Conservation of Structure and Mechanism in Primary and

Conservation of Structure and Mechanism in Primary andSecondary Transporters Exemplified by SiaP, a Sialic AcidBinding Virulence Factor from Haemophilus influenzae*

Received for publication, April 11, 2006, and in revised form, May 11, 2006 Published, JBC Papers in Press, May 15, 2006, DOI 10.1074/jbc.M603463200

Axel Muller‡, Emmanuele Severi§, Christopher Mulligan§, Andrew G. Watts‡1, David J. Kelly¶, Keith S. Wilson‡,Anthony J. Wilkinson‡2, and Gavin H. Thomas§3

From the ‡Structural Biology Laboratory, Department of Chemistry and §Department of Biology, University of York,York YO10 5YW, United Kingdom and ¶Department of Molecular Biology and Biotechnology, University of Sheffield,Firth Court, Western Bank, Sheffield, S10 2TN, United Kingdom

Extracytoplasmic solute receptors (ESRs) are important com-ponents of solute uptake systems in bacteria, having been stud-ied extensively as parts of ATP binding cassette transporters.Herein we report the first crystal structure of an ESR proteinfrom a functionally characterized electrochemical ion gradient-dependent secondary transporter. This protein, SiaP, formspartof a tripartite ATP-independent periplasmic transporter spe-cific for sialic acid inHaemophilus influenzae. Surprisingly, thestructure reveals anoverall topology similar toATPbinding cas-sette ESR proteins, which is not apparent from the sequence,demonstrating that primary and secondary transporters canshare a common structural component. The structure of SiaP inthe presence of the sialic acid analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid reveals the ligand bound in a deep cav-ity with its carboxylate group forming a salt bridge with a highlyconserved Arg residue. Sialic acid binding, which obeys simplebimolecular association kinetics as determined by stopped-flowfluorescence spectroscopy, is accompanied by domain closureabout a hinge region and the kinking of an �-helix hinge com-ponent.The structureprovides insight into the evolution,mech-anism, and substrate specificity of ESR-dependent secondarytransporters that are widespread in prokaryotes.

The uptake of solutes into bacterial cells is critical for growthand survival in every environment and is catalyzed by a varietyof different protein-mediated transport systems. One common

feature of uptake systems, including the well studiedATP bind-ing cassette (ABC)4 transporters, is an extracytoplasmic solutereceptor (ESR) (often also known as a periplasmic-binding pro-tein), which captures the substrate for the transporter anddelivers it to the membrane subunits (1). Structures have beendetermined formany ESR proteins fromABC transporters, andtheir mechanism of ligand binding is so well established thatthese proteins are now being used in a host of biotechnologicalapplications (2).The ABC systems are examples of primary active transport-

ers, so called because they hydrolyze ATP directly to energizetransport (3). These differ from another major grouping, thesecondary active transporters, like Escherichia coli lactose per-mease, so-called because many of them use the membranepotential to energize uptake and direct ATP hydrolysis is notinvolved. The use of an ESR protein, which endows the trans-porter with high affinity for its substrate, was for a long timebelieved to be exclusive to the ABC transporters, but biochem-ical studies of a C4-dicarboxylate uptake system fromRhodobacter capsulatus led to the discovery of a novel family ofESR-dependent secondary transporters, the so called tripartiteATP-independent periplasmic (TRAP) transporters (4–8).These transporters contain two membrane protein compo-nents, the larger of which contains 12 predicted transmem-brane helices. This subunit is a member of the ion transportersuperfamily (6, 9) and probably forms the translocation chan-nel. The smallermembrane component of four transmembranehelices has an unknown but essential function (7). Microbialgenome sequencing has revealed that the TRAP transportersare widespread in the prokaryotic world (10), and known sub-strates now include sialic acid, ectoine, 2,3-diketo-L-gulonate,and pyruvate in addition to C4-dicarboxylates (11–14). Wehave recently characterized the sialic acid TRAP transporterfrom the human pathogen Haemophilus influenzae and dem-onstrated that this transporter is essential for uptake of sialicacid (Neu5Ac) in this bacterium. Neu5Ac is an important host-acquired molecule that is used by the bacterium to modify its

* The work described here was funded by the European Commission as Struc-tural Proteomics in Europe (SPINE), Contract QLG2-CT-2002-00988 underthe Research Technological Development (RTD) program “Quality of Lifeand Management of Living Resources” and by a grant from the UnitedKingdom Biotechnology and Biological Science Research Council (toG. H. T. and D. J. K.). The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

The atomic coordinates and structure factors (code 2CEY (unliganded structure)and 2CEX (Neu5Ac2en structure)) have been deposited in the Protein DataBank, Research Collaboratory for Structural Bioinformatics, Rutgers Univer-sity, New Brunswick, NJ (http://www.rcsb.org/).

