Conservation of Structure and Mechanism in Primary and Secondary Transporters Exemplified by SiaP, a Sialic Acid Binding Virulence Factor from Haemophilus influenzae
2006; Elsevier BV; Volume: 281; Issue: 31 Linguagem: Inglês
10.1074/jbc.m603463200
ISSN1083-351X
AutoresAxel Müller, Emmanuele Severi, Christopher Mulligan, Andrew G. Watts, David J. Kelly, Keith S. Wilson, Anthony J. Wilkinson, Gavin H. Thomas,
Tópico(s)Antibiotic Resistance in Bacteria
ResumoExtracytoplasmic solute receptors (ESRs) are important components of solute uptake systems in bacteria, having been studied extensively as parts of ATP binding cassette transporters. Herein we report the first crystal structure of an ESR protein from a functionally characterized electrochemical ion gradient dependent secondary transporter. This protein, SiaP, forms part of a tripartite ATP-independent periplasmic transporter specific for sialic acid in Haemophilus influenzae. Surprisingly, the structure reveals an overall topology similar to ATP binding cassette ESR proteins, which is not apparent from the sequence, demonstrating that primary and secondary transporters can share a common structural component. The structure of SiaP in the presence of the sialic acid analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid reveals the ligand bound in a deep cavity with its carboxylate group forming a salt bridge with a highly conserved Arg residue. Sialic acid binding, which obeys simple bimolecular association kinetics as determined by stopped-flow fluorescence spectroscopy, is accompanied by domain closure about a hinge region and the kinking of an α-helix hinge component. The structure provides insight into the evolution, mechanism, and substrate specificity of ESR-dependent secondary transporters that are widespread in prokaryotes. Extracytoplasmic solute receptors (ESRs) are important components of solute uptake systems in bacteria, having been studied extensively as parts of ATP binding cassette transporters. Herein we report the first crystal structure of an ESR protein from a functionally characterized electrochemical ion gradient dependent secondary transporter. This protein, SiaP, forms part of a tripartite ATP-independent periplasmic transporter specific for sialic acid in Haemophilus influenzae. Surprisingly, the structure reveals an overall topology similar to ATP binding cassette ESR proteins, which is not apparent from the sequence, demonstrating that primary and secondary transporters can share a common structural component. The structure of SiaP in the presence of the sialic acid analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid reveals the ligand bound in a deep cavity with its carboxylate group forming a salt bridge with a highly conserved Arg residue. Sialic acid binding, which obeys simple bimolecular association kinetics as determined by stopped-flow fluorescence spectroscopy, is accompanied by domain closure about a hinge region and the kinking of an α-helix hinge component. The structure provides insight into the evolution, mechanism, and substrate specificity of ESR-dependent secondary transporters that are widespread in prokaryotes. The uptake of solutes into bacterial cells is critical for growth and survival in every environment and is catalyzed by a variety of different protein-mediated transport systems. One common feature of uptake systems, including the well studied ATP binding cassette (ABC) 4The 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-acetylneuraminic acid; dNeu5Ac, 2-deoxy-β-N-acetylneuraminic acid; ESR, extracytoplasmic solute receptor; SiaP, sialic acid-binding protein; SeMet, selenomethionine.4The 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-acetylneuraminic acid; dNeu5Ac, 2-deoxy-β-N-acetylneuraminic acid; ESR, extracytoplasmic solute receptor; SiaP, sialic acid-binding protein; SeMet, selenomethionine. transporters, is an extracytoplasmic solute receptor (ESR) (often also known as a periplasmic-binding protein), which captures the substrate for the transporter and delivers it to the membrane subunits (1Wilkinson