Interaction with Type IV Pili Induces Structural Changes in the Bacterial Outer Membrane Secretin PilQ
2005; Elsevier BV; Volume: 280; Issue: 19 Linguagem: Inglês
10.1074/jbc.m411603200
ISSN1083-351X
AutoresRichard F. Collins, Stephan A. Frye, Seetha V. Balasingham, Robert C. Ford, Tone Tønjum, Jeremy P. Derrick,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoType IV pili are cell surface organelles found on many Gram-negative bacteria. They mediate a variety of functions, including adhesion, twitching motility, and competence for DNA uptake. The type IV pilus is a helical polymer of pilin protein subunits and is capable of rapid polymerization or depolymerization, generating large motor forces in the process. Here we show that a specific interaction between the outer membrane secretin PilQ and the type IV pilus fiber can be detected by far-Western analysis and sucrose density gradient centrifugation. Transmission electron microscopy of preparations of purified pili, to which the purified PilQ oligomer had been added, showed that PilQ was uniquely located at one end of the pilus fiber, effectively forming a "mallet-type" structure. Determination of the three-dimensional structure of the PilQ-type IV pilus complex at 26-Å resolution showed that the cavity within the protein complex was filled. Comparison with a previously determined structure of PilQ at 12-Å resolution indicated that binding of the pilus fiber induced a dissociation of the "cap" feature and lateral movement of the "arms" of the PilQ oligomer. The results demonstrate that the PilQ structure exhibits a dynamic response to the binding of its transported substrate and suggest that the secretin could play an active role in type IV pilus assembly as well as secretion. Type IV pili are cell surface organelles found on many Gram-negative bacteria. They mediate a variety of functions, including adhesion, twitching motility, and competence for DNA uptake. The type IV pilus is a helical polymer of pilin protein subunits and is capable of rapid polymerization or depolymerization, generating large motor forces in the process. Here we show that a specific interaction between the outer membrane secretin PilQ and the type IV pilus fiber can be detected by far-Western analysis and sucrose density gradient centrifugation. Transmission electron microscopy of preparations of purified pili, to which the purified PilQ oligomer had been added, showed that PilQ was uniquely located at one end of the pilus fiber, effectively forming a "mallet-type" structure. Determination of the three-dimensional structure of the PilQ-type IV pilus complex at 26-Å resolution showed that the cavity within the protein complex was filled. Comparison with a previously determined structure of PilQ at 12-Å resolution indicated that binding of the pilus fiber induced a dissociation of the "cap" feature and lateral movement of the "arms" of the PilQ oligomer. The results demonstrate that the PilQ structure exhibits a dynamic response to the binding of its transported substrate and suggest that the secretin could play an active role in type IV pilus assembly as well as secretion. To establish either a commensal or pathogenic relationship with its human host, Neisseria meningitidis develops specific cell-cell contacts and, to this end, exploits a diverse arsenal of adhesive proteins located on the cell surface (1Heckels J.E. Clin. Microbiol. Rev. 1989; 2: S66-S73Crossref PubMed Google Scholar, 2Potts W.J. Saunders J.R. Mol. Microbiol. 1988; 2: 647-653Crossref PubMed Scopus (48) Google Scholar). During tissue colonization many pathogenic Gram-negative bacteria display long adhesive fibers, termed type IV pili, from the cell surface that mediate cellular attachment to epithelial tissue receptors (3Giron J.A. Ho A.S.Y. Schoolnik G.K. Science. 