1 Current address: Dept. of Pharmacy and Pharmacology, University of Bath,BA2 7AY, UK.

2 To whom correspondence may be addressed. Tel.: 44-1904-328261; E-mail:[email protected].

3 To whom correspondence may be addressed. Tel.: 44-1904-328268; E-mail:[email protected].

4 The abbreviations used are: ABC, ATP binding cassette; TRAP transporter,tripartite ATP-independent periplasmic transporter; Neu5Ac, sialic acid,N-acetylneuraminic acid; Neu5Ac2en, 2,3-didehydro-2-deoxy-N-acetyl-neuraminic acid; dNeu5Ac, 2-deoxy-�-N-acetylneuraminic acid; ESR, extra-cytoplasmic solute receptor; SiaP, sialic acid-binding protein; SeMet,selenomethionine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 31, pp. 22212–22222, August 4, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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lipopolysaccharide to make it appear as “self” and evade theinnate immune response (15). Deletion of the TRAP trans-porter results in loss of lipopolysaccharide sialylation andserum resistance in H. influenzae Rd (13), a phenotype alsoobserved recently in non-typeable strains of H. influenzae (16)and in the related animal pathogen Pasteurella multocida (17).These were the first reports of TRAP transporters having a rolein virulence and highlight the importance of a greater under-standing of the function and mechanism of these systems inprokaryotes.The sialic acid-binding protein SiaP is a member of the DctP

protein family, named after the first characterized TRAP ESRprotein that binds C4-dicarboxylates (4). This is the major fam-ily of ESR proteins found in TRAP transport systems (10).Given the potential importance of TRAP transporters in thebiology of prokaryotes but the paucity of information on them,we solved the structure of SiaP at 1.7 Å in an unliganded formand also at 2.2 Å in complex with the sialic acid analogue, 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (Neu5Ac2en).Our study provides important new information on sialic acidtransport and insight into the function and evolution of thisnovel family of ESR-dependent secondary transporters.

EXPERIMENTAL PROCEDURES

Expression and Purification of SiaP—The SiaP protein waspurified from E. coli using a modification of the methodsdescribed in Severi et al. (13). Cells of E. coli BL21(DE3) pLysSpGTY3 were grown in 5 ml of LB (Lennox broth) for 6 h,washed in M9 minimal medium (18), and used to inoculate 50ml M9 minimal medium for overnight growth at 37 °C. Thiswas used to inoculate 1 liter of M9 minimal medium at 25 °C.Cells were allowed to grow to an A650 of 0.3–0.4 before induc-ing expression with 1 mM isopropyl 1-thio-�-D-galactopyrano-side followed by overnight incubation. Cells were washed inice-cold 50 mM Tris-HCl, pH 8, incubated in SET buffer (0.5 Msucrose, 5 mM EDTA in 50 mM Tris-HCl, pH 8, 600 �g/mllysozyme) for 1 h at 30 °C, and the periplasmic fractionwas thenclarified by centrifugation and dialyzed against 50 mM Tris-HCl, pH 8, containing 1.5 M (NH4)2SO4. SiaP was purified byfast protein liquid chromatography using a hydrophobic inter-action column followed by size exclusion chromatographyusing a G75-Sepharose column as described previously (13).Protein concentration was determined from the absorbance at280 nm using a molar absorption coefficient for SiaP of 23840M�1 cm�1. The correct cleavage of the signal peptide (first 23amino acids) and the absence of pre-bound Neu5Ac were con-firmed by electrospraymass spectrometry (13). For preparationof the selenomethionine (SeMet) derivative of SiaP, the proteinwas expressed from a 1-liter culture as described (19) and puri-fied to �95% homogeneity using a single anion exchange step(Mono Q).Crystallization—For crystallization, SiaP was concentrated

to 30 mg/ml in 20 mM Tris-HCl, pH 8, 150 mM NaCl in thepresence or absence of 5mMNeu5Ac2en and 5mMzinc acetate.Crystallization experiments utilized the vapor diffusionmethod and aMOSQUITOnanoliter dispensing robot to set upsitting drops. Three crystal forms were analyzed. Form 1 crys-tals belonging to space group P21212 were grown from drops

made up of 150 nl of SeMet-substituted SiaP and 150 nl of 100mMTris-HCl, pH 8.0, 20% polyethylene glycol 6000, and 10mMzinc acetate. Form 2 crystals of native SiaP belonging to spacegroup I222 grew under identical conditions. Form 3 crystalsbelonging to space group C2 were grown from drops made upfrom 150 nl of 100 mM Tris-HCl, pH 8.5, 0.2 M magnesiumchloride, and 25%polyethylene glycol 3350. Even though SiaP isnot zinc-dependent, no crystals appeared in the absence of thismetal.Data Collection and Structure Solution—Three-wavelength

data were collected from the Form 1 crystals together with sin-gle-wavelength data from the Form 2 and 3 crystals at the Euro-pean Synchrotron Radiation Facility, Grenoble, on beamlineBM14 (Table 1). The Form 1 SeMet crystals diffracted to 2.6 Åwith high values ofRmerge in the outer ranges. Although the databeyond 2.9 Å were very weak, they proved to be essential forsuccessful phasing. Before data collection, it had been expectedthat the native and SeMet crystals would be isomorphous andthat the isomorphous and anomalous components could becombined in the phasing procedure. Unfortunately this provednot to be so.The programs SHELXC and SHELXD (20) readily found 14

