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• W2059486580 abstract "Article26 May 2005free access Structural basis of chaperone–subunit complex recognition by the type 1 pilus assembly platform FimD Mireille Nishiyama Mireille Nishiyama Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland Search for more papers by this author Reto Horst Reto Horst Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, SwitzerlandPresent address: Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Search for more papers by this author Oliv Eidam Oliv Eidam Biochemisches Institut, Universität Zürich, Zürich, Switzerland Search for more papers by this author Torsten Herrmann Torsten Herrmann Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland Search for more papers by this author Oleksandr Ignatov Oleksandr Ignatov Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland Search for more papers by this author Michael Vetsch Michael Vetsch Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland Search for more papers by this author Pascal Bettendorff Pascal Bettendorff Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland Search for more papers by this author Ilian Jelesarov Ilian Jelesarov Biochemisches Institut, Universität Zürich, Zürich, Switzerland Search for more papers by this author Markus G Grütter Markus G Grütter Biochemisches Institut, Universität Zürich, Zürich, Switzerland Search for more papers by this author Kurt Wüthrich Kurt Wüthrich Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland Search for more papers by this author Rudi Glockshuber Corresponding Author Rudi Glockshuber Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland Search for more papers by this author Guido Capitani Corresponding Author Guido Capitani Biochemisches Institut, Universität Zürich, Zürich, Switzerland Search for more papers by this author Mireille Nishiyama Mireille Nishiyama Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland Search for more papers by this author Reto Horst Reto Horst Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, SwitzerlandPresent address: Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Search for more papers by this author Oliv Eidam Oliv Eidam Biochemisches Institut, Universität Zürich, Zürich, Switzerland Search for more papers by this author Torsten Herrmann Torsten Herrmann Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland Search for more papers by this author Oleksandr Ignatov Oleksandr Ignatov Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland Search for more papers by this author Michael Vetsch Michael Vetsch Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland Search for more papers by this author Pascal Bettendorff Pascal Bettendorff Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland Search for more papers by this author Ilian Jelesarov Ilian Jelesarov Biochemisches Institut, Universität Zürich, Zürich, Switzerland Search for more papers by this author Markus G Grütter Markus G Grütter Biochemisches Institut, Universität Zürich, Zürich, Switzerland Search for more papers by this author Kurt Wüthrich Kurt Wüthrich Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland Search for more papers by this author Rudi Glockshuber Corresponding Author Rudi Glockshuber Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland Search for more papers by this author Guido Capitani Corresponding Author Guido Capitani Biochemisches Institut, Universität Zürich, Zürich, Switzerland Search for more papers by this author Author Information Mireille Nishiyama1, Reto Horst1, Oliv Eidam2, Torsten Herrmann1, Oleksandr Ignatov1, Michael Vetsch1, Pascal Bettendorff1, Ilian Jelesarov2, Markus G Grütter2, Kurt Wüthrich1, Rudi Glockshuber 1 and Guido Capitani 2 1Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland 2Biochemisches Institut, Universität Zürich, Zürich, Switzerland *Corresponding authors: Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, 8093 Zürich, Switzerland. Tel.: +41 1 633 6819; Fax: +41 1 633 1036; E-mail: [email protected] Institut, Universität Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland. Tel.: +41 1 635 5587; Fax: +41 1 635 6834; E-mail: [email protected] The EMBO Journal (2005)24:2075-2086https://doi.org/10.1038/sj.emboj.7600693 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Adhesive type 1 pili from uropathogenic Escherichia coli are filamentous protein complexes that are attached to the assembly platform FimD in the outer membrane. During pilus assembly, FimD binds complexes between the chaperone FimC and type 1 pilus subunits in the periplasm and mediates subunit translocation to the cell surface. Here we report nuclear magnetic resonance