Structure of the nonameric bacterial amyloid secretion channel

Abstract

Various strains of bacteria are able to produce a unique