of the 16 expected Se atoms in the asymmetric unit of the crys-tal using the combined MAD signal. However, the resulting2.6-Å resolution map was difficult to interpret, and simpleapplication of the ARP/wARP suite (21) produced amodel witha large number of disconnected peptides and scarcely any of thesequence docked into the density. The program RESOLVE (22)produced a model consisting of about half of the protein back-bone but still with very few side chains docked successfully. Thebreakthrough came via the use of the experimental phase prob-ability distributions in terms of theHendrickson-Lattman coef-ficients as restraints during the ARP/wARP-REFMAC rebuild-ing giving a model with more than 570 of the expected 612residues and with most of the side chain correctly assigned.This model was used for molecular replacement with the Form2 native data, and MOLREP (23) subsequently provided anessentially complete model using ARP/wARP-REFMAC. TheForm 2 crystal structure was in turn used as a search model inmolecular replacement calculations with the Form 3 crystaldata in the program MOLREP, leading to the identification offour molecules in the asymmetric unit. For three of these mol-ecules, A, C, and D, the maps were of satisfactory quality. Itbecame apparent that relative domain movements had takenplace in molecule B because the calculated maps satisfactorilycovered only the N-terminal domain I. A mask was, therefore,applied to the 3.5 molecules which fitted the maps well, andfurther calculations using the programMOLREP using the car-boxyl domain II as a search model completed molecularreplacement. The model was refined by iterative cycles of REF-MAC (24) interspersed with manual modeling in COOT (25).Refinement statistics for the Form 2 and Form 3 structures aregiven in Table 1. Coordinates and structure factors have beendeposited with the Protein Data Bank (unliganded structure,2CEY; Neu5Ac2en structure, 2CEX).Steady-state and Stopped-flow Fluorescence Spectroscopy—

Steady-state protein fluorescence studies were performed aspreviously described (13) unless specifically outlined in the

Structure of a Sialic Acid-binding Protein, SiaP

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text. The Kd values were determined from at least four titra-tions, except that for 2-deoxy-�-N-acetylneuraminic acid(dNeu5Ac), which was determined from three titrations.Stopped-flow kinetic measurements were made using anApplied Photophysics sequential stopped-flow spectrofluo-rimeter (slit width � 1 nm) using an excitation wavelength of280 nm and monitoring the fluorescence emission above 305nm (the emission maximum of SiaP occurs at 310 nm (13)).All reactions were performed using 1 �M SiaP (final concen-tration) at 20 °C in 50mMTris-HCl, pH 8, containing 100mMNaCl. Neu5Ac binding to SiaP was monitored under pseudo-first-order conditions using at least a 4-fold excess ofNeu5Ac over purified SiaP. One thousand data points wererecorded over the course of each reaction, and at least sixruns were averaged for each measurement. Kinetic traceswere analyzed using the Pro-K software supplied by AppliedPhotophysics Ltd. The reactions were rapid and monophasicand were fitted to a single-exponential consistent with a sim-ple one-step equilibrium process (26, 27).

P � L -|0k1

k�1

PL (Eq. 1)

The kobs obtained by a fitting of the traces was plotted in Sig-maPlot, fromwhich the dependence of kobs onNeu5Ac concen-tration was determined.Examination of ligand binding to SiaP usingmass-spectrom-

etry was performed as described previously (13). The Neu5Acderivatives used in this work were prepared as described previ-ously for sialyl amide (28) and for dNeu5Ac (29).Sequence Analysis and Bioinformatics—Sequences of TRAP

ESR proteins and other components have been collected into

the TRAP-DB data base,5 which contains sequences of �1000TRAP transporter proteins from bacteria and Archaea. Thesequences selected for a multiple sequence alignment werehomologues of SiaP, a member of the DctP family of TRAPESRs, that are encoded within operons containing the genes forthe two membrane components of the transporter (either asseparate genes or a single fused gene as in siaQM fromH. influ-enzae). These 248 sequences were aligned using ClustalX, andthe percentage sequence conservation of the residues present inH. influenzae Rd SiaP was calculated in Excel after exportingthe ClustalX alignment into BioEDIT.

RESULTS

The SiaP Structure Is a Variation of a Typical ESR Fold—The structure of SiaP was solved to 1.7 Å spacing by MADphasing of a SeMet derivative crystal (Form 1). The structureof the Form 2 crystal, which diffracted to higher resolution,was then solved by molecular replacement (Table 1). Therefined model contains all 306 residues of SiaP and 307 watermolecules. SiaP has two �/� domains connected by threesegments of the polypeptide and separated by a large cleft(Fig. 1A). Domain I, encompassing residues 1–124 and 213–252, contains a 5-stranded �-sheet against which are packedsix �-helices. The strand order is �2-�1-�3-�10-�4, withstrand �10 running anti-parallel to the other four strands(Fig. 2). Domain II contains residues 125–212 and 253–306and has a 6-stranded �-sheet surrounded by 3 �-helices (Fig.1A and Fig. 2). Here the sheet topology is �7-�6-�8-�5-�9-�11 with strand �5 running anti-parallel to the otherstrands. Residues 280–306 at the C terminus of the molecule

