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• W2121956307 abstract "Gram-negative pathogens commonly exhibit adhesive pili on their surfaces that mediate specific attachment to the host. A major class of pili is assembled via the chaperone/usher pathway. Here, the structural basis for pilus fiber assembly and secretion performed by the outer membrane assembly platform—the usher—is revealed by the crystal structure of the translocation domain of the P pilus usher PapC and single particle cryo-electron microscopy imaging of the FimD usher bound to a translocating type 1 pilus assembly intermediate. These structures provide molecular snapshots of a twinned-pore translocation machinery in action. Unexpectedly, only one pore is used for secretion, while both usher protomers are used for chaperone-subunit complex recruitment. The translocating pore itself comprises 24 β strands and is occluded by a folded plug domain, likely gated by a conformationally constrained β-hairpin. These structures capture the secretion of a virulence factor across the outer membrane of Gram-negative bacteria. Gram-negative pathogens commonly exhibit adhesive pili on their surfaces that mediate specific attachment to the host. A major class of pili is assembled via the chaperone/usher pathway. Here, the structural basis for pilus fiber assembly and secretion performed by the outer membrane assembly platform—the usher—is revealed by the crystal structure of the translocation domain of the P pilus usher PapC and single particle cryo-electron microscopy imaging of the FimD usher bound to a translocating type 1 pilus assembly intermediate. These structures provide molecular snapshots of a twinned-pore translocation machinery in action. Unexpectedly, only one pore is used for secretion, while both usher protomers are used for chaperone-subunit complex recruitment. The translocating pore itself comprises 24 β strands and is occluded by a folded plug domain, likely gated by a conformationally constrained β-hairpin. These structures capture the secretion of a virulence factor across the outer membrane of Gram-negative bacteria. The chaperone/usher (CU) pathway is responsible for the assembly of a major class of adhesive fibers on the outer membrane (OM) of a diverse group of Gram-negative bacteria, including important human and animal pathogens (Sauer et al., 2004Sauer F.G. Remaut H. Hultgren S.J. Waksman G. Fiber assembly by the chaperone-usher pathway.Biochim. Biophys. Acta. 2004; 1694: 259-267Crossref PubMed Scopus (159) Google Scholar). These filamentous extracellular organelles, called pili or fimbriae, form a class of virulence factors responsible for specific host recognition and attachment, invasion, and biofilm formation. CU pilus biogenesis encompasses the ordered noncovalent polymerization of periplasmic chaperone-bound pilus subunits at an OM assembly platform termed the usher (Figure 1) (Sauer et al., 2004Sauer F.G. Remaut H. Hultgren S.J. Waksman G. Fiber assembly by the chaperone-usher pathway.Biochim. Biophys. Acta. 2004; 1694: 259-267Crossref PubMed Scopus (159) Google Scholar). The usher catalyzes polymerization and facilitates the translocation of folded subunits across the bacterial OM in the absence of accessory proteins and in a process independent of cellular ATP or the proton motive force (Jacob-Dubuisson et al., 1994Jacob-Dubuisson F. Striker R. Hultgren S.J. Chaperone-assisted self-assembly of pili independent of cellular energy.J. Biol. Chem. 1994; 269: 12447-12455PubMed Google Scholar). Type 1 and P pili have served as model systems for the elucidation of the CU biosynthetic pathway. All uropathogenic Escherichia coli produce type 1 pili that are responsible for attachment, invasion, and establishment of biofilms in the bladder (Wright et al., 2007Wright K.J. Seed P.C. Hultgren S.J. Development of intracellular bacterial communities of uropathogenic Escherichia coli depends on type 1 pili.Cell. Microbiol. 