Structure of the Bundle-forming Pilus from Enteropathogenic Escherichia coli
2005; Elsevier BV; Volume: 280; Issue: 48 Linguagem: Inglês
10.1074/jbc.m508099200
ISSN1083-351X
AutoresStéphanie Ramboarina, P.J.L. Fernandes, Sarah J. Daniell, Suhail A. Islam, P. J. Simpson, Gad Frankel, Frank P. Booy, Michael S. Donnenberg, Stephen Matthews,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoBundle-forming pili (BFP) are essential for the full virulence of enteropathogenic Escherichia coli (EPEC) because they are required for localized adherence to epithelial cells and auto-aggregation. We report the high resolution structure of bundlin, the monomer of BFP, solved by NMR. The structure reveals a new variation in the topology of type IVb pilins with significant differences in the composition and relative orientation of elements of secondary structure. In addition, the structural parameters of native BFP filaments were determined by electron microscopy after negative staining. The solution structure of bundlin was assembled according to these helical parameters to provide a plausible atomic resolution model for the BFP filament. We show that EPEC and Vibriocholerae type IVb pili display distinct differences in their monomer subunits consistent with data showing that bundlin and TcpA cannot complement each other, but assemble into filaments with similar helical organization. Bundle-forming pili (BFP) are essential for the full virulence of enteropathogenic Escherichia coli (EPEC) because they are required for localized adherence to epithelial cells and auto-aggregation. We report the high resolution structure of bundlin, the monomer of BFP, solved by NMR. The structure reveals a new variation in the topology of type IVb pilins with significant differences in the composition and relative orientation of elements of secondary structure. In addition, the structural parameters of native BFP filaments were determined by electron microscopy after negative staining. The solution structure of bundlin was assembled according to these helical parameters to provide a plausible atomic resolution model for the BFP filament. We show that EPEC and Vibriocholerae type IVb pili display distinct differences in their monomer subunits consistent with data showing that bundlin and TcpA cannot complement each other, but assemble into filaments with similar helical organization. A wide variety of pathogenic bacteria express fimbriae or pili, surface appendages that in many cases allow the microbe to attach to host epithelia. This attachment is often the first step toward colonization of their preferred host niche. The most important classes of pili are the chaperone-usher family and the type IV fimbriae. Type IV pili are produced by a vast array of important human pathogens including Vibriocholerae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Legionella pneumophila, Salmonella enterica serovar Typhi, and enteropathogenic Escherichia coli (EPEC). 3The abbreviations used are: EPECenteropathogenic E. coliBFPbundle-forming piliEMelectron microscopyFTfourier transformationTMVtobacco mosaic virusPDBProtein Data Bank. A number of important phenotypes are associated with the expression of type IV pili including auto-aggregation (1Bieber D. Ramer S.W. Wu C.Y. Murray W.J. Tobe T. Fernandez R. Schoolnik G.K. Science. 1998; 280: 2114-2118Crossref PubMed Scopus (382) Google Scholar, 2Kirn T.J. Lafferty M.J. Sandoe C.M. Taylor R.K. Mol. Microbiol. 2000; 35: 896-910Crossref PubMed Scopus (164) Google Scholar, 3Park H.S. Wolfgang M. van Putten J.P. Dorward D. Hayes S.F. Koomey M. Mol. Microbiol. 2001; 42: 293-307Crossref PubMed Scopus (30) Google Scholar), adherence (4Doig P. Sastry P.A. Hodges R.S. Lee K.K. Paranchych W. Irvin R.T. Infect. Immun. 