Structural Evidence That the 32-Kilodalton Lipoprotein (Tp32) of Treponema pallidum Is an l-Methionine-binding Protein

Abstract

A structure-to-function approach was undertaken to gain insights into the potential function of the 32-kDa membrane lipoprotein (Tp32) of Treponema pallidum, the syphilis bacterium. The crystal structure of rTp32 (determined at a resolution of 1.85 Å) shows that the organization of rTp32 is similar to other periplasmic ligand-binding proteins (PLBPs), in that it consists of two α/β domains, linked by two crossovers, with a binding pocket between them. In the pocket, a molecule of l-methionine was detected in the electron density map. Residues from both domains interact with the ligand. One of the crossover regions is comprised of a 310-helix, a feature not typical in other ligand-binding proteins. Sequence comparison shows strong similarity to other hypothetical methionine-binding proteins. Together, the data support the notion that rTp32 is a component of a periplasmic methionine uptake transporter system in T. pallidum. A structure-to-function approach was undertaken to gain insights into the potential function of the 32-kDa membrane lipoprotein (Tp32) of Treponema pallidum, the syphilis bacterium. The crystal structure of rTp32 (determined at a resolution of 1.85 Å) shows that the organization of rTp32 is similar to other periplasmic ligand-binding proteins (PLBPs), in that it consists of two α/β domains, linked by two crossovers, with a binding pocket between them. In the pocket, a molecule of l-methionine was detected in the electron density map. Residues from both domains interact with the ligand. One of the crossover regions is comprised of a 310-helix, a feature not typical in other ligand-binding proteins. Sequence comparison shows strong similarity to other hypothetical methionine-binding proteins. Together, the data support the notion that rTp32 is a component of a periplasmic methionine uptake transporter system in T. pallidum. Although syphilis is one of the oldest recognized sexually transmitted diseases, it remains among the most poorly understood. This primarily is a consequence of the fact that the etiological agent, Treponema pallidum, cannot be cultivated continuously in vitro (1Norris S.J. Microbiol. Rev. 1993; 57: 750-779Crossref PubMed Google Scholar). Hence, classical approaches for discerning T. pallidum membrane biology, which could reveal key aspects of its enigmatic parasitic strategy, are largely constrained (2Radolf J.D. Trends Microbiol. 1994; 2: 307-311Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 3Radolf J.D. Mol. Microbiol. 1995; 16: 1067-1073Crossref PubMed Scopus (79) Google Scholar). It is now generally accepted that lipoproteins are important integral membrane proteins of T. pallidum (4Chamberlain N.R. Brandt M.E. Erwin A.L. Radolf J.D. Norgard M.V. Infect. Immun. 1989; 57: 2872-2877Crossref PubMed Google Scholar, 5Haake D.A. Microbiology. 2000; 146: 1491-1504Crossref PubMed Scopus (150) Google Scholar); in fact, lipoproteins represent about 3% of the total coding capacity of the treponemal genome (6Fraser C.M. Norris S.J. Weinstock G.M. White O. Sutton G.G. Dodson R. Gwinn M. Hickey E.K. Clayton R. Ketchum K.A. Sodergren E. Hardham J.M. McLeod M.P. Salzberg S. Peterson J. Khalak H. Richardson D. Howell J.K. Chidambaram M. Utterback T. McDonald L. Artiach P. Bowman C. Cotton M.D. Fujii C. Garland S. Hatch B. Horst K. Roberts K. Sandusky M. Weidman J. Smith H.O. Venter J.C. Science. 1998; 281: 375-388Crossref PubMed Scopus (854) Google Scholar). In other bacteria, lipoproteins have importance as virulence factors, modular components of ATP-binding cassette (ABC) 1The abbreviations used are: ABC, ATP-binding cassette; ESI-MS, electrospray ionization-mass spectrometry; HSQC, heteronuclear single quantum coherence; LAOBP, lysine-, arginine-, ornithine-binding protein; QBP, glutamine-binding protein; MUT, methionine uptake transporter; PLBPs, periplasmic ligand-binding proteins; r.m.s., root mean square. transporters, receptors, protective immune targets, and pro-inflammatory agonists that contribute significantly to innate immune responses (5Haake D.A. Microbiology. 2000; 146: 1491-1504Crossref PubMed Scopus (150) Google Scholar, 7Hayashi S. Wu H.C. J. Bioenerget. Biomem. 1990; 22: 451-471Crossref PubMed Scopus (428) Google Scholar, 8Sutcliff I.C. Russell R.R.B. J. Bacteriol. 