The Staphylococcus aureus Siderophore Receptor HtsAUndergoes Localized Conformational Changes to EncloseStaphyloferrin A in an Arginine-rich Binding Pocket*□S

Received for publication, December 22, 2009, and in revised form, January 24, 2010 Published, JBC Papers in Press, February 10, 2010, DOI 10.1074/jbc.M109.097865

Jason C. Grigg‡1,2, John D. Cooper§1, Johnson Cheung§1, David E. Heinrichs§, and Michael E. P. Murphy‡3

From the ‡Department of Microbiology and Immunology, Life Sciences Institute, The University of British Columbia, Vancouver,British Columbia V6T 1Z3, Canada and the §Department of Microbiology and Immunology, University of Western Ontario, London,Ontario N6A 5C1, Canada

Staphylococcus aureus uses several efficient iron acquisitionstrategies to overcome iron limitation. Recently, the geneticlocus encoding biosynthetic enzymes for the iron chelatingmol-ecule, staphyloferrin A (SA), was determined. S. aureus synthe-sizes and secretes SA into its environment to scavenge iron. Themembrane-anchored ATP binding cassette-binding protein,HtsA, receives the ferric-chelate for import into the cell.Recently, we determined the apoHtsA crystal structure, the firstsiderophore receptor from Gram-positive bacteria to be struc-turally characterized. Herein we present the x-ray crystal struc-ture of the HtsA-ferric-SA complex. HtsA adopts a class IIIbinding protein fold composed of separate N- and C-terminaldomains bridged by a single �-helix. Recombinant HtsA canefficiently sequester ferric-SA from S. aureus culture superna-tants where it is bound within the pocket formed between dis-tinct N- and C-terminal domains. A basic patch composedmainly of six Arg residues contact the negatively charged sid-erophore, securing it within the pocket. The x-ray crystal struc-tures from two different ligand-bound crystal forms were de-termined. The structures represent the first structuralcharacterization of an endogenous �-hydroxycarboxylate-typesiderophore-receptor complex.One structure is in anopen formsimilar to apoHtsA, whereas the other is in a more closed con-formation. The conformational change is highlighted by iso-lated movement of three loops within the C-terminal domain, adomain movement unique to known class III binding proteinstructures.

Iron is an essential component of many biological systemsplaying roles in most forms of life (1). Despite its abundance inbiological systems, free iron is scarce in most environments,

especially those encountered by pathogenic bacteria (2, 3).However, iron-complexes are abundant in the human body,and bacterial pathogens use multiple distinct strategies toacquire these iron stores. Iron in the human body is primarilylocatedwithin iron-shuttling glycoproteins, such as transferrin,in the iron storage protein, ferritin, and as the central atom inhememoieties from numerous proteins, including hemoglobinand cytochromes (4). Because the human body contains suffi-cient amounts of complexed iron to support bacterial growth,the ability to utilize these iron sources offers a significant selec-tive advantage to a pathogen.Staphylococcus aureus is one of themost commonly acquired

bacterial infectious agents in hospitals and a significant sourceof infection in community acquired infections of medical con-cern (5). Its prevalence combined with escalating antibioticresistance within hospital and community isolated strains hasmade understanding S. aureus pathogenesis pertinent to com-bating infection (6). This successful bacterial pathogen invadesand causes disease in most human body tissues due to its enor-mous repertoire of virulence factors (7–9).Iron uptake systems are paramount to survival in any envi-

ronment, but the diversity of S. aureus iron uptake systemslikely contributes to its ability to thrive in most mammaliantissues. S. aureus possesses the iron-regulated surface determi-nant heme transport system (10). This system has been wellcharacterized, and ligand-bound structures are determined formost components of the system (11–16). S. aureus can alsogrow on holo-transferrin or lactoferrin as an iron source, whichis likelymediated by specific binding on the cell surface (17, 18).S. aureus is able to synthesize and utilize siderophores, smalliron chelating molecules (for a recent review of siderophoreuptake in S. aureus, see Ref 19). Uptake of exogenous hydrox-amate-type siderophores occurs via the Fhu transport machin-ery (20–25). S. aureus synthesizes two siderophores of knownstructure: staphyloferrin A (SA)4 and staphyloferrin B (SB)(26–30). Biosynthesis and transport of SB in S. aureus is medi-ated by proteins encoded by the sbn (biosynthesis) and sir(transport) operons (31). Independent work from two groupsidentified the locus encoding the SA biosynthetic enzymes (32,33). Beasley et al. (32) further demonstrate that htsABC, situ-

* This work was supported by Canadian Institutes of Health Research Oper-ating Grants MOP-49597 and MOP-38002 (to M. E. P. M. and D. E. H.,respectively).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. 1 and 2.

The atomic coordinates and structure factors (codes 3LHS and 3LI2) have beendeposited in the Protein Data Bank, Research Collaboratory for Structural Bioin-formatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

1 Recipients of Natural Sciences and Engineering Research Council CanadaGraduate Scholarships.

2 Supported by a Michael Smith Foundation for Health Research Junior Grad-uate Trainee Award.

3 To whom correspondence should be addressed. Tel.: 604-822-0254; Fax:604-822-6041; E-mail: Michael.Murphy@ubc.ca.

4 The abbreviations used are: SA, staphyloferrin A; SB, staphyloferrin B;Fe-SA, ferric-bound staphyloferrin A; HPLC, high performance liquidchromatography.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 15, pp. 11162–11171, April 9, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

11162 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 15 • APRIL 9, 2010

by guest on February 11, 2018http://w

ww

.jbc.org/D

ownloaded from

ated adjacent to the SA biosynthetic locus in the S. aureusgenome, encodes an ABC transporter required for ferric-SA(Fe-SA) import. HtsABC was previously implicated in hemetransport (34), and the possibility of a dual role in heme uptakehas not been excluded.Recently, we presented the crystal structure of apoHtsA (32),

the first structure of a siderophore receptor protein from aGram-positive bacterium. The structure is a two-domain pro-tein with a single �-helix bridging the two domains, similar torelated metal and metal compound ABC transporter-associ-ated binding proteins (35, 36). Herein, we describe structures ofthe HtsA-Fe-SA complex from two different crystal forms. Thestructures are the first for an �-hydroxycarboxylate-type sid-erophore-receptor complex and provide insights into ligandrecognition and the conformational change required for pro-ductive interaction with the permease (HtsBC).

EXPERIMENTAL PROCEDURES

Cloning and Protein Expression—Recombinant HtsA wasproduced by cloning htsA from S. aureus Newman genomicDNA into pET28a. The HtsA expression construct wasdesigned to exclude the N-terminal secretion signal and lipida-tion site (residues 1–20) and 17 additional N-terminal residues(21–37) that were omitted from the design because of predicteddisorder (37). pET28a-htsA (to express residues 38–327) wastransferred into Escherichia coli strain ER2566. Cultures weregrown in 2�YTmedia at 30 °C to an optical density of�0.8. Atthis point the culture temperature was shifted to 25 °C, andprotein expression was induced with 0.4 mM isopropyl 1-thio-�-D-galactopyranoside. Induced cells were incubated for anadditional 16 h. Cells were resuspended in 50 mM Tris, pH 8.0,100 mM NaCl and disrupted using an EmulsiFlex-C5 homoge-nizer (Avestin). His6-tagged protein was purified using a His-Trap HP column (GE Healthcare) in the same resuspensionbuffer and eluted with a 0–500 mM imidazole gradient. His6-HtsA was dialyzed into 50 mMHEPES, pH 7.8, and the His6 tagwas cleaved by thrombin digestion (1:500 mass ratio HtsA:thrombin). HtsA was further purified by cation exchange chro-matography (Source 15S, GE Healthcare) in 50 mMHEPES, pH7.8, and eluted with a 0–500 mM NaCl gradient. Protein sam-ples were dialyzed into 20 mM Tris, pH 8.0, for crystallization.