2007; 9: 2230-2241Crossref PubMed Scopus (224) Google Scholar, Justice et al., 2006Justice S.S. Hunstad D.A. Hultgren S.J. Filamentation by Escherichia coli subverts innate immunity during urinary tract infection.Proc. Natl. Acad. Sci. USA. 2006; 103: 19884-19889Crossref PubMed Scopus (208) Google Scholar). P pili are produced by pyelonephritic strains of E. coli and are required for colonization of the kidney (Roberts et al., 1994Roberts J.A. Marklund B.I. Ilver D. Haslam D. Kaack M.B. Baskin G. Louis M. Mollby R. Winberg J. Normark S. The Gal(alpha 1–4)Gal-specific tip adhesin of Escherichia coli P-fimbriae is needed for pyelonephritis to occur in the normal urinary tract.Proc. Natl. Acad. Sci. USA. 1994; 91: 11889-11893Crossref PubMed Scopus (303) Google Scholar). P and type 1 pili are complex extended fibers composed of two distinct subassemblies: (1) a distal tip fibrillum measuring 2 nm in diameter that is connected to a (2) rigid helical rod of 6.8 nm diameter (Jones et al., 1995Jones C.H. Pinkner J.S. Roth R. Heuser J. Nicholes A.V. Abraham S.N. Hultgren S.J. FimH adhesin of type 1 pili is assembled into a fibrillar tip structure in the Enterobacteriaceae.Proc. Natl. Acad. Sci. USA. 1995; 92: 2081-2085Crossref PubMed Scopus (331) Google Scholar, Kuehn et al., 1992Kuehn M.J. Heuser J. Normark S. Hultgren S.J. P pili in uropathogenic E. coli are composite fibres with distinct fibrillar adhesive tips.Nature. 1992; 356: 252-255Crossref PubMed Scopus (254) Google Scholar). At their distal end, type 1 and P pilus tips contain the FimH and PapG adhesin, respectively, targeting the bacteria to mannose-containing receptors on the bladder epithelium (FimH) or to Gal-α(1-4)-Gal-containing glycolipid receptors on the kidney epithelium (PapG) (Choudhury et al., 1999Choudhury D. Thompson A. Stojanoff V. Langermann S. Pinkner J. Hultgren S.J. Knight S.D. X-ray structure of the FimC-FimH chaperone-adhesin complex from uropathogenic Escherichia coli.Science. 1999; 285: 1061-1066Crossref PubMed Scopus (500) Google Scholar, Dodson et al., 2001Dodson K.W. Pinkner J.S. Rose T. Magnusson G. Hultgren S.J. Waksman G. Structural basis of the interaction of the pyelonephritic E. coli adhesin to its human kidney receptor.Cell. 2001; 105: 733-743Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). In type 1 pilus tips, FimH is followed by one copy each of the FimG and FimF subunits (Figure 1A, right) (Hahn et al., 2002Hahn E. Wild P. Hermanns U. Sebbel P. Glockshuber R. Haner M. Taschner N. Burkhard P. Aebi U. Muller S.A. Exploring the 3D molecular architecture of Escherichia coli type 1 pili.J. Mol. Biol. 2002; 323: 845-857Crossref PubMed Scopus (180) Google Scholar, Jones et al., 1995Jones C.H. Pinkner J.S. Roth R. Heuser J. Nicholes A.V. Abraham S.N. Hultgren S.J. FimH adhesin of type 1 pili is assembled into a fibrillar tip structure in the Enterobacteriaceae.Proc. Natl. Acad. Sci. USA. 1995; 92: 2081-2085Crossref PubMed Scopus (331) Google Scholar, Saulino et al., 2000Saulino E.T. Bullitt E. Hultgren S.J. Snapshots of usher-mediated protein secretion and ordered pilus assembly.Proc. Natl. Acad. Sci. USA. 2000; 97: 9240-9245Crossref PubMed Scopus (75) Google Scholar). In P pili, PapG is attached via the PapF adaptor subunit to a tip fibrillum formed by a PapE homopolymer, itself attached to the pilus rod via the PapK adapter subunit (Figure 1A, left) (Kuehn et al., 1992Kuehn M.J. Heuser J. Normark S. Hultgren S.J. P pili in uropathogenic E. coli are composite fibres with distinct fibrillar adhesive tips.Nature. 1992; 356: 252-255Crossref PubMed Scopus (254) Google Scholar). The pilus rod consists of a homopolymer of over 1000 copies of the FimA or PapA subunits in type 1 and P pili, respectively (Hahn et al., 2002Hahn E. Wild P. Hermanns U. Sebbel P. Glockshuber R. Haner M. Taschner N. Burkhard P. Aebi U. Muller S.A. Exploring the 3D molecular architecture of Escherichia coli type 1 pili.J. Mol. Biol. 2002; 323: 845-857Crossref PubMed Scopus (180) Google Scholar, Kuehn et al., 1992Kuehn M.J. Heuser J. Normark S. Hultgren S.J. P pili in uropathogenic E. coli are composite fibres with distinct fibrillar adhesive tips.Nature. 