and X-ray protein structures of the N-terminal substrate recognition domain of FimD (FimDN) before and after binding of a chaperone–subunit complex. FimDN consists of a flexible N-terminal segment of 24 residues, a structured core with a novel fold, and a C-terminal hinge segment. In the ternary complex, residues 1–24 of FimDN specifically interact with both FimC and the subunit, acting as a sensor for loaded FimC molecules. Together with in vivo complementation studies, we show how this mechanism enables recognition and discrimination of different chaperone–subunit complexes by bacterial pilus assembly platforms. Introduction A wide variety of pathogenic bacteria possess adhesive surface organelles (‘pili’) that mediate binding to host tissue. These highly oligomeric, filamentous protein complexes are anchored to the outer bacterial membrane (Jones et al, 1995). Type 1 pili from uropathogenic Escherichia coli strains are required for bacterial attachment to mannose units of the glycoprotein receptor uroplakin Ia on the surface of urinary epithelium cells, and thus mediate the first critical step in the infection process (Mulvey et al, 1998; Zhou et al, 2001). In addition, type 1 pili are responsible for bacterial invasion and persistence in target cells (Baorto et al, 1997; Martinez et al, 2000). The quaternary structure of type 1 pili is characterized by a 6.9-nm wide pilus rod consisting of a right-handed, helical array of 500–3000 copies of the most abundant structural subunit FimA, and a linear tip fibrillum composed of the adhesin FimH and several copies of the subunits FimG and FimF (Jones et al, 1995; Hahn et al, 2002) (Figure 1). Figure 1.Schematic model of type 1 pilus assembly by the chaperone–usher pathway. The periplasmic chaperone FimC forms stoichiometric complexes with the newly translocated pilus subunits (FimA, FimG, FimF, FimH). In these complexes, FimC donates its G1 donor strand to the individual subunits, thereby completing the immunoglobulin-like fold of the subunits. FimC–subunit complexes diffuse to the assembly platform (usher) FimD, which specifically recognizes FimC–subunit complexes via its periplasmic, N-terminal segment of residues 1–139. Subsequently, FimC is released to the periplasm, and the subunit is delivered to the translocation pore of FimD, where it is supposed to interact with the previously incorporated subunit via donor strand exchange. The pilus rod, composed of FimA subunits, assembles into its helical quaternary structure on the cell surface. IM, inner membrane; OM, outer membrane. Download figure Download PowerPoint Biogenesis of type 1 pili is governed by the chaperone–usher pathway (Thanassi and Hultgren, 2000; Sauer et al, 2004). The assembly machinery is composed of two specialized classes of proteins: a periplasmic chaperone and an outer membrane assembly platform, which is also referred to as the usher. The periplasmic type 1 pilus chaperone FimC forms stoichiometric complexes with pilus subunits, catalyzes their folding, and transports them to the assembly platform FimD in the outer membrane (Jones et al, 1993; Vetsch et al, 2004) (Figure 1). The X-ray structure of the FimC–FimH complex (Choudhury et al, 1999) as well as the structures of the related chaperone–subunit complexes PapD–PapK (Sauer et al, 1999), PapD–PapE (Sauer et al, 2002), and Caf1M–Caf1 (Zavialov et al, 2003) have shown that pilus subunits have an incomplete immunoglobulin-like fold that lacks the seventh, C-terminal β-strand (referred to hereafter as ‘pilin fold’). In chaperone–subunit complexes, the missing β-strand is provided by a polypeptide segment of the chaperone, the ‘donor strand’, which is inserted parallel to the sixth strand of the subunit (Choudhury et al, 1999; Sauer et al, 1999, 2002; Zavialov et al, 2003). In the assembled pilus, an N-terminal extension of about 15 residues, preceding the pilin fold, acts as the donor strand and complements the pilin fold of the adjacent pilus subunit (Sauer et al, 2002; Zavialov et al, 2003). In this way, each subunit provides its own donor strand to the preceding subunit and accepts a donor strand from the following subunit. In contrast to the chaperone–subunit complexes, the orientation of the inserted donor strand in the pilus is antiparallel to the sixth β-strand of the preceding subunit. Structure comparison of a chaperone-bound subunit and the same subunit in complex with another subunit indicates that a conformational transition of the pilin fold occurs upon exchange of the donor strand during subunit assembly (Sauer et al, 2002; Zavialov et al, 2003). It has been proposed that this conformational change is the driving force for pilus assembly (Sauer et