1991; 254: 710-713Crossref PubMed Scopus (493) Google Scholar, 4Hobbs M. Mattick J.S. Mol. Microbiol. 1993; 10: 233-243Crossref PubMed Scopus (306) Google Scholar, 5Strom M.S. Lory S. Annu. Rev. Microbiol. 1993; 47: 565-596Crossref PubMed Scopus (411) Google Scholar). Pili are also involved in several other bacterial processes, including bacterial auto-agglutination (6Swanson J. Kraus S.J. Gotschlich E.C. J. Exp. Med. 1971; 134: 886-906Crossref PubMed Scopus (167) Google Scholar), variation of target tissue specificity (7Jönsson A.B. Ilver D. Falk P. Normark S. Mol. Microbiol. 1994; 13: 403-416Crossref PubMed Scopus (82) Google Scholar), and natural competence for DNA uptake (8Zhang Q.Y. Deryckere D. Lauer P. Koomey M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5366-5370Crossref PubMed Scopus (92) Google Scholar, 9Frøholm L.O. Jyssum K. Bøvre M. Acta Pathol. Microbiol. Scand. 1973; 81: 525-537Google Scholar). Recent data have demonstrated that type IV pili are retractile and that this retraction process is responsible for twitching motility of neisserial cells on solid and mucosal surfaces (10McBride M.J. Annu. Rev. Microbiol. 2001; 55: 49-75Crossref PubMed Scopus (280) Google Scholar, 11Merz A.J. So M. Sheetz M.P. Nature. 2000; 407: 98-102Crossref PubMed Scopus (604) Google Scholar). Type IV pili are long (>1–5 μm), thin (60–70 Å), mechanically strong polymeric fibers containing 500–2000 subunits of the major pilin protein, PilE (12Parge H.E. Forest K.T. Hickey M.J. Christensen D.A. Getzoff E.D. Tainer J.A. Nature. 1995; 378: 32-38Crossref PubMed Scopus (393) Google Scholar). The assembly of pilin into pili, as well as pilus disassembly, is controlled by a complex interacting apparatus of up to 30 proteins (13Tønjum T. Koomey M. Gene (Amst.). 1997; 192: 155-163Crossref PubMed Scopus (90) Google Scholar, 14Alm R.A. Mattick J.S. Gene (Amst.). 1997; 192: 89-98Crossref PubMed Scopus (132) Google Scholar, 15Stone K.D. Zhang H.Z. Carlson L.K. Donnenberg M.S. Mol. Microbiol. 1996; 20: 325-337Crossref PubMed Scopus (170) Google Scholar), which is similar to the apparatus for secretion of proteins of the general (type II) secretory pathway (16Pugsley A.P. Microbiol. Rev. 1993; 57: 50-108Crossref PubMed Google Scholar, 17Nunn D. Trends Cell Biol. 1999; 9: 402-408Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar).Prior to pilus assembly PilE is processed through the cytoplasmic membrane by the pre-pilin peptidase PilD, removing a hydrophobic leader peptide and methylating the N-terminal amino acid en route (13Tønjum T. Koomey M. Gene (Amst.). 1997; 192: 155-163Crossref PubMed Scopus (90) Google Scholar). At this stage PilE is located in the periplasm but is thought to remain tethered to the inner membrane by a single transmembrane α-helix located at its N terminus (18Forest K.T. Tainer J.A. Gene (Amst.). 1997; 192: 165-169Crossref PubMed Scopus (82) Google Scholar). It is hypothesized that this phenomenon creates a "pool" of accessible pilin subunits associated with the inner membrane. Several other neisserial proteins, termed pseudopilins or minor pilins, share homology within their N-terminal regions with the PilE subunit and are also required for correct pilus expression, although they are expressed at low levels compared with PilE (19Aas F.E. Lovold C. Koomey M. Mol. Microbiol. 2002; 46: 1441-1450Crossref PubMed Scopus (57) Google Scholar). A number of these minor pilins are thought to be integrated into the growing pilus (20Winther-Larsen H.C. Hegge F.T. Wolfgang M. Hayes S.F. van Putten J.P.M. Koomey M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15276-15281Crossref PubMed Scopus (98) Google Scholar). An adhesin protein (PilC) has been shown to bind to the host epithelial receptors CD 46 (21Rudel T. van Putten J.P.M. Gibbs C.P. Haas R. Meyer T.F. Nature. 