1990; 58: 124-130Crossref PubMed Google Scholar, 5Donnenberg M.S. Giron J.A. Nataro J.P. Kaper J.B. Mol. Microbiol. 1992; 6: 3427-3437Crossref PubMed Scopus (249) Google Scholar, 6Stone B.J. Abu Kwaik Y. Infect. Immun. 1998; 66: 1768-1775Crossref PubMed Google Scholar), twitching motility (7Merz A.J. So M. Sheetz M.P. Nature. 2000; 407: 98-102Crossref PubMed Scopus (621) Google Scholar, 8Bradley D.E. Can. J. Microbiol. 1980; 26: 146-154Crossref PubMed Scopus (242) Google Scholar), biofilm formation (9O'Toole G.A. Kolter R. Mol. Microbiol. 1998; 30: 295-304Crossref PubMed Scopus (2053) Google Scholar), horizontal gene transmission (10Seifert H.S. Ajioka R.S. Marchal C. Sparling P.F. So M. Nature. 1988; 336: 392-395Crossref PubMed Scopus (131) Google Scholar, 11Yoshida T. Furuya N. Ishikura M. Isobe T. Haino-Fukushima K. Ogawa T. Komano T. J. Bacteriol. 1998; 180: 2842-2848Crossref PubMed Google Scholar, 12Chen I. Dubnau D. Front. Biosci. 2003; 8: s544-s556Crossref PubMed Scopus (16) Google Scholar), cellular invasion (13Donnenberg M.S. Bacterial Invasiveness. 1996; Vol. 209: 79-98Crossref Scopus (7) Google Scholar, 14Zhang X.L. Tsui I.S. Yip C.M. Fung A.W. Wong D.K. Dai X. Yang Y. Hackett J. Morris C. Infect. Immun. 2000; 68: 3067-3073Crossref PubMed Scopus (124) Google Scholar), and virulence (1Bieber D. Ramer S.W. Wu C.Y. Murray W.J. Tobe T. Fernandez R. Schoolnik G.K. Science. 1998; 280: 2114-2118Crossref PubMed Scopus (382) Google Scholar, 15Herrington D.A. Hall R.H. Losonsky G. Mekalanos J.J. Taylor R.K. Levine M.M. J. Exp. Med. 1988; 168: 1487-1492Crossref PubMed Scopus (531) Google Scholar, 16Farinha M.A. Conway B.D. Glasier L.M.G. Ellert N.W. Irvin R.T. Sherburne R. Paranchych W. Infect. Immun. 1994; 64: 4118-4123Crossref Google Scholar). enteropathogenic E. coli bundle-forming pili electron microscopy fourier transformation tobacco mosaic virus Protein Data Bank. The bundle-forming pili (BFP) of EPEC is an excellent model for the study of type IV pili. BFP are required for the characteristic localized adherence of EPEC to epithelial cells (5Donnenberg M.S. Giron J.A. Nataro J.P. Kaper J.B. Mol. Microbiol. 1992; 6: 3427-3437Crossref PubMed Scopus (249) Google Scholar, 17Giron J.A. Ho A.S.Y. Schoolnik G.K. Science. 1991; 254: 710-713Crossref PubMed Scopus (497) Google Scholar), for auto-aggregation and for full virulence in volunteers (1Bieber D. Ramer S.W. Wu C.Y. Murray W.J. Tobe T. Fernandez R. Schoolnik G.K. Science. 1998; 280: 2114-2118Crossref PubMed Scopus (382) Google Scholar). The 14-gene bfp operon contains all of the genes specifically required for BFP biogenesis (18Stone K.D. Zhang H.Z. Carlson L.K. Donnenberg M.S. Mol. Microbiol. 1996; 20: 325-337Crossref PubMed Scopus (172) Google Scholar). Mutagenesis studies have shown that all but one of these genes is necessary to produce functional BFP (19Anantha T.P. Stone K.D. Donnenberg M.S. J. Bacteriol. 2000; 182: 2498-2506Crossref PubMed Scopus (55) Google Scholar, 20Hwang J.W. Bieber D. Ramer S.W. Wu C.Y. Schoolnik G.K. J. Bacteriol. 2003; 185: 6695-6701Crossref PubMed Scopus (28) Google Scholar, 21Schreiber W. Stone K.D. Strong M.A. DeTolla L.J. Hoppert M. Donnenberg M.S. Microbiology-Sgm. 2002; 148: 2507-2518Crossref PubMed Scopus (18) Google Scholar). The first gene of the bfp operon, bfpA, encodes prebundlin, which upon cleavage yields a 19-kDa protein that is the only known structural component of BFP (5Donnenberg M.S. Giron J.A. Nataro J.P. Kaper J.B. Mol. Microbiol. 