5 C. Mulligan, P. Bryant, and G. H. Thomas, unpublished information.

TABLE 1

Form I Form II Form IIIData collection at BM14Wavelength (Å) 0.97907 0.97921 0.90777 0.97624 0.97624Resolution range (Å)/highest resolutionshell

50.0–2.63/2.72–2.63 50.0–2.70/2.80–2.70 50.0–2.63/2.72–2.63 50.0–1.70/1.76–1.70 50.0–2.20/2.28–2.20

Space group P21212 P21212 P21212 I222 C2Unit-cell parameters (Å) a � 46.08 a � 45.78 a � 46.13 a � 46.76 a � 131.45

b � 103.36 b � 103.46 b � 103.67 b � 102.51 b � 88.70c � 199.10 c � 198.77 c � 199.40 c � 202.65 c � 115.91

� � 105.33°Number of unique reflections, overall/outer shell

28,863/2,562 26,676/2,434 28,271/2,192 41,133/460 49,350/826

Completeness (%), overall/outer shell 98.5/88.4 99.2/93.2 96.4/75.6 77.4/8.8 75.7/12.7Redundancy, overall/outer shell 6.6/5.1 6.5/4.9 6.0/4.1 5.1/1.1 3.2/1.3I/�(I ), overall/outer shell 10.1/1.5 10.7/1.0 8.8/0.8 12.20/1.33 10.7/1.1Rmerge (%), overall/outer shell 14.6/70.8 14.1/84.8 17.0/97.4 11.4/96.5 9.7/56.0

Refinement and model statisticsR-factora/R-freeb 0.19/0.24 0.20/0.28Reflections (working/free) 38,727/2,091 46,872/2,390Outer shell R-factor/R-freeb 50.0/70.0 36.4/57.3Molecules/asymmetric unit 1 4Number of protein non hydrogen atoms 2711 10040Number of Zn2� atoms 2.5 6Number of water molecules 307 467

Root mean square deviation from targetcBond lengths (Å) 0.023 0.006Bond angles (°) 1.944 0.899Average B-factor (Å2) 30.6 27.7Ramachandran plotd 93.5/5.8/0.7/0 91.8/8.0/0.2/0

aR-factor � ��Fo� � �Fc�/��Fo� where Fo and Fc are the observed and calculated structure factor amplitudes, respectively.bR-free is the R-factor calculated with 5% of the reflections chosen at random and omitted from refinement.c Root mean square deviation of bond lengths and bond angles from ideal geometry.d Percentage of residues in most-favored/additionally allowed/generously allowed/disallowed regions of the Ramachandran plot, according to PROCHECK.




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form a pair of �-helices that fold across the base of the mol-ecule and pack against both domains. A striking feature ofthe structure is the long helix, �9, which spans the breadth ofthe molecule (Fig. 1).

A DALI search revealed the existence of a large number ofstructures with similarity to SiaP. The highest scoring match(Z � 8.6; with 112 of 309 C� atoms aligning with a root meansquare deviation of 2.9 Å) is the periplasmic glycine-betaineESR protein from anABC transporter (PDB 1R9L).Many othermatches were found to other ABC ESR proteins, LysR-typetranscription factors and eukaryotic glutamate receptors. It isimmediately apparent from an examination of the domaintopologies that SiaP is a type II ESR protein (Fig. 2). These arecharacterized by a domain dislocation of one of the�-strands ineach of the �-sheets (30).The Ligand-bound SiaP Protein Adopts a Closed Conforma-

tion—To investigate the structural basis for ligand binding, wegrew crystals of SiaP in the presence of Neu5Ac and selectedanalogues (13). Analysis of a third crystal form (Form 3), grownin the presence of one of these sialic acid analogues, revealedthe presence of four molecules in the asymmetric unit. Theligand, Neu5Ac2en, was clearly defined in the electron densitymaps for molecule B after molecular replacement (Fig. 1B);however, molecules A, C, and D were unliganded. Superposi-tion of 297 equivalent C� atoms of molecules A, C, and D byleast squares minimization methods gives pairwise root mean

FIGURE 2. Schematic diagram of the topology of SiaP (A) in comparisonwith an ancestral type II ESR protein (B) (53). The N-terminal domain is inlight gray, and the C-terminal domain is in black. Features that distinguish SiaPare in dark gray. Unfilled circles are 310 helices. This diagram adopts the style ofFukami-Kobayashi (53), where the hinge �-strands are drawn as part of the�-sheets. The �4 and �5 schematic elements for SiaP actually form a singleextended �-strand that extends across both domains but has been displayedas two elements in the schematic to be consistent with the numberingscheme for the type II ESR.

FIGURE 1. Overall structure of the unliganded (A) and Neu5Ac2en-bound (B) forms of SiaP and the superposition by least squares minimization proceduresapplied to the positions of the C� atoms of domain I (C ) in an orientation to illustrate the domain closure around the ligand. The ribbon diagrams were drawnusing CCP4MG (52), and the ligand is colored by atom type. D, surface representation of the Neu5Ac2en structure viewed looking down into the binding cleft.The ligand is in blue, and domain I is on the left, whereas domain II is on the right.