A.J. Verschueren K.H.G. Holland I.B. Cole S.P.C. Kuchler K. Higgins C.F. ABC Proteins: From Bacteria to Man. Academic Press, London2003: 187-207Google Scholar). Structures have been determined for many ESR proteins from ABC transporters, and their mechanism of ligand binding is so well established that these proteins are now being used in a host of biotechnological applications (2Dwyer M.A. Hellinga H.W. Curr. Opin. Struct. Biol. 2004; 14: 495-504Crossref PubMed Scopus (262) Google Scholar).The ABC systems are examples of primary active transporters, so called because they hydrolyze ATP directly to energize transport (3Davidson A.L. Chen J. Annu. Rev. Biochem. 2004; 73: 241-268Crossref PubMed Scopus (484) Google Scholar). These differ from another major grouping, the secondary active transporters, like Escherichia coli lactose permease, so-called because many of them use the membrane potential to energize uptake and direct ATP hydrolysis is not involved. The use of an ESR protein, which endows the transporter with high affinity for its substrate, was for a long time believed to be exclusive to the ABC transporters, but biochemical studies of a C4-dicarboxylate uptake system from Rhodobacter capsulatus led to the discovery of a novel family of ESR-dependent secondary transporters, the so called tripartite ATP-independent periplasmic (TRAP) transporters (4Shaw J.G. Hamblin M.J. Kelly D.J. Mol. Microbiol. 1991; 5: 3055-3062Crossref PubMed Scopus (44) Google Scholar, 5Hamblin M.J. Shaw J.G. Kelly D.J. Mol. Gen. Genet. 1993; 237: 215-224Crossref PubMed Scopus (31) Google Scholar, 6Rabus R. Jack D.L. Kelly D.J. Saier Jr., M.H. Microbiology. 1999; 145: 3431-3445Crossref PubMed Scopus (82) Google Scholar, 7Wyborn N.R. Alderson J. Andrews S.C. Kelly D.J. FEMS Microbiol. Lett. 2001; 194: 13-17Crossref PubMed Google Scholar, 8Forward J.A. Behrendt M.C. Wyborn N.R. Cross R. Kelly D.J. J. Bacteriol. 1997; 179: 5482-5493Crossref PubMed Google Scholar). These transporters contain two membrane protein components, the larger of which contains 12 predicted transmembrane helices. This subunit is a member of the ion transporter superfamily (6Rabus R. Jack D.L. Kelly D.J. Saier Jr., M.H. Microbiology. 1999; 145: 3431-3445Crossref PubMed Scopus (82) Google Scholar, 9Prakash S. Cooper G. Singhi S. Saier Jr., M.H. Biochim. Biophys. Acta. 2003; 1618: 79-92Crossref PubMed Scopus (93) Google Scholar) and probably forms the translocation channel. The smaller membrane component of four transmembrane helices has an unknown but essential function (7Wyborn N.R. Alderson J. Andrews S.C. Kelly D.J. FEMS Microbiol. Lett. 2001; 194: 13-17Crossref PubMed Google Scholar). Microbial genome sequencing has revealed that the TRAP transporters are widespread in the prokaryotic world (10Kelly D.J. Thomas G.H. FEMS Microbiol. Rev. 2001; 25: 405-424Crossref PubMed Google Scholar), and known substrates now include sialic acid, ectoine, 2,3-diketo-l-gulonate, and pyruvate in addition to C4-dicarboxylates (11Tetsch L. Kunte H.J. FEMS Microbiol. Lett. 2002; 211: 213-218Crossref PubMed Google Scholar, 12Grammann K. Volke A. Kunte H.J. J. Bacteriol. 2002; 184: 3078-3085Crossref PubMed Scopus (139) Google Scholar, 13Severi E. Randle G. Kivlin P. Whitfield K. Young R. Moxon R. Kelly D. Hood D. Thomas G.H. Mol. Microbiol. 2005; 58: 1173-1185Crossref PubMed Scopus (106) Google Scholar, 14Thomas G.H. Southworth T. Leon-Kempis M.R. Leech A. Kelly D.J. Microbiology. 2006; 152: 187-198Crossref PubMed Scopus (44) Google Scholar). We have recently characterized the sialic acid TRAP transporter from the human pathogen Haemophilus influenzae and demonstrated that this transporter is essential for uptake of sialic acid (Neu5Ac) in this bacterium. Neu5Ac is an important hostacquired molecule that is used by the bacterium to modify its lipopolysaccharide to make it appear as “self” and evade the innate immune response (15Hood D.W. Makepeace K. Deadman M.E. Rest R.F. Thibault P. Martin A. Richards J.C. Moxon E.R. Mol. Microbiol. 