans, Streptococcus pyogenes, and Bacillus subtilis) as well as FhuD and BtuF from E. coli. This multiple sequence alignment was manually adjusted using the three-dimensional structural alignment between E. coli FhuD and BtuF proteins as a guide. We reasoned that the FhuD structures from Gram-positive organisms would diverge in the same regions as the E. coli FhuD and BtuF structures, and therefore gaps that had been inserted into structurally conserved regions of E. coli BtuF and FhuD were moved into regions where the two structures diverged. The final alignment is shown in Fig. 1.The folds of E. coli BtuF and FhuD are similar, but we found that SAXS data from FhuD2 showed a better agreement with E. coli FhuD than with BtuF. Therefore, we used the coordinates of E. coli FhuD (4.Clarke T.E. Ku S.Y. Dougan D.R. Vogel H.J. Tari L.W. Nat. Struct. Biol. 2000; 7: 287-291Crossref PubMed Scopus (104) Google Scholar) to model the structure of FhuD2. Based on the alignment in Fig. 1, the sequence of FhuD2 was threaded through the coordinates of E. coli FhuD. This initial model was submitted to Swiss-Model (21.Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9472) Google Scholar) for optimization. Manual adjustments were made to the model using the program O (20.Jones T.A. Bergdoll M. Kjeldgaard M. Bugg C.F. Ealick S.E. Crystallographic and Modeling Methods in Molecular Design. Springer-Verlag, New York1990: 189-195Google Scholar), and it was subjected to one round of conjugant gradient minimization in CNS (22.Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16930) Google Scholar), with harmonically restrained CA positions. The final model has good stereochemistry, with only one residue falling just outside of allowed Ramachandran regions.RESULTSFunctional Characterization of Conserved Residues in FhuD2—Our previous molecular genetic studies showed that the fhuD2 gene product participates in the transport of a variety of iron(III)-hydroxamate complexes in S. aureus (8.Sebulsky M.T. Heinrichs D.E. J. Bacteriol. 2001; 183: 4994-5000Crossref PubMed Scopus (86) Google Scholar). Searches of the data bases, which now include many completed genome sequences, indicate that FhuD2 is representative of a large family of conserved proteins that are present in both Gram-positive and Gram-negative bacteria. Of the homologs in Gram-positive bacte" @default.
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• W2163871231 title "The Role of FhuD2 in Iron(III)-Hydroxamate Transport in Staphylococcus aureus" @default.
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