Recombinant SfaB and SfaD (SA synthetases) were requiredto produce staphyloferrin A in vitro. The sfaB and sfaD codingregions were amplified from S. aureusNewman genomic DNAand cloned into pET28a(�) for overexpression in E. coli BL21(DE3). Cultures were grown in Luria broth (Difco) supple-mented with 30 �g/ml kanamycin at 37 °C to an optical densityof �0.9. Isopropyl 1-thio-�-D-galactopyranoside (500 �M) wasadded, and cultures were incubated an additional 18 h at roomtemperature with shaking. Cells were harvested by centrifuga-tion at 15,000� g and resuspended in binding buffer consistingof 50 mM HEPES, pH 7.4, 500 mM NaCl, 10 mM imidazole andpassed through a French pressure cell at 1500 p.s.i. Cell lysatewas centrifuged at 15,000 � g to remove unbroken cells anddebris before the supernatant was subjected to additional cen-trifugation at 150,000� g for 60min to precipitate the insolublematerial. The soluble fraction was then applied to a 1-ml His-Trap nickel affinity column (GE Healthcare) equilibrated with

binding buffer, and the His6-tagged proteins were eluted with agradient of 0–80% elution buffer over 20 column volumes (elu-tion buffer consisted of 50 mM HEPES buffer, pH 7.4, 500 mM

NaCl, 500 mM imidazole). Proteins were then dialyzed into 50mM HEPES, pH 7.4, 150 mM NaCl, and 10% glycerol at 4 °C.Protein purity was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then frozen (at �80 °C)and stored as 100-�l aliquots. The protein yields for SfaB andSfaD were 6.8 and 25 mg/liter of culture, respectively.Staphyloferrin A Enrichment from Culture Supernatant—

Staphyloferrin A was enriched from concentrated S. aureusculture supernatants as previously described (32). For use incrystallization, FeCl3 was added to concentrated siderophoreextracts to a final concentration of 5mM, added in 3-fold excessto recombinant HtsA and incubated at room temperature for�30 min. Protein solutions were then passed over a SephadexG-50 (GE Healthcare) column and concentrated to 25 mg/ml.Staphyloferrin A Synthesis and Purification—Using recombi-

nant SfaB and SfaD, staphyloferrin A biosynthesis reactionswere set up as previously described (33) and incubated for 12 h.The staphyloferrin A reaction was centrifuged in an Amicon�Ultra-0.5 10k filter column (Millipore) at 14,000 � g for 15 minto remove enzymes. The filtrate was then supplemented with 3mMFeCl3 and centrifuged at 18m000� g to remove precipitate.50 �l of the solution was then injected onto a Waters xTerraC18 reversed-phase 5-�m column (150 mm x 2.1 mm) on aBeckmanSystemGoldHPLCequippedwith a photodiode arraydetector. Samples were run at 0.2 ml/min using a step gradientas previously described (33). Solvent A was 10 mM tetrabu-tylammonium phosphate, pH 7.3, in HPLC grade water(Fisher), and solvent B was 100% acetonitrile (Fisher). Datawere analyzed using the 32 Karat Software Version 8.0 system,and peaks were monitored at 340 nm. The peak correspondingto Fe-SA eluted at 17min andwas collected. Collected fractionswere then vacuum-centrifuged to dryness and resuspended indeionized water, and the concentration of iron was determinedusing atomic absorption spectroscopy (see below) before use influorescence titration experiments with HtsA.Determination of Ferric-StaphyloferrinAConcentration—Atomic

absorption spectrometry was used to determine the concentra-tion of iron in HPLC-purified Fe-SA samples. The concentra-tion of iron was used to determine the concentration of staphy-loferrin A by assuming a 1:1 molar ratio in the Fe-SA complex.Sampleswere diluted in 1Mnitric acid before being drawnby anSPS 5 sample preparation system (autosampler) into a VarianAA240 atomic absorption spectrometer. Absorbance wasdetected by a iron/manganese hallow cathode lamp, whichemits at 248.3 nm specific for iron detection. Absorbance datawere analyzed and compared with a linear calibration curvebased on known iron standards in ppm. Iron standards werediluted in 1 M nitric acid from an atomic absorption spectrom-eter certified 1000 ppm � 1% stock (Fisher). Calibration stand-ards were separately analyzed first before iron-siderophoresamples.Fluorescence Spectroscopy—Fluorescence titration experi-

ments were performed at room temperature using recombi-nant HtsA (15 nM) in 50 mM HEPES, pH 7.4, in a Fluorolog-3spectrofluorometer (ISA Instruments). The excitation and

Staphyloferrin A-bound HtsA

APRIL 9, 2010 • VOLUME 285 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 11163

by guest on February 11, 2018http://w

ww

.jbc.org/D

ownloaded from

emission slits were set at 2.1 and 6.3 nm, respectively. The exci-tation and emission wavelengths were set at 280 and 334 nm,respectively. Titration experiments were performed on twoseparate occasions, each time in triplicate, and the valuesreported are an average of all data sets. Dissociation constants(Kd) and relevant parameters were calculated by fitting thefluorescence titration data for Fe-SA (across a concentrationrange between 0.22 and 226 nM ligand) to a one-site bindingmodel accounting for ligand depletion. Data were analyzed bynonlinear regression analysis using the solver tool add-in fromMicrosoft Excel software, as described previously (22, 23).Multiple Sequence Alignments—A BLAST (38) search of the

NCBI non-redundant protein data base identified manyhomologous proteins. The top 100 hits (E values �7 � 10�31)were exported from NCBI and filtered to remove sequencesabove 80% identity. The 34 remaining sequences were alignedusing the program T-coffee (39, 40). The alignment was man-ually adjusted and visualized using Jalview (41).Crystallization—HtsA was exposed to ferrated concentrated

spent culture supernatant from S. aureus grown under ironrestriction to form the HtsA-staphyloferrin A complex. Twodifferent conditions yielded diffraction quality holoproteincrystals. Crystal form 1 was grown in hanging drop plates bymicroseeding with apoHtsA crystals grown as previouslydescribed (32). The crystals formed in 0.1MHEPES, pH7.0, 24%Jeffamine ED-2001. The crystals were frozen in the same bufferwith 26% Jeffamine ED-2001 and 15% glycerol. Crystal form 2grew in sitting drop plates containing a 1:1 ratio of proteinsample to well solution (0.05 M zinc acetate, 20% polyethyleneglycol 3350). The crystal was frozen in well solution supple-mented with 20% ethylene glycol.Structure Solution and Analysis—X-ray data for crystal form

1 were collected at the Stanford Synchrotron Radiation Light-source on beamline 9-2 at 1.00 Å wavelength. Data were pro-cessed to 1.3 Å resolution using HKL2000 (42). The proteincrystallized in the P21 space group with one molecule in theasymmetric unit. The siderophore-bound HtsA structure wassolved by molecular replacement using MolRep (43) from theCCP4 program suite (44) with the previously describedapoHtsA structure (PDB entry 3EIW) as the searchmodel (32).The structure was edited using COOT (45) and refined withRefmac5 (46). Data collection and refinement statistics areshown in Table 1.X-ray data for crystal form 2 were collected at the Canadian