1992; 356: 252-255Crossref PubMed Scopus (254) Google Scholar). Fiber assembly requires a periplasmic chaperone (FimC and PapD for type 1 and P pili, respectively) that interacts with each pilus subunit, providing a platform onto which the subunits fold (Barnhart et al., 2000Barnhart M.M. Pinkner J.S. Soto G.E. Sauer F.G. Langermann S. Waksman G. Frieden C. Hultgren S.J. PapD-like chaperones provide the missing information for folding of pilin proteins.Proc. Natl. Acad. Sci. USA. 2000; 97: 7709-7714Crossref PubMed Scopus (145) Google Scholar, Bann et al., 2004Bann J.G. Pinkner J.S. Frieden C. Hultgren S.J. Catalysis of protein folding by chaperones in pathogenic bacteria.Proc. Natl. Acad. Sci. USA. 2004; 101: 17389-17393Crossref PubMed Scopus (37) Google Scholar, Vetsch et al., 2004Vetsch M. Pourger C. Spirig T. Grauschopf U. Weber-Ban E.U. Glockshuber R. Pilus chaperones represent a new type of protein-folding catalyst.Nature. 2004; 431: 329-333Crossref PubMed Scopus (84) Google Scholar). Previous crystal structures of chaperone-subunit and subunit-subunit complexes have established a unifying mechanism for subunit stabilization and polymerization by the CU pathway (Choudhury et al., 1999Choudhury D. Thompson A. Stojanoff V. Langermann S. Pinkner J. Hultgren S.J. Knight S.D. X-ray structure of the FimC-FimH chaperone-adhesin complex from uropathogenic Escherichia coli.Science. 1999; 285: 1061-1066Crossref PubMed Scopus (500) Google Scholar, Sauer et al., 1999Sauer F.G. Futterer K. Pinkner J.S. Dodson K.W. Hultgren S.J. Waksman G. Structural basis of chaperone function and pilus biogenesis.Science. 1999; 285: 1058-1061Crossref PubMed Scopus (322) Google Scholar, Sauer et al., 2002Sauer F.G. Pinkner J.S. Waksman G. Hultgren S.J. Chaperone priming of pilus subunits facilitates a topological transition that drives fiber formation.Cell. 2002; 111: 543-551Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, Zavialov et al., 2003Zavialov A.V. Berglund J. Pudney A.F. Fooks L.J. Ibrahim T.M. MacIntyre S. Knight S.D. Structure and biogenesis of the capsular F1 antigen from Yersinia pestis: preserved folding energy drives fiber formation.Cell. 2003; 113: 587-596Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Pilus subunits share a common noncanonical immunoglobulin (Ig)-like fold characterized by the absence of its C-terminal β strand (Figure 1B). After translocation of the newly synthesized pilus subunit across the inner membrane into the periplasm, the cognate periplasmic chaperone “donates” part of a β strand for the intermolecular complementation of the incomplete Ig fold of the pilus subunit, a process termed donor-strand complementation (Figure 1B, topology diagram at bottom) (Choudhury et al., 1999Choudhury D. Thompson A. Stojanoff V. Langermann S. Pinkner J. Hultgren S.J. Knight S.D. X-ray structure of the FimC-FimH chaperone-adhesin complex from uropathogenic Escherichia coli.Science. 1999; 285: 1061-1066Crossref PubMed Scopus (500) Google Scholar, Sauer et al., 1999Sauer F.G. Futterer K. Pinkner J.S. Dodson K.W. Hultgren S.J. Waksman G. Structural basis of chaperone function and pilus biogenesis.Science. 1999; 285: 1058-1061Crossref PubMed Scopus (322) Google Scholar). Polymerization of the subunits at the bacterial OM occurs through a similar fold complementation mechanism, which involves the donation of the N-terminal extension (Nte) peptide of an incoming pilus subunit to complement the incomplete Ig fold of the previously assembled subunit in the growing pilus fiber (Figure 1B, topology diagram at top) (Sauer et al., 2002Sauer F.G. Pinkner J.S. Waksman G. Hultgren S.J. Chaperone priming of pilus subunits facilitates a topological transition that drives fiber formation.Cell. 2002; 111: 543-551Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, Zavialov et al., 2003Zavialov A.V. Berglund J. Pudney A.F. Fooks L.J. Ibrahim T.M. MacIntyre S. Knight S.D. Structure and biogenesis of the capsular F1 antigen from Yersinia pestis: preserved folding energy drives fiber formation.Cell. 2003; 113: 587-596Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). This process of donor-strand exchange, in which the chaperone donor strand is exchanged for another subunit's Nte, occurs at the OM usher (FimD and PapC for type 1 and P pilus subunits, respectively) (Figure 1B). In sharp contrast to the wealth of information available on chaperone-subunit and subunit-subunit interactions, little is known of the structure or mechanism of action of the usher (Nishiyama et al., 2005Nishiyama M. Horst R. Eidam O. Herrmann