1995; 373: 357-359Crossref PubMed Scopus (249) Google Scholar) and C4B (22Blom A.M. Rytkonen A. Vasquez P. Lindahl G. Dahlback B. Jonsson A.B. J. Immunol. 2001; 166: 6764-6770Crossref PubMed Scopus (49) Google Scholar). PilC also plays a role in the assembly process and a minor pilin, PilV, has been implicated in its functional presentation (20Winther-Larsen H.C. Hegge F.T. Wolfgang M. Hayes S.F. van Putten J.P.M. Koomey M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15276-15281Crossref PubMed Scopus (98) Google Scholar). Furthermore, type IV pilus retraction in Neisseria appears to be regulated by PilC (23Morand P. Bille E. Morelle S. Eugene E. Beretti J. Wolfgang M. Meyer T.F. Koomey M. Nassif X. EMBO J. 2004; 23: 2009-2017Crossref PubMed Scopus (93) Google Scholar). Other essential components for the biogenesis and expression of type IV pili include a cytoplasmic protein, PilF, and a polytopic inner membrane protein, PilG, of unknown functions (24Tønjum T. Freitag N.E. Namork E. Koomey M. Mol. Microbiol. 1995; 16: 451-464Crossref PubMed Scopus (126) Google Scholar, 25Darzins A. Russell M.A. Gene (Amst.). 1997; 192: 109-115Crossref PubMed Scopus (73) Google Scholar). PilF, PilT, and PilU contain a consensus ATP binding motif, are homologues of GspE (a member of the AAA chaperone/mechanico-enzyme family) and presumably drive pilus assembly/disassembly by ATP hydrolysis (26Herdendorf T.J. McCasli D.R. Forest K.T. J. Bacteriol. 2002; 184: 6465-6471Crossref PubMed Scopus (54) Google Scholar, 27Maier B. Potter L. So M. Seifert H.S. Sheetz M.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16012-16017Crossref PubMed Scopus (301) Google Scholar). The cytoplasmic protein PilT is dispensable for pilus assembly but essential for retraction (28Wolfgang M.H.-S.P. Hayes S.F. van Putten J. P.M. Koomey M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14973-14978Crossref PubMed Scopus (122) Google Scholar). Despite individual characterization of some of these components, their function and coordinate regulation in pilus assembly, extrusion, and retraction are poorly understood.The branch point between the assembly and retraction of pili through the outer membrane occurs at the PilQ oligomer. PilQ is an antigenically conserved, abundant outer membrane protein that is essential for meningococcal pilus expression (29Tønjum T. Caugant D.A. Dunham S.A. Koomey M. Mol. Microbiol. 1998; 29: 975-986Crossref PubMed Scopus (57) Google Scholar). It is a member of the general secretory pathway secretin superfamily, members of which translocate a variety of macromolecules across the outer membrane (16Pugsley A.P. Microbiol. Rev. 1993; 57: 50-108Crossref PubMed Google Scholar, 29Tønjum T. Caugant D.A. Dunham S.A. Koomey M. Mol. Microbiol. 1998; 29: 975-986Crossref PubMed Scopus (57) Google Scholar, 30Bitter W. Koster M. Latjinhouwers M. de Cock H. Tommassen J. Mol. Microbiol. 1998; 27: 209-219Crossref PubMed Scopus (191) Google Scholar). Double knock-out mutants of pilT and pilQ form long, membrane-covered pilus-like structures that fill the periplasm (31Wolfgang M. van Putten J.P.M. Hayes S.F. Dorward D. Koomey M. EMBO J. 2000; 19: 6408-6418Crossref PubMed Scopus (206) Google Scholar). PilQ subunits form a heat- and SDS-stable complex, which requires the PilP lipoprotein for functional oligomer assembly (32Drake S. Sandstedt S.A. Koomey M. Mol. Microbiol. 1997; 23: 657-668Crossref PubMed Scopus (118) Google Scholar). The N-terminal part of neisserial PilQ contains two to seven copies of unique octapeptides, termed small basic repeats (29Tønjum T. Caugant D.A. Dunham S.A. Koomey M. Mol. Microbiol. 