Light Source on beam line 08ID-1 using a wavelength of0.97934 Å. Data were processed to 2.2 Å usingMosflm (47) andScala (48). Indexing suggested an apparent space group ofC2221. The structure was solved by molecular replacement,refined, and edited as described for crystal form 1. However,poor statistics, namely high values and large discrepanciesbetween Rwork and Rfree values despite good density maps inaddition to near identical values for a and c cell dimensions,suggested the space group was P21 (a � 52.28, b � 148.60, c �52.27, � � 117.1°) with twinning. Several cases of twinning inP21 by the operator l, -k, h have been recently described (49–52). The revised solution in P21 contained twomolecules in theasymmetric unit andwas refined using Refmacwith amplitude-based twin refinement (46). A twinning fraction of 0.49 (twin-

ning operator l, -k,h) dramatically improvedRwork (0.21 to 0.18)and Rfree (0.29 to 0.24). Themodel was further refinedwith TLSparameters (53, 54) to a final Rwork and Rfree of 16.5 and 21.6,respectively.Staphyloferrin A coordinates were generated using the pro-

gram Sketcher from the CCP4 Program Suite (44). The centraliron was identified in the electron density by a large peak in thedifference map. Fe-SA was modeled into the structures afterfitting the protein backbone before adding waters. Fe-SA wasmodeled into the closed structure at full occupancy and has anaverage B-factor of 28.5 Å2. The open conformation was mod-eled with Fe-SA at 0.70 occupancy, as determined to minimizedensity peaks in an Fo � Fcmap at the iron atom. Fe-SA refinedwith an average B-factor of 34.6 Å2. Data collection and finalrefinement statistics are shown in Table 1. Figures were gener-ated with PyMol (55).

RESULTS

Affinity of HtsA for Staphyloferrin A—Our previous studiesshowed that HtsABC was required for SA utilization in S. au-reus (32). HtsA, a class III substrate binding protein, is tetheredto the extracellular face of the cytoplasmic membrane via anN-terminal lipidation and functions as the receptor for staphy-loferrin A. Changes in the intrinsic fluorescence of recombi-nantHtsA (lacking signal peptide) that occur upon ligand bind-ing were examined to determine substrate affinity. Saturatingconcentrations of HPLC-purified ferric-SA resulted in an aver-age 52.5% reduction in fluorescence emission. The dissociationconstant (Kd) of HtsA and Fe-SA was in the low nM range butcould not be accurately determined because the concentrationof HtsA required to see fluorescence change (15 nM) was �15-fold greater than the KD (supplemental Fig. S1). The specificityof HtsA for Fe-SA was demonstrated by the fact that ferric-staphyloferrin B (staphyloferrin B was synthesized as described(32) and HPLC-purified in a similar fashion to SA) failed to

TABLE 1X-ray data collection and refinement statistics

Fe-SA-HtsA open Fe-SA-HtsA closed

Data collectionaResolution range (Å) 50-1.30 (1.35-1.30) 50-2.20 (2.32-2.20)Space group P21 P21Unit cell dimensions (Å) a � 44.56, b � 43.52, a � 52.28, b � 148.60,

c � 75.32, � � 100.6° c � 52.27, � � 117.1°Unique reflections 70,082 35,958Completeness (%) 99.2 (97.7) 99.2 (99.2)Average I/�I 19.6 (3.1) 10.8 (4.1)Redundancy 3.5 (3.1) 4.0 (4.0)Rmerge 0.056 (0.355) 0.085 (0.309)

RefinementRwork (Rfree) 15.3 (18.6) 16.5 (21.6)B-factors (Å2)All atoms 16.6 37.1Protein 15.0 37.1Staphyloferrin A 24.3 25.5Water 28.0 38.1

r.m.s.d.b bond length (Å) 0.013 0.013Ramachandran plot,

% residuesIn most-favorable

region92.5 86.7

In disallowed regions 0.0 0.0a Values in parentheses represent the highest resolution shell.b r.m.s.d., root mean square deviation.

Staphyloferrin A-bound HtsA

11164 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 15 • APRIL 9, 2010

by guest on February 11, 2018http://w

ww

.jbc.org/D

ownloaded from

quench the intrinsic fluorescence of HtsA. These data are inagreement with previously published biological data (31, 32).Overall Structure of Open and Closed HtsA—HtsA possesses

distinct N- and C-terminal domains joined by a single �-helix(56, 57) (Fig. 1A). The apoHtsA structure (PDB entry 3EIW(32)) overlays with the Fe-SA-bound crystal form 1 (open) andcrystal form 2 (closed) HtsA structures with an root meansquare deviation over all C� atoms of 0.5 and 1.6 Å, respectively(Fig. 1B). Unlike the typical interdomain movement character-istic of many �-sheet bridged binding proteins, the N- and

C-terminal domain cores overlay well with the apo structure.Relative to the apo structure, the open holo structure does notundergo significant domain movement (hinged motion of lessthan 2°) Instead, the predominant structural difference is centeredat three loops at the surfaceof theC-terminal domain.These threeloops are composed of residues 201–208 (Loop201–208),228–258 (Loop228–258), and 265–271 (Loop265–271) (Fig. 1B).In the open structures, residues in the loops display elevatedB-factors relative to core residues (Fig. 1C), whereas in theclosed structure, the B-factors are more similar (Fig. 1D).Loop228–258 undergoes the largest structural change in theclosed protein with C� atoms moving as much as 12.1 Å (Tyr-239) across the binding pocket relative to the apo or open struc-tures. The large loop movement is accommodated by a slightunwinding of the �-helix230–239 preceding the loop. Tyr-239 islocated at the C-terminal end of the�-helix230–239 and is trans-lated from the central portion of the C-terminal domain intothe binding pocket to form anH-bond to the lateral side of Fe-SA.A second Tyr, Tyr-244, is shifted across the pocket, forming aH-bond (2.5 Å) to the Phe-146 main chain carbonyl of the N-ter-minal domain. The loop movement also creates several intrado-main H-bonds. Lys-238 forms a H-bond to Tyr-212 (3.0 Å).Hydrophobic contacts are also created by the loop movement.Leu-240 forms a hydrophobic contact with Phe-146, again bridg-ing the N- and C-terminal domains. Pro-243 forms a stackedhydrophobic interactionwithTyr-244 (3.8Å), potentially stabiliz-ing the interdomain contacts facilitated by Tyr-244.In the closed structure, two Zn2�-mediated crystal contacts

are formed between symmetry related C-terminal domains ofeach molecule. One site involves two metals bound by His-266and Lys-270 from chain A and Glu-250, His-251, and Asp-254from a symmetry-related chain B. The other site involves thesame five residues, this time with the Glu-250, His-251, andAsp-254 from Chain A and His-266 and Lys-270 from a sym-metry-related chain B. Zn2� is present in the crystallizationbuffer and has been modeled into both sites. Because thesecrystal contacts involve residues from the loops with the largeststructural changes, it is feasible that these crystal contactsinduce a conformational change in holo-HtsA. Given the addi-tional Fe-SA and interdomain HtsA contacts formed, a morelikely explanation is that the closed conformer seen is biologi-cally relevant and the crystal contacts simply form between twostable, closed structures.Structure of Staphyloferrin A—SA is synthesized from two

molecules of citrate forming amide bonds with the aminogroups of a D-ornithine, forming N2,N5-di-(l-oxo-3-hydroxy-3,4-dicarboxylbutyl)-D-ornithine (Fig. 2A) (28). Electron den-sity for Fe-SA is present in the binding pocket formed betweenthe N- and C-terminal domains in both the open and closedforms of HtsA. Electron density is present for the completeFe-SAmolecule in the closed structure, butweak density for theornithine backbone and additional density around one terminalcarboxylate group is apparent in the open structure (Fig. 2B).Two waters have been modeled into the extra density but donot completely account for the extra positive peaks. The ligandis likely afforded additional flexibility in the absence of interac-tion by Loop228–258 that moves across the pocket in the closedstructure.