1999; 33: 679-692Crossref PubMed Scopus (160) Google Scholar). Deletion of the TRAP transporter results in loss of lipopolysaccharide sialylation and serum resistance in H. influenzae Rd (13Severi E. Randle G. Kivlin P. Whitfield K. Young R. Moxon R. Kelly D. Hood D. Thomas G.H. Mol. Microbiol. 2005; 58: 1173-1185Crossref PubMed Scopus (106) Google Scholar), a phenotype also observed recently in non-typeable strains of H. influenzae (16Allen S. Zaleski A. Johnston J.W. Gibson B.W. Apicella M.A. Infect. Immun. 2005; 73: 5291-5300Crossref PubMed Scopus (74) Google Scholar) and in the related animal pathogen Pasteurella multocida (17Steenbergen S.M. Lichtensteiger C.A. Caughlan R. Garfinkle J. Fuller T.E. Vimr E.R. Infect. Immun. 2005; 73: 1284-1294Crossref PubMed Scopus (60) Google Scholar). These were the first reports of TRAP transporters having a role in virulence and highlight the importance of a greater understanding of the function and mechanism of these systems in prokaryotes.The sialic acid-binding protein SiaP is a member of the DctP protein family, named after the first characterized TRAP ESR protein that binds C4-dicarboxylates (4Shaw J.G. Hamblin M.J. Kelly D.J. Mol. Microbiol. 1991; 5: 3055-3062Crossref PubMed Scopus (44) Google Scholar). This is the major family of ESR proteins found in TRAP transport systems (10Kelly D.J. Thomas G.H. FEMS Microbiol. Rev. 2001; 25: 405-424Crossref PubMed Google Scholar). Given the potential importance of TRAP transporters in the biology of prokaryotes but the paucity of information on them, we solved the structure of SiaP at 1.7 Å in an unliganded form and 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 acid transport and insight into the function and evolution of this novel family of ESR-dependent secondary transporters.EXPERIMENTAL PROCEDURESExpression and Purification of SiaP—The SiaP protein was purified from E. coli using a modification of the methods described in Severi et al. (13Severi E. Randle G. Kivlin P. Whitfield K. Young R. Moxon R. Kelly D. Hood D. Thomas G.H. Mol. Microbiol. 2005; 58: 1173-1185Crossref PubMed Scopus (106) Google Scholar). Cells of E. coli BL21(DE3) pLysS pGTY3 were grown in 5 ml of LB (Lennox broth) for 6 h, washed in M9 minimal medium (18Neidhardt F.C. Bloch P.L. Smith D.F. J. Bacteriol. 1974; 119: 736-747Crossref PubMed Google Scholar), and used to inoculate 50 ml M9 minimal medium for overnight growth at 37 °C. This was 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 inducing expression with 1 mm isopropyl 1-thio-β-d-galactopyranoside followed by overnight incubation. Cells were washed in ice-cold 50 mm Tris-HCl, pH 8, incubated in SET buffer (0.5 m sucrose, 5 mm EDTA in 50 mm Tris-HCl, pH 8, 600 μg/ml lysozyme) for 1 h at 30 °C, and the periplasmic fraction was then clarified by centrifugation and dialyzed against 50 mm Tris-HCl, pH 8, containing 1.5 m (NH4)2SO4. SiaP was purified by fast protein liquid chromatography using a hydrophobic interaction column followed by size exclusion chromatography using a G75-Sepharose column as described previously (13Severi E. Randle G. Kivlin P. Whitfield K. Young R. Moxon R. Kelly D. Hood D. Thomas G.H. Mol. Microbiol. 2005; 58: 1173-1185Crossref PubMed Scopus (106) Google Scholar). Protein concentration was determined from the absorbance at 280 nm using a molar absorption coefficient for SiaP of 23840 m-1 cm−1. The correct cleavage of the signal peptide (first 23 amino acids) and the absence of pre-bound Neu5Ac were confirmed by electrospray mass spectrometry (13Severi E. Randle G. Kivlin P. Whitfield K. Young R. Moxon R. Kelly D. Hood D. Thomas G.H. Mol. Microbiol. 