1998; 29: 975-986Crossref PubMed Scopus (57) Google Scholar). The outer membrane protein Omp85 has also been shown to affect the assembly of the PilQ oligomer into the outer membrane (33Voulhoux R. Bos M.P. Geurtsen J. Mols M. Tommassen J. Science. 2003; 299: 262-265Crossref PubMed Scopus (575) Google Scholar). Images of isolated bacterial secretins have been recorded by transmission electron microscopy, and "donut-like" ring projections surrounding a central cavity have been observed (34Bitter W. Tommassen J. Trends Microbiol. 1999; 7: 4-6Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 35Thanassi D.G. J. Mol. Microbiol. Biotechnol. 2002; 4: 11-20PubMed Google Scholar), with dimensions that vary considerably among secretins and appear to be related to the specific translocated substrate. The three-dimensional (3-D) 1The abbreviations used are: 3-D, three-dimensional; 2-D, two-dimensional; TEM, transmission electron microscopy; BSA, bovine serum albumin; CTF, contrast transfer function; Ni-NTA, nickel-nitrilotriacetic acid.1The abbreviations used are: 3-D, three-dimensional; 2-D, two-dimensional; TEM, transmission electron microscopy; BSA, bovine serum albumin; CTF, contrast transfer function; Ni-NTA, nickel-nitrilotriacetic acid. structure of the PilQ oligomer from N. meningitidis has been determined using single particle averaging, initially on samples prepared in conventional negative stain (36Collins R.F. Ford R.C. Kitmitto A. Olsen R. Tonjum T. Derrick J.P. J. Bacteriol. 2003; 185: 2611-2617Crossref PubMed Scopus (71) Google Scholar) and, more recently, at the higher resolution of 12 Å using cryo-negative stain (37Collins R.F. Frye S.A. Kitmitto A. Ford R.C. Tønjum T. Derrick J.P. J. Biol. Chem. 2004; 279: 39750-39756Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The structure is dominated by a large central cavity, which is closed at both ends by "plug" and "cap" features, and four "arm-like" features, which form the sides. The PilQ oligomer exhibits 4-fold rotational symmetry with 12-fold quasi-symmetry (37Collins R.F. Frye S.A. Kitmitto A. Ford R.C. Tønjum T. Derrick J.P. J. Biol. Chem. 2004; 279: 39750-39756Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). It is hard to rationalize this unusual structure as a passive pore within the outer membrane, acting as a conduit for a pilus fiber already assembled in the periplasm. We demonstrate here that type IV pili, purified from meningococci, spontaneously interact with the meningococcal PilQ oligomer in a highly specific fashion. Substantial conformational changes occur in the cap and arm portions of the PilQ oligomer upon association with type IV pili. These findings suggest that PilQ does not function solely as a passive pore for passage of pili through the outer membrane but is capable of a dynamic response upon interaction with the secreted substrate.MATERIALS AND METHODSBacterial Strains and Constructs—Meningococcal strain M1080 was grown overnight on 5% blood agar in an atmosphere containing 5% CO2 before harvesting. The fragments encoding the full-length, N-terminal and central portions of the PilQ gene were cloned into the vector pQE-30 (Qiagen, Germany), and the C-terminal portion of the pilQ gene was cloned into vector pET28-b(+) (Novagen). Both vectors encode a polyhistidine tag. All proteins were overexpressed in Escherichia coli ER2566 (New England Biolabs) (see Table I). The recombinant PilQ proteins were expressed in E. coli as inclusion bodies and were subsequently solubilized using 8 m urea in phosphate-buffered saline. The recombinant proteins were affinity-purified using Ni-NTA-agarose (Qiagen, Germany) under denaturing conditions in 8 m urea. After immediate buffer exchange on a PD10 column (Amersham Biosciences) into 50 mm NaH2PO4 (pH 