al, 2002; Zavialov et al, 2003). This hypothesis is further supported by the observations that pilus assembly is independent of ATP and of an electrochemical gradient (Jacob-Dubuisson et al, 1994), and that subunit–subunit complexes are thermodynamically more stable than chaperone–subunit complexes (Vetsch et al, 2004). The type 1 pilus assembly platform FimD is a multifunctional outer membrane protein of 833 residues (Klemm and Christiansen, 1990), which not only anchors the pilus to the cell surface but also recognizes FimC–subunit complexes in the periplasm and mediates translocation of folded subunits through the outer membrane (Saulino et al, 1998, 2000). In spite of the fundamental role of assembly platforms in pilus biogenesis, no structural information on the atomic level is available to date. Based on electron microscopy data on its P pilus homologue PapC, FimD is supposed to form a pore of about 2 nm diameter into the outer membrane (Saulino et al, 2000; Li et al, 2004). This pore size would be wide enough for translocation of individual folded subunits from the periplasm to the cell surface, but not for translocation of the helical pilus rod, which appears to attain its final quaternary structure only on the cell surface (Bullitt and Makowski, 1995). In a previous study, we showed that FimD possesses an N-terminal periplasmic domain, FimDN, comprising residues 1–139. FimDN is soluble in the absence of detergents, folds autonomously, and specifically binds FimC–subunit complexes with micromolar affinities although FimC or pilus subunits alone are not recognized (Nishiyama et al, 2003). In accordance with these data, the recognition site of chaperone–subunit complexes in PapC was also localized to the N-terminal 124 residues (Ng et al, 2004). Nevertheless, it has been discussed controversially whether the N-terminal chaperone–subunit-binding region of pilus assembly platforms is an independent periplasmic domain (Harms et al, 1999; Nishiyama et al, 2003), or belongs to the porin-like β-barrel transmembrane domain of FimD (Henderson et al, 2004; Ng et al, 2004). Here we report nuclear magnetic resonance (NMR) and X-ray protein structures that provide snapshots of the initial step of pilus formation at the site of the assembly platform, that is, the chaperone–subunit recognition domain of an assembly platform before and after binding of a chaperone–subunit complex. The NMR structure of isolated FimDN reveals that this domain consists of a flexible, N-terminal ‘tail’ (residues 1–24), a structured ‘core’ (residues 25–125) with a novel polypeptide fold, and a potential hinge segment (residues 126–135) that connects the structured core to the transmembrane region of FimD. The most remarkable feature of FimDN is its flexible N-terminal tail, which adopts a defined conformation only upon binding to the complex between FimC and the pilin domain of FimH (FimHP, residues 158–279 of FimH), as revealed by the 1.8 Å crystal structure of the ternary FimDN–FimC–FimHP complex. The structural data, in conjunction with biochemical experiments and in vivo complementation studies, suggest a mechanism in which the assembly platform utilizes its flexible N-terminal segment 1–24 to accomplish specific recognition of different chaperone–subunit complexes. Results and discussion The NMR solution structure of free FimDN reveals a previously unknown fold with mobile chain ends Initial NMR experiments with FimDN(1–139) (residues 1–139 of FimD) showed that this construct is susceptible to N-terminal degradation when incubated for several days at 25°C and at a concentration of 1 mM, most likely due to minute protease contaminations. Edman sequencing and mass spectrometry revealed nonspecific N-terminal degradation of FimDN(1–139) with cleavage after residues Leu9, Ala10, Gln13, and Ser20 (data not shown). Moreover, measurement of [15N,1H]NOEs showed that the segment 1–24 of the polypeptide chain is flexibly disordered (Supplementary Figure S1). We then incubated the ternary complex formed by FimDN(1–139), FimC, and the pilin domain of FimH (FimHP) under identical conditions. In the complex, we observed specific and quantitative cleavage of FimDN(1–139) at a single site close to the C-terminus (Ala125), but no N-terminal degradation was observed. Comparison of the thermal stabilities of FimDN(1–139) and its truncated variants FimDN(25–139) and FimDN(1–125) at pH 7.4, which were monitored by the far-UV circular dichroism signal at 218 nm, showed identical transition midpoints (Tm) of 67.6±0.5°C for all the constructs (Supplementary Table S1). Combined with the aforementioned NMR data, the thermal denaturation data show that the segment 25–125 of FimDN(1–139) adopts