Structural Basis for Hemoglobin Capture by Staphylococcusaureus Cell-surface Protein, IsdH□S

Received for publication, July 29, 2011, and in revised form, August 16, 2011 Published, JBC Papers in Press, September 14, 2011, DOI 10.1074/jbc.M111.287300

Kaavya Krishna Kumar‡1,2, David A. Jacques‡1,3, Gleb Pishchany§, Tom Caradoc-Davies¶, Thomas Spirig�,G. Reza Malmirchegini�, David B. Langley‡, Claire F. Dickson**, Joel P. Mackay‡, Robert T. Clubb�, Eric P. Skaar§,J. Mitchell Guss‡, and David A. Gell**4

From the ‡From the School of Molecular Bioscience, University of Sydney, New South Wales 2006, Australia, the §Department ofPathology, Microbiology and Immunology, Vanderbilt University Medical School, Nashville, Tennessee 37232, the ¶AustralianSynchrotron, Clayton, Victoria 3168, Australia, the �Department of Chemistry and Biochemistry, UCLA, Los Angeles, California90095, and the **Menzies Research Institute, University of Tasmania, Tasmania 7000, Australia

Background: Bacteria need iron from the host to establish infection.Results: We report the first structure of hemoglobin bound to a bacterial protein and show that targeted disruption of thisinteraction can reduce Staphylococcus aureus growth when hemoglobin is the sole iron source.Conclusion: Physical capture of hemoglobin is important for iron uptake by S. aureus.Significance: Hemoglobin receptors may be targets for new antibacterial agents.

Pathogens must steal iron from their hosts to establish infec-tion. In mammals, hemoglobin (Hb) represents the largest res-ervoir of iron, and pathogens express Hb-binding proteins toaccess this source. Here, we show how one of the commonestand most significant human pathogens, Staphylococcus aureus,captures Hb as the first step of an iron-scavenging pathway. Thex-ray crystal structure of Hb bound to a domain from the Isd(iron-regulated surface determinant) protein, IsdH, is the firststructure of a Hb capture complex to be determined. Surfacemutations in Hb that reduce binding to the Hb-receptor limitthe capacity of S. aureus to utilize Hb as an iron source, suggest-ing that Hb sequence is a factor in host susceptibility to infec-tion. The demonstration that pathogens make highly specificrecognition complexes withHb raises the possibility of develop-ing inhibitors of Hb binding as antibacterial agents.

Bacteria that cause human disease must acquire iron fromtheir host to establish infection (1). Host strategies to restrictiron availability therefore constitute a formof innate immunity.Most iron is sequestered within cells, and serum iron is kept atan extremely low concentration (�10�24 M) by the iron trans-port protein transferrin (2). A related protein, lactoferrin, ispresent in exocrine solutions such as tears and milk and is alsoreleased from neutrophils at sites of infection (3). Iron uptake

and homeostasis are even tuned toward anemia in chronicinfection by the hormone hepcidin (4). In cases where serumiron levels are either artificially or genetically elevated, there aredramatic increases in levels of bacteremia (5).Bacteria in all iron-restricted environments, including the

human body, employ a variety of generic mechanisms to scav-enge this essential element. Thesemechanisms include the pro-duction of siderophores, small (typically�1000Da) soluble fac-tors that bind with very high affinity (�10�22–10�50 M) toFe(III). Siderophores are subsequently reabsorbed by the bac-teria in the iron-charged form (6). Alternatively, iron-reducingcompounds can be released into the environment to generatesoluble Fe(II) that is absorbed through the FeoB permease,escaping the requirement for metabolically expensive sidero-phore production (7).Naturally, the rich iron reservoirs found in mammals and

other animals has driven evolution of iron-scavenging mecha-nisms that are specific to the pathogen-host relationship. Per-haps the most distinguishing feature of the mammalian ironprofile is that�75% of the total body-iron is locked up in heme,a cyclic organic compound (porphyrin) with central square-planar Fe(II/III) ion coordinated by four nitrogen atoms. Thisiron pool is considered largely inaccessible to siderophores (6),and pathogens have therefore evolved specialized pathwaysdedicated to heme uptake. As with all forms of iron, heme isretained tightly sequestered into host proteins at all times.Hb isthe most abundant heme protein in humans, accounting for�70% of total body iron, making it a particularly attractive ironsource for invading microbes.Highly pathogenic species such as Staphylococcus aureus

secrete hemolysins (8) to release Hb into the serum where it isaccessible to the bacteria. S. aureus displays a preference forheme as an iron source (9) and can grow on Hb as the solesource of iron (10, 11). Utilization ofHb in S. aureus ismediatedby Isd proteins (12, 13), which are also found in a large numberof other Gram-positive pathogens (14, 15). The Isd pathway inS. aureus comprises nine proteins IsdA-I (12).Of these, the four

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental “Experimental Procedures,” Table 1, Figs. 1– 6, and addi-tional references.

The atomic coordinates and structure factors (code 3SZK) have been deposited inthe Protein Data Bank, Research Collaboratory for Structural Bioinformatics,Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

1 Both authors contributed equally to this work.2 Supported by a University of Sydney international scholarship.3 Supported by an Australian Institute of Nuclear Science and Engineering

postgraduate research award.4 Supported by an Australia Research Council research fellowship. To whom

correspondence should be addressed: Menzies Research Institute Tasma-nia, 17 Liverpool St., Hobart, TAS 7000, Australia. Tel.: 612-6226-4608;E-mail: david.gell@utas.edu.au.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 44, pp. 38439 –38447, November 4, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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proteins IsdA, IsdB, IsdC, and IsdH are expressed on the bac-terial cell-surface, anchored through a C-terminal covalentlinkage to the peptidoglycan cell wall. Notably, the surfaceexposed Isd proteins are the most highly up-regulated genes inresponse to iron starvation in S. aureus (16) and Bacillusanthracis (17). IsdB and IsdH are Hb-binding proteins (10, 11).IsdA and IsdC are heme-binding proteins that cooperate withIsdB/H through an unknown mechanism to transfer heme in aunidirectional manner to the membrane-associated lipopro-tein IsdE (18–20). A dedicated ABC transporter complex (IsdDand IsdF) transfers heme from IsdE into the S. aureus cyto-plasm, where the porphyrin macrocycle is cleaved by a hemeoxygenase complex (IsdG and IsdI), releasing its iron (21).Genetic inactivation of IsdA (22), IsdB (11, 22, 23), IsdH (24,