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square positional deviations in the range 0.4–0.8 Å, similar tothose seen when these structures are superposed on the coor-dinates from the Form 2 crystal (0.5–1.0 Å). However, super-position onto the liganded molecule B gave much larger devia-tions in the range of 2.5–3.1 Å. The superposition was muchbetter when the individual domains were overlaid (0.4–0.9 Å).These comparisons suggest that a rigid body domain move-ment accompanies ligand binding, as is apparent in Fig. 1C.Quantitative comparison of these structures using DynDom(31) reveals that this movement can be described by a rotationof 25–31o about a hinge that runs close to the peptide bondsconnecting residues Thr-127–Arg-128, Ile-211—Leu-212, andGlu-254—Lys-255.Although the majority of the conformational changes can be

attributed to the rigid body rotation about the hinge, a smallregion near the surface of the ligand binding cleft in Domain II(Ala-186—Tyr-197) makes an additional movement beyondthat caused by the hinge bending, which results in the reorien-tation of Phe-170 to form a stacking interaction against the sideof the sugar ring. Interestingly, the hinge bending observed inthe ligand-bound form also results in the kinking of the �-hel-ical component of the hinge.The ligand-bound molecule B in the Form 3 crystal con-

tains a single molecule of Neu5Ac2en, which is consistentwith the 1:1 stoichiometry of binding for Neu5Ac2en andNeu5Ac determined by electrospraymass spectrometry (13).Neu5Ac2en differs from the physiological ligand Neu5Ac inthat it contains a C2 C3 double bond that introduces partialplanarity into the sugar ring (its structure is drawn in Fig. 5).The ligand is bound in a pocket formedby the twodomains, andits carboxylate group forms a salt bridge to Arg-147 and a polarinteraction with Asn-187, both in the C-terminal domain (Fig.3, A and B). A salt bridging interaction is also made with Arg-127, which is in the hinge region. Unusually, the carboxylate inNeu5Ac2en is almost perpendicular to rather than planar withthe ring. The glycerol group ofNeu5Ac2en appears to form twohydrogen bonds to Glu-67. There is an additional contactbetweenAsn-10 and the carbonyl oxygen of theN-acetyl group.The ligand is almost completely buried, with only 32 Å2 of its435-Å2 surface area accessible to the solvent (Fig. 1D).SiaP Binds Neu5Ac by a Simple Bimolecular Association—

Wewished to determine the mechanism of binding of Neu5Acby SiaP using pre-steady-state kinetics and specifically testwhether the data supported previous kinetic schemes proposedfor other TRAP and ABC ESR proteins. Although ligand bind-ing to a number of ESR proteins appears to follow monophasickinetics, the mechanism of binding can be distinguished basedon the dependence of the observed rate constant (kobs) on theconcentration of ligand. In ABC ESR proteins the lineardependence of kobs on the concentration of ligand implies thatligand binding occurs by a single-step, bimolecular mechanismand that the ESR is predominantly in an open unliganded con-formation before undergoing fast closure upon ligand binding(26, 27, 32). However, analysis of ligand binding to the TRAPESR DctP revealed a kinetic behavior not seen in ABC ESRs inthat kobs decreased in a hyperbolicmannerwith increasing con-centrations of ligands (33, 34). This unusual behavior was inter-preted as the consequence of a pre-isomerization process of the

protein from a closed unliganded conformation to an openunliganded formbefore ligand binding. Using stopped-flow flu-orescence spectroscopy under pseudo-first order conditions,we observed an enhancement in fluorescence after the additionof Neu5Ac that could be fitted to a single exponential equation(Fig. 4A). The observed rate constant (kobs) increased linearlywith the Neu5Ac concentration (Fig. 4B), which suggests thatSiaP binds Neu5Ac using a similar mechanism to ABC-ESRproteins.From the gradient of the plot of kobs versus ligand concentra-

tion in Fig. 4B, we calculated the value of k1 for the process as3.5 � 0.1 � 107 M�1 s�1. We were not able to determine a k�1from this plot as the intercept of the line was too close to zeroand, hence, cannot be reliably inferred from a linear plot (35).However, we used steady-state fluorescence spectroscopy tocalculate a Kd of 58 � 5 nM (Fig. 4C) for Neu5Ac binding to theprotein under identical conditions to those used in the pre-steady state analysis (20 °C in the presence of 100 mM NaCl).This is about 2-fold lower than the valuewedetermined at 37 °Cwith no salt (138 � 6 nM (data not shown), which is similar tothe value of 120 � 6 nM reported previously (13)). From thisvalue of theKd forNeu5Acbindingwewere able to calculate thek-1 to be 2.03 s�1. The k1 and k-1 values determined for Neu5Acbinding to SiaP are in the range of those determined for cognateligand binding to a number of ESR proteins from ABC trans-porters (26, 27). The unusual binding mechanism observed forDctP has been observed with another TRAP ESR protein,RRC01191, from R. capsulatus. However, the data from thisstudy using SiaP and those using the E. coli TRAP ESR proteinYiaO (14) suggest that this is not a conserved property of theTRAP ESRs.The Carboxylate Group of Neu5Ac Is Essential for High Affin-