FIGURE 1. The overall structure of the HtsA-staphyloferrin A complex.A, the open structure (crystal form 1) of HtsA is shown as a schematic coloredin a gradual color change from the N terminus (blue) to the C terminus (red).Staphyloferrin A is shown in the binding pocket as sticks with carbon, nitro-gen, oxygen, and iron shown in gray, blue, red, and orange, respectively.B, shown is the overlay of open holoHtsA (blue, crystal form 1) and closedholoHtsA (red, crystal form 2) and apoHtsA (green, PDB entry 3EIW). The struc-ture is rotated �90° relative to A to look into the binding pocket. Backbonesare shown as tubes. Staphyloferrin A is shown in the binding pocket as in A.C, shown is a B-factor tube diagram of open-HtsA. Regions of increasing B-fac-tor are shown with larger diameter and coloring from blue (low) to red (high).D, shown is a B-factor tube diagram of closed-HtsA. The structure is colored asin C.

Staphyloferrin A-bound HtsA

APRIL 9, 2010 • VOLUME 285 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 11165

by guest on February 11, 2018http://w

ww

.jbc.org/D

ownloaded from

The SA structure has the ornithine C� atom in the R-config-uration. The two chiral centers of the citrate components aremodeled into the density for both structures with the moreburied citrate in the S configuration and the more solvent-ex-posed citrate in the R configuration (Figs. 2, B and C). Thecitrates on either side of the iron bind as mirror images of oneanother. The chirality at the citrate carbons are in line withpredictions from an early characterization of SA, suggesting thechiral centers were likely R,S (28). However, subsequent find-ings by a different group based on the CD spectra of model

compounds suggested the chiralcenters were S,S (26). Modeling ofSA with the S,S configuration doesnot fit the observed electron densityas well in either the open or closedstructures. Modeling in the secondS configuration clearly strains thesiderophore providing poor fit tothe density for O1, O2, and O3groups (see supplemental Fig. S2for a comparison of S,S and S,Rmodels).Ferric iron is coordinated by an

oxygen atom from each of �-hy-droxy,�-carboxyl-substituted car-boxylates from the two citrate con-stituents. SA coordinates Fe3� withdistorted octahedral geometry andligand bond lengths of 2.0–2.2 Å.The ligand bond angles are dis-torted from perfect octahedralgeometry ranging from 75° to 101°(Fig. 2, B and C).Siderophore Bound in the Open

Conformation of HtsA—The basicpatch previously suggested as theputative Fe-SA binding site contrib-utes the majority of siderophorecontacts (32). In the open structure,five Arg residues at the pocket sur-face form direct H-bonds to oxygenatoms of Fe-SA (Fig. 3A and Table2). Arg-104 and Arg-126 formH-bonds to Fe-SA. Arg-299 is mod-eled in two conformations, bothwithin H-bonding distance of theFe-SA ornithine hydroxyl group.Arg-304 and Arg-306 forms anadditional H-bond to the terminalcarboxylate group of the more bur-ied terminus. His-209 forms anadditional H-bond to the hydroxylof the ornithine component, andfour ordered water molecules aremodeled interacting with the orni-thine carboxylate and the carbonylas well as two carboxylates from oneof the citrate moieties (Fig. 3A and

Table 2). The B-factors of regions of the siderophore backboneare dependent on solvent accessibility. The more buried citrategroup has the lowest B-factors (carbon atoms ranging from14–18Å2). The carbon atomB-factors of the ornithine increasefrom 20 to 36 Å2, moving from the inner citrate group to thefully solvent-exposed ornithine carboxylate. The outermost cit-rate group carbons have B-factors in the 24–30 Å2 range.Siderophore Bound in theClosedConformation ofHtsA—The

major difference in HtsA-Fe-SA interactions between the openand closed conformations occur due to additional contacts

FIGURE 2. The structure and chirality of staphyloferrin A. A, a linear schematic of the staphyloferrin Amolecule is shown. Stereochemistry at the three chiral centers is indicated. Atoms that directly interact with theiron are numbered according to Konetschny-Rapp et al. (28). B, shown is the conformation of staphyloferrin Afrom the open HtsA-SA structure. Extra density can be seen at the distal end of the terminal carboxylate group(group 3 from 2A). C, shown is the conformation of SA from the closed HtsA-SA structure. The omit Fo � Fc mapsare contoured at 2.5 and 3.5 � for B and C, respectively. Atoms are indicated by their element symbols.

FIGURE 3. Staphyloferrin A in the HtsA binding pocket. A, shown is the open HtsA binding pocket. Residuesforming direct contacts with SA (cyan) are shown as sticks with carbon, nitrogen, oxygen, and iron shown asgreen, blue, red, and orange, respectively. Hydrogen bonds are indicated by dashed lines. Residues are num-bered according to full-length HtsA. B, the closed HtsA binding pocket shown colored as in A.

Staphyloferrin A-bound HtsA

11166 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 15 • APRIL 9, 2010

by guest on February 11, 2018http://w

ww

.jbc.org/D

ownloaded from

from two of the loops undergoing large conformationalchanges. Relative to the open conformation, Tyr-239 C� trans-lates 12.1 Å in Loop228–258 and forms a H-bond with the car-bonyl group of the less buried citrate. Additionally, the transla-tion of Loop201–208 orients Lys-203 and Arg-83 to formH-bonds with the Fe-SA carboxylate groups (Fig. 3B and Table2). Despite these additional contacts, Fe-SA is boundwithin thebinding pocket of the closedHtsA in a similar orientation to theopen form; however, it is shifted slightly deeper into the pocket(Fig. 3B). The result of the conformational changes is significantocclusion of solvent from the Fe-SA molecule, reducing the33.0% solvent exposure in the open structure to 14.5% inthe closed structure (as determined with AREAIMOL (44)).The distribution of siderophore backboneB-factors is similar tothat of the open conformation. The lowest B-factors are asso-ciated with the more buried citrate group (15–20 Å2), a broadrange is observed in the ornithine group (25–40 Å2), and theouter citrate group has elevated B-factors (27–30 Å2).In an overlay of the open and closed protein structures there

is an �1.5 Å average atom displacement over all 34 Fe-SAatoms. The half of Fe-SA located closest to the exterior of theHtsA binding pocket undergoes the largest displacement withan average shift of�2.1 Å into the pocket. Only slight intramo-lecular atomic displacements occur within Fe-SA with meanatom displacements relative to the protein core of �0.5 Å(Fig. 2B).Several of the additional key protein-Fe-SA contacts identi-

fied in the open structure are similar to those in the closedstructure; however, H-bond-lengths are altered in many cases(Fig. 3B and Table 2). In total, six Arg residues in the Arg-richregion form direct contacts with Fe-SA. Arg-86, Arg-104, Arg-126, Arg-299, Arg-304, and Arg-306 all form H-bonds to Oatoms in the citrate moieties of Fe-SA (Fig. 3B and Table 2).Also similar to the open structure, His-209 forms a H-bond tothe carbonyl group on the ornithine component of Fe-SA.Multiple Sequence Alignments—A BLAST (38) search of the