25), IsdG, or IsdI (26) reduces the ability of S. aureus to causeinfections in mice, confirming the Isd system as an importantvirulence mechanism. It has been demonstrated that immuni-zation with IsdA, IsdB or IsdH antigens (24, 25, 27–29) oradministration of purified antibodies to IsdA, IsdB, IsdC, andIsdH (22) confers some protection from infection in variousanimal models. These studies suggest that blocking the Isdpathway of heme/iron uptake could have therapeutic benefit inhuman infections.To transfer the heme group from Hb to the bacterial mem-

brane, the cell wall-anchored proteins IsdA, IsdB, IsdC, andIsdH each possess one �130-residue NEAT (near iron trans-porter) domain that binds to heme (30–34). Extraction of hemefrom Hb relies upon a physical interaction of Hb with the Hbreceptors IsdB and IsdH (20). IsdB and IsdH contain, respec-tively, one or two variantNEATdomains that possessHb-bind-ing activity but do not bind to heme (see Fig. 1A) (10, 35–37).The structural basis for heme binding by NEAT domains iscomparatively well understood (30–34), but the molecularmechanism of Hb recognition has remained elusive and is thesubject of the current investigation.Many pathogens express Hb-binding proteins, but the

molecular details underlying Hb recognition are unknown inevery case. Here, we show that the first NEAT domain fromIsdH (IsdHN1) binds to a site on the �-chain of Hb (�Hb) anddetermine the x-ray crystal structure of the IsdHN1�Hb com-plex. Using mutant forms of Hb that are defective in IsdHN1binding, we demonstrate that physical capture of Hb byS. aureus is important for the utilization of this iron source.

EXPERIMENTAL PROCEDURES

Protein Preparation—The DNA sequence encoding theIsdHN1 (IsdH residues 86–229) and IsdHN2 (residues 321–467)domains from S. aureus strain TCH1516 were cloned intopET15b (Novagen) for expression with an N-terminal His6 tag.The proteins were expressed and purified as described previ-ously (35) to yield a final product with the additional N-termi-nal sequence MGSSHHHHHHSSGLVPRGSHM. Nativehuman Hb A was prepared and separated into its constituent�Hband�Hbchains as described previously (38).Hb,�Hb, and�Hb were maintained in the carbonmonoxy-liganded stateduring purification and subsequent analysis, unless otherwisespecified.

Recombinant human Hb (rhHb)5 was produced in Esche-richia coli strain BL21(DE3) from the pHb0.0 plasmid, a gift ofDr. John Olson (39). pHUG21 (a gift of Dr. Doug Henderson)harboring the Plesiomonas shigelloides heme transport systemwas co-transformed to enhance rHb expression (40). Theheme transport system was induced by iron restriction with 50�g/ml of the iron chelator ethylenediamine-di-(o-hydroxyphe-nyl acetic acid) (EDDHA, LGC Standards GmbH). rHb expres-sion was performed at 16 °C overnight, and bacteria were lysedby passage through a French press twice at 1200 psi. Hb waspurified in a single step over nickel-nitrilotriacetic acid beads(Qiagen) by virtue of interactions with naturally occurring Hisresidues on the surface of Hb and dialyzed twice against PBS.Substitution mutations within the Hb genes were generatedusing PCR-based mutagenesis and confirmed by sequencing.For protein crystallography, native Hb purified in the car-

bonmonoxy-liganded state was converted to the oxygenatedform by passing a pure stream of oxygen over a protein solutionheld on ice and illuminated with a focused beam from a 50-watthalogen lamp. The oxy-Hb was converted to met-Hb by addi-tion of excess potassium ferricyanide in 20 mM sodium phos-phate, pH 7.0. The reaction was monitored to completion byUV-visible spectroscopy, at which point the Hb protein wasisolated over SephadexG-25.Met-Hbwas combinedwith puri-fied IsdHN1 in a 1:1 molar ratio (with respect to �Hb��Hbdimers) for crystallization.Light Scattering—Samples were separated on a Superose 12

column (GE Healthcare) equilibrated in 20 mM sodium phos-phate, 150 mM sodium chloride, pH 7.0, with in-line MALS(mini-DAWN; Wyatt Technology Corp., Santa Barbara, CA)and refractive index (Optilab differential refractometer, WyattTechnology Corp.) measurements. The refractive index incre-ment with respect to mass concentration (dn/dc) was taken tobe 0.19 ml g�1 for all proteins/complexes.Isothermal Titration Calorimetry (ITC)—ITC was carried

out on a MicroCal iTC200 instrument. All components weredialyzed into 20mM sodiumphosphate, pH 7.5, 150mM sodiumchloride. Multiple injections of 1.1 �l were performed at 150-sintervals, with continuous stirring at 20 °C. The volume of thereaction cell was 350 �l. Heats of dilution were determinedfrom injections of protein solution into buffer and subtractedfrom the experimental data.UV-visible Spectroscopy—Changes in heme coordination of

�Hb (20 �M) were measured by UV-visible absorption spec-troscopy (Shimadzu UV1800) at 30 °C in the presence/absenceof NEAT domains in 20 mM sodium phosphate, 10 �M diethyl-enetriaminepenta-acetic acid, pH 7.0.Structure Determination—Crystals were prepared by hang-

ing-drop vapor diffusion at 293 K inwhich 2�l protein solution(10.9 mg/ml) was mixed with 2 �l of precipitant (0.2 M potas-sium thiocyanate, 0.1 M Bis-tris propane, pH 7.5, 20% PEG).Crystals of 150–200 �m appeared within 2 days. Seeding wasdone to grow diffraction quality crystals. The crystals were

5 The abbreviations used are: rhHb, recombinant human Hb; rHb, recombi-nant Hb; ITC, isothermal titration calorimetry; PDB, Protein Data Bank;EDDHA, ethylenediamine-di-(o-hydroxyphenyl acetic acid); SEC, sizeexclusion chromatography.