1992; 6: 3427-3437Crossref PubMed Scopus (249) Google Scholar). Prebundlin is associated with the inner membrane with its 12-amino acid N-terminal leader sequence in the cytoplasm and the C-terminal, globular head domain located in the periplasm. Prebundlin is processed into mature bundlin after removal of the N-terminal leader sequence by the prepilin peptidase, BfpP (22Zhang H.Z. Lory S. Donnenberg M.S. J. Bacteriol. 1994; 176: 6885-6891Crossref PubMed Google Scholar). It has also been shown that DsbA, a periplasmic oxidoreductase stabilizes the globular head domain by catalyzing the formation of a well conserved disulfide bond in the C-terminal part of bundlin (23Zhang H.Z. Donnenberg M.S. Mol. Microbiol. 1996; 21: 787-797Crossref PubMed Scopus (119) Google Scholar). Single substitutions of the two cysteine residues to serine are detrimental for pilus biogenesis and EPEC cell adhesion (23Zhang H.Z. Donnenberg M.S. Mol. Microbiol. 1996; 21: 787-797Crossref PubMed Scopus (119) Google Scholar). The conserved disulfide bond is a common feature of type IV pili (24Strom M.S. Lory S. Annu. Rev. Microbiol. 1993; 47: 565-596Crossref PubMed Scopus (413) Google Scholar). In addition, type IV pili share sequence similarity in the fifty N-terminal amino acids, which have been assumed to form an extended, hydrophobic N-terminal helix that promotes the assembly of type IV fimbriae (25Craig L. Taylor R.K. Pique M.E. Adair B.D. Arvai A.S. Singh M. Lloyd S.J. Shin D.S. Getzoff E.D. Yeager M. Forest K.T. Tainer J.A. Mol. Cell. 2003; 11: 1139-1150Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). As BFP from EPEC represents an attractive system to study the assembly of type IVb pili, we have investigated the structure of the major subunit of BFP filaments, bundlin, by NMR. Here we report the high resolution structure of the 18.3-kDa globular head domain from bundlin and reveal a new variation in the topology of type IVb pilins. In combination with EM data collected on native BFP we were able to propose an atomic resolution model for the type IVb filament structure. NMR Spectroscopy—The 18.3-kDa 15N-13C-labeled recombinant bundlin domain was expressed and purified as described previously (26Ramboarina S. Fernandes P. Simpson P. Frankel G. Donnenberg M. Matthews S. J. Biomol. NMR. 2004; 29: 427-428Crossref PubMed Scopus (6) Google Scholar). NMR samples of bundlin (350 μl) were prepared at a concentration of about 0.5 mm into 20 mm sodium phosphate buffer pH 5.2, 0.1% NaN3, and 3-(trimethylsilyl)-propionic acid for referencing 1H, 15N, and 13C chemical shifts. NMR data were acquired at 303 K on a 500 MHz four-channel Bruker DRX500 spectrometer equipped with a z-shielded gradient triple resonance cryoprobe. Sequence-specific backbone and side chain assignments for bundlin were determined previously (26Ramboarina S. Fernandes P. Simpson P. Frankel G. Donnenberg M. Matthews S. J. Biomol. NMR. 2004; 29: 427-428Crossref PubMed Scopus (6) Google Scholar). Three-dimensional 500 MHz 1H-15N and 800 MHz 1H-13C NOESY-HSQC data were collected with a mixing time of 100 ms. Spectra were processed with NMRPipe (27Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11570) Google Scholar) and analyzed using NMRview (28Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2678) Google Scholar). 15N relaxation rate measurements were performed as previously described (29Kay L.E. Torchia D.A. Bax A. Biochemistry. 1989; 28: 8972-8979Crossref PubMed Scopus (1794) Google Scholar, 30Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Formankay J.