T. Ignatov O. Vetsch M. Bettendorff P. Jelesarov I. Grutter M.G. Wuthrich K. et al.Structural basis of chaperone-subunit complex recognition by the type 1 pilus assembly platform FimD.EMBO J. 2005; 24: 2075-2086Crossref PubMed Scopus (99) Google Scholar). OM ushers selectively recruit chaperone-bound subunits to the OM, facilitate their ordered polymerization, and translocate the nascent fiber across the OM. They comprise four predicted domains: an N-terminal periplasmic domain (∼125 residues), a central β-barrel domain (predicted residues ∼135 to ∼640) interrupted by a middle domain (predicted residues ∼220 to ∼325), and a C-terminal periplasmic domain (∼170 residues) (Figure 1C) (Capitani et al., 2006Capitani G. Eidam O. Grutter M.G. Evidence for a novel domain of bacterial outer membrane ushers.Proteins. 2006; 65: 816-823Crossref PubMed Scopus (20) Google Scholar, Ng et al., 2004Ng T.W. Akman L. Osisami M. Thanassi D.G. The usher N terminus is the initial targeting site for chaperone-subunit complexes and participates in subsequent pilus biogenesis events.J. Bacteriol. 2004; 186: 5321-5331Crossref PubMed Scopus (69) Google Scholar, Nishiyama et al., 2003Nishiyama M. Vetsch M. Puorger C. Jelesarov I. Glockshuber R. Identification and characterization of the chaperone-subunit complex-binding domain from the type 1 pilus assembly platform FimD.J. Mol. Biol. 2003; 330: 513-525Crossref PubMed Scopus (63) Google Scholar, Saulino et al., 1998Saulino E.T. Thanassi D.G. Pinkner J.S. Hultgren S.J. Lombardo M.J. Roth R. Heuser J. Ramifications of kinetic partitioning on usher-mediated pilus biogenesis.EMBO J. 1998; 17: 2177-2185Crossref PubMed Scopus (127) Google Scholar, Thanassi et al., 2002Thanassi D.G. Stathopoulos C. Dodson K. Geiger D. Hultgren S.J. Bacterial outer membrane ushers contain distinct targeting and assembly domains for pilus biogenesis.J. Bacteriol. 2002; 184: 6260-6269Crossref PubMed Scopus (67) Google Scholar). Chaperone-subunit recruitment occurs via the usher's N-terminal domain (Ng et al., 2004Ng T.W. Akman L. Osisami M. Thanassi D.G. The usher N terminus is the initial targeting site for chaperone-subunit complexes and participates in subsequent pilus biogenesis events.J. Bacteriol. 2004; 186: 5321-5331Crossref PubMed Scopus (69) Google Scholar, Nishiyama et al., 2005Nishiyama M. Horst R. Eidam O. Herrmann T. Ignatov O. Vetsch M. Bettendorff P. Jelesarov I. Grutter M.G. Wuthrich K. et al.Structural basis of chaperone-subunit complex recognition by the type 1 pilus assembly platform FimD.EMBO J. 2005; 24: 2075-2086Crossref PubMed Scopus (99) Google Scholar). This domain discriminately recognizes different chaperone-subunit complexes by directly binding to the N-terminal domain of the periplasmic chaperone and part of the chaperone-bound subunit (Nishiyama et al., 2005Nishiyama M. Horst R. Eidam O. Herrmann T. Ignatov O. Vetsch M. Bettendorff P. Jelesarov I. Grutter M.G. Wuthrich K. et al.Structural basis of chaperone-subunit complex recognition by the type 1 pilus assembly platform FimD.EMBO J. 2005; 24: 2075-2086Crossref PubMed Scopus (99) Google Scholar). Subunit polymerization has been shown to be a concerted process, with a new subunit's Nte binding at an initiation site (called the P5 pocket) on chaperone-bound subunits and gradually displacing the chaperone donor strand (Remaut et al., 2006Remaut H. Rose R.J. Hannan T.J. Hultgren S.J. Radford S.E. Ashcroft A.E. Waksman G. Donor strand exchange in chaperone-assisted pilus assembly proceeds through a concerted beta strand displacement mechanism.Mol. Cell. 2006; 22: 831-842Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, Vetsch et al., 2006Vetsch M. Erilov D. Moliere N. Nishiyama M. Ignatov O. Glockshuber R. Mechanism of fibre assembly through the chaperone-usher pathway.EMBO Rep. 2006; 7: 734-738Crossref PubMed Scopus (48) Google Scholar). How the usher facilitates this process or what parts of the usher are involved is unknown. Cryo-electron microscopy (cryo-EM) images of the PapC usher reconstituted in E. coli lipids reveal that ushers form dimeric channels in the OM, compatible in size with the secretion of folded subunits (Li et al., 2004Li H. Qian L. Chen Z. Thibault D. Liu G. Liu T. Thanassi D.G. The outer membrane usher forms a twin-pore secretion complex.J. Mol. Biol. 2004; 344: 1397-1407Crossref PubMed Scopus (58) Google Scholar). Complementation studies in PapC loss-of-function mutants confirm OM ushers to be functional as dimers (So and Thanassi, 2006So S.S. Thanassi D.G. Analysis of the requirements for pilus biogenesis at the outer membrane usher and the function of the usher C-terminus.Mol. Microbiol. 