7.5), the proteins were purified by anion exchange chromatography using a Resource Q column (Amersham Biosciences) before dialysis against 50 mm NaH2PO4 (pH 7.5). The recombinant OpaD and HmbR proteins from N. meningitidis strain MC58 were expressed in E. coli as inclusion bodies, solubilized with 6 m guanidine HCl, refolded by rapid dilution, and purified by ion exchange and size exclusion chromatography. Recombinant OpcA was obtained as described previously (38Prince S.M. Feron C. Janssens D. Lobet Y. Achtman M. Kusecek B. Bullough P.A. Derrick J.P. Acta Crystallogr. 2001; D57: 1164-1166Google Scholar). The plasmid pMF121 was generously provided by Matthias Frosch (39Frosch M. Schultz E. Glenncalvo E. Meyer T.F. Mol. Microbiol. 1990; 4: 1215-1218Crossref PubMed Scopus (43) Google Scholar) and used to generate a capsule negative mutant of N. meningitidis strain M1080 expressing truncated lipooligosaccharide. The pilE-negative Neisseria gonorrhoeae N400 was generously provided by Mike Koomey (31Wolfgang M. van Putten J.P.M. Hayes S.F. Dorward D. Koomey M. EMBO J. 2000; 19: 6408-6418Crossref PubMed Scopus (206) Google Scholar). Genomic DNA from this strain was used to generate a pilE-negative N. meningitidis M1080 strain.Table IProperties of recombinant PilQ and native PilE proteins used in this studyGene productAmino acid position in M1080 PilQLengthMasspIresidueskDaFull-lengthaRecombinant PilQ constructs were prepared using the pilQ gene from N. meningitidis strain M1080 (accession number AJ564200).25-76174980.69.34N-terminal25-35436338.79.77C-terminal352-76142045.58.32Central region218-47827630.68.98PilENAbNA, not applicable.16217.25.66a Recombinant PilQ constructs were prepared using the pilQ gene from N. meningitidis strain M1080 (accession number AJ564200).b NA, not applicable. Open table in a new tab Purification of Native and Histidine Tag PilQ Oligomer—The native PilQ oligomer was isolated from meningococci using a purification procedure described previously, employing the detergent Zwittergent 3–10 (36Collins R.F. Ford R.C. Kitmitto A. Olsen R. Tonjum T. Derrick J.P. J. Bacteriol. 2003; 185: 2611-2617Crossref PubMed Scopus (71) Google Scholar). For the generation of PilQ histidine-tagged in the N-terminal small basic repeat region, splicing by overlapping extension PCR and non-selective transformation of the PCR product into N. meningitidis strain M1080 were employed. The native histidine tag PilQ oligomer was isolated from meningococcal outer membranes as previously described (36Collins R.F. Ford R.C. Kitmitto A. Olsen R. Tonjum T. Derrick J.P. J. Bacteriol. 2003; 185: 2611-2617Crossref PubMed Scopus (71) Google Scholar).Purification of Meningococcal Type IV Pilus Fibers—Type IV pili were purified from the meningococcal cell surface using ammonium sulfate precipitation of a shearing fraction (40Brinton C.C. Bryan J. Dillon J.-A. Guerina N. Jacobson L.J. Labik A. Lee S. Levine A. Lim S. McMichael J. Polen S. Rogers K. To A. C.-C. To S. C.M. Brooks G.E. Gotschlich E.C. Homes K.H. Sawyer W.D. Young F.E. Immunobiology of Neisseria gonorrhoeae. American Society for Microbiology Press, Washington, D. C.1978: 155-178Google Scholar). Meningococcal cells derived from four heavily streaked Petri dishes were vortexed for 1 min in 10 ml of 0.15 m ethanolamine buffer (pH 10.5), and cellular debris was removed by centrifugation at 14,000 × g for 15 min. Pilus fibers were precipitated at room temperature for 30 min by addition of onetenth volume of ammonium sulfate-saturated 0.15 m ethanolamine buffer and collected by centrifugation at 14,000 × g for 15 min. Pili were subsequently washed twice with 50 mm Tris-buffered saline.SDS-PAGE and Far-Western Assay—1–2 μg of the purified recombinant PilQ proteins, purified native