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (2013) Google Scholar) on a 800 MHz Varian Inova spectrometer. For the measurement of T1, ten experiments were performed with relaxation delays of 8.6, 9.7, 193, 795, 996, 1493, 2000, 193, and 996 ms. Ten experiments were also performed for the measurement of T2 with relaxation delays of 8.5, 17.1, 34.1, 59.7, 76.8, 93.8, 119.4, 145.0, 17.1, and 76.8 ms. For T1 and T2, two experiments were repeated with identical delays to estimate the noise level. The heteronuclear 1H-15N NOEs were measured from two experiments with and without proton saturation. All the 1H-15N HSQC spectra were processed with NMRview. Rates and errors were fitted as implemented in the NMRview5 software version. Structure Calculations—Secondary structure elements in bundlin were first identified using the chemical shift-based, dihedral angle prediction software TALOS (31Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2738) Google Scholar). A manual analysis of 15N and 13C NOESY-HSQC spectra allowed the backbone NOEs characteristic of secondary structures to be identified. Additional long distance NOEs were assigned between side chain protons of residues Ile47 and Tyr104. The manually assigned NOEs were used to start the automated, iterative, assignment procedure ARIA 1.2 (32Nilges M. O'Donoghue S.I. Prog. NMR. Spectrosc. 1998; 32: 107-139Abstract Full Text PDF Scopus (224) Google Scholar, 33Linge J.P. O'Donoghue S.I. Nilges M. Methods in Enzymology: Nuclear Magnetic Resonance of Biological Macromolecules, Part B,Vol. 339. Academic Press, San Diego, CA2001: 71-90Google Scholar), which employed the structure calculation program CNS (34Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar). Hydrogen bonds were measured from H2O/D2O exchange experiments and extracted from 1H-15N HSQC spectra recorded every hour over 24 h. The disulfide bridge between Cys116 and Cys166 (numbering referenced to bundlin) was included in the calculations. 100 structures were imposed in the final iteration, and 15 lowest energy structures with no distance and dihedral angle violations greater than 0.2 Å and 5°, respectively were retained and water-refined in ARIA. A summary of NMR-derived restraints and statistics on the ensemble of structures is shown in TABLE ONE.TABLE ONEStructural statistics on the 50 best NMR structures (PDB: 1zwt)Structural restraints (from Aria1.2*)Ambiguous NOEs aDerived using Aria 1.2, and no distance violation greater than 0.5 Å occurred in the final ensemble.577Unambigous NOEs aDerived using Aria 1.2, and no distance violation greater than 0.5 Å occurred in the final ensemble.2277Hydrogen bonds38ϕ and ψ dihedral angles bDerived from TALOS, and no violation greater than 5° occurred in the final ensemble.87Deviations from idealized geometryBonds (Å)0.0004 ± 0.0001Angles (degrees)0.55 ± 0.03Impropers (degrees)1.73 ± 0.10Root mean square deviations (Å)Backbone (all residues)1.09 ± 0.19Heavy atoms (all residues)1.49 ± 0.18Backbone on secondary structure0.79 ± 0.14Heavy atoms on secondary structure1.04 ± 0.14Ramachandran plot (from Procheck NMR)Most favored regions77%Additionally allowed regions18%Generously allowed regions3%Disallowed regions2%a Derived using Aria 1.2, and no distance violation greater than 0.5 Å occurred in the final ensemble.b Derived from TALOS, and no violation greater than 5° occurred in the final ensemble. Open table in a new tab Bacterial Culture—50 μl of EPEC bacterial culture, wild-type strain E2348/69, were used to seed growth at a 1:100 dilution in Hepes-modified Dulbecco's modified essential medium and grown for 3 h, without shaking at 37 °C. Bacteria were collected by centrifugation at 4000 × g for 5 min at room temperature, and the pellet was resuspended in 50 μl of phosphate-buffered saline. The bacterial suspension was diluted 2-fold in phosphate-buffered saline, and TMV was added at 10 μgml-1 as a calibration control. Bacteria were negatively stained using a modified version of Valentine's technique. 