ity Binding to SiaP—The clear interaction between the carbox-ylate group of Neu5Ac2en and the side chains of Arg-147/Arg-127/Asn-187 in the structure of SiaP suggests that thecarboxylate is important for binding to SiaP. To probe the sig-nificance of this interaction we investigated ligand binding by aderivative of Neu5Ac in which the carboxylate was replaced byan amide (sialyl amide). This ligand bound weakly to SiaP, asjudged by tyrosine fluorescence spectroscopy, with a Kd of243 � 28 �M, which is around 2000-fold higher than that forNeu5Ac (138 nM). The ligand binding properties of other vari-ants of Neu5Ac have been described (13) (Fig. 5), where theN-acetyl group is altered or removed, a lactose group is added atC2, or the C2 position is dehydrated resulting in partial ringflattening (Neu5Ac2en). However, the change of the carboxy-late for an amide gives by far the greatest decrease in affinity,suggesting that this functional group of the ligand is the mostimportant for binding to SiaP.The differences between Neu5Ac and Neu5Ac2en are dehy-

dration of C2 C3 and the partial flattening of the ring (see Fig.5). To determine the contribution of the hydroxyl group at C2,we investigated the binding of dNeu5Ac by SiaP. dNeu5Acretains the chair conformation of the ring seen in Neu5Ac buthas lost the hydroxyl at C2 (Fig. 5). It binds with aKd of 34� 2.5�M, which is similar to that reported for Neu5Ac2en (20 � 3.8�M (13)), suggesting that the lower affinity of SiaP forNeu5Ac2en relative to Neu5Ac is primarily caused by the loss




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of the hydroxyl on C2. This suggests that the natural ligands forSiaP are sialic acids with a free hydroxyl group on C2 and notconjugated forms.To further investigate the contributions of the carboxylate

andN-acetyl groups, we tested the binding of 4-acetylaminocy-

clohexane carboxylic acid to SiaP. This molecule contains botha carboxylate and an N-acetyl group in analogous positions tothe natural ligand Neu5Ac and also adopts a chair conforma-tion similar to Neu5Ac (Fig. 5). We were unable to detect bind-ing of this compound to SiaP using either tyrosine fluorescence

FIGURE 3. A, stereo view of the electron density (contoured at 1. 5�) for Neu5Ac2en and residues involved in the coordination of this ligand in SiaP. B, Ligplotrepresentation of the interactions between Neu5Ac2en and the protein.




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spectroscopy or electrospray mass spectroscopy (data notshown). Combined, these results suggest that the carboxylateandN-acetyl groups are essential, but not sufficient, for the highaffinity binding of Neu5Ac to SiaP.Sequence Analysis of the TRAP ESR Proteins in Light of the

SiaP Structure—Multiple sequence alignment of SiaP with itsseven most similar homologues, all of which are encoded inoperons with genes for sialometabolism, revealed strong con-servation of residues involved in coordination of the carboxy-

late of the Neu5Ac2en (Arg-147, Asn-187, Arg-127) as well asPhe-170 that stacks against the ligand (Fig. 6). The other resi-dues in domain I that bind the glycerol moiety (Glu-67 andAsp-49) are conserved in 7 of 8 sequences; however, Asn-10,which bonds to theN-acetyl group ofNeu5Ac2en is very poorlyconserved, being replaced by either glutamine, valine, or thre-onine in the other 7 sequences. This suggests that the positionof theN-acetyl group ofNeu5Ac2en in the structure is probablynot exactly the same as inNeu5Acor that this component of theinteraction between the protein and ligand is not dependent ona conserved residue in this position. Indeed in other proteins forwhich structures of complexes with both Neu5Ac andNeu5Ac2en are known, the binding sites are identical, but theconformation of the ligands is different (36, 37). Given that theaffinity of SiaP for Neu5Ac is 200-fold greater than forNeu5Ac2en, it would follow that the analogue is presumablynot bound in exactly the same conformation as the physiologi-cal ligand.We next expanded the analysis to a wider set of TRAP ESR

that are (i) homologous toDctP and (ii) whose genes are locatedadjacent to genes for the membrane subunits of a TRAP trans-porter. This set contained 248 proteins that bind a range ofdifferent ligands. The analysis revealed that the most highlyconserved residues fall in domain II of the protein (Fig. 7). Themost highly conserved residue is Arg-147 (present in 98% of thesequences) that forms a salt bridge to the carboxylate group ofNeu5Ac2en in SiaP. The region directly preceding Arg-147 isthe most highly conserved region in the family (Asp-140 is 92%conserved, Gly-143 is 95% conserved, and Lys-145 is 86% con-served), suggesting that the correct positioning of Arg-147within �6 is critical for function of the TRAP ESRs. Addition-ally, highly conserved residues pack against this region fromabove (Gly-162, 92% conserved) and below (Asp-183, 92% con-served). None of the other residues implicated in coordinatingthe Neu5Ac2en is conserved to this extent across the wholeTRAP ESR family. It should also be noted that there is a regionof conserved charge on the surface of domain II that is unusualin that it is not seen with ABC ESRs. In domain I there are onlytwo residues that are well conserved, both of which are glycines(Gly-34 is 90% conserved, and Gly-59 is 91% conserved). Thesesit at turns in the domain after �-helices and probably playimportant roles in maintaining the overall structure of thedomain.A comparison of the residues conserved in the SiaP group and

the larger alignment of all TRAP ESRs revealed an additionalregion of SiaP that is very highly conserved within the sialic bind-ingESRclusterbutnotoutsideof this (Fig. 6).This is the�6and�5regions, which are adjacent to each other in the structure ofdomain II and form a face on the surface of the protein that couldhave a role in specific recognition of the membrane subunits ofthese particular transporters.