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cryoprotected with 30% glycerol and flash-cooled in a coldnitrogen stream (100 K). X-ray diffraction data were collectedin-house using copper K� x-ray produced by a Rigaku 007HFrotating-anode generator with Osmic Varimax optics andrecorded on a MAR345 image plate (Marresearch GmBH).Data were collected over a phi-range of 180° and were

indexed and scaled using MOSFLM (41) and Scalepack (42).Structure was solved by molecular replacement using PHASER(43), which gave a unique solution when using �Hb or �Hb(PDB code 1IRD) as independent search models. The structurewas refined using REFMAC5 (44), withmanual map inspectionand model building performed in COOT (45). The quality ofthe model was regularly checked for steric clashes, incorrectstereochemistry, and rotamer outliers using MolProbity (46).Bacterial Strains and Growth Conditions—All experiments

were carried out with S. aureus strain Newman (47) or withmutants generated in its background. All cultures were inocu-lated from a single colony and grown overnight (�20 h) in 5 mlof RPMI medium (Thermo) supplemented with 1% casaminoacids in 15-ml conical tubes at 37 °C with shaking at 180 rota-tions per minute (rpm). The isogenic variant in which the Hbreceptors IsdB and IsdH have been deleted (�isdBH) has beendescribed previously (11).S. aureus Growth Curves—Single colonies of S. aureus were

inoculated into RPMI medium plus casamino acids, supple-mented with 0.5 mM EDDHA and grown overnight. One ml ofovernight cultures was normalized to A600 of 3.0, and bacteriawere sedimented (9000 � g, 3 min) and resuspended in 1 ml ofNRPMIwith 0.5mMEDDHA.NRPMIwas prepared in advanceby treating RPMI plus casamino acids with Chelex 100 (Sigma)according to themanufacturer’s recommendations and supple-menting the resulting ion-deficientmediumwith 25mMZnCl2,25 �M MnCl2, 100 �M CaCl2, and 1 �M MgCl2. The resultingsuspension of S. aureus was subcultured (1:100) into 1 ml ofNRPMI with 0.5 mM EDDHA and rHb at 2.5 �g/ml. One-mlcultures were incubated at 37 °C in 15-ml conical tubes withshaking at 180 rpm.A600 measurements were taken at the indi-cated time points by mixing 20-�l aliquots of the culture with180 �l of PBS in 96-well plates. The graphs represent a mean ofthree independent experiments. Error bars represent S.D.;asterisks denote values upon mutant rHb supplementationsignificantly different from values upon wild type rHb supple-mentation at the same time point (Student’s two-tailed t test,p � 0.05).

RESULTS

First and Second NEAT Domains of IsdH Bind to �Hb Chainof Hb—To investigate the molecular mechanism of Hb recog-nition by S. aureus, we produced the Hb-binding domains,IsdHN1 and IsdHN2 (Fig. 1A), in an E. coli expression system.Recombinant IsdHN1 and IsdHN2 are pure monomers as deter-mined by SEC (Fig. 1B) and Rayleigh light scattering (supple-mental Table 1). Both NEAT domains bind to native adult HbA, shifting the SEC elution peak to an earlier elution time (Fig.1C, solid line). Formation of a protein complex was confirmedby light scattering measurements (supplemental Fig. 1). Todetermine the binding sites on Hb, we first separated the con-stituent�Hb and�Hb chains.Mixing of IsdHN1 or IsdHN2with

purified �Hb, at a 1:1 molar ratio, lead to the formation of adistinct protein complex (Fig. 1D), with a molecular weightclose to that expected for a heterodimer (supplemental Fig. 1).The binding affinity for the IsdHN1:�Hb was 100 nM, deter-mined by isothermal titration calorimetry (ITC, supplementalFig. 2). Interaction between IsdHN1 and �Hb could not bedetected using SEC (Fig. 1E), light scattering or ITC (supple-mental Figs. 1 and 2). Some evidence of complex formation wasdetected uponmixing equimolar quantities of IsdHN2 and�Hb,but themixture retained a substantial proportion of freemono-mers suggesting a weaker interaction (Fig. 1E). Thus, theIsdHN1/N2 domains show a clear preference for �Hb binding,although it is conceivable that interaction of IsdHN2 with �Hbmight also be significant in vivo. In the absence of heme, theresulting apo-�Hbchain is no longer competent to bind IsdHN1(supplemental Fig. 3), suggesting that Hb is released from theHb receptor following heme removal.IsdHN1/N2 Do Not Induce Large-scale Changes in Heme

Pocket Structure of �Hb—To determine whether IsdHN1 orIsdHN2 domains induce structural changes that might promoteheme release, we employed UV-visible absorption spectros-

FIGURE 1. The N-terminal NEAT domains of IsdH bind to Hb through the�Hb chain. A, the S. aureus Hb receptors IsdH and IsdB possess NEAT domainsthat bind to Hb (stippled) or heme (gray). Amino acid sequence identity isindicated for the pairs of NEAT domains joined by dashed lines. C-terminalsortase cleavage sites are indicated. B, elution profiles of purified IsdHN1 andIsdHN2 on SEC. C, SEC traces for free Hb A (dashed lines) or Hb A mixed with anequimolar quantity of IsdHN1 or IsdHN2 (solid lines, equimolar with respect totetrameric HbA). A shift in the SEC elution peak to smaller elution volumeindicates the formation of a protein complex. D and E, SEC traces for �Hb/�Hbalone (dashed lines) or mixed with an equimolar quantity of IsdHN1 or IsdHN2(solid lines, equimolar with respect to �Hb or �Hb monomers). Refractometervoltage is proportional to protein concentration. Light scattering dataaccompanying B–E are shown in supplemental Fig. 1.