2 μl of the bacterial/TMV suspension were pipetted between a thin carbon film and its mica support (1 mm2). The carbon film was washed by partially floating onto water in a teflon block and then floated off completely on 1% uranyl acetate stain. The carbon film was then collected on a lacey grid (Ted Pella) from below and viewed in an FEI CM200 TEM operating at 120 kV. Images of bacteria expressing BFP were recorded in low-dose mode (using Kodak SO163 film) at a magnification of ×36,000 with the first zero of the contrast transfer function in the range of 18-22 Å. Image Analysis—Suitable micrographs were selected based on electron optical parameters (such as appropriate defocus, stigmation, and absence of drift) and were digitized using a Nikon8000 Coolscan densitometer at 6.35 μm/pixel. All computational analysis was carried out using IMAGIC software (35vanHeel M. Harauz G. Orlova E.V. Schmidt R. Schatz M. J. Struct. Biol. 1996; 116: 17-24Crossref PubMed Scopus (1049) Google Scholar). Two-dimensional Projection Image—Individual filaments were divided into segments of 350 Å in length, which were then normalized, band-pass filtered, masked, aligned, and summed to obtain a higher contrast, average two-dimensional projection image. In addition, larger areas containing multiple, naturally aligned BFP were selected, aligned, and summed, as above. Fourier Transformation—The FTs of the individual pili, the summed projection image and individual and summed areas of BFP were calculated and compared. All transforms resulted in common layer lines. The distance between the layer lines was measured to determine the axial repeat. A lattice was drawn on the diffraction pattern resulting from the summed FTs of individual BFP, which incorporated the major reflections. Bessel orders were calculated from the spacing of the reflections from the meridional line, as described under "Results." Computational Assembly of Filament—A space-filling fiber was built based on the NMR structure of the bundlin monomer and the helical parameters and measurements derived from the EM data. First, the 22-amino acid N-terminal helix was restored to the bundlin monomer. The orientation of the bundlin monomer was then carefully optimized by analogy with the type IV pilus models (36Craig L. Pique M.E. Tainer J.A. Nat. Rev. Microbiol. 2004; 2: 363-378Crossref PubMed Scopus (571) Google Scholar). The long N-terminal α-helix of the bundlin monomer was first oriented roughly parallel to the central axis of the pilus with a slight orientation of about 10° of the monomer with respect to the central axis to avoid clashes in the BFP filament. Furthermore, the bundlin monomer was oriented such that the N-terminal helix was positioned inside the helical strand, forming the innermost layer of the helical fiber. Hlxbuild software (situs.biomachina.org) was then used to assemble a single helix six subunits long. The helical rise was set to 22 Å; the radial displacement of each chain from the helical axis (x-offset in hlxbuild) was optimized to 23 Å, which gave an outer diameter close to that measured by EM (∼75 Å) and avoided subunit clashes in the final structure. The resulting helical strand was then duplicated by opening two further copies using Swiss-Pdb (37Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9590) Google Scholar) to form a three-stranded helical fiber. Each copy was then translated by 44 and 88 Å, respectively. The helical strands then were merged to create the final representation. Coordinates Deposition—Coordinates for the ensemble of NMR structures have been deposited at the Protein Data Bank under the accession code 1zwt. Tables of NMR assignments and restraints have been deposited in the BioMagResBank in Madison, WI under the accession code 6003. Description of the Structure of Bundlin—Recombinant, double-labeled 15N-13C-mature bundlin lacking the hydrophobic N-terminal 22 residues was expressed and purified as described previously (26Ramboarina S. Fernandes P. Simpson P. Frankel G. Donnenberg M. Matthews S. J. Biomol. NMR. 2004; 29: 427-428Crossref PubMed Scopus (6) Google Scholar). Backbone resonance and side chain assignments were obtained using standard triple resonance NMR experiments (38Sattler M. Schleucher J. Griesinger C. Prog. NMR Spectrosc. 