DISCUSSION

The widespread occurrence of TRAP transporters in pro-karyotes, including pathogens, suggests that they have impor-tant functions in the biology of these organisms, and this paperprovides the first structural information for a component of afunctionally characterized TRAP transporter. One of the most

FIGURE 4. Investigation of the presteady-state kinetics of Neu5Acbinding to SiaP monitored using stopped-flow fluorescence spectros-copy. A, trace SiaP (1 �M) pushed against buffer (flat line) and against 8 �M

Neu5Ac. The binding data have been fit to a single exponential equation.B, plot of the kobs of the association between SiaP (1 �M) and Neu5Acversus the concentration of Neu5Ac under pseudo-first order conditions.The points on the graph are the averages from the three independenttitrations. The k1 was determined from the slope of the line of best fit andaveraged from three independent sets of titrations. C, representativesteady-state fluorescence titration of SiaP with Neu5Ac under identicalconditions used in the pre-steady-state experiments.




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unusual features of TRAP ESR proteins reported in the litera-ture has come from kinetic data that suggest the protein pre-dominates in a closed conformation even in the absence ofligand (33). This is different from all other ESR proteins forwhich binding data are available, implying amechanistic differ-ence betweenTRAPESRproteins andABCESRproteins.How-ever, the kinetics of SiaP andE. coliYiaO (14) are similar toABCESR proteins in that in the absence of ligand they predominatein an open conformation. The unusual properties of DctP andRRC01191 suggest that a subset of the TRAPESRs have evolvedto adopt a closed conformation in the absence of ligand but thatthis is not an overriding feature of a TRAP ESR.In accordancewithABCESRs, we suggest thatNeu5Ac bind-

ing to SiaP is initiated by the interaction of the carboxylategroup of the ligand and the conserved Arg residue (Arg-147 inSiaP) in domain II of the protein. In SiaP, the full coordinationof the carboxylate also includes an interaction with Arg-127,which is within the hinge region, and the formation of thisinteraction could be involved in triggering the hinge bending ofthe protein, as has been proposed for the E. colimaltose-bind-ing protein (MBP) (38). Once the hinge bending has occurred,the domain I interactionswith the ligand can also form, keepingthe ligand bound tightly. This mechanism is possible for manyor all of the TRAP ESR proteins given the conservation of theArg residues and the presence of a carboxylate within all char-acterized ligands of TRAP transporters.A unique feature of SiaP compared with ABC ESRs is the

presence of a “mixed hinge” consisting of two �-strands and an�-helix. The hinge region in typical ABC ESR proteins com-

prises 2 or 3 short �-strands, e.g. GlnH, whereas the morerecently described structures of family 9 ESRs and siderophorebinding ESRs contain a single long inflexible �-helix (39–41).In SiaP the hinge �-helix is 35 amino acid residues in length,similar to the �-helical hinge in the family 9 ESRs, but it ispositioned toward the C terminus of the protein rather thanbetween the two domains as in the cluster 9 proteins. In thesiderophore binding ESR proteins that contain a single �-helixhinge, there is only a relatively small movement of domains Iand II upon ligand binding, and the ligand sits in a shallowgroove formed by the two domains rather than being deeplyburied between the domains. However, in SiaP there is signifi-cant bending upon ligand binding that is similar to that seen inESRs with hinges composed entirely of �-strands. To accom-modate this hinge bending, a kink is induced in this �-helix inSiaP that will result in an altered surface of this region afterligand binding. This is expected to be an energetically unfavor-able event and perhaps functions as a switch to hold the proteinin either the open or closed conformation.Other structures of proteins that bind sialic acid are known.

A conserved arginine is a common theme among proteins thathave diverse structures and biological functions (42–44). Themost studied sialic acid-binding proteins are the neuramini-dases that contain a characteristic arginine triad that coordi-nates the carboxylate group (45–47). SiaP is similar in using atriad of residues to coordinate the carboxylate group butachieves this using two arginine residues and one asparagine, aconserved structural motif that appears to be important forhigh affinity binding. Functionally SiaP is more similar to the

FIGURE 5. Chemical structures and Kd values of sialic acid (Neu5Ac) and related analogues used in this study and in Severi et al. (13). Neu5Gc,N-glycolylneuraminic acid; KDN, 2-keto-3-deoxy-D-glycero-D-galactonononic acid.




