Structural basis of chaperone-subunit complex recognition by the type 1 pilus assembly platform FimD

Abstract

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 (FimD(N)) before and after binding of a chaperone-subunit complex. FimD(N) 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 FimD(N) 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.

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.

Figure 2

NMR studies on FimD_N. (A) Polypeptide backbone of FimD_N(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 FimD_N(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 FimD_N(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 FimD_N upon binding to FimC–FimH_P. Δδ_Av is the weighted average of the ¹⁵N and ¹H chemical shifts, (Pellecchia et al, 1999). (F) Heteronuclear [¹⁵N,¹H]NOE measurements of FimD_N(1–139) in the FimD_N–FimC–FimH_P ternary complex. Values between 0.5 and 1 indicate well-structured parts of the protein; values<0.5 manifest increased flexibility.

Figure 3

X-ray structure of the ternary FimD_N(1–125)–FimC–FimH_P complex. (A) Ribbon diagram of the ternary complex, with FimD_N(1–125) depicted in green, FimC in cyan and the pilin domain FimH_P in yellow. The G₁ donor strand of FimC is colored in blue. A black dashed line indicates residues 10–18 of FimD_N, for which no electron density was observed. The N- and C-termini of FimD_N are labeled in green. (B) Close-up view of the hydrophobic contacts between Phe8 of the N-terminal FimD_N tail (green) and residues from FimC (cyan) and FimH_P (yellow). The final 2mFo−DFc electron density map is contoured at 1σ level. (C) Stereo representation of the tail interface. Residues from FimD_N, in stick model, are shown in green. The molecular surfaces of FimC (slate-grey) and FimH_P (light yellow) are shown in semitransparent mode. Residues contributing to the FimC and FimH_P surfaces and interacting with FimD_N are shown in more intense color: cyan for FimC and yellow for FimH_P 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 FimD_N core 25–125. Some hydrogen bonds between FimC and the FimD_N core are depicted as thin dashed lines. Color coding is as in (A). The figure was prepared with Pymol (www.pymol.org).

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 (FimD_N(1–125)) and cyan (FimC). Residues of FimD_N(1–125) interacting (5.0 Å distance cutoff) with FimC and FimH_P are indicated with cyan and yellow triangles, respectively. Residues interacting with both FimC and FimH_P are indicated with black triangles. FimC residues involved in contacts (5.0 Å distance cutoff) with FimD_N(1–125) are indicated with green triangles. The alignment was generated using CLUSTAL W (Thompson et al, 1994) and displayed with ALSCRIPT (Barton, 1993).

Figure 5

Analysis of amino-acid replacements and deletions in FimD, FimD_N(1–139), and replacements in FimC with respect to type 1 pilus biogenesis in vivo and formation of ternary FimD_N(1–139)–FimC–FimH_P 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 FimD_N(1–139) and FimC variants to form the ternary complex as well as the ability of FimC variants to bind FimH_P in vitro are indicated as ‘yes' (+) or ‘no' (–). ND, not determined. (B) Analytical gel filtration at pH 7.4 and 25°C, probing the effect of mutations in FimD_N(1–139) or FimC on the formation of the FimD_N(1–139)–FimC–FimH_P complex.