NCBI non-redundant protein data base identified proteinswith

greater than 30% sequence identity. The list of sequences is ofproteins from Gram-positive and Gram-negative bacteria.Because several staphylococcal species produce SA (28, 29), thebest matches correspond to homologous receptor proteins inStaphylococcus species aureus, epidermidis, warneri, haemo-lyticus, capitis, saprophyticus, and hominus with amino acidsequence identities of �80%. Three proteins were identifiedfrom Bacillus sp.: (i) YfmC (�42% identity), annotated as a fer-ric citrate-binding protein (58), (ii) YhfQ (�37% identity), anunknown siderophore-binding protein (58, 59), and (iii) YfiY(�30% identity), the binding protein for the siderophoreschizokinen (60). Another interesting homolog identified wasthe ferric citrate-binding protein, FecB, found in many orga-nisms but probably best studied in E. coli (E. coli FecB, �35%identity).An alignment of 41 of the homologous sequences was made

(for a subset of sequences, see Fig. 4). The HtsA sequence con-tains an �8-residue insertion relative to most homologoussequences in the alignment around Tyr-239, in Loop228–258,which undergoes the largest structural change and forms adirect Fe-SA contact in the closed structure (Figs. 1B and 4).This suggests the structural change will not be seen in manyhomologous structures and may be an adaptation for entrap-ment of specific substrates.The residues contacting Fe-SA are conserved to varying

degrees (Arg-86 (22/41), Arg-104 (37/41), Arg-126 (36/41),His-209 (30/41), Tyr-239 (9/41), Arg-299 (21/41), Arg-304 (20/34, 6 Lys), Arg-306 (37/41)), although in many cases the aminoacid substitutions are conservative. Furthermore, two con-served Glu residues, Glu-110 (39/41, 2 Asp) and Glu-250 (38/41, 2 Asp, 1 Ser), are located such that they could form saltbridges between the N- and C-terminal domains of HtsA andconservedArg residues on theABC transporter permease com-ponents (HtsBC) to mediate protein docking. Interestingly,Glu-250 is located on an �-helix that shifts �2.8 Å toward thedomain interface in the closed structure. Amodel of the HtsBCpermease based on the BtuF structure (PDB entry 2qi9 (61))suggests themovement ofGlu-250 brings it toward a conservedArg-74 of HtsB or Arg56 of HtsC, which may mediate differen-tiation of ligand-bound or free HtsA.Several functionally interesting residues are highly conserved

(Fig. 4). Gly-102 (41/41) and Pro-107 (39/41) occur on eitherside of the conserved Fe-SA-interacting Arg-104, orienting thearginine into the binding pocket. His-127 (40/41) forms aH-bond to themain chainO of Arg-126 and likely stabilizes theloop containing SA-interacting Arg-126. Four sequential resi-dues, Ile-137—Thr-140, are conserved in most sequences andoccur on a tightly turning surface loop. Tyr-150 (35/41) forms aH-bond between the N-terminal domain and the His-177 (33/41) N� from the domain-spanning �-helix, which would con-tribute to interdomain stability. Trp-302 (41/41) is locatedclose to two Fe-SA-ligands (Arg-304 and Arg-306) but isdirected into the core of the protein where it forms severalhydrophobic contacts in addition to a H-bond from its N�1group to Glu-317. Trp-302 burial likely imparts stability to theloop and anchors the ligand binding Arg residues. Similar tothe N-terminal linkage, a domain linking interaction betweenthe C-terminal domain and the bridging �-helix is mediated by

TABLE 2Fe-SA-HtsA bond distances (Å)

HtsA atom-Fe-SA atoma Bond distanceFe-SA-HtsA open Fe-SA-HtsA closed

ÅArg-86 N-O2 NAb 3.1Arg-104 N1, N2-O3, O3 2.9, 3.0 2.8, 3.0Arg-126 N-O3 3.5 3.5Arg-126 N1, N2-O3, O3 2.8, 3.1 2.7, 3.1Lys-203 O-ornithine carboxylate NAb 2.9His-209 N�2-citrate carbonyl 2.8 2.7Tyr-239 O-ornithine N, O1 NAb 2.8, 2.6Arg-299 N�-O2 3.0 or 2.7c NAb

Arg-299 N-O2, O2 2.8, 3.6 or 3.1, 3.3c 2.7, 3.1Arg-299 N-citrate carbonyl 2.9 or NAb,c NAb

Arg-304 N�-O2 3.4 3.4Arg-304 N�-O2 3.3 3.1Arg-304 NH-O2 3.2 3.5Arg-306 N-O3 2.8 2.4Water-ornithine carboxylate 2.9 NAb

Water-citrate carbonyl 3.0 NAb

Water-O2 3.1 NAb

Water-O3 3.3 3.0a Fe-SA atoms are numbered according to atom labels in Fig. 2A.b NA, not applicable because residues contact not present in structure.c Refers to each of the two conformations modeled for Arg-299 in open structure.

Staphyloferrin A-bound HtsA

APRIL 9, 2010 • VOLUME 285 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 11167

by guest on February 11, 2018http://w

ww

.jbc.org/D

ownloaded from

a H-bond formed between Glu-312 (32/41) and Arg-173 inboth the open and closed conformations. However, in theclosed conformation, Arg-173models in two orientations, bothbonding and non-bonding.

DISCUSSION

The crystal structures of Fe-SA-bound HtsA in the open andclosed forms have enabled identification of residues that inter-act with Fe-SA and demonstrated an unprecedented mode ofligand entrapment not previously observed in this family ofbinding proteins. Several x-ray crystal structures of related classIII binding proteins possessing similar structural folds havebeen determined in both apo and holo forms. BtuF (E. coli B12uptake) (56, 62), TroA (E. coli Zn2� uptake) (63, 64), FhuD(E. coli ferrichrome uptake) (65, 66), and ShuT (Shigella dysen-teriae heme uptake) (67) all display little to no conformationalchange between the apo and holo forms (�4° hinge movementand small intradomain atom displacement).

A few recent examples demonstrate that a larger interdo-main movement is possible. The apo structure of E. coli FitEwas recently presented with both open and closed conforma-tions found within the four molecules of the asymmetric unit.Within each domain FitE undergoes little conformationalchange (0.4–0.75 Å root mean square deviation for C� atoms).However, the domain-bridging �-helix undergoes a significanthinged motion (�18.5°) (68). The structure of the Bacillus sub-tilis bacillibactin receptor (FeuA) undergoes a hinged move-ment of�20° between the apo (PDBentry 2phz) andholo struc-tures (69). Consistent with these crystal structures, moleculardynamics simulations of FhuD predict a greater hinge motion(�6°) than what is observed between the apo and holo struc-tures (2°) (66).HtsA undergoes significant conformational changes upon

Fe-SA binding. However, the conformational changes do notmirror the rigid interdomain movement seen in the FitE orFeuA structures or FhuD simulations. Instead, the large confor-

FIGURE 4. HtsA sequence alignments. A subset of the 34 non-redundant sequences (�80% sequence identity) is shown aligned. Residues are numbered asin full-length HtsA. Sequences are identified by the protein name or identifier followed by an underscore, and the first uppercase letter of the genus namefollowed by the first three letters of the species name. Arrows indicate HtsA residues that directly interact with SA.