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copy as a sensitive probe of heme coordination chemistry.These studies revealed that IsdHN1/N2 had no substantial effecton �Hb heme pocket structure or heme release over a period of8 h (Fig. 2A). Small absorbance changes were observed in allsamples consistent with slow autooxidation. After 48 h thespectrum of IsdHN1:�Hb contained distinct peaks at 630, 500,and 405 nm (Fig. 2B, solid line), characteristic of the auto-oxi-dation product, met-Hb (48). The sample of free �Hb con-tained precipitate and following filtration, yielded a spectrumindicative of a mixture of heme-ligand states (Fig. 2B, dashedline). Our interpretation is that met-�Hb forming in the free�Hb sample was being lost as precipitate (met-�Hb is wellknown to be highly unstable) and that IsdHN1 was stabilizingmet-�Hb. ITC revealed that IsdHN1 binds carbonmonoxy-�Hbormet-�Hbwith the same affinity (supplemental Fig. 2). Thesedata indicate that IsdHN1 and IsdHN2 capture Hb, independentof ligand/oxidation state, but do not induce conformationalchanges in Hb that cause heme release. Hence, rapid hemerelease must rely upon recruitment of the heme acceptordomain of IsdH.Crystal Structure of IsdHN1�Hb Complex—To reveal the

molecularmechanismofHbbinding, IsdHN1was crystallized incomplexwithmet-Hb, and x-ray diffraction datawere collectedto a resolution of 3.0Å (Table 1). Initial phaseswere obtained bymolecular replacement using �Hb or �Hb (PDB code 1IRD) asindependent search models. In the crystal structure, IsdHN1binds to one �Hb��Hb dimer through a site on the �Hb chain(Fig. 3A). There are two independent IsdHN1�Hb complexes inthe crystallographic asymmetric unit. The IsdHN1 and �Hbsubunits are identical in the two complexes (C� coordinatesoverlay with a root mean square displacement of 0.35 Å, sup-plemental Fig. 4). Electron density in one of the�Hb subunits is

weak, possibly due to whole domain movement. The welldefined�Hb��Hbdimer is very similar in structure to the nativeHb dimer (C� displacement of�1 Å rootmean square) with nodramatic conformational change being induced by IsdHN1(supplemental Fig. 4). The dimeric form of Hb appears to havebeen selected by the crystallization process, as IsdHN1 does notoverlap with the Hb dimer-tetramer interface. Indeed, molec-ular weightmeasurements obtained for the complex in solutionwere consistent with IsdHN1 bound to a mixture of Hb dimersand tetramers (supplemental Table 1). In any case, the met-Hbdimer-tetramer interaction is relativelyweak (Kd� 30�M) (49);hence, the�Hb��Hbdimer observed in our structure is likely tobe a physiologically relevant species in plasma.IsdHN1��Hb Interface—The IsdHN1 domain is a nine-

stranded distorted �-barrel with an immunoglobulin-like fold.The conformation is very similar to the heme-binding NEATdomains from IsdA, IsdB, and IsdC (30–34) but with an addi-tional N-terminal helix loop. The binding site for Hb is formedfrom loops at one end of the�-barrel (Fig. 3A), designated loops2, 4, 6, and 8 based on the conserved NEAT domain fold. Loop2 of IsdHN1 contains an unusual string of five consecutive aro-matic residues (Tyr-125, Tyr-126, His-127, Phe-128, and Phe-129; Fig. 3, B and C), which are crucial for Hb binding (36). Anumber of these aromatic side chains make van derWaals con-tacts with non-polar surfaces on�Hb and are partially (Tyr-125andPhe-128) or completely (Tyr-126 andPhe-129) buried fromsolvent. Loops 4 and 6 of IsdHN1 account for the majority ofelectrostatic interactions with �Hb (Fig. 3C). Overall, the �Hb-interacting face of IsdHN1 displays a negative electrostaticpotential that complements a surface with positive potential on�Hb. Of particular note, the positively charged �-amino groupof �Hb Lys-11 protrudes into an electronegative pocket onIsdHN1 (Fig. 3D) lined with hydroxyl and carbonyl groups fromSer-130, Tyr-126, Asn-151, and Thr-152 (Fig. 3C).A comparison with the NMR structure of the free IsdHN1

domain (Fig. 4, A and B) (36) reveals a substantial conforma-tional change in IsdHN1 loops 2 and 8 upon complex formation.Loop 8 requires a considerable movement of �8–10 Å toaccommodate �Hb. More strikingly, residues 123–129 of loop2 are disordered in free IsdHN1 (36) and undergo a transition toan ordered helical conformation upon interaction with Hb. Inthe absence of �Hb, it appears that the aromatic side chains inloop 2 cannot bury sufficient surface area to attain a stable fold.In the helical conformation Gln-124 and His-127 in loop 2make specific intramolecular H-bonding interactions with theunderlying �-sheet (Fig. 4C). Remarkably, the Hb-binding siteon IsdHN1 occupies the same face of the NEAT fold that is usedto bind heme in other NEAT domains (Fig. 4D). We infer fromthis that structural properties of the NEAT fold might predis-pose this site to ligand interactions.Mutations in �Hb Abrogate Binding to IsdHN1/N2—To gain

insight into the ligand binding specificity of NEATdomains, wecompared the IsdHN1-binding surface of �Hb (Fig. 5A) to thesurface of�Hb, which does not bind IsdHN1 (Fig. 5B). Themostoutstanding difference between these two surfaces is a substi-tution of Lys-11 in �Hb and for Thr-12 in �Hb. Interestingly,Lys-11 is conserved across �Hb sequences from distantlyrelatedmammalian species (Fig. 5C). To test the role of Lys-11

FIGURE 2. Spectral analysis of the IsdHN1/N2��Hb complexes. A, oxygen-ated �Hb was incubated with or without IsdHN1/N2 at 30 °C, and visibleabsorption spectra were recorded at 0, 0.5, 1, 1.5, 2, 2.5, 3.5, 5.0, and 8.0 h.Arrows indicate the direction of spectral change. B, after 48 h, the spectrum ofIsdHN1��Hb contained peaks at 405, 500, and 630 nm, characteristic ofmet-�Hb.