type IV pilus fibers, neisserial outer membrane proteins (OpaD, OpcA, and HmbR), and BSA were separated by SDS-PAGE, and the proteins were transferred onto a Hybond-C extra nitrocellulose blotting membrane (Amersham Biosciences) in blotting buffer (25 mm Tris-HCl, 190 mm glycine, 20% methanol, pH 8.9). For the far-Western analysis, the membrane was briefly washed twice with cold renaturing buffer (10 mm Tris-HCl, 100 mm NaCl, 0.5% BSA, 0.25% gelatin, 0.2% Triton X-100, 5 mm 2-mercaptoethanol, pH 7.5), and the proteins were renatured by overnight incubation at 4 °C in the same buffer. For the detection of type IV pilus fiber binding to PilQ fragments, the membrane was incubated for 3 h with purified pilus fibers in renaturing buffer and washed in Tris-buffered saline (pH 7.5) (41Fouassier L. Yun C.C. Fitz J.G. Doctor R.B. J. Biol. Chem. 2000; 275: 25039-25045Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The pilus fiber-overlaid membrane was then exposed to anti-PilE polyclonal rabbit antiserum at a dilution of 1:1000. For immunoblotting of purified protein, anti-PilQ and type IV pilus-specific rabbit polyclonal antisera were used at dilutions of 1:2000 and 1:1000, respectively. Procedures for SDS-PAGE, immunoblotting, and antisera production have been described previously (24Tønjum T. Freitag N.E. Namork E. Koomey M. Mol. Microbiol. 1995; 16: 451-464Crossref PubMed Scopus (126) Google Scholar, 29Tønjum T. Caugant D.A. Dunham S.A. Koomey M. Mol. Microbiol. 1998; 29: 975-986Crossref PubMed Scopus (57) Google Scholar). For the detection of PilQ fragments binding to PilE from type IV pilus fibers, the membrane was incubated for 3 h with purified PilQ fragments and exposed to anti-PilQ polyclonal rabbit antiserum at a dilution of 1:1000.Density Gradient Centrifugation—Sucrose density gradients were performed as described by Pugsley and Possot (42Pugsley A.P. Possot O. Mol. Microbiol. 1993; 10: 665-674Crossref PubMed Scopus (49) Google Scholar). Gradients with a final volume of 12 ml were prepared with a Hoefer SG 50 gradient maker by using sucrose concentrations of 60 and 28% (w/v), respectively, in Tris/EDTA buffer (20 mm Tris-HCl, 2 mm EDTA, pH 7.5). 20 μg of the PilQ oligomer, purified as described above, with or without type IV pilus fibers or control proteins (purified OpaD or BSA), was mixed in Tris/EDTA buffer and incubated on ice for 30 min in 200-μl final volume. The samples were applied to the top of the gradients and centrifuged for 18 h at 4 °C and 245,000 × g. Fractions were collected from the bottom of the gradients and subjected to immunoblotting using rabbit anti-PilQ and anti-type IV pilus antisera. Parts of these fractions were also subjected to enzyme-linked immunosorbent assay detection using anti-PilQ and anti-PilE antisera. Anti-rabbit IgG, coupled with alkaline phosphatase, was employed as the secondary antibody and detected using para-nitrophenylphosphate as a substrate. The enzyme-linked immunosorbent assay reactivity was measured from absorption at 405 nm in a Wallac Victor2 plate reader (PerkinElmer Life Sciences). The sucrose concentration was determined from the refractive index (Rf) of corresponding gradient fractions measured with a refractometer from Precision Instruments (Bellingham and Stanley, London, UK).Formation of PilQ-Type IV Pilus Fiber Complex and Sample Preparation for TEM—5-μl samples of PilQ oligomer (100 μg/ml in 10 mm Tris-HCl, pH 7.5, 10 mm NaCl, 5 mm EDTA, and 0.1% (w/v) Zwittergent 3–10) were incubated with 5-μl samples of pilus fibers (100 μg/ml) for 30–60 min at room temperature. 10-μl aliquots of the PilQ oligomerpilus fiber incubation were adsorbed to freshly glow discharged carboncoated copper grids (No. 400) prior to negative staining.2-D Image Analysis of Type IV Pili—Non-overlapping lengths of type IV pili were selected into 200-Å2 boxes (43Mu X.Q. Egelman E.H. Bullitt E. J. Bacteriol. 