Staphyloferrin A-bound HtsA

11168 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 15 • APRIL 9, 2010

by guest on February 11, 2018http://w

ww

.jbc.org/D

ownloaded from

mational changes are isolated to specific regions within theC-terminal domain. These conformational changes allowHtsAto clamp around Fe-SA, providing additional contacts to thesiderophore as well as interdomain contacts that may facilitatethe slight hinge closing motion. Similar isolated conforma-tional changes of this magnitude have not previously beenobserved upon ligand binding in class III binding proteins.The ligand-dependent conformational changes in class III

binding proteins likely affect docking to the permease compo-nent of the ABC transporter, thereby providing a means of dis-criminating between the ligand-bound versus ligand-freereceptor. The BtuCD-F complex crystal structure has beendetermined, demonstrating that the binding of BtuF to BtuC ismediated by salt bridges between Glu-72 and Glu-202 fromBtuF and Arg-56 and Arg-295 from BtuB (61). Alignments andsubsequent site-directed mutagenesis of two similar class IIIbinding proteins, E. coli FecB (70) and S. aureus FhuD2 (22),suggest that similar Glu-Arg salt bridges mediate docking asvariants affect ligand transport but not ligand binding. Becausedocking is mediated by salt bridge formation, even minimalhinged motion would allow discrimination of open and closedconformations. The closed HtsA structure illustrates an alter-native mechanism of discrimination of the ligand-bound formof the receptor. Themovement of Loop228–258 to enclose Fe-SAalters the placement of the homologous Glu-250, which likelymediates salt-bridge formation with the permease HtsBC.The HtsA-Fe-SA complex is the first complex of an �-hy-

droxycarboxylate-type siderophore bound to its cognate bind-ing protein, and to our knowledge this study reports the firstexamination of affinity between an �-hydroxycarboxylate sid-erophore and its receptor. TheKd of HtsA for Fe-SA, in the lownM range, provides the explanation for how HtsA, whenexposed to ferrated S. aureus culture supernatant, was able tocomplex and crystallize with Fe-SA. The combination of a largenumber of charge interactions and receptor closures aroundthe siderophore afford specificity to the interaction of SA withHtsA. Many siderophore-binding proteins and transportershave broad specificity for siderophores of a similar type; how-ever, the Hts system is highly specific. SB is also an �-hydroxy-carboxylate-type siderophore (30), yet the Hts system cannottransport sufficient amounts for growth (32). The only com-mon constituent is a single citric acid component. Staphylo-ferrin B is also composed of a L-2,3-diaminopropionic acid,1,2-diaminoethane, and a succinic semialdehyde (30). Thespecificity of the Hts system for one negatively charged �-hy-droxycarboxylate siderophore over another is likely a reflectionof the specific ionic contacts formed in the closed structure thatwould not properly accommodate staphyloferrin B. Althoughthe advantage gained by this specificity is unclear, it could betailored to the low concentrations the siderophores expected tobe present in serum at the point of infection.The affinity of HtsA for Fe-SA is within the range of several

outer membrane siderophore receptors in Gram-negative bac-teria. Compared with other class III substrate binding proteins,the binding affinity of HtsA for SA is greater than the presentedBacillus sp. receptors and orders of magnitude stronger thanE. coli FhuD (Table 3). These apparent differences in affinitymay be a reflection of the extent and type of residues involved in

siderophore binding or may result from differences in confor-mational changes or tryptophan masking that can affect fluo-rescence emission. The disparity between binding affinities forthe E. coli hydroxamate-binding protein, FhuD, and HtsAcould reflect the differences in specificity. FhuD is a receptor fora broad array of ferric hydroxamates, so the binding pocketsacrifices affinity for diversity, where HtsA is specialized for SAalone.The chemical characteristics of siderophore binding pockets

of receptors varies with the class of the cognate siderophore.Ferric complexes of hydroxamate-type siderophores are gener-ally hydrophobic. Crystal structures of the outer membranereceptor FhuA (71) and the binding protein FhuD (65, 72) fromthe E. coli hydroxamate uptake system have been determined.Siderophore binding in both structures is primarily mediatedthrough hydrophobic contacts. Similarly, in Pseudomonasaeruginosa, receptors for the largely hydrophobic siderophoreferric chelates, pyoverdin (FpvA) (73) and pyochelin (FptA),have binding sites that are primarily composed of hydrophobicand aromatic residues (74).Catecholate-type siderophore ferric chelates generally have a

net negative charge, and the receptor binding residues oftenmirror the net charge. Ferric enterobactin carries a �3 netcharge, so not surprisingly, the binding pockets of the E. coliouter membrane receptor, FepA (75), and the Campylobacterjejuni-binding protein, CeuE (76), contain several positivelycharged residues. In CeuE, the net negative charge is balancedby three Arg residues, and although the binding residues inFepA could not be definitively identified, an Arg-rich bindingsite was found (75, 76). Interestingly, a hydrophobic patch wasidentified in FepA and shown by mutagenesis data to contrib-ute to the affinity for ferric enterobactin (77). The structures ofthe catecholate receptors FeuA (Bacillus cereus) and YclQ(B. subtilis), which bind ferric-bacillibactin and ferric-petrobactin, respectively, represent the only other Gram-posi-tive bacterial siderophore receptor structures determined todate. Recently, a FeuA-ferric-bacillibactin crystal structure wasdetermined (69). Ferric-bacillibactin carries a net negativecharge that is neutralized by three basic residues, Lys-84, Lys-105, and Arg-180, that interact with deprotonated catecholateoxygen atoms (69). Gln-181 and Gln-215 also form directH-bonds to bacillibactin. Furthermore, the YclQ ligand binding

TABLE 3Dissociation constants (Kd) for receptor-ferric-siderophorecomplexes

Protein-siderophore (organism)a Kd Reference

nMHtsA-staphyloferrin A (S. aureus) Low nM This StudyFeuA-bacillibactin (B. cereus) 19 60FeuA-enterobactin (B. cereus) 12 60FatB-3,4-DHB (B. cereus) 1.2 60FatB-petrobactin (B. cereus) 127 60FpuA-petrobactin (B. cereus) 175 60YfiY-schizokinen (B. cereus) 34 60YxeB-desferroximine (B. cereus) 18 60YclQ-petrobactin (B. subtilis) 113 78FhuD-ferric hydroxamates (E. coli) 300–7900 82FpvA*-pyoverdin (P. aeruginosa) 0.37 83FhuA*-ferrichrome (P. aeruginosa) 0.65 83FptA*-pyochelin (P. aeruginosa) 0.54 83

a Proteins listed are class III ligand-binding proteins, except for those with asterisks,which are outer membrane receptor proteins in Gram-negative bacteria.

Staphyloferrin A-bound HtsA

APRIL 9, 2010 • VOLUME 285 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 11169

by guest on February 11, 2018http://w

ww

.jbc.org/D

ownloaded from

cleft contains a series of conserved positively charged residues(Arg-104, Arg-192, Arg-236, and Try-275) that are predicted tointeract with a negatively charged petrobactin ligand (78).Crystal structures of mammalian siderocalin bound to ferric

complexes of catecholate-type siderophores have been deter-mined (79, 80). The crystal structures show that two Lys resi-dues and a single Argmediate binding to the negatively chargedcatecholates. Recently, it was established that electrostaticinteractions between ferric siderophores and siderocalin arethe prime determinants of binding affinity (81).Analogous to these examples of charged siderophores, Fe-SA

carries a net negative charge that is neutralized by six Arg res-idues in HtsA. This large number of positively charged residuesis expected to favor tight binding to a compound that would bepresent in low concentrations in the environment. The abun-dance of positive electrostatic potential, in concert with theocclusion of the binding site upon closing, likely determinesthe specificity of the transporter for Fe-SA. Furthermore, theextensive protein-siderophore contacts that enclose the smallsiderophore likely serve to discriminate between Fe-free andFe-bound SA.In summary, we have demonstrated that the S. aureus ABC

transporter-associated binding protein HtsA binds Fe-SA andundergoes conformational changes upon binding involving avery small scale hingemotion and relatively largemovements atloops in the C-terminal domain to enclose the ligand. Further-more, binding is mediated primarily by six Arg, a Tyr, and a Hisresidue in the binding pocket. The coordinating residues arewell conserved in several proteins, suggesting that a similarmeans of coordinationmay be utilized in both siderophore andexogenous ferric citrate uptake pathways from a broad range ofbacteria.