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in IsdH recognition, we produced recombinant Hb (rHb)carrying a Lys-11 to Thr mutation in the �Hb chain(rHb(�K11T)). A second mutant (rHb(��18,19)) was pro-duced to test the effect of removing the binding site for one

of the IsdHN1 loop 2 aromatic residues (Tyr-125) by short-ening the linker between �Hb helices A and B. Thirdly,rHb(�A5E) was generated in response to the natural varia-tion in this position across �Hb sequences from different

TABLE 1Crystallographic data collection and refinement statistics

Data collectionSpace group P212121Cell dimensions a � 65.880, b � 123.206, and c � 143.933 Å; � �, and �� 90°Resolution (Å) 50.00–3.01 (3.16–3.01)Rmerge 0.244 (0.785)I/�I 8.6 (2.3)Completeness (%) 98.9 (92.8)Redundancy 6.9 (6.5)

RefinementResolution (Å) 3.01No. of reflections 21,420Rwork/Rfree 0.245/0.275No. of atomsProtein 6434Ligand/ion 172

B-factorsProtein 22.7Ligand/ion 22.5

Root mean square deviationsBond lengths (Å) 0.005Bond angles 0.698°

Ramachandran plotFavored (%) 95.2Disallowed (%) 0.0PDB code 3SZK

FIGURE 3. The structure of the IsdHN1�Hb complex. A, IsdHN1 (yellow) binds to the �Hb subunit (orange) of the �Hb:�Hb dimer. B, stereo view of the 2Fo � Fcelectron density map contoured at 1� (mesh) for portions of IsdHN1 loop 2 (green), IsdHN1 loop 4 (cyan), and helix A of �Hb (orange). C, aromatic residues fromIsdHN1 loop 2 (green) pack against �Hb. Loops 4 and 6 of IsdHN1 (cyan) account for the majority of polar and H-bonding interactions (dashed lines). D, the�Hb-contacting surface of IsdHN1 colored by electrostatic potential (positive, blue; negative, red; supplemental “Experimental Procedures”) showing thebinding pocket for �Hb Lys-11.

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species (Fig. 5C). Reciprocal mutations were also introducedinto the �Hb chains (Fig. 5C).Wild type and mutant rHb samples were purified and con-

verted to the stable cyanomet form (50). All proteins had nor-mal UV-visible spectra (data not shown) and eluted in onemajor peak from SEC, at the expected mobility (supplementalFig. 5), indicating no substantial defect in folding or �Hb-�Hbsubunit interactions. The individual SEC traces for rHb andIsdHN1 are overlaid in Fig. 5D, upper panel. Mixing of thesecomponents lead to formation of the expected IsdHN1�Hb com-plex (Fig. 5D, lower panel, black). Strikingly, rHb(�K11T)showed no detectable interaction with IsdHN1, yielding twoseparate elution peaks (Fig. 5D, blue). Similarly, rHb(�K11T)failed to interact significantly with IsdHN2 (Fig. 5E, blue),strongly suggesting that IsdHN1 and IsdHN2 bind a commonsite on Hb. The reverse mutation in �Hb, rHb(�T12K), had nodetectable effect on IsdHN1 binding (Fig. 5D, red). However,rHb(�T12K) experienced a larger than normal shift in SECretention time when mixed with IsdHN2 (Fig. 5E, red), suggest-ing that the intrinsic weak binding to wild type �Hb subunitsmight be enhanced by the �T12K mutation. The �A5E muta-tion partially inhibited binding to IsdHN1 but not IsdHN2 andrHb(��G18,A19) also displayed reduced binding to IsdHN1 butwas not tested against IsdHN2 (Fig. 5F and supplemental Fig. 5).In summary, both IsdHN1 and IsdHN2 are likely to bind Hbthrough the same site on �Hb but display some differences in

sequence specificity that could potentially provide greater tol-erance to species variation in the Hb sequence.IsdHN1-�Hb Interaction Is Important for S. aureusGrowth on

Hb as Sole Iron Source—To investigate the importance ofNEAT-Hb interactions for iron uptake from Hb, we grewS. aureus strain Newman onmedium containing Hb as the soleiron source. When supplemented with rHb, a culture ofS. aureus grew over an incubation time of 40 h as shown in Fig.5G (black). In contrast Hb carrying �Hbmutations that inhibitIsdHN1 and/or IsdHN2 binding supported a significantly slowerrate of culture growth. An S. aureus strain carrying deletions ofboth Hb receptors, IsdB and IsdH (�isdBH), failed to replicateon wild type or mutant Hb (Fig. 5H), confirming that theIsdB/H Hb receptors are required for iron uptake from Hb inthis assay. Compared with isdBH, the residual growth ofS. aureusNewman onmutant Hb suggested that our individual�Hbmutations are not sufficient to completely abrogate inter-actions with intact IsdH and/or IsdB. This could be due to avid-ity affects arising from the multidomain structure of IsdB/H,binding of Hb through �Hb chains, or because the mutationsthat inhibit binding to IsdH have more limited effects on theIsdB-Hb interaction. In any case, our results demonstrate thatcapture ofHb throughNEATdomain receptors is an importantstep that allows S. aureus to utilize iron from Hb.

DISCUSSION

Direct binding of host iron proteins to bacterial cell-surfacereceptors is a widespread strategy to capture iron, but despitetheir importance, these interactions are still poorly understoodat themolecular level. For example, the structures of transferrinreceptors present on the surface of pathogenic species from thegeneraNeisseria and Pasteurella have only recently been deter-mined (51, 52) and only then in the absence of the transferrinligand. The importance of Hb-binding proteins is emphasizedby their widespread distribution across bacterial and protozoanpathogens. In Gram-negative bacteria, TonB-dependent hemetransporters in the bacterial outer membrane act as Hb recep-tors in Haemophilus (53), Neisseria (54), Pasteurella (55), Por-phyromonas (56), and Helicobacter (57) species. In Gram-pos-itive bacteria, the IsdB/H proteins from S. aureus are the mostwell documented Hb receptors, but cell-surface proteins fromStreptococcus pyogenes (58) Streptococcus equi (59) Bacilluscereus (60), andCorynebacterium diphtheriae (61) are reportedto bind Hb. In addition to the surface-bound receptors,secreted hemophores produced by Gram-negative (62) andGram-positive (63, 64) bacteria may transiently interact withHb to facilitate heme transfer. The IsdHN1�met-Hb complex isthe first Hb receptor complex for which the structure has beendetermined. It reveals a highly specific protein-protein interac-tion, raising the possibility that inhibitors to this interactionmight be developed. If this is so, the diversity of Hb-bindingfunctions described above may provide restriction pointsthrough which to inhibit colonization of the host by a range ofpathogens.Loops 2 and 4 account for the majority of IsdHN1-Hb inter-

actions and are largely conserved in IsdHN2 and IsdBN1 (sup-plemental Fig. 6), suggesting that all three NEAT domains bindHbA in a similar way. In each case, loop 2 contains an aromatic-