2005; 58: 1173-1185Crossref PubMed Scopus (106) Google Scholar). For preparation of the selenomethionine (SeMet) derivative of SiaP, the protein was expressed from a 1-liter culture as described (19Ducros V.M. Lewis R.J. Verma C.S. Dodson E.J. Leonard G. Turkenburg J.P. Murshudov G.N. Wilkinson A.J. Brannigan J.A. J. Mol. Biol. 2001; 306: 759-771Crossref PubMed Scopus (82) Google Scholar) and purified 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 the presence or absence of 5 mm Neu5Ac2en and 5 mm zinc acetate. Crystallization experiments utilized the vapor diffusion method and a MOSQUITO nanoliter dispensing robot to set up sitting drops. Three crystal forms were analyzed. Form 1 crystals belonging to space group P21212 were grown from drops made up of 150 nl of SeMet-substituted SiaP and 150 nl of 100 mm Tris-HCl, pH 8.0, 20% polyethylene glycol 6000, and 10 mm zinc acetate. Form 2 crystals of native SiaP belonging to space group I222 grew under identical conditions. Form 3 crystals belonging to space group C2 were grown from drops made up from 150 nl of 100 mm Tris-HCl, pH 8.5, 0.2 m magnesium chloride, and 25% polyethylene glycol 3350. Even though SiaP is not zinc-dependent, no crystals appeared in the absence of this metal.Data Collection and Structure Solution—Three-wavelength data were collected from the Form 1 crystals together with single-wavelength data from the Form 2 and 3 crystals at the European Synchrotron Radiation Facility, Grenoble, on beamline BM14 (Table 1). The Form 1 SeMet crystals diffracted to 2.6 Å with high values of Rmerge in the outer ranges. Although the data beyond 2.9 Å were very weak, they proved to be essential for successful phasing. Before data collection, it had been expected that the native and SeMet crystals would be isomorphous and that the isomorphous and anomalous components could be combined in the phasing procedure. Unfortunately this proved not to be so.TABLE 1Form IForm IIForm IIIData collection at BM14Wavelength (Å)0.979070.979210.907770.976240.97624Resolution range (Å)/highest resolution shell50.0-2.63/2.72-2.6350.0-2.70/2.80-2.7050.0-2.63/2.72-2.6350.0-1.70/1.76-1.7050.0-2.20/2.28-2.20Space groupP21212P21212P21212I222C2Unit-cell parameters (Å)a = 46.08a = 45.78a = 46.13a = 46.76a = 131.45b = 103.36b = 103.46b = 103.67b = 102.51b = 88.70c = 199.10c = 198.77c = 199.40c = 202.65c = 115.91β = 105.33°Number of unique reflections, overall/outer shell28,863/2,56226,676/2,43428,271/2,19241,133/46049,350/826Completeness (%), overall/outer shell98.5/88.499.2/93.296.4/75.677.4/8.875.7/12.7Redundancy, overall/outer shell6.6/5.16.5/4.96.0/4.15.1/1.13.2/1.3I/σ(I), overall/outer shell10.1/1.510.7/1.08.8/0.812.20/1.3310.7/1.1Rmerge (%), overall/outer shell14.6/70.814.1/84.817.0/97.411.4/96.59.7/56.0Refinement and model statisticsR-factoraR-factor = ∑||Fo| - |Fc||/∑|Fo| where Fo and Fc are the observed and calculated structure factor amplitudes, respectively./R-freebR-free is the R-factor calculated with 5% of the reflections chosen at random and omitted from refinement.0.19/0.240.20/0.28Reflections (working/free)38,727/2,09146,872/2,390Outer shell R-factor/R-freebR-free is the R-factor calculated with 5% of the reflections chosen at random and omitted from refinement.50.0/70.036.4/57.3Molecules/asymmetric unit14Number of protein non hydrogen atoms271110040Number of Zn2+ atoms2.56Number of water molecules307467Root mean square deviation from targetcRoot mean square deviation of bond lengths and bond angles from ideal geometry.Bond lengths (Å)0.0230.006Bond angles (°)1.9440.899Average B-factor (Å2)30.627.7Ramachandran plotdPercentage of residues in most-favored/additionally allowed/generously allowed/disallowed regions of the Ramachandran plot, according to PROCHECK.93.5/5.8/0.7/091.8/8.0/0.2/0a R-factor = ∑||Fo| - |Fc||/∑|Fo| where Fo and Fc are the observed and calculated structure factor amplitudes, respectively.b R-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. Open table in a new tab The programs SHELXC and SHELXD (20Schneider T.R. Sheldrick G.M. Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 1772-1779Crossref PubMed Scopus (1574) Google Scholar) readily found 14 of the 16 expected Se atoms in the asymmetric unit of the crystal using the combined MAD signal. However, the resulting 2.6-Å resolution map was difficult to interpret, and simple application of the ARP/wARP suite (21Perrakis A. Harkiolaki M. Wilson K.S. Lamzin V.S. Acta Crystallogr. D Biol. Crystallogr. 2001; 57: 1445-1450Crossref PubMed Scopus (459) Google Scholar) produced a model with a large number of disconnected peptides and scarcely any of the sequence docked into the density. The program RESOLVE (22Terwilliger T.C. Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 38-44Crossref PubMed Scopus (593) Google Scholar) produced a model consisting of about half of the protein backbone but still with very few side chains docked successfully. The breakthrough came via the use of the experimental phase probability distributions in terms of the Hendrickson-Lattman coefficients as restraints during the ARP/wARP-REFMAC rebuilding giving a model with more than 570 of the expected 612 residues and with most of the side chain correctly assigned. This model was used for molecular replacement with the Form 2 native data, and MOLREP (23Vagin A. Teplyakov A. Acta Crystallogr. D Biol. Crystallogr. 2000; 56: 1622-1624Crossref PubMed Scopus (690) Google Scholar) subsequently provided an essentially complete model using ARP/wARP-REFMAC. The Form 2 crystal structure was in turn used as a search model in molecular replacement calculations with the Form 3 crystal data in the program MOLREP, leading to the identification of four molecules in the asymmetric unit. For three of these molecules, A, C, and D, the maps were of satisfactory quality. It became apparent that relative domain movements had taken place in molecule B because the calculated maps satisfactorily covered only the N-terminal domain I. A mask was, therefore, applied to the 3.5 molecules which fitted the maps well, and further calculations using the program MOLREP using the carboxyl domain II as a search model completed molecular replacement. The model was refined by iterative cycles of REF-MAC (24Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13779) Google Scholar) interspersed with manual modeling in COOT (25Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22815) Google Scholar). Refinement statistics for the Form 2 and Form 3 structures are given in Table 1. Coordinates and structure factors have been deposited 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 as previously described (13Severi E. Randle G. Kivlin P. Whitfield K. Young R. Moxon R. Kelly D. Hood D. Thomas G.H. Mol. Microbiol. 2005; 58: 1173-1185Crossref PubMed Scopus (106) Google Scholar) unless specifically outlined in the text. The Kd values were determined from at least four titrations, except that for 2-deoxy-β-N-acetylneuraminic acid (dNeu5Ac), which was determined from three titrations. Stopped-flow kinetic measurements were made using an Applied Photophysics sequential stopped-flow spectrofluorimeter (slit width = 1 nm) using an excitation wavelength of 280 nm and monitoring the fluorescence emission above 305 nm (the emission maximum of SiaP occurs at 310 nm (13Severi E. Randle G. Kivlin P. Whitfield K. Young R. Moxon R. Kelly D. Hood D. Thomas G.H. Mol. Microbiol. 2005; 58: 1173-1185Crossref PubMed Scopus (106) Google Scholar)). All reactions were performed using 1 μm SiaP (final concentration) at 20 °C in 50 mm Tris-HCl, pH 8, containing 100 mm NaCl. Neu5Ac binding to SiaP was monitored under pseudofirst-order conditions using at least a 4-fold excess of Neu5Ac over purified SiaP. One thousand data points were recorded over the course of each reaction, and at least six runs were averaged for each measurement. Kinetic traces were analyzed using the Pro-K software supplied by Applied Photophysics Ltd. The reactions were rapid and monophasic and were fitted to a single-exponential consistent with a simple one-step equilibrium process (26Miller D.M. II I Olson J.S. Quiocho F.A. J. Biol. Chem. 1980; 255: 2465-2471Abstract Full Text PDF PubMed Google Scholar, 27Miller D.M. II I Olson J.S. Pflugrath J.W. Quiocho F.A. J. Biol. Chem. 1983; 258: 13665-13672Abstract Full Text PDF PubMed Google Scholar).P+L⇄k-1k1PL(Eq. 1) The kobs obtained by a fitting of the traces was plotted in SigmaPlot, from which the dependence of kobs on Neu5Ac concentration was determined.Examination of ligand binding to SiaP using mass-spectrometry was performed as described previously (13Severi E. Randle G. Kivlin P. Whitfield K. Young R. Moxon R. Kelly D. Hood D. Thomas G.H. Mol. Microbiol. 