2006; 60: 364-375Crossref PubMed Scopus (38) Google Scholar). The predicted central β-barrel domain is likely to form the translocation channel, but the roles of the predicted middle domain or the periplasmically located C-terminal domain in usher function are unclear. The latter is believed to participate in activating the usher for translocation in a process that is initiated by the chaperone-adhesin complex (So and Thanassi, 2006So S.S. Thanassi D.G. Analysis of the requirements for pilus biogenesis at the outer membrane usher and the function of the usher C-terminus.Mol. Microbiol. 2006; 60: 364-375Crossref PubMed Scopus (38) Google Scholar). Understanding the complex multistep processing of CU pilus subunits into a functional, membrane-anchored filamentous organelle has been hampered by the lack of high-resolution structural information on the usher and complexes thereof. Here, we present the crystal structure of the PapC translocation channel and the cryo-EM structure of the closely related FimD usher bound to the translocating FimC:F:G:H quaternary tip complex. These structures capture a twinned-pores usher transporter in action and provide an integrated model of the CU-mediated subunit assembly mechanism. To gain a structural understanding of pilus biogenesis at the OM, we solved the structure of the translocation channel of the PapC usher. The presence of the flexible periplasmic domains in the mature 809-residue PapC prevented growth of crystals of sufficient quality for structure determination of the full-length protein. Through limited proteolysis, we identified a stable 55 kDa fragment (residues 130–640) corresponding to the predicted OM translocation domain and the recently identified middle domain (Capitani et al., 2006Capitani G. Eidam O. Grutter M.G. Evidence for a novel domain of bacterial outer membrane ushers.Proteins. 2006; 65: 816-823Crossref PubMed Scopus (20) Google Scholar). As expected, PapC130–640 localized to the OM and the integrity of the β-barrel domain was confirmed by a heat-modifiable mobility shift on SDS-PAGE, typical of folded OM β-barrel proteins (results not shown). Two crystal forms were obtained. The structure of PapC130–640 was determined at 3.4 Å resolution using the multiwavelength anomalous dispersion phasing method on F222 crystals and refined against 3.2 Å resolution data from crystals in the C2 space group to a final R and free R factor of 25.9% and 29.6%, respectively (see Experimental Procedures, Figure S1, and Table S1). It encompasses the full translocation pore consisting of a kidney-shaped, 24-stranded β-barrel (residues 146–635), ∼45 Å in height and with outer and inner dimensions of 65 Å by 45 Å and 45 Å by 25 Å, respectively (Figures 2A, 2B, and 2C). OM ushers therefore correspond to the largest single-protein β-pores observed to date (previously determined β-pore structures contain 22 β strands at most). The β-barrel closes in an end-to-end fashion and positions the N and C termini on the periplasmic side of the membrane. The N- and C-terminal globular domains will thus be juxtaposed and reside in the periplasm, consistent with their role in chaperone-subunit recruitment and adhesin-induced pore activation (Ng et al., 2004Ng T.W. Akman L. Osisami M. Thanassi D.G. The usher N terminus is the initial targeting site for chaperone-subunit complexes and participates in subsequent pilus biogenesis events.J. Bacteriol. 