FIGURE 4. The ligand-interaction sites in Hb-binding and heme-bindingNEAT domains share some common features. A, the NMR structure of freeIsdHN1 (PDB code 2H3K) contains a disordered loop 2 (36). B, when bound toHb, loop 2 of IsdHN1 adopts an �-helical conformation. C, in the �-helicalconformation, loop 2 makes H-bonding interactions with the underlying�-sheet. D, NEAT domains have evolved to bind Hb or heme ligands using asimilar face of the NEAT domain fold. Shown here, for comparison withIsdHN1, is the heme-binding NEAT domain from IsdC in complex with Zn pro-toporphyrin IX (PDB 2K78) (33). Green shading indicates regions that areaffected by conformational exchange in the ligand-free states of IsdHN1 andIsdC.

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rich sequence and is predicted to adopt a similar �-helicalstructure, with conservedGln andHis residues interactingwiththe underlying �-sheet (supplemental Fig. 6). In IsdHN1, Tyr-126 and Ser-130 form part of the binding pocket for �Hb Lys-11, and these residues are conserved in IsdHN2 and IsdBN1.Similarly, side chains from IsdHN1 loop 2 involved in H-bond-ing to Lys-11 are conserved in IsdHN2 and IsdBN1. Thus, it islikely that the preference for binding to �Hb observed forIsdHN1 and IsdHN2 will also hold true for IsdB. This, along withthe fact that Lys-11 is highly conserved in �Hb sequences from

many mammalian species, argues that targeting the �Hb sub-unit is of mechanistic importance.The transfer of heme from met-Hb to the isolated heme

acceptor domains of IsdH/B is governed by the simple dissoci-ation of heme from met-Hb (20, 35) and is 2–3 orders of mag-nitude slower than heme transfer to the full-lengthHb receptor(20). Thus, physical interaction between Hb and the Hb recep-tors is crucial for activating heme transfer. Structural and UV-visible spectroscopy data obtained for IsdHN1/N2 argue that thisenhancement in heme transfer rate does not arise from a con-

FIGURE 5. Mutation of IsdH binding site in �Hb restricts Hb utilization by S. aureus. A, the surface of �Hb, colored by electrostatic potential (positive, blue;negative, red) and �Hb-contacting loops from IsdHN1 (same orientation as Fig. 3C). B, a model showing the surface of �Hb (PDB 1GZX) overlaid with the�Hb-binding residues of IsdHN1. Dashed ovals mark substantial differences between the molecular surfaces of �Hb and �Hb. C, helix A sequences of �Hb and�Hb from divergent mammalian species, showing �Hb residues that make H-bonds (stars) or other contacts (dots) with IsdHN1. D, upper panel, SEC elutiontraces for IsdHN1 (solid curve) and recombinant wild type Hb (rHb, dashed curve). Lower panel, SEC elution traces for mixtures of IsdHN1 with rHb (black),rHb(�T12K) (red), or rHb(�K11T) (blue). E, SEC elution traces showing interactions of IsdHN2 with rHb and rHb mutants. F, summary of binding data for rHbmutants, indicating the fraction of unbound IsdHN1 in chromatographic separations (supplemental Fig. 5). G, growth of S. aureus strain Newman with rHb orrHb mutants as the sole iron source. H, growth of S. aureus �isdBH under conditions as in G.

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formational change in Hb that promotes heme release. Instead,we favor an alternative model whereby binding to Hb throughthe IsdHN1/N2 or IsdBN1 domains enhances the rate of produc-tive encounters between Hb chains and the IsdHN3 or IsdBN2heme acceptor domains. One potential role for the specificbinding of IsdB/H to �Hb subunits might be to control thesequential release of heme from the �Hb and �Hb chains byorienting the heme acceptor domains in a particular way.�Hb��Hb dimers can loose heme from one subunit (semi-Hb)and retainmuchof their native structure (65), potentially allow-ing them to remain associated with theHb receptor until all theheme is removed. IsdHN1 does not bind to free apo-�Hb, sug-gesting that the IsdH�Hb complex would dissociate followingcomplete heme removal from Hb.NEAT domains with the characteristic aromatic loop 2

sequence of IsdHN1 are restricted to the genomes of S. aureusand the related Staphylococcus lugdunensis, suggesting that theHb-binding mechanism described here is restricted to theseorganisms. However, Hb-binding function has been reportedfor other NEAT domain containing proteins, including IlsAfrom B. cereus (60), IsdX1 from Bacillus anthrasis (63) and Shrfrom S. pyogenes (66), suggesting that NEAT domains haveadapted to make different interactions with Hb or thatsequences outside the NEAT domains are responsible for theHb-binding activity of these proteins.Concerted folding and binding is proposed to allow recogni-

tion of diverse molecular targets by a single protein (67), and itis possible that, being an opportunistic pathogen, folding andbinding of IsdH might help S. aureus to accommodate some ofthe natural sequence variation present in Hbs from differenthost species. IsdH also binds directly to haptoglobin, a mam-malian protein that is unrelated in sequence or structure to Hb(10, 25, 35). Aromatic residues in IsdHN1 loop 2 are required forbinding toHbor haptoglobin (35), and it will nowbe interestingto establish whether folding and binding has a role in accom-modating these different ligands. The function of haptoglobinis to sequester Hb that is released by continuous physiologicallevels of hemolysis, hence the Hb�haptoglobin complex repre-sents an alternative route through which S. aureus might cap-ture Hb.Among Isd proteins, folding and binding is not limited to

IsdH. AnNMR study of the heme-binding NEAT domain fromIsdC revealed that loop 2 (Fig. 4D) undergoes significant con-formational dynamics in the absence of ligand (33) consistentwith a folding-and-binding event. Here, loop flexibility mayfacilitate insertion of the hydrophobic heme molecule “under”loop 2. In contrast, loop 2 from the heme-binding NEATdomain of IsdA adopts a helical conformation in crystal struc-tures of the heme-free and heme-bound forms. The structure ofIsdA has not been determined in solution, and it is conceivablethat a folded loop 2 conformation might be stabilized duringcrystallization. Although a more extensive study into the gen-eral role of conformational dynamics in NEAT-ligand interac-tions is required, it is tempting to speculate that structural flex-ibility in an ancestral NEAT domain may have played a role inthe remarkable adaptation of this domain to binding such vastlydifferent molecular targets as heme and Hb protein.