1999; 34: 93-158Abstract Full Text Full Text PDF Scopus (1390) Google Scholar). A correlation time of about 11.5 ns was calculated from 15N relaxation data (T1/T2 ratios along the sequence shown in Fig. 1A), which is consistent with that of the 18.3 kDa bundlin monomer. Overall 15N relaxation data for the backbone amide of bundlin reveal a well ordered structure with very few highly mobile regions (Fig. 1). Interestingly, resonance assignments residues were not achieved for Lys167, Asn168, and Thr169 as signals were not observed in the NMR spectra (numbering referenced to bundlin). Furthermore, high T1/T2 fluctuations are observed for the region between residues 162 and 173 without a decrease of 1H-15N NOE (Fig. 1A). This is indicative of conformational exchange for the protein backbone in this region as is also evidenced by 1H-1H NOE cross-peaks of much lower intensity than those exhibited by the ordered parts of the protein. The ensemble of 15 low energy structures with neither distance nor dihedral violations more than 0.2 Å or 5°, respectively is shown in Fig. 1B. The three-dimensional structure of the globular head domain of bundlin is composed of a seven-stranded mixed parallel and antiparallel β-sheet surrounded by four α-helices and two 310-helices (Fig. 1C). The backbone root mean square deviation is 0.79 ± 0.14 Å for the secondary structure elements and 1.09 ± 0.19 Å for all atoms (TABLE ONE). The four helices encompass residues Lys32-Tyr51 (α1-C), Leu60-Asn67 (α2), Lys113-Ala120 (α3), and Thr158-Ala165 (α4) with α3 and α4 conjoined by the disulfide bond between Cys116-Cys166. The loop region (52-59) connecting α1 and α2 displays a higher degree of flexibility in bundlin structure than the rest of the molecule as shown by a decrease of 1H-15N NOEs within this region (Fig. 1A). In contrast, the second loop (68-80) connecting α 2 to the β-sheet possesses a short 310-helix from Asp73-Tyr75 that is rigidly associated with the rest of the secondary structure. The β-strands are defined by residues Lys81-Thr84 (β1), Glu90-Ala96 (β2), Tyr104-Thr109 (β3), Lys129-Val133 (β4), Ser143-Gly145 (β5), Ala151-Ser154 (β6), and Asn173-Met179 (β7). The first three β-strands do not form the β-meander that is usually observed in type IV pilins (see below) (25Craig L. Taylor R.K. Pique M.E. Adair B.D. Arvai A.S. Singh M. Lloyd S.J. Shin D.S. Getzoff E.D. Yeager M. Forest K.T. Tainer J.A. Mol. Cell. 2003; 11: 1139-1150Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 39Hazes B. Sastry P.A. Hayakawa K. Read R.J. Irvin R.T. J. Mol. Biol. 2000; 299: 1005-1017Crossref PubMed Scopus (118) Google Scholar, 40Keizer D.W. Slupsky C.M. Kalisiak M. Campbell A.P. Crump M.P. Sastry P.A. Hazes B. Irvin R.T. Sykes B.D. J. Biol. Chem. 2001; 276: 24186-24193Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 41Xu X.F. Tan Y.W. Lam L. Hackett J. Zhang M.J. Mok Y.K. J. Biol. Chem. 2004; 279: 31599-31605Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Instead they are arranged in a complex mixed β-sheet topology, with the following strand order β2/β3/β1 (Fig. 1D). This arrangement is unusual and results principally from hydrophobic interactions