a stable tertiary structure, independent of whether or not the terminal chain segments are present. Based on these data, we decided to perform a NMR structure determination of the N- and C-terminally truncated protein fragment FimDN(25–125) (Figure 2A and B; Table I). The scaffold of the tertiary structure is formed by a three-stranded, antiparallel β-sheet (β1–β3) consisting of residues 31–39, 42–53, and 60–62, and a two-stranded, antiparallel β-sheet (β4 and β5) comprising residues 101–105 and 110–114, respectively. The two β-sheets are connected by a peptide segment comprising a single-turn 310-helix (residues 76–78), and the α-helices α1 (residues 66–72) and α2 (residues 93–96). The invariant cysteine pair (Cys63 and Cys90; cf. Figure 4) forms a disulfide bond stabilizing this peptide segment. A second 310-helix (residues 117–119) is located close to the C-terminus. The helices α1 and α2 are packed tightly against the β-sheets, with Met72 of α1 in direct contact with Met44 and Leu39 of β2 and Leu113 of β5, and Leu93 of α2 in contact with Ala102 of β4 and Leu113 of β5. The helices α1 and α2 pack at an angle of 50° to each other, with pronounced hydrophobic interactions between Leu69 and Leu93. A comparison of the structure of FimDN(25–125) with the structures deposited in the Protein Data Bank (PDB) (Berman et al, 2000) using the DALI server (Holm and Sander, 1998) identified no structural homologues. The two structurally most closely related proteins, PDB entries 1SFO and 1T0Y, exhibited Z-scores of 2.1 and 2.0, respectively, with r.m.s.d. values for the Cα atoms of 3.8 and 3.6 Å over 51 and 58 aligned residues, respectively. This shows that FimDN(25–125) represents a previously unknown polypeptide fold. In addition, the NMR data confirm that FimDN(25–125) forms a self-folding periplasmic domain that precedes the transmembrane domain of FimD, and they are in clear-cut contrast with models predicting that the N-terminal region of the assembly platform belongs to the β-barrel transmembrane domain (Henderson et al, 2004; Ng et al, 2004). Figure 2.NMR studies on FimDN. (A) Polypeptide backbone of FimDN(25–125) represented by a bundle of 20 energy-minimized DYANA conformers. Selected positions along the polypeptide chain are identified with sequence positions. (B) Ribbon drawing of one of the 20 energy-minimized conformers. β1–β5 and α1–α2 indicate five β-strands and two α-helices, respectively. The disulfide bridge Cys63–Cys90 is drawn in yellow. The chain ends are identified by the letters N and C. (C) NMR structure of FimDN(25–139) represented by a bundle of 20 energy-minimized DYANA conformers showing only the polypeptide backbone. The chain ends are identified by the letters N and C. The C-terminal residues 125–139 are shown in magenta. (D) Close-up view of the surface of one of the 20 energy-minimized conformers of FimDN(25–139). Relative to (C), the structure has been rotated by approximately 90° about a vertical axis. The backbone of the C-terminal stretch 125–139 is drawn in magenta, and the side chain of Trp133 is indicated in red. Those side chains which show long-range NOE connectivities with Trp133 are drawn in bronze. In total, 14 long-range upper-distance limits between Trp133 and the rest of the protein (shown in cyan) define the position of the aromatic ring of Trp133. (E) Chemical shift variations of FimDN upon binding to FimC–FimHP. ΔδAv is the weighted average of the 15N and 1H chemical shifts, (Pellecchia et al, 1999). (F) Heteronuclear [15N,1H]NOE measurements of FimDN(1–139) in the FimDN–FimC–FimHP ternary complex. Values between 0.5 and 1 indicate well-structured parts of the protein; values<0.5 manifest increased flexibility. Download figure Download PowerPoint Figure 3.X-ray structure of the ternary FimDN(1–125)–FimC–FimHP complex. (A) Ribbon diagram of the ternary complex, with FimDN(1–125) depicted in green, FimC in cyan and the pilin domain FimHP in yellow. The G1 donor strand of FimC is colored in blue. A black dashed line indicates residues 10–18 of FimDN, for which no electron density was observed. The N- and C-termini of FimDN are labeled in green. (B) Close-up view of the hydrophobic contacts between Phe8 of the N-terminal FimDN tail (green) and residues from FimC (cyan) and FimHP (yellow). The final 2mFo−DFc electron density map is contoured at 1σ level. (C) Stereo representation of the tail interface. Residues from FimDN, in stick model, are shown in green. The molecular surfaces of FimC (slate-grey) and FimHP (light yellow) are shown in semitransparent mode. Residues contributing to the FimC and FimHP surfaces and interacting with FimDN are shown in more intense color: cyan for FimC and yellow