1995; 177: 1123-1128Crossref PubMed Google Scholar, 9Becker P.S. Akins D.R. Radolf J.D. Norgard M.V. Infect. 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Bacteriol. 2004; 186: 2303-2308Crossref PubMed Scopus (17) Google Scholar), existing gene sequences and/or BLAST search information have not been fruitful for predicting the functions of treponemal lipoproteins. As an alternative approach to understanding the peculiar membrane biology of T. pallidum (2Radolf J.D. Trends Microbiol. 1994; 2: 307-311Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 3Radolf J.D. Mol. Microbiol. 1995; 16: 1067-1073Crossref PubMed Scopus (79) Google Scholar, 15Cox D.L. Chang P. McDowall A. Radolf J.D. Infect. Immun. 1992; 60: 1076-1083Crossref PubMed Google Scholar), we recently embarked on a structural biology initiative that seeks to garner structural insights into putative functions for a number of the T. pallidum membrane lipoproteins. To this end, x-ray crystallographic studies were performed on Tp32 (gene number Tp0821). In the course of these studies, l-methionine was detected within a putative ligand-binding protein-like cleft. This has led us to conclude that Tp32 likely serves as a periplasmic receptor for l-methionine, probably lipid-linked to the outer leaflet of the T. pallidum cytoplasmic membrane. These findings add to our knowledge of the functions of treponemal membrane lipoproteins. The importance of Tp32 as a receptor for l-methionine, as well as its unique features, is discussed. Construction of His6-tagged rTp32—To create a non-lipidated version of recombinant Tp32 (rTp32) in Escherichia coli, a fragment encoding amino acids 2-245 (residue 1 is the post-translationally modified N-terminal cysteine) was amplified by PCR using a genomic DNA template isolated from T. pallidum. The PCR primers were 5′-acgcGGATCCACTCAGGTGAAGGATGAAACGGTGG-3′ and 5′-atccAAGCTTTTACAAAGCAGGCGCCACCTCC-3′. The forward primer contained both an acgc overhang and a BamHI site (bold); the reverse primer contained an atcc overhang and a HindIII site (bold). PCR was performed using TaKaRa Ex Taq DNA polymerase with an annealing temperature of 55 °C. The amplification product was directionally cloned into the pProEX HTb vector (Invitrogen) that adds an N-terminal His6 tag followed by a Tobacco Etch Virus protease cleavage site. Ligations were transformed into E. coli XL1-Blue competent cells (Stratagene), and colonies that tested positive by restriction digest were sequenced for verification. Expression and Purification of rTp32—E. coli XL1-Blue containing the cloned tp32 gene fusion was grown at 37 °C in LB medium containing 100 μg/ml of ampicillin until the cell density reached an OD600 of 0.6. The culture was then shifted to 30 °C and induced for 5 h with 600 μm isopropyl-1-thio-β-d-galactopyranoside. Cells were harvested by centrifugation and lysed at room temperature for 30 min using 50 ml/liter culture of B-PER II (Pierce). The resulting suspension was spun at 27,000 × g for 15 min to pellet the cell debris. rTp32 was isolated from the supernatant by immobilization on a Ni2+-affinity column and was eluted using 20 mm Tris-HCl (pH 8.5), 200 mm NaCl, 200 mm imidazole. Protein buffer was exchanged with 20 mm Hepes (pH 7.5), 100 mm NaCl, 15 mm β-mercaptoethanol, 2 mm octyl-β-glucoside (buffer A) by ultrafiltration using an Amicon device with a 10,000 molecular weight (MW) size exclusion. The concentrated protein was then applied to a HiLoad 16/60 Superdex 75 gel filtration column that was preequilibrated in buffer A, using an Äkta fast performance liquid chromatography (FPLC) system (Amersham Biosciences). The column was run at a flow rate of 0.2 ml/min. Subsequent to elution, peak fractions were analyzed by SDS-PAGE. At this stage, protein was pure to apparent homogeneity (i.e. >95%). Fractions containing purified rTp32 were pooled and concentrated in buffer A to 30-35 mg/ml for crystallization. The oligomeric state of the purified protein was determined by size exclusion chromatography, using a calibration curve made with protein standards (Amersham Biosciences). For the production of selenomethionine (SeMet)-labeled protein, the expression vector was transformed into the methionine auxotroph E. coli B834 (Novagen) and grown in the presence of LB and 95% M9 medium supplemented with 125 mg/liter each of adenine, uracil, thymine, and