between Ile61 from β1, and Pro86, Leu108, and Leu111, which are located in the β1/β2-loop, β3 and β3/α3-loop, respectively. Helix α3 connects β3 to β4, which forms an antiparallel pairing with neighboring strands, β6 and β7. β4 is followed by a short 310-helix (Glu138-Asn140) and the β5-strand, which lies antiparallel to β6. In summary, all seven strands form a twisted contiguous surface arranged in the order β2, β3, β1, β7, β4, β6, and β5 (Fig. 1, C and D). The β-sheet region of bundlin is flanked by the four α-helices on three sides. The α1-C helix is positioned across the concave face of the sheet by hydrophobic interactions between Ile47/Tyr104, Thr44/Tyr104, and Ala43/Phe87. Residues Ile47, Tyr51, Tyr57, Leu60, Ile64, Val93, and Pro95 delineate a hydrophobic platform located between the first two helices and the first part of the β-sheet. Significant hydrophobic interactions involving residues Ile47, Tyr51, Tyr57, and Tyr104 lead to a well-defined orientation for α2 of 90° relative to α1-C. The long loop, 68-80, following α2 also adopts a fixed orientation (Fig. 1, C and D), mediated by hydrophobic interactions between residues Leu65, Pro72, Tyr75, and Tyr105. Two important hydrophobic cores appear to stabilize the protein. The first hydrophobic core comprising residues within β1, β3/α3-loop, α3, β4, α4, and β7, is delineated by Ile83, Leu111, Ala114, Leu119, Tyr131, Val143, Ala165, and Tyr177. Another cluster of hydrophobic residues, located on the opposite face, encompasses residues Phe102, Tyr105, Ile135, Ile141, Phe144, Ala152, and Phe178. As a result the well ordered β4/β5-loop is found close to the β2/β3-loop in the bundlin structure (Fig. 1, C and D). Residues forming the hydrophobic cores are highly conserved among the different EPEC strains. Analysis of BFP by Electron Microscopy—BFP expressed by wild-type EPEC were viewed after negative staining (Fig. 2, A and B). The majority of BFP was expressed in early stage growth and formed closely associated swathes of pili emanating from the bacterial membrane. Clearly discernable individual pili were chosen for further analysis. Short sections corresponding ∼350 Å in length were selected, band-pass filtered, normalized, aligned, and summed, resulting in the two-dimensional-averaged structure shown in Fig. 2C. The image clearly shows a repeating diagonal pattern of protein densities (white) along the pilus, which is indicative of a helical structure. From this averaged projection image, the pili are estimated to have a diameter of about 75 Å. From this structure, the pitch of the dominant helix was estimated to be 44 Å, as shown in Fig. 2C. Fourier transforms were calculated from the two-dimensional projection image, from individual pili and also from larger areas selected from the naturally occurring aligned bundles of pili. All Fourier transforms have major layer lines in common. The FT shown in Figs 3, A and B is derived from the sum of transforms calculated from individual pili and was used to deduce the helical parameters of BFP. All pili were centered prior to calculation of their FT. This summed pattern was used as the layer lines are most clearly represented. A two-dimensional lattice was drawn on the FT to intercept the major reflections (Fig. 3B). The distances between these reflections and the equatorial line were then measured, as shown. Three main reflections are visible with spacings of 1/88 Å, 1/44 Å, and 1/22 Å on layer lines 4, 8, and 12, respectively. The distance between layer lines was measured as 1/175 Å, which corresponds to the axial repeat defined by the predicted 1-start helix. The 1/22 Å meridional spacing was only visible in some FTs, but defines the axial rise of the subunits as 