for FimHP residues, respectively. Residues from the G1 donor strand of FimC contributing to the molecular surface appear in blue. (D) Stereo representation of the interface between FimC and the folded FimDN core 25–125. Some hydrogen bonds between FimC and the FimDN core are depicted as thin dashed lines. Color coding is as in (A). The figure was prepared with Pymol (www.pymol.org). Download figure Download PowerPoint Table 1. Input for the structure calculation and characterization of the energy-minimized NMR structures of FimDN(25–139) and FimDN(25–125) Quantitya FimDN(25–139) FimDN(25–125) NOE upper distance limits 2928 2953 Dihedral angle constraints 94 94 Residual target function (Å2) 1.57±0.33 1.20±0.43 Residual NOE violations Number ⩾0.1 Å 31±6 (24–45) 22±5 (5–29) Maximum (Å) 0.14±0.01 (0.12–0.15) 0.14±0.01 (0.12–0.17) Residual dihedral angle violations Number ⩾2.5 deg 0±1 (0–2) 1±1 (0–3) Maximum (deg) 1.82±1.12 (0.35–4.54) 2.89±1.32 (1.57–7.44) Amber energies (kcal/mol) Total −4323.89±76.56 −4129.35±55.45 Van der Waals −327.64±13.24 −292.04±16.14 Electrostatic −4948.89±74.37 −4673.08±52.16 R.m.s.d. from ideal geometry Bond lengths (Å) 0.0078±0.0001 0.0079±0.0002 Bond angles (deg) 2.035±0.044 2.022±0.067 R.m.s.d. to the mean coordinates (Åb) bb (35–120) 0.43±0.06 (0.33–0.54) 0.40±0.06 (0.28–0.54) ha (35–120) 0.74±0.07 (0.62–0.95) 0.74±0.06 (0.67–0.90) Ramachandran plot statisticsc Most favored regions (%) 72 71 Additional allowed regions (%) 25 26 Generously allowed regions (%) 2 2 Disallowed regions (%) 1 1 a Except for the three top entries, the average value for the 20 energy-minimized conformers with the lowest residual DYANA target function values and the standard deviation among them are given. For the residual violations and the r.m.s.d. values, the range from the minimum to the maximum value is given in parentheses. b bb indicates the backbone atoms N, Cα, Cγ; ha stands for ‘all heavy atoms’. The numbers in parentheses indicate the residues for which the r.m.s.d. was calculated. c As determined by PROCHECK (Laskowski et al, 1993). In order to study the role of the segment 126–139, which is supposed to connect FimDN(25–125) to the transmembrane domain of FimD (according to a topology prediction program by Martelli et al (2002), residue 138 is the first residue of a transmembrane β-barrel of FimD), we further solved the NMR structure of FimDN(25–139) (Figure 2C). Except for the additional C-terminal residues, the structure of FimDN(25–139) is in very close agreement with that of FimDN(25–125), with an r.m.s.d. of 1.0 Å for the Cα atoms of the residues 30–120. Interestingly, the segment 126–135 is not disordered in FimDN(25–139), even though it does not adopt a regular secondary structure (Figure 2C). Although the residues 136–139 show negative [15N,1H]NOE values indicative of high-frequency internal motions, those of the residues 121–135 are positive, suggesting rotational tumbling with an effective rotational correlation time similar to that for overall tumbling of the globular domain (Supplementary Figure S1). In addition, we identified a network of long-range NOEs connecting side-chain protons of Trp133 with Val49, Leu64, Thr65, Gln68, and Met72 of the globular domain. These NOEs define a unique position of the aromatic ring of Trp133 in a binding pocket on the surface of the folded domain FimDN(25–125) (Figure 2D). We interpret these observations in terms of a rapid, intramolecular association/dissociation equilibrium between the domain FimDN(25–125) and the segment 126–135. The fact that FimDN(25–139) and FimDN(25–125) have identical Tm values indicates that the C-terminal segment 126–135 dissociates in a spectroscopically silent fashion from the folded core of FimDN, presumably at a temperature below the observed Tm value (Supplementary Table S1). As will be discussed below, there are indications that the intramolecular association/dissociation equilibrium between the domain FimDN(25–125) and the polypeptide segment 126–135 might be related to a spatial rearrangement of residues 1–125 relative to the transmembrane domain of FimD when chaperone–subunit complexes are bound. X-ray structure determination of the FimDN(1–125)–FimC–FimHP ternary complex The search for optimal crystallization conditions of a ternary complex between FimDN, FimC, and a bound pilus subunit led us to use protein constructs without disordered segments that might impair crystallization. We therefore investigated the requirement of the flexible segment 1–24 and the C-terminal region 126–139 of FimD for the recognition of FimC–subunit complexes. In addition, we used the C-terminal pilin domain of FimH (FimHP, residues 158–279 of FimH) instead