2002; 184: 4868-4874Crossref PubMed Scopus (26) Google Scholar), and the resulting single particles were translationally and rotationally aligned using referencefree algorithms as implemented in SPIDER (44Frank J. Radermacher M. Penczek P. Zhu J. Ladjadj M. Leith A. J. Struct. Biol. 1996; 116: 190-199Crossref PubMed Scopus (1799) Google Scholar). Aligned particles (n = 2906) were grouped using correspondence analysis and hierarchical descendant clustering, and a 2-D projection map was generated from the major class average (n = 450). The helical pitch of fibers was determined by boxing out areas of long and straight pili from high contrast CTF-corrected micrographs recorded at 3- to 4-μm defocus. These data were then used to generate fast Fourier transforms in the 2-D crystallographic package CRISP (45Hovmoller S. Ultramicroscopy. 1992; 41: 121-135Crossref Scopus (347) Google Scholar). The resulting fast Fourier transforms were noisy because of the inherent low contrast of pilus fibers, but a characteristic helical cross-diffraction pattern and first order layer lines could be indexed (46Hahn E. Wild P. Schraner E.M. Bertschinger H.U. Haner M. Muller S.A. Aebi U. J. Struct. Biol. 2000; 132: 241-250Crossref PubMed Scopus (17) Google Scholar) allowing the range of helical pitch of the pili to be determined.Negative Staining—Samples were adsorbed to freshly glow discharged carbon-coated grids (No. 300–400) as previously described (64Holzenburg A. Shepherd F.H. Ford R.C. Micron. 1994; 25: 447-451Crossref Scopus (24) Google Scholar). Grids were placed carbon film downward on 10-μl droplets of complexed sample or PilQ (∼100 μg/ml) for 2–3 min. Grids were then immediately placed on a 10-μl droplet of 4% (w/v) uranyl acetate or a 10-μl droplet of 6% (w/v) ammonium molybdate (pH 6.8)/1% (w/v) trehalose for 30 s and then briefly blotted onto double-layered Whatman 50 filter paper.Ni-NTA-Gold Labeling—PilQ-type IV pilus complexes were prepared as described above. 20 μl of the PilQ-type IV pilus complex was incubated with 3.3 μl (1 nm) of Ni-NTA-nanogold (Nanoprobes) for 18 h at room temperature. Samples were then negatively stained with 6% (w/v) ammonium molybdate (pH 6.8)/1% (w/v) trehalose.Image Analysis and Volume Calculations of the PilQ-Type IV Pilus Complex Using Negative Stain and the Random Conical Tilt Method—Table II summarizes all additional information pertinent to TEM low dose data collection, micrograph scanning, and volume back projection used in this study. The PilQ oligomer was found to interact with pili of different fiber lengths, and, as with free pili, we observed that there was an intrinsic tendency for fibers to bend along their length. To calculate an accurate 3-D structure of the PilQ-type IV pilus complex, allowance was made for the bending observed along the pilus fiber length by treating the complex as a single particle, selected into a box 342 × 342 Å, with a determined center at the PilQ-pilus interface. The CTF of each micrograph was examined, and appropriate CTF phase corrections were applied to the entire particle data-set. Aligned particles were grouped using correspondence analysis and hierarchical ascendant classification, and a 2-D projection map was generated from the major class average. 3-D volumes of interacting PilQ-pilus complexes were subsequently calculated using the random conical reconstruction method (47Frank J. Three-dimensional electron microscopy of macromolecular assemblies. Plenum Press, San Diego1996Crossref Google Scholar, 48Frank J. Penczek P. Grassucci R. Srivastav S. J. Cell Biol. 1991; 115: 597-605Crossref PubMed Scopus (137) Google Scholar, 49Penczek P. Radermacher M. Frank J. Ultramicroscopy. 