Acknowledgments—We thank Michael Tiedemann for assistancewith the atomic absorption spectrometer, Woo Cheol Lee and AngeleArrieta for significant technical assistance, and Anson Chan andCatherine Gaudin for critical reading of this manuscript. Portions ofthis research were carried out at the Canadian Light Source, which issupported by the Natural Science and Engineering Research Council,the National Research Council, the Canadian Institutes of HealthResearch, and the University of Saskatchewan and the Stanford Syn-chrotron Radiation Laboratory, a national user facility operated byStanford University on behalf of the United States Dept. of Energy,Office of Basic Energy Sciences. The SSRL Structural Molecular Biol-ogy Program is supported by the Dept. of Energy, Office of Biologicaland Environmental Research and by the National Institutes ofHealth, National Center for Research Resources, Biomedical Technol-ogy Program, and the National Institute of GeneralMedical Sciences.

REFERENCES1. Wandersman, C., and Delepelaire, P. (2004) Annu. Rev. Microbiol. 58,

611–6472. Radtke, A. L., and O’Riordan, M. X. (2006) Cell Microbiol. 8, 1720–17293. Ratledge, C., and Dover, L. G. (2000) Annu. Rev. Microbiol. 54, 881–9414. Stojiljkovic, I., and Perkins-Balding, D. (2002)DNACell Biol. 21, 281–2955. Weems, J. J. (2001) Postgrad. Med. 110, 24–26, 29–31, 35–366. Klevens, R. M., Morrison, M. A., Nadle, J., Petit, S., Gershman, K., Ray, S.,

Harrison, L. H., Lynfield, R., Dumyati, G., Townes, J. M., Craig, A. S., Zell,E. R., Fosheim,G. E.,McDougal, L. K., Carey, R. B., and Fridkin, S. K. (2007)

JAMA 298, 1763–17717. Clarke, S. R., and Foster, S. J. (2006) Adv. Microb. Physiol. 51, 187–2248. Foster, T. J. (2005) Nat. Rev. Microbiol. 3, 948–9589. Lowy, F. D. (1998) N. Engl. J. Med. 339, 520–53210. Mazmanian, S. K., Skaar, E. P., Gaspar, A. H., Humayun, M., Gornicki, P.,

Jelenska, J., Joachmiak, A., Missiakas, D. M., and Schneewind, O. (2003)Science 299, 906–909

11. Grigg, J. C., Vermeiren, C. L., Heinrichs, D. E., and Murphy, M. E. (2007)Mol. Microbiol. 63, 139–149

12. Grigg, J. C., Vermeiren, C. L., Heinrichs, D. E., and Murphy, M. E. (2007)J. Biol. Chem. 282, 28815–28822

13. Sharp, K. H., Schneider, S., Cockayne, A., and Paoli, M. (2007) J. Biol.Chem. 282, 10625–10631

14. Villareal, V. A., Pilpa, R. M., Robson, S. A., Fadeev, E. A., and Clubb, R. T.(2008) J. Biol. Chem. 283, 31591–31600

15. Watanabe, M., Tanaka, Y., Suenaga, A., Kuroda, M., Yao, M., Watanabe,N., Arisaka, F., Ohta, T., Tanaka, I., and Tsumoto, K. (2008) J. Biol. Chem.283, 28649–28659

16. Lee,W. C., Reniere, M. L., Skaar, E. P., andMurphy, M. E. P. (2008) J. Biol.Chem. 283, 30957–30963

17. Clarke, S. R., Wiltshire, M. D., and Foster, S. J. (2004)Mol. Microbiol. 51,1509–1519

18. Taylor, J. M., and Heinrichs, D. E. (2002)Mol. Microbiol. 43, 1603–161419. Beasley, F. C., and Heinrichs, D. E. (2010) J. Inorg. Biochem. 104, 282–28820. Sebulsky, M. T., and Heinrichs, D. E. (2001) J. Bacteriol. 183, 4994–500021. Sebulsky, M. T., Hohnstein, D., Hunter, M. D., and Heinrichs, D. E. (2000)

J. Bacteriol. 182, 4394–440022. Sebulsky, M. T., Shilton, B. H., Speziali, C. D., and Heinrichs, D. E. (2003)

J. Biol. Chem. 278, 49890–4990023. Sebulsky, M. T., Speziali, C. D., Shilton, B. H., Edgell, D. R., and Heinrichs,

D. E. (2004) J. Biol. Chem. 279, 53152–5315924. Speziali, C. D., Dale, S. E., Henderson, J. A., Vines, E. D., and Heinrichs,

D. E. (2006) J. Bacteriol. 188, 2048–205525. Cabrera, G., Xiong, A., Uebel, M., Singh, V. K., and Jayaswal, R. K. (2001)

Appl. Environ. Microbiol. 67, 1001–100326. Drechsel, H., and Winkelmann, G. (2005) Biometals 18, 75–8127. Haag, H., Fiedler, H. P., Meiwes, J., Drechsel, H., Jung, G., and Zahner, H.

(1994) FEMS Microbiol. Lett. 115, 125–13028. Konetschny-Rapp, S., Jung, G., Meiwes, J., and Zahner, H. (1990) Eur.

J. Biochem. 191, 65–7429. Meiwes, J., Fiedler, H. P., Haag, H., Zahner, H., Konetschny-Rapp, S., and

Jung, G. (1990) FEMS Microbiol. Lett. 55, 201–20530. Drechsel, H., Freund, S., Nicholson, G., Haag, H., Jung, O., Zahner, H., and

Jung, G. (1993) Biometals 6, 185–19231. Cheung, J., Beasley, F. C., Liu, S., Lajoie, G. A., and Heinrichs, D. E. (2009)

Mol. Microbiol. 74, 594–60832. Beasley, F. C., Vines, E. D., Grigg, J. C., Zheng, Q., Liu, S., Lajoie, G. A.,

Murphy, M. E., and Heinrichs, D. E. (2009)Mol. Microbiol. 72, 947–96333. Cotton, J. L., Tao, J., and Balibar, C. J. (2009) Biochemistry 48, 1025–103534. Skaar, E. P., Humayun, M., Bae, T., DeBord, K. L., and Schneewind, O.