2005; 58: 1173-1185Crossref PubMed Scopus (106) Google Scholar). The Neu5Ac derivatives used in this work were prepared as described previously for sialyl amide (28Brossmer R. Holmquist L. Hoppe-Seyler's Z. Physiol. Chem. 1971; 352: 1715-1719Crossref PubMed Scopus (30) Google Scholar) and for dNeu5Ac (29Schmid W. Christian R. Zbiral E. Tetrahedron Lett. 1988; 29: 3643-3646Crossref Scopus (61) Google Scholar).Sequence Analysis and Bioinformatics—Sequences of TRAP ESR proteins and other components have been collected into the TRAP-DB data base, 5C. Mulligan, P. Bryant, and G. H. Thomas, unpublished information. which contains sequences of >1000 TRAP transporter proteins from bacteria and Archaea. The sequences selected for a multiple sequence alignment were homologues of SiaP, a member of the DctP family of TRAP ESRs, that are encoded within operons containing the genes for the two membrane components of the transporter (either as separate genes or a single fused gene as in siaQM from H. influenzae). These 248 sequences were aligned using ClustalX, and the percentage sequence conservation of the residues present in H. influenzae Rd SiaP was calculated in Excel after exporting the ClustalX alignment into BioEDIT.RESULTSThe SiaP Structure Is a Variation of a Typical ESR Fold—The structure of SiaP was solved to 1.7 Å spacing by MAD phasing of a SeMet derivative crystal (Form 1). The structure of the Form 2 crystal, which diffracted to higher resolution, was then solved by molecular replacement (Table 1). The refined model contains all 306 residues of SiaP and 307 water molecules. SiaP has two α/β domains connected by three segments of the polypeptide and separated by a large cleft (Fig. 1A). Domain I, encompassing residues 1-124 and 213-252, contains a 5-stranded β-sheet against which are packed six α-helices. The strand order is β2-β1-β3-β10-β4, with strand β10 running anti-parallel to the other four strands (Fig. 2). Domain II contains residues 125-212 and 253-306 and has a 6-stranded β-sheet surrounded by 3 α-helices (Fig. 1A and Fig. 2). Here the sheet topology is β7-β6-β8-β5-β9-β11 with strand β5 running anti-parallel to the other strands. Residues 280-306 at the C terminus of the molecule form a pair of α-helices that fold across the base of the molecule and pack against both domains. A striking feature of the structure is the long helix, α9, which spans the breadth of the molecule (Fig. 1).FIGURE 2Schematic diagram of the topology of SiaP (A) in comparison with an ancestral type II ESR protein (B) (53Fukami-Kobayashi K. Tateno Y. Nishikawa K. J. Mol. Biol. 1999; 286: 279-290Crossref PubMed Scopus (164) Google Scholar). The N-terminal domain is in light gray, and the C-terminal domain is in black. Features that distinguish SiaP are in dark gray. Unfilled circles are 310 helices. This diagram adopts the style of Fukami-Kobayashi (53Fukami-Kobayashi K. Tateno Y. Nishikawa K. J. Mol. Biol. 1999; 286: 279-290Crossref PubMed Scopus (164) Google Scholar), where the hinge β-strands are drawn as part of the β-sheets. The β4 and β5 schematic elements for SiaP actually form a single extended β-strand that extends across both domains but has been displayed as two elements in the schematic to be consistent with the numbering scheme for the type II ESR.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A DALI search revealed the existence of a large number of structures with similarity to SiaP. The highest scoring match (Z = 8.6; with 112 of 309 Cα atoms aligning with a root mean square deviation of 2.9 Å) is the periplasmic glycine-betaine ESR protein from an ABC transporter (PDB 1R9L). Many other matches were found to other ABC ESR proteins, LysR-type transcription factors and eukaryotic glutamate receptors. It is immediately apparent from an examination of the domain topologies that SiaP is a type II ESR protein (Fig. 2). These are characterized by a domain dislocation of one of the β-strands in each of the β-sheets (30Fukami-Kobayashi K. Tateno Y. Nishikawa K. Mol. Biol. Evol. 2003; 20: 267-277Crossref PubMed Scopus (46) Google Scholar).The Ligand-bound SiaP Protein Adopts a Closed Conformation—To investigate the structural basis for ligand binding, we grew crystals of SiaP in the presence of Neu5Ac and selected analogues (13Severi E. Randle G. Kivlin P. Whitfield K. Young R. Moxon R. Kelly D. Hood D. Thomas G.H. Mol. Microbiol. 