2004; 186: 5321-5331Crossref PubMed Scopus (69) Google Scholar, Nishiyama et al., 2003Nishiyama M. Vetsch M. Puorger C. Jelesarov I. Glockshuber R. Identification and characterization of the chaperone-subunit complex-binding domain from the type 1 pilus assembly platform FimD.J. Mol. Biol. 2003; 330: 513-525Crossref PubMed Scopus (63) Google Scholar, Saulino et al., 1998Saulino E.T. Thanassi D.G. Pinkner J.S. Hultgren S.J. Lombardo M.J. Roth R. Heuser J. Ramifications of kinetic partitioning on usher-mediated pilus biogenesis.EMBO J. 1998; 17: 2177-2185Crossref PubMed Scopus (127) Google Scholar, Thanassi et al., 2002Thanassi D.G. Stathopoulos C. Dodson K. Geiger D. Hultgren S.J. Bacterial outer membrane ushers contain distinct targeting and assembly domains for pilus biogenesis.J. Bacteriol. 2002; 184: 6260-6269Crossref PubMed Scopus (67) Google Scholar). The predicted middle domain (residues 257–332) is formed by a long sequence between strands β6 and β7 and consists of a six-stranded, β sandwich fold (strands βA–βF; Figure 2C). The domain is positioned laterally inside the β-barrel pore (Figure 2B). As a result, the middle domain, hereafter referred to as the plug domain, completely occludes the luminal volume of the translocation pore, preventing passage of solutes or periplasmic proteins across the channel in its nonactivated form (Figures 2A and 2B). Globular plug or cork domains have also been observed in bacterial siderophore transporters (Ferguson et al., 1998Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide.Science. 1998; 282: 2215-2220Crossref PubMed Scopus (648) Google Scholar). Unlike these, however, the PapC plug domain is inserted into the loop connecting two β strands (strands 6 and 7) rather than positioned at the N-terminal extremity of the β-barrel. The plug domain is held in place by a β-hairpin (strands β5 and β6, hereafter referred to as the β5-6 hairpin, Figures 2A, 2B, and 2C) that folds in from the barrel wall into the channel lumen. The inward curvature of the β5-6 hairpin creates a large gap in the side of the β-barrel extending well into the part submerged in the OM bilayer (Figure 2B), a feature unprecedented in previously known OM β-barrel structures. The point where the β5-6 hairpin breaks out of the barrel coincides with a bulge (Ser209-Arg210) in the adjacent strand β4 (Figure 3A). This bulge disrupts the local secondary structure interaction with the hairpin strand β5 and, in combination with a charged residue (Glu228) in β5 that is pointing into the nonpolar bilayer, pushes the β5-6 hairpin into the barrel lumen, leaving the unusual gap in the lining of the transmembrane barrel (Figure 2B). In addition, the luminal part of the β5-6 hairpin is capped from the extracellular side by the only helix in the structure, the α1 helix (residues 448–465). Inside the β-barrel, the β5-6 hairpin interacts with the inner surface of the channel and helix α1 through a patch of hydrophobic interactions formed by Ile231, Phe236 on the hairpin, Trp239, Phe357, and Val377 on the barrel wall, and Met451 on the helix (Figure 3B). In addition, the β5-6 hairpin forms two electrostatic interaction networks that bridge the plug domain with the channel wall (Glu361-Lys339-Asp234-Arg303) and α1 helix (Arg237-Glu467-Arg305-Asp323) and help position the plug domain laterally inside the translocation channel (Figure 3B). Given the conservation in sequence and predicted topologies in usher proteins, the translocator pore structure of the PapC usher can be considered a prototype for all bacterial ushers (sequence alignment; Figure S2).Figure 3Stereo View of the β-Hairpin InteractionsShow full caption(A) Close-up of the site of inward orientation of the β5-6 hairpin. Residues in the β4 bulge and hairpin involved in the hairpin curvature are shown in stick representation. Color scheme is according to Figure 1.(B) Close-up of the β5-6 hairpin interactions with the plug domain, helix α1, and the channel wall. Residues involved in the interaction are shown in stick representation.(C) Charge interactions between the plug domain and the β-barrel, viewed from the periplasmic side of the membrane. Side chains of neighboring residues are labeled by residue number.