(2004) Science 305, 1626–162835. Claverys, J. P. (2001) Res. Microbiol. 152, 231–24336. Tam, R., and Saier, M. H., Jr. (1993)Microbiol. Rev. 57, 320–34637. Ward, J. J., Sodhi, J. S.,McGuffin, L. J., Buxton, B. F., and Jones, D. T. (2004)

J. Mol. Biol. 337, 635–64538. Altschul, S. F., Gish,W.,Miller,W.,Myers, E.W., and Lipman, D. J. (1990)

J. Mol. Biol. 215, 403–41039. Notredame, C., Higgins, D. G., and Heringa, J. (2000) J. Mol. Biol. 302,

205–21740. Poirot, O., O’Toole, E., and Notredame, C. (2003) Nucleic Acids Res. 31,

3503–350641. Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M., and Barton,

G. J. (2009) Bioinformatics 25, 1189–119142. Otwinowski, Z., and Minor, W. (1997)Methods Enzymol. 276, 307–32643. Vagin, A., and Teplyakov, A. (1997) J. Appl. Crystallogr. 30, 1022–102544. CCP4 (1994) Acta Crystallogr. D Biol. Crystallogr. 50, 760–76345. Emsley, P., and Cowtan, K. (2004)Acta Crystallogr. D Biol. Crystallogr. 60,

2126–2132

Staphyloferrin A-bound HtsA

11170 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 15 • APRIL 9, 2010

by guest on February 11, 2018http://w

ww

.jbc.org/D

ownloaded from

46. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997)Acta Crystallogr.D Biol. Crystallogr. 53, 240–255

47. Leslie, A. G.W. (1992) Joint CCP4 and ESF-EACMBNewsletter on ProteinCrystallography, No. 26, Daresbury Laboratory, Warrington, UK

48. Evans, P. (1997) Joint CCP4 and ESF-EACMBNewsletter on Protein Crys-tallography., No. 33, pp. 22–24

49. Hamdane, D., Lechauve, C., Marden, M. C., and Golinelli-Pimpaneau, B.(2009) Acta Crystallogr. D Biol. Crystallogr. 65, 388–392

50. Heitmann, D., and Einsle, O. (2008) Acta Crystallogr. D Biol. Crystallogr.64, 993–999

51. Sultana, A., Alexeev, I., Kursula, I., Mantsala, P., Niemi, J., and Schneider,G. (2007) Acta Crystallogr. D Biol. Crystallogr. 63, 149–159

52. Wittmann, J. G., and Rudolph, M. G. (2007) Acta Crystallogr. D Biol.Crystallogr. 63, 744–749

53. Painter, J., and Merritt, E. A. (2006) J. Appl. Crystallogr. 39, 109–11154. Painter, J., and Merritt, E. A. (2006) Acta Crystallogr. D Biol. Crystallogr.

62, 439–45055. DeLano, W. L. (2002) The PyMOL Molecular Graphics System, DeLano

Scientific LLC, San Carlos, CA56. Borths, E. L., Locher, K. P., Lee, A. T., and Rees, D. C. (2002) Proc. Natl.

Acad. Sci. U.S.A. 99, 16642–1664757. Quiocho, F. A., and Ledvina, P. S. (1996)Mol. Microbiol. 20, 17–2558. Ollinger, J., Song, K. B., Antelmann, H., Hecker, M., and Helmann, J. D.

(2006) J. Bacteriol. 188, 3664–367359. Baichoo, N., Wang, T., Ye, R., and Helmann, J. D. (2002) Mol. Microbiol.

45, 1613–162960. Zawadzka, A. M., Abergel, R. J., Nichiporuk, R., Andersen, U. N., and

Raymond, K. N. (2009) Biochemistry 48, 3645–365761. Hvorup, R. N., Goetz, B. A., Niederer, M., Hollenstein, K., Perozo, E., and

Locher, K. P. (2007) Science 317, 1387–139062. Karpowich, N. K., Huang, H. H., Smith, P. C., and Hunt, J. F. (2003) J. Biol.

Chem. 278, 8429–843463. Lee, Y. H., Deka, R. K., Norgard, M. V., Radolf, J. D., and Hasemann, C. A.

(1999) Nat. Struct. Biol. 6, 628–63364. Lee, Y. H., Dorwart, M. R., Hazlett, K. R., Deka, R. K., Norgard, M. V.,

Radolf, J. D., and Hasemann, C. A. (2002) J. Bacteriol. 184, 2300–230465. Clarke, T. E., Ku, S. Y., Dougan, D. R., Vogel, H. J., and Tari, L. W. (2000)

Nat. Struct. Biol. 7, 287–29166. Krewulak, K. D., Shepherd, C. M., and Vogel, H. J. (2005) Biometals 18,

375–38667. Ho, W. W., Li, H., Eakanunkul, S., Tong, Y., Wilks, A., Guo, M., and

Poulos, T. L. (2007) J. Biol. Chem. 282, 35796–3580268. Shi, R., Proteau, A., Wagner, J., Cui, Q., Purisima, E. O., Matte, A., and

Cygler, M. (2009) Proteins 75, 598–60969. Peuckert, F., Miethke, M., Albrecht, A. G., Essen, L. O., and Marahiel,

M. A. (2009) Angew. Chem. Int. Ed. Engl. 48, 7924–792770. Braun, V., and Herrmann, C. (2007) J. Bacteriol. 189, 6913–691871. Ferguson, A. D., Braun, V., Fiedler, H. P., Coulton, J. W., Diederichs, K.,

and Welte, W. (2000) Protein Sci. 9, 956–96372. Clarke, T. E., Braun, V., Winkelmann, G., Tari, L. W., and Vogel, H. J.

(2002) J. Biol. Chem. 277, 13966–1397273. Cobessi, D., Celia, H., Folschweiller, N., Schalk, I. J., Abdallah, M. A., and

Pattus, F. (2005) J. Mol. Biol. 347, 121–13474. Cobessi, D., Celia, H., and Pattus, F. (2005) J. Mol. Biol. 352, 893–90475. Buchanan, S. K., Smith, B. S., Venkatramani, L., Xia, D., Esser, L., Palnitkar,

M., Chakraborty, R., van der Helm, D., and Deisenhofer, J. (1999) Nat.Struct. Biol. 6, 56–63

76. Muller, A., Wilkinson, A. J., Wilson, K. S., and Duhme-Klair, A. K. (2006)Angew. Chem. Int. Ed Engl. 45, 5132–5136

77. Annamalai, R., Jin, B., Cao, Z., Newton, S. M., and Klebba, P. E. (2004) J.Bacteriol. 186, 3578–3589

78. Zawadzka, A. M., Kim, Y., Maltseva, N., Nichiporuk, R., Fan, Y.,Joachimiak, A., and Raymond, K. N. (2009) Proc. Natl. Acad. Sci. U.S.A.106, 21854–21859

79. Goetz, D. H., Holmes, M. A., Borregaard, N., Bluhm, M. E., Raymond,K. N., and Strong, R. K. (2002)Mol. Cell 10, 1033–1043

80. Holmes,M.A., Paulsene,W., Jide, X., Ratledge, C., and Strong, R. K. (2005)Structure 13, 29–41

81. Hoette, T. M., Abergel, R. J., Xu, J., Strong, R. K., and Raymond, K. N.(2008) J. Am. Chem. Soc. 130, 17584–17592

82. Rohrbach, M. R., Braun, V., and Koster, W. (1995) J. Bacteriol. 177,7186–7193

83. Hoegy, F., Celia, H., Mislin, G. L., Vincent, M., Gallay, J., and Schalk, I. J.(2005) J. Biol. Chem. 280, 20222–20230

Staphyloferrin A-bound HtsA

APRIL 9, 2010 • VOLUME 285 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 11171

by guest on February 11, 2018http://w

ww

.jbc.org/D

ownloaded from

MurphyJason C. Grigg, John D. Cooper, Johnson Cheung, David E. Heinrichs and Michael E. P.

PocketConformational Changes to Enclose Staphyloferrin A in an Arginine-rich Binding

Siderophore Receptor HtsA Undergoes LocalizedStaphylococcus aureusThe

doi: 10.1074/jbc.M109.097865 originally published online February 10, 20102010, 285:11162-11171.J. Biol. Chem. 

  10.1074/jbc.M109.097865Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

  http://www.jbc.org/content/suppl/2010/02/10/M109.097865.DC1

  http://www.jbc.org/content/285/15/11162.full.html#ref-list-1

This article cites 80 references, 26 of which can be accessed free at

by guest on February 11, 2018http://w

ww

.jbc.org/D

ownloaded from