The IsdHN1�Hb structure reveals a highly specific recogni-tion interface that allows S. aureus to capture heme directlyfrom the richest iron source in the mammalian host. The Hb-receptor mechanism establishes a direct conduit for the trans-fer of heme groups from host Hb into the bacterial cytoplasm.In this regard, it contrastswith strategies such as siderophore orprotease secretion, which mobilize iron into the extracellularmilieu where it can be intercepted by competing bacterial pop-ulations. The highly specific nature of the IsdH-Hb interactionsuggests that sequence variation in the IsdB/H-binding face ofHb could influence susceptibility to S. aureus infection, asdescribed recently for mice and humans (68). Determining thestructure of an Hb-capture complex now raises the excitingpossibility of mimicking the NEAT-interacting face of �Hb as astrategy for developing new anti-virulence treatments forS. aureus infection.

Acknowledgment—Data supporting this project, but not included inthe final manuscript, were collected theMX2 beamline at the Austra-lian Synchrotron.

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Supplementary information

The structural basis for hemoglobin capture by the Staphylococcus aureus cell-surface protein,

IsdH

Kaavya Krishna Kumar1,#, David A. Jacques1,#, Gleb Pishchany2, Tom Caradoc-Davies3, Thomas

Spirig4, G. Reza Malmirchegini4, David B. Langley1, Claire F. Dickson5, Joel P. Mackay1, Robert T.

Clubb4, Eric P. Skaar2, J. Mitchell Guss1 and David A. Gell5

1School of Molecular Bioscience, University of Sydney, NSW 2006, Australia 2Department of Microbiology and Immunology, Vanderbilt University Medical School, Nashville, TN

37232, USA 3Australian Synchrotron, Clayton, Vic 3168, Australia 4Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, USA 5Menzies Research Institute, University of Tasmania, TAS 7000, Australia

Supplementary Figures

Supplementary Figure 1 Light scattering data showing that IsdHN1 and IsdHN2 bind to the αHb chain

of Hb, supplementary to Figure 1B-E. Elution profiles from size-exclusion chromatography (SEC, solid

lines; refractive index ∝ protein concentration) and molecular weights calculated from in-line Rayleigh

light scattering data (dots, ordinate axis scale at right hand side). Free IsdHN1/N2 is shown in red, globin

(HbA, αHb or βHb) is shown in blue and the IsdHN1/N2:globin complex is shown in black. Free HbA,

αHb and βHb samples each contain a mixture of oligomeric states but elutes as a single peak in SEC

due to rapid exchange between these states (Supplementary Materials and Methods). Traces shown in

same panel were obtained using the same hardware setup. In all, data were obtained from a number of

different chromatography systems and elution volumes may vary between panels.

Supplementary Figure 2 Isothermal titration calorimetry (a) Titration of IsdHN1 (150 µM in syringe)

into carbonmonoxy(CO)-liganded αHb (10 µM in cell). The upper pane shows the difference in power

applied to the sample and reference cells to maintain constant temperature. Negative values indicate the

interaction is exothermic. The lower panel shows the binding curve generated by non-linear least

squares fitting to a single-site binding model. The thermodynamic parameters obtained were:

association constant KA = 11.3 ± 3 × 106 M-1 (average ± 1 standard deviation from three different

experiments); stoichiometry n = 0.94 ± 0.04; apparent enthalpy ΔHapp = −8.8 ± 0.9 kcal mol-1. (b)

Titration of IsdHN1 (150 µM in syringe) into CO-βHb (10 µM as monomer, in cell). (c) Titration of

IsdHN1 (100 µM in syringe) into met-αHb (10 µM in cell). The thermodynamic parameters obtained

were: KA = 11.1 ± 1 × 106 M-1 (average ± 1 standard deviation from two different experiments);

stoichiometry n = 0.91 ± 0.007; apparent enthalpy ΔHapp = −14.8 ± 1.4 kcal mol-1.

Supplementary Figure 3 IsdHN1 does not bind to isolated apo-αHb. The free apo-αHb is highly

unstable and is lost on the column under the conditions employed, unless it forms a stable complex

with a binding partner. IsdHN1 elutes in the same peak with (black) or without (red) addition of apo-

αHb, indicating that these proteins do not make a stable complex. Apo-αHb is stabilized through

interaction with the αHb-stabilizing protein (AHSP), allowing the apo-αHb:AHSP complex to be

eluted (green). AHSP induces a native-like structure in apo-αHb (1) allowing a ternary IsdHN1:apo-

αHb:AHSP complex to be eluted (maroon). IsdHN1 did not interact directly with AHSP (not shown).

Supplementary Figure 4

αHb(1) βHb(1)

IsdHN1(1)

IsdHN1(2)αHb(2)

βHb(2)

a

b

c

αHb

βHbIsdHN1

d αHbαHb

βHb

Supplementary Figure 4 Structure of the IsdHN1:HbA complex and comparison with HbA. (a) Two

independent IsdHN1:HbA complexes are present in the crystallographic asymmetric unit. In one

complex the αHb, βHb and IsdHN1 subunits are colored as in Figure 3. The second complex is colored

magenta. (b) A superposition of the two complexes from a. (c) A superposition of one αHb:βHb dimer

from a (blue, orange) with the tight αHb:βHb dimer in native Hb (magenta, PDB 2dn3). The overlay

was performed to minimize the deviation in βHb coordinates (0.5 Å RMS over 140 Cα atoms). (d) An

overlay of the αHb subunits alone from c gives a displacement of 1 Å RMS over 140 Cα atoms.

Supplementary Figure 5 The effect of Hb mutations on IsdHN1/N2 binding. SEC elution traces for

IsdHN1, IsdHN2, recombinant Hb (rHb) and rHb mutants as labeled (dashed lines). SEC elution traces

for mixtures of IsdHN1/N2 with rHb and rHb mutants, mixed at a 1:1 molar ratio (calculated on the basis

of αHb:βHb dimers) are shown in solid lines.