2005; 58: 1173-1185Crossref PubMed Scopus (106) Google Scholar). Analysis of a third crystal form (Form 3), grown in the presence of one of these sialic acid analogues, revealed the presence of four molecules in the asymmetric unit. The ligand, Neu5Ac2en, was clearly defined in the electron density maps for molecule B after molecular replacement (Fig. 1B); however, molecules A, C, and D were unliganded. Superposition of 297 equivalent Cα atoms of molecules A, C, and D by least squares minimization methods gives pairwise root mean square positional deviations in the range 0.4-0.8 Å, similar to those seen when these structures are superposed on the coordinates from the Form 2 crystal (0.5-1.0 Å). However, superposition onto the liganded molecule B gave much larger deviations in the range of 2.5-3.1 Å. The superposition was much better when the individual domains were overlaid (0.4-0.9 Å). These comparisons suggest that a rigid body domain movement accompanies ligand binding, as is apparent in Fig. 1C. Quantitative comparison of these structures using DynDom (31Hayward S. Lee R.A. J. Mol. Graph. Model. 2002; 21: 181-183Crossref PubMed Scopus (247) Google Scholar) reveals that this movement can be described by a rotation of 25-31° about a hinge that runs close to the peptide bonds connecting residues Thr-127-Arg-128, Ile-211—Leu-212, and Glu-254—Lys-255.Although the majority of the conformational changes can be attributed to the rigid body rotation about the hinge, a small region near the surface of the ligand binding cleft in Domain II (Ala-186—Tyr-197) makes an additional movement beyond that caused by the hinge bending, which results in the reorientation of Phe-170 to form a stacking interaction against the side of the sugar ring. Interestingly, the hinge bending observed in the ligand-bound form also results in the kinking of the α-helical component of the hinge.The ligand-bound molecule B in the Form 3 crystal contains a single molecule of Neu5Ac2en, which is consistent with the 1:1 stoichiometry of binding for Neu5Ac2en and Neu5Ac determined by electrospray mass spectrometry (13Severi E. Randle G. Kivlin P. Whitfield K. Young R. Moxon R. Kelly D. Hood D. Thomas G.H. Mol. Microbiol. 2005; 58: 1173-1185Crossref PubMed Scopus (106) Google Scholar). Neu5Ac2en differs from the physiological ligand Neu5Ac in that it contains a C2 C3 double bond that introduces partial planarity into the sugar ring (its structure is drawn in Fig. 5). The ligand is bound in a pocket formed by the two domains, and its carboxylate group forms a salt bridge to Arg-147 and a polar interaction 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 in Neu5Ac2en is almost perpendicular to rather than planar with the ring. The glycerol group of Neu5Ac2en appears to form two hydrogen bonds to Glu-67. There is an additional contact between Asn-10 and the carbonyl oxygen of the N-acetyl group. The ligand is almost completely buried, with only 32 Å2 of its 435-Å2 surface area accessible to the solvent (Fig. 1D).FIGURE 5Chemical structures and Kd values of sialic acid (Neu5Ac) and related analogues used in this study and in Severi et al. (13Severi E. Randle G. Kivlin P. Whitfield K. Young R. Moxon R. Kelly D. Hood D. Thomas G.H. Mol. Microbiol. 2005; 58: 1173-1185Crossref PubMed Scopus (106) Google Scholar). Neu5Gc, N-glycolylneuraminic acid; KDN, 2-keto-3-deoxy-d-glycero-d-galactonononic acid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3A, stereo view of the electron density (contoured at 1. 5σ) for Neu5Ac2en and residues involved in the coordination of this ligand in SiaP. B, Ligplot representation of the interactions between Neu5Ac2en and the protein.View Large Image
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