22 Å. The distances between the major reflections and the meridional line were also measured (d) and the Bessel order (n) was then calculated using the equation n = 2πRr-2, where r = 1/d and r is the radius of the filament. The Bessel orders describe two helical families comprising a 6-start helix (layer line 4) with a pitch of 88 Å and a 3-start helix (layer line 8) with a pitch of 44 Å. It is notable that the main reflection consistently seen on all the calculated FTs corresponds to the 3-start helix, which appears to be the dominant feature. This 3-start helix, with an axial rise of 22 Å and pitch of 44 Å thus has six subunits per turn, which is to be compared with the 3-start 45-Å helix with six subunits per turn described for the Tcp of V. cholerae (25Craig L. Taylor R.K. Pique M.E. Adair B.D. Arvai A.S. Singh M. Lloyd S.J. Shin D.S. Getzoff E.D. Yeager M. Forest K.T. Tainer J.A. Mol. Cell. 2003; 11: 1139-1150Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). All spacings were measured using the 1/23 Å-1 third layer line of TMV as a calibration standard. Bundlin Structure Provides New Insights into the Type IV Family—The NMR structure of bundlin, the major subunit of BFP from EPEC, adopts an α/β fold composed of four α-helices that surround a platform formed by a seven-stranded, twisted, contiguous β-sandwich. Our study reveals a new topology for the mixed parallel and antiparallel β-strands arranged in the following order β2, β3, β1, β7, β4, β6, and β5. Although common characteristics can be observed between bundlin and other type IV pilin structures, the new structural features reported here for bundlin have not been described for other members of the type IV pilin family. The type IV pilin family is divided in two groups, types IVa and IVb, distinguished by the length of the pre-pilin leader sequence and the size of the pilin protein (36Craig L. Pique M.E. Tainer J.A. Nat. Rev. Microbiol. 2004; 2: 363-378Crossref PubMed Scopus (571) Google Scholar). The first high resolution structures of pilin proteins were those from the type IVa class, GC from N. gonorrhoeae (42Parge H.E. Forest K.T. Hickey M.J. Christensen D.A. Getzoff E.D. Tainer J.A. Nature. 1995; 378: 32-38Crossref PubMed Scopus (400) Google Scholar), PAK and K122-4 from P. aeruginosa (39Hazes B. Sastry P.A. Hayakawa K. Read R.J. Irvin R.T. J. Mol. Biol. 2000; 299: 1005-1017Crossref PubMed Scopus (118) Google Scholar, 40Keizer D.W. Slupsky C.M. Kalisiak M. Campbell A.P. Crump M.P. Sastry P.A. Hazes B. Irvin R.T. Sykes B.D. J. Biol. Chem. 2001; 276: 24186-24193Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The type IVa pilin structure is characterized by an extended N-terminal α-helix and a globular head domain that is folded into an α-β roll configuration (39Hazes B. Sastry P.A. Hayakawa K. Read R.J. Irvin R.T. J. Mol. Biol. 2000; 299: 1005-1017Crossref PubMed Scopus (118) Google Scholar, 40Keizer D.W. Slupsky C.M. Kalisiak M. Campbell A.P. Crump M.P. Sastry P.A. Hazes B. Irvin R.T. Sykes B.D. J. Biol. Chem. 2001; 276: 24186-24193Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 42Parge H.E. Forest K.T. Hickey M.J. Christensen D.A. Getzoff E.D. Tainer J.A. Nature. 1995; 378: 32-38Crossref PubMed Scopus (400) Google Scholar). The globular head domain is well conserved and characterized by a four-stranded antiparallel continuous β-meander among all the type IVa pilins (36Craig L. Pique M.E. Tainer J.A. Nat. Rev. Microbiol. 2004; 2: 363-378Crossref PubMed Scopus (571) Google Scholar). The structures of TcpA from V. cholerae and PilS from S. typhi from the type IVb group have been determined recently by x-ray diffraction (25Craig L
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