1992; 40: 33-53Crossref PubMed Scopus (399) Google Scholar, 50Ruprecht J. Nield J. Prog. Biophys. Mol. Biol. 2001; 75: 121-164Crossref PubMed Scopus (67) Google Scholar) executed using the software packages SPIDER and WEB. The same procedures have been described previously in detail for the 3-D structure calculation of the PilQ oligomer (36Collins R.F. Ford R.C. Kitmitto A. Olsen R. Tonjum T. Derrick J.P. J. Bacteriol. 2003; 185: 2611-2617Crossref PubMed Scopus (71) Google Scholar).Table IIElectron microscopy and image analysis information for PilQ-type IV pilus complexMicroscopePhilips Tecnai 10:TEM low dose modeOperating voltage100 kVMicrograph filmKodak SO-163Calibrated magnification×42,010ScannerUMAX Photolook 3000: 256 greyscaleScan increment15.9 μmSpecimen level (Å/pixel)3.8Tilt pair angles0° and 50-55°Micrograph pairs used27Defocus range-1.5 to -3.0 μmMallet projections used for back projection570 (Total = 607)Back projection reconstruction sphere radius114-133 ÅVolumes used2Euler angle relationship between volumes (ϕ, θ, and ψ)18.6°, 0.0°, and -18.7°Volume orientation correlation coefficient0.92Spatial resolution of merged volume (36Collins R.F. Ford R.C. Kitmitto A. Olsen R. Tonjum T. Derrick J.P. J. Bacteriol. 2003; 185: 2611-2617Crossref PubMed Scopus (71) Google Scholar, 47Frank J. Three-dimensional electron microscopy of macromolecular assemblies. Plenum Press, San Diego1996Crossref Google Scholar)26 Å Open table in a new tab Symmetry Analysis—Density map correlation coefficients were calculated by rotation of the unsymmetrized 3-D map about the long axis of the pilus fiber using MAPROT (51Stein P.E. Boodhoo A. Armstrong G.D. Cockle S.A. Klein M.H. Read R.J. Structure. 1994; 2: 45-57Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar), and the density map correlation coefficient was calculated using OVERLAPMAP (52Branden C.I. Jones T.A. Nature. 1990; 343: 687-689Crossref Scopus (253) Google Scholar), both from the CCP4 suite (53CCP4 Acta Crystallogr. 1994; D50: 760-763Google Scholar).RESULTSThe Meningococcal PilQ Monomer Interacts Directly and Specifically with Pili—PilQ from N. meningitidis is required for pilus biogenesis (29Tønjum T. Caugant D.A. Dunham S.A. Koomey M. Mol. Microbiol. 1998; 29: 975-986Crossref PubMed Scopus (57) Google Scholar) but has not been shown to form a direct interaction with its secreted substrate. We employed far-Western analysis to assess the association between purified type IV pili and recombinant fragments of PilQ, which covered the N-terminal, central, and C-terminal portions of the protein (Fig. 1A and Table I). The binding of three other neisserial outer membrane proteins to type IV pili were also examined as controls, specifically the adhesins OpcA and OpaD, and the heme receptor HmbR. For the far-Western blot analysis (Fig. 1B, gel a), the membrane was probed with pilus protein before detection with polyclonal anti-PilE antibody. A clear interaction of PilE with full-length PilQ was apparent (lane 2), and interactions with the N- and C-terminal fragments were also evident (lanes 3 and 5) (Fig. 1B, gel a). However, no PilE binding was found to the central portion of PilQ (lane 4) or to the control proteins OpcA, OpaD, HmbR, and bovine serum albumin (lanes 6–9). For the far-Western analysis, denatured PilQ fragments in the SDS-PAGE gel were blotted onto the supporting membrane and a "renaturation" step was introduced, which involved incubating the membrane overnight. When the experiment was repeated without this renaturation step the PilQ-pilus interaction was much weaker, suggesting that the conformation adopted by the PilQ proteins was less
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