guanosine nucleotide, 2.5 mg/liter thiamine, 4 mg/liter d-biotin, 20 mm glucose, 2 mm MgSO4, 50 mg/liter each of 19 l-amino acids (excluding methionine), and 50 mg/liter l-selenomethionine (Sigma). For 15N/13C-Met incorporation, E. coli containing the expression vector was grown in M9 minimal medium supplemented with 1 mg/liter of 15NH4Cl (Cambridge as the and 1 mm (Sigma). Protein was from a at F. L. T. 1995; PubMed Scopus Google Scholar). The molecular weight of rTp32 was determined by electrospray ionization-mass Protein N-terminal was performed by in an protein and at the were used for protein of and the production of purified protein was at room temperature for h in buffer A containing was by ultrafiltration in an cell using a 10,000 size exclusion and with of the Protein was by into 1 of buffer A at a rate of ml/min. protein was from the protein by centrifugation at × g for 20 The protein was purified by gel filtration chromatography and concentrated for were performed on a using F. S. J. A. J. 1995; PubMed Scopus Google for data and J. 1994; PubMed Scopus Google for data were on μm in buffer A at °C. Protein and rTp32 was by G.M. P. J. M. T. D. 1998; PubMed Scopus Google using were used to of The of rTp32 were with containing 5 of protein at a of mg/ml in buffer A and 5 of (pH at 20 °C. of a of rTp32 were the in In to a a of and was to the crystal that the of the was were to a of and then in liquid and liquid data were at 100 using from of the at the The data were and in the Z. PubMed Scopus Google Scholar). were on a crystal of the of rTp32 to a of and on a rTp32 crystal 1.85 on the crystallographic data are in for the protein were by the with the J. D. 1999; PubMed Scopus Google using data on the of rTp32 at a to the of The resolution used was The and the a of of was using the D. PubMed Scopus Google Scholar, D. PubMed Scopus Google and with the M. A. PubMed Scopus Google Scholar). At this stage, electron density for a l-selenomethionine was at the between the two domains in of the data from the crystal of The data electron density for a The was using the and in version G.M. P. J. M. T. D. 1998; PubMed Scopus Google Scholar). for were and were The was using and J. Mol. 1992; Google Scholar). were the protein was and The of rTp32 and a molecule of shows the for proteins were using the L. C. 1994; PubMed Scopus Google Scholar). The similarity 2 is to be was between rTp32 and the hypothetical protein an of the protein was used as the of the to of the periplasmic protein as by the of were apparent T. C. J. Mol. 1995; PubMed Scopus Google Scholar). For II of rTp32 a of with one of the domains of the lysine-, arginine-, and ornithine-binding and in are for the resolution of the is the of the for h and the mean of the of protein for is the as that for it is with a of the structure in this that not been used for a modified in are for the resolution of the is the of the for h and the mean of the of The for is the as that for it is with a of the structure in this that not been used for a modified Davis J.M. Richardson Richardson PubMed Scopus Google Scholar). in a of Tp32 was as a fusion protein with an N-terminal His6 tag and amino The expression of this fusion protein in E. coli in the production of for were of culture pure at 4 °C in 20 mm Hepes (pH 7.5), 100 mm NaCl, 15 mm β-mercaptoethanol, recombinant rTp32 The was by the of 2 mm followed by for h at room chromatography that rTp32 was rTp32 eluted from the column as a single peak with a molecular of about This is in with the determined by that the recombinant protein as a in However, the on the of our of rTp32 is the of the purified N-terminal that the of amino of rTp32 been The heteronuclear single quantum of rTp32 peak and that rTp32 was well and for crystallization. of rTp32—To insights into the function of we determined its crystal structure at a resolution of 1.85 rTp32 two domains a α/β with both domains of similar The 55 from structural in the two domains be with a of The structure of each is comprised of a which consists of a by on both this the In each one is and is II The of and In to two to the of the II has a and two of its For the of this is as one a feature of this is that it is at both The protein two between the domains, and II is of a The total at the between the two domains is molecule of l-methionine was within the electron density within a pocket between