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sialic acid binding Ig-like lectin (Siglec) molecules found oneukaryotic cell surfaces that bind sialic acids but do not modifythem. Siglecs have general roles in adhesion and signaling (48)and bind sialic acid in a surface groove of a V-set immunoglob-ulin-like fold with only one face of the Neu5Ac in contact withthe protein. There are multiple interactions between the pro-tein and ligand, including a salt bridge between the carboxylateand an invariant arginine (49). Similarly, the structure of theNeu5Ac-bound lectin domain of theVibrio cholerae neuramin-

idase reveals the ligand bound in a shallow cleft such that onlythe anomeric oxygen and the O9 of the glycerol side chain arenot involved in interactions with the protein (50). This domainbinds Neu5Ac with a Kd of �30 �M, which is relatively lowaffinity compared with SiaP (13, 50).After ligand binding, the ESR must interact with the mem-

brane subunits of the transporter, and given the similarity instructure and ligand binding mechanisms between SiaP andABC ESRs there must be some similarity in how the ligand is

FIGURE 6. Multiple sequence alignment of H. influenzae SiaP and 7 related TRAP ESR proteins that are likely to form components of a sialic acidtransporter. The genes for all eight of these sequences are encoded in operons encoding sialometabolic genes. The regions indicated by �4 and �5 form asingle extended �-strand that is part of both domains but is labeled as two �-strands for consistency with Fig. 2 and text.




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“delivered” to the membrane subunits. However, uptake via aTRAP transporter is not coupled toATPbinding andhydrolysisevents but rather to the symport of a coupling ion (there isevidence for both H� and Na� ions being the coupling ions forTRAP transporters) (8, 10, 54), and so it is likely that there willbe differences in the mechanism by which ESR opening andligand release is coupled to movement of the ligand across themembrane.Sequence analysis of the sialic acid cluster of TRAP ESRs

supports the hypothesis of a direct interaction between the ESRand the translocation pore, as in ABC transporters. Thus, aparticularlywell conserved face of SiaP formedby the�6 and�5region (Fig. 6) is in an analogous position on the surface of thedomains as is seen in ABC ESRs like MBP. Importantly thisregion is not conserved in the larger alignment of TRAP ESRs,supporting the idea that it confers specificity of interactionwiththe cognate membrane subunit of the transporter.The structure of SiaP also provides insight into the evolution

of the TRAP transporters due to its structural similarity to anancestral type II ESR. This suggests that an ancestral type II ESRwas recruited to work with an ancestral secondary transporterof the ion transporter superfamily and that over time itssequence diverged from ABC ESR proteins beyond the level ofdetection. During this divergence, the TRAP ESRs have addedadditional sequence to the ancestral type II sequence includingthe �-helix that forms the mixed hinge, an extra �-strand indomain II, and two extra helices that interact with the addi-tional helices found in domain I.Although the DctP family of ESRs are used in the majority of

TRAP transporters, we defined a different family of ESRs calledthe TAXI family (InterPro family IPR011852) that are found ina small number of uncharacterized TRAP transporters (10).Fortuitously, the structure of a protein thatwe believe is a TAXIESR has been solved as part of a structural genomics project,although this was not recognized by the authors (55). This ESRfrom Thermus thermophilus is encoded by a gene (TTHA1157)adjacent to the gene for a fused TRAP membrane subunit(TTHA1158) and, therefore, is very likely to be a genuine com-

ponent of a TAXI-TRAP transporter. The structure revealedthat the ESR bound glutamate/glutamine and that it is clearly atype II ESR, although interestingly, it binds the amino acidligand using a completely different set of residues to the ABC-type ESRs like GlnH. Finally, the recent structure of the BugDprotein from Bordetella pertussis provides additional supportfor the widespread nature of the type II ESR fold for use withsecondary transporters (51). This protein of unknown functionis not encoded alongside genes for a transporter. However, it ishomologous to BctA, a component of a tripartite tricarboxylatetransporter, which forms a second smaller family of ESR-de-pendent secondary transporters. Again, this structure has atype II ESR fold but coordinates its ligand (aspartate) using anunusual set of interactions; in fact, in this structure the carbox-ylate of the aspartate is coordinated solely by water molecules.In summary, the structure of SiaP provides insight into a high

affinity binding site for sialic acid and in combination withbioinformatics reveals the importance of the Arg/carboxylateinteraction in all TRAP transporters. The additional findingthat SiaP is a type II ESR supports the hypothesis that TRAPtransporters have evolved from ancestral secondary transport-ers via the recruitment of an ancestral type II ESR to specificallycatalyze uptake of organic anionswith high affinity and that thisappears to have been a common feature of the evolution of therelated TAXI TRAP and also the tripartite tricarboxylate trans-porter families of ESR-dependent secondary transporters.

Acknowledgments—We thank the European Synchrotron RadiationFacility, Grenoble, for excellent data collection facilities. We alsothank the participants of the SPINEmeeting (York, July 11–15, 2005),which greatly assisted in the solution of the structure and Prof.Colin Kleanthous for comments on the manuscript.

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Kelly, Keith S. Wilson, Anthony J. Wilkinson and Gavin H. ThomasAxel Müller, Emmanuele Severi, Christopher Mulligan, Andrew G. Watts, David J.

Haemophilus influenzaeTransporters Exemplified by SiaP, a Sialic Acid Binding Virulence Factor from

Conservation of Structure and Mechanism in Primary and Secondary

doi: 10.1074/jbc.M603463200 originally published online May 15, 20062006, 281:22212-22222.J. Biol. Chem.

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