Supplementary Figure 6 One region of a structure-based sequence alignment of the seven NEAT

domains from S. aureus IsdA,B,C,H. Sequences are grouped based on their target ligand: heme (upper

four sequences) or Hb (lower three sequences). Residues that participate in ligand interactions from

experimentally determined structures (see main article introduction) are highlighted white-on-black.

Residues from IsdHN2 and IsdHN3 predicted from the current work to be involved in Hb-binding are

show black-on-grey. Residues likely to be involved in intra-molecular H-bonding interactions (Fig. 4C)

are joined by dashed lines and are conserved across Hb-binding, but not heme-binding, NEAT domains.

Supplementary Table 1 Molecular weights (Mw) for IsdHN1 and IsdHN2 complexes measured by

light scattering in-line with SEC

Sample

(Mw from sequence,

kDa)

Column load (µg

in 0.2 ml)

Mw (kDa); average ± 1

SD for (n)

measurements

100 19.0 ± 0.4 (3) IsdHN1

(19.0) 200 19.4 ± 0.1 (2)

100 20.81± 0.24 (2) IsdHN2

(19.4) 200 20.38± 0.41 (4)

100 45 ± 5 (6) Hb

(64.5, as tetramer) 200 59 ± 1.3 (4)

100 16.9 ± 0.5 (2) αHb

(15.7) 200 19.1 ± 0.1 (2)

70 54 ± 3 (5) IsdHN1:Hb

200 78 ± 2 (3)

IsdHN2:Hb 200 74.25± 2.2 (4)

100 32 ± 0.5 (2) IsdHN1:αHb

200 38 ± 1.4 (4)

IsdHN2:αHb 200 36.6 ± 0.83 (3)

Supplemental Experimental Procedures

Light scattering

Samples were separated on a Superose 12 column (GE Healthcare) and eluting protein fractions were

monitored by in-line light scattering (LS) and refractive index (RI) measurements. LS intensity was

measured at three angles (41.5˚, 90.0˚ and 138.5˚) using a mini-DAWN (Wyatt Technology Corp.,

Santa Barbara CA) equipped with a 690 nm laser. Voltages for the three detectors were normalized

using BSA monomer (Sigma) as a standard isotropic scatterer. Absolute LS intensity was calibrated

once against a standard sample of HPLC grade toluene. Protein concentrations were obtained from in-

line RI measurements (Optilab differential refractometer, Wyatt Technology Corp.), calibrated against

standard salt solutions and the refractive index increment with respect to mass concentration (dn/dc)

was taken to be 0.19 ml g-1 for all proteins/complexes. Weight-average molecular weight (Mw) of the

solute was calculated for every 5 µl volume across each eluting peak using the Rayleigh-Debye-Gans

scattering model for a dilute polymer solution, as implemented by the ASTRA software (Wyatt

Technology Corp.)(2).

Hb samples were prepared in the CO-liganded form. Under the SEC conditions, Hb A is a mixture of

dimer and tetramer species (3,4). Similarly, αHb and βHb undergo well-characterized self-association

processes. Mw measurements by LS made over a wide range of loading concentrations (not shown)

were consistent with the previously characterized weak monomer-dimer exchange of αHb (5,6) and

monomer-tetramer exchange of βHb (7); (5). In each case only a single peak is observed in SEC, due to

rapid exchange (on the timescale of the separation) between the different self-association states, and the

elution time of the peak is concentration-dependent.

Electrostatics calculation

Atom charges and radii were calculated using the CHARMM27 forcefield (8) and PDB2PQR (9), with

optimized hydrogen bonding networks, and titration states at pH 7 assigned using PROPKA (10).

Electrostatic potential maps were calculated in the presence of 150 mM monovalent ions by solving the

Poisson-Boltzmann equation using the software, Adaptive Poisson-Boltzmann Solver (APBS) (11).

Electrostatic contact surfaces were visualized by coloring the van der Waals surface according to the

electrostatic potential at the solvent accessible surface (determined using a 1.4 Å probe) in the range ±

5 kbTe (where kb is Boltzmann’s constant, T = 310 K, e is the charge of an electron) using PyMOL (12).

Supplementary References

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71 7. Philo, J. S., Lary, J. W., and Schuster, T. M. (1988) J Biol Chem 263(2), 682-689 8. MacKerell, A. D., Bashford, D., Bellott, Dunbrack, R. L., Evanseck, J. D., Field, M. J., Fischer,

S., Gao, J., Guo, H., Ha, S., Joseph-McCarthy, D., Kuchnir, L., Kuczera, K., Lau, F. T. K., Mattos, C., Michnick, S., Ngo, T., Nguyen, D. T., Prodhom, B., Reiher, W. E., Roux, B., Schlenkrich, M., Smith, J. C., Stote, R., Straub, J., Watanabe, M., Wiorkiewicz-Kuczera, J., Yin, D., and Karplus, M. (1998) The Journal of Physical Chemistry B 102(18), 3586-3616

9. Dolinsky, T. J., Nielsen, J. E., McCammon, J. A., and Baker, N. A. (2004) Nucleic Acids Research 32(suppl 2), W665-W667

10. Li, H., Robertson, A. D., and Jensen, J. H. (2005) Proteins 61, 704-721 11. Baker, N. A., Sept, D., Joseph, S., Holst, M. J., and McCammon, J. A. (2001) Proc Natl Acad

Sci U S A 98(18), 10037-10041 12. Schrodinger, LLC. (2010) The PyMOL Molecular Graphics System, Version 1.1r1. In.

Mackay, Robert T. Clubb, Eric P. Skaar, J. Mitchell Guss and David A. GellThomas Spirig, G. Reza Malmirchegini, David B. Langley, Claire F. Dickson, Joel P.

Kaavya Krishna Kumar, David A. Jacques, Gleb Pishchany, Tom Caradoc-Davies,Protein, IsdH

Cell-surfaceStaphylococcus aureusStructural Basis for Hemoglobin Capture by

doi: 10.1074/jbc.M111.287300 originally published online September 14, 20112011, 286:38439-38447.J. Biol. Chem. 

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