of full-length FimH, because the structure of the FimC–FimH complex (Choudhury et al, 1999) had revealed that FimC interacts exclusively with FimHP. Moreover, the interaction between FimC and FimHP through donor strand complementation is representative for all FimC–pilus subunit complexes (Choudhury et al, 1999), and the lectin domain is not required for recognition of the FimC–FimH complex by FimDN(1–139) (Nishiyama et al, 2003). Taking these facts into account, we tested the ability of the truncated FimDN variants FimDN(12–139), FimDN(25–139), and FimDN(1–125) to bind the FimC–FimHP complex in vitro. Analytical gel filtration revealed that residues 1–24 are strictly required for the formation of the FimDN–FimC–FimHP ternary complex (Figure 5B), although they are disordered in the NMR structure of isolated FimDN(1–139) (Supplementary Figure S1). The requirement of segment 1–24 was confirmed by the observation that deletion of residues 1–12 or residues 1–24 in full-length FimD completely abolished the ability of plasmid-encoded FimD to restore type 1 pilus formation in an E. coli fimD deletion strain (W3110ΔfimD) (Figure 5A). In contrast, residues 126–139 in FimDN are not needed for the formation of the ternary complex in vitro, since the variant FimDN(1–125) exhibits the same affinity towards the FimC–FimHP complex as full-length FimDN(1–139) (Supplementary Table S2). Figure 4.Multiple sequence alignments of N-terminal domains of assembly platforms (A) and periplasmic chaperones (B). Sequences are identified by their SWISS-PROT IDs. Residue numbering refers to mature FimD (A) and FimC (B). Identical residues are boxed in red, conserved ones are highlighted in yellow. Secondary structure elements derived from the X-ray structure of the ternary complex are shown in green (FimDN(1–125)) and cyan (FimC). Residues of FimDN(1–125) interacting (5.0 Å distance cutoff) with FimC and FimHP are indicated with cyan and yellow triangles, respectively. Residues interacting with both FimC and FimHP are indicated with black triangles. FimC residues involved in contacts (5.0 Å distance cutoff) with FimDN(1–125) are indicated with green triangles. The alignment was generated using CLUSTAL W (Thompson et al, 1994) and displayed with ALSCRIPT (Barton, 1993). Download figure Download PowerPoint Based on these results, we crystallized the ternary complex between FimDN(1–125), FimC, and FimHP, and obtained two different crystal forms, A and B, with space groups P63 and P212121, respectively. The structure of the ternary complex was solved with data collected from a single crystal of form B at 1.8 Å resolution through molecular replacement based on the structure of the FimC–FimH complex (Choudhury et al, 1999). Structure refinement resulted in R-factor and free R-factor values of 0.19 and 0.22, respectively (Table II). The final model encompasses residues 1–205 of FimC, residues 158–279 of FimHP, and residues 1–9 and 19–125 of FimDN(1–125) (Figure 3). Residues 10–18 of FimDN(1–125) were not included in the model due to missing electron density. The lack of electron density in this region was confirmed by computation of a simulated-annealing omit map. As the FimDN segment 10–18 is also disordered in the electron density map obtained from crystal form A, the lack of density in this region appears to be an intrinsic property of the FimDN–FimC–FimHP complex. The nature of the structural disorder was further investigated by measurements of heteronuclear [15N,1H]NOEs for FimDN in the ternary complex (Figure 2F), which showed that the effective rotational correlation time of the residues 10–18 is significantly shorter than that for the structured parts of the protein. Interestingly, the residues Asn5 and Arg7 have positive [15N,1H]NOE values, which is an indication that these residues get immobilized upon complex formation. Figure 5.Analysis of amino-acid replacements and deletions in FimD, FimDN(1–139), and replacements in FimC with respect to type 1 pilus biogenesis in vivo and formation of ternary FimDN(1–139)–FimC–FimHP complexes in vitro. (A) Yeast agglutination assays, probing the formation of functional type 1 pili through agglutination with yeast cells. The E. coli strains W3110ΔfimD and W3110ΔfimC were transformed with expression plasmids carrying the indicated FimD and FimC variants, respectively. Agglutination intensities are indicated as (−) no agglutination, (±) weak or (+) strong. The ability of FimDN(1–139) and FimC variants to form the ternary complex as well as the ability of FimC variants to bind FimHP in vitro are indicated as ‘yes’ (+) or ‘no’ (–). ND, not determined. (B) Analytic" @default.
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