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Close-up of the site of inward orientation of the β5-6 hairpin. Residues in the β4 bulge and hairpin involved in the hairpin curvature are shown in stick representation. Color scheme is according to Figure 1. (B) Close-up of the β5-6 hairpin interactions with the plug domain, helix α1, and the channel wall. Residues involved in the interaction are shown in stick representation. (C) Charge interactions between the plug domain and the β-barrel, viewed from the periplasmic side of the membrane. Side chains of neighboring residues are labeled by residue number. Available structural and biochemical evidence demonstrates OM ushers function as dimers organized in twinned pores (Li et al., 2004Li H. Qian L. Chen Z. Thibault D. Liu G. Liu T. Thanassi D.G. The outer membrane usher forms a twin-pore secretion complex.J. Mol. Biol. 2004; 344: 1397-1407Crossref PubMed Scopus (58) Google Scholar, So and Thanassi, 2006So S.S. Thanassi D.G. Analysis of the requirements for pilus biogenesis at the outer membrane usher and the function of the usher C-terminus.Mol. Microbiol. 2006; 60: 364-375Crossref PubMed Scopus (38) Google Scholar). Both the C2 and F222 crystal forms of PapC130–640 obtained in this study contain similar dimers formed by a crystallographic two-fold axis (Figure 2D). These dimers closely correspond in size and conformation to the twinned pores observed by negative stain EM of full-length PapC (Figure S3) and by cryo-EM imaging on single particles of detergent-solubilized full-length PapC and 2D crystals of PapC reconstituted in E. coli lipids (Li et al., 2004Li H. Qian L. Chen Z. Thibault D. Liu G. Liu T. Thanassi D.G. The outer membrane usher forms a twin-pore secretion complex.J. Mol. Biol. 2004; 344: 1397-1407Crossref PubMed Scopus (58) Google Scholar). The dimer interface in the 3D crystals is formed by the flat side of the kidney-shaped β-barrel, encompassing strands β11–β20 (residues 397–573; Figure 2D). However, contrary to what is seen in other oligomerizing OM proteins, the two PapC protomers in the dimer interface make little direct contacts with each other (side chains are typically between 4 and 6 Å apart; Figure 2D). Instead, the electron density at the dimer interface in the PapC130–640 crystals reveals that the interface between protomers is mediated by several detergent molecules. Likely, the extensive detergent wash steps (see Experimental Procedures) preceding PapC130–640 crystallization resulted in the exchange of interface lipids for detergent molecules. In this respect, it is interesting to note the two usher protomers in the PapC130–640 crystals (average Cα-Cα distance of 15 Å) are in somewhat closer proximity than seen in the lipid-reconstituted 2D crystals and in the FimD2:C:F:G:H complex (Cα-Cα distance of 27 Å; see below). Presumably the loss of lipids in the former results in the two protomers being closer to one another. Finally, since the PapC130–640 construct used for crystallization lacks the N- and C-terminal periplasmic domains, it cannot be excluded that parts of these domains are involved in maintaining the usher dimer integrity. To understand the mechanism of pilus subunit translocation by the OM usher, we isolated pilus assembly intermediates by capturing type 1 pilus tip fibers during secretion through the FimD usher. FimD was expressed in bacteria together with the FimH adhesin, FimG and FimF tip subunits, and a His6-tagged FimC chaperone (FimCHis). By not providing the FimA helical rod subunit to the assembly system, pilus production stalls after incorporation of the last tip subunit FimF, resulting in production of quasihomogeneous FimD:tip complexes. Affinity and gel filtration chromatography of the detergent-solubilized OM fraction yielded the type 1 pilus tip complex as a single major peak in the chromatography profile (Figure 4A). Gel electrophoresis and Coomassie blue staining show all expected components (FimD:C:F:G:H) to be present in the complex (Figure 4B). Densitometry of the Coomassie-stained gel suggested a molar ratio for FimD:C:F:G:H of approximately 2:2:1:1:1, calculated by setting FimH equal to 1 mol (Figure 4B). However, since FimC adsorbs Coomassie blue dye twice as strongly as FimH, as demonstrated previously with dissolved 3D crystals of 1:1 FimC:FimH complex (Knight et al., 1997Knight S. Mulvey M. Pinkner J. Crystallization and preliminary X-ray diffraction studies of the FimC-FimH chaperone-adhesin complex from Escherichia coli.Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 207-210Crossref" @default.
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