the two domains The of this molecule was using two the size and of the electron density was with the selenomethionine of rTp32 density in an at the of a in selenomethionine amino acids were in the we the methionine from the of the expression The binding of l-methionine to rTp32 to be the electron density in the binding pocket of binding by other amino acids and a of other are in the E. coli domains in the binding of the l-methionine The pocket be into two binding and The of these is well to binding an l-methionine, at has amino and and a The binding of the pocket is with and these in to the amino and of the l-methionine for the on the methionine is by the and The binding of the pocket a of and The and with the of the on this of the pocket not to the methionine to the One a the of its to the in the methionine The amino pocket of rTp32 to be well to the expression of the protein in the of E. coli, l-methionine a of other for binding to the amino binding The l-methionine is to the This is by the fact that the amino not from rTp32 in which l-methionine was this notion is the that were of the l-methionine The for this binding are from the The of the amino binding pocket is to that of of the is in with the the between and the the It be to other amino acids could to this methionine-binding it was determined that l-methionine was to purified were undertaken to To this end, the protein was to in followed by rTp32 in this was then analyzed by A of the and of rTp32 that protein as by peak and an in the number of in the of the In of peak between the and proteins that of the protein the l-methionine ligand. The protein is as by the fact that the of 1 mm l-methionine to the protein in an that was to the protein the of in studies of binding to rTp32 to search for proteins that Tp32 is a of a of proteins the periplasmic binding protein-like This other proteins that amino as the lysine-, arginine-, ornithine-binding protein and the protein J. K. S. J. 1993; Full Text PDF PubMed Google Scholar, N. S. 1994; PubMed Scopus Google Scholar). proteins function as receptor components of transporter H. 1998; PubMed Scopus Google that to from the to the These structural with studies that Tp32 has amino to a protein from E. coli, Z. Chang M.S. Microbiol. PubMed Scopus Google Scholar). is the methionine receptor for a transporter the methionine uptake transporter amino comparison between and Tp32 shows that the two of the proteins are about In the that are in with the methionine are The exception is of The in is a The of rTp32 to l-methionine and the of similarity between and Tp32 us to conclude that the function of Tp32 likely is to as the methionine receptor a at the cytoplasmic membrane of T. pallidum. The of similarity between Tp32 and to of the the that the l-methionine are well in this The is This is to the fact that at of this methionine-binding It is both of methionine to T. pallidum the methionine for of to the of this to is other of periplasmic ligand-binding proteins with amino acids are N. S. 1994; PubMed Scopus Google Scholar, J. 1994; Full Text PDF PubMed Google Scholar, J. J. Mol. 1998; PubMed Scopus Google Scholar). are LAOBP, and the glutamine-binding protein The of these is similar to that of In to the of the with of about in of rTp32 and is in between the two of the In two from the of are not in have that these interact with the components of the LAOBP, and J. K. S. J. 1993; Full Text PDF PubMed Google Scholar, J. J. Mol. 1998; PubMed Scopus Google Scholar). the of the rTp32 has a that is in The is between and an of rTp32 that the of and as well as a This feature is to a of on the protein A. K.A. B. PubMed Scopus Google Scholar). The of the and the this of the protein a for the that with the component of the treponemal of of potential are and of positive potential are The used for the potential was to This was using A. K.A. B. PubMed Scopus Google between rTp32 and the other periplasmic binding proteins in the In LAOBP, and QBP, the regions have are In one of the regions a This is unique among the of the of The regions of other R.K. Norgard M.V. Radolf J.D. 1999; PubMed Scopus Google J. J. Mol. 1998; PubMed Scopus Google Scholar), a in this has been in methionine binding components of the of the and of the other periplasmic amino binding proteins that the two domains with to one J. K. S. J. 1993; Full Text PDF PubMed Google Scholar, J. J. Mol. 1998; PubMed Scopus Google Scholar). The presence of the that are to It be to the structure of the in to the of in this The which were using the protein between the amino binding pocket of rTp32 and of the other amino binding proteins. the l-methionine to rTp32 is to the amino acids to the other proteins The of the l-methionine the that the in the other The of the amino binding pocket of rTp32 from of the other amino binding and the of and within the pocket are These in the of the amino similarity recently that Tp32 has with a hypothetical protein from Z. Chang M.S. Microbiol. PubMed Scopus Google have that protein is a methionine binding of the of The structural similarity between and rTp32 is the proteins have have an of a in its The protein has an with This a and an that on the of In to the protein to a a The and amino of the methionine within the amino binding pocket of are similar to in and the amino binding of the two proteins are two of the pocket are between the two proteins. The is in the binding of the pocket, rTp32 is by a in The is in the binding of the rTp32 which a to the amino of the l-methionine, has been in with an In other amino between the two proteins have the of the binding of the pocket in These the N-terminal of the it that of the proteins by Z. Chang M.S. Microbiol. PubMed Scopus Google as methionine-binding proteins These that at of the that of rTp32 is poorly in the methionine-binding proteins is a amino at this in to of methionine in the binding and pallidum is among the that cannot be cultivated in This has of for key of the enigmatic of the discerning the functions of its membrane proteins. Although genome has Tp32 as a cell lipoprotein (6Fraser C.M. Norris S.J. Weinstock G.M. White O. Sutton G.G. Dodson R. Gwinn M. Hickey E.K. Clayton R. Ketchum K.A. Sodergren E. Hardham J.M. McLeod M.P. Salzberg S. Peterson J. Khalak H. Richardson D. Howell J.K. Chidambaram M. Utterback T. McDonald L. Artiach P. Bowman C. Cotton M.D. Fujii C. Garland S. Hatch B. Horst K. Roberts K. Sandusky M. Weidman J. Smith H.O. Venter J.C. 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This the to within the of the with the that rTp32 was its were This membrane in T. pallidum, is to the of lipoproteins as one of two for to cell for the of a O. Microbiol. Mol. Rev. 1999; PubMed Google Scholar). As one from of the system in (i.e. S. E. A. Microbiol. 2004; PubMed Scopus Google Scholar). T. pallidum, its membrane membrane that of to the peculiar membrane biology of this enigmatic (2Radolf J.D. Trends Microbiol. 1994; 2: 307-311Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 3Radolf J.D. Mol. Microbiol. 1995; 16: 1067-1073Crossref PubMed Scopus (79) Google Scholar, 15Cox D.L. Chang P. McDowall A. Radolf J.D. Infect. Immun. 1992; 60: 1076-1083Crossref PubMed Google Scholar). of genome information that T. pallidum likely transporter of which to the transporter The of T. pallidum to key to have been in the to its by proteins for and In this T. pallidum to the that be in of with the exception of which methionine to which is important for D.A. M.S. 2004; PubMed Scopus Google Scholar). Subsequent to our of the structure of Z. Chang M.S. Microbiol. PubMed Scopus Google that the T. pallidum (Tp32) were of E. coli and and likely were of the However, most other which to as Z. Chang M.S. Microbiol. PubMed Scopus Google Scholar), the treponemal are not are the gene to tp32 with tp32 other of (6Fraser C.M. Norris S.J. Weinstock G.M. White O. Sutton G.G. Dodson R. Gwinn M. Hickey E.K. Clayton R. Ketchum K.A. Sodergren E. Hardham J.M. McLeod M.P. Salzberg S. Peterson J. Khalak H. Richardson D. Howell J.K. Chidambaram M. Utterback T. McDonald L. Artiach P. Bowman C. Cotton M.D. Fujii C. Garland S. Hatch B. Horst K. Roberts K. Sandusky M. Weidman J. Smith H.O. Venter J.C. Science. 1998; 281: 375-388Crossref PubMed Scopus (854) Google Scholar). Tp32 as the methionine-binding protein likely with and to methionine to the of T. pallidum. Although these putative to be the of Tp32 to a protein 5 and be for with (i.e. crystallographic studies of the treponemal system in the state reveal the and that and with methionine in T. pallidum. our crystal structure data support the notion that Tp32 is an of E. coli and Tp32 is a of the Z. Chang M.S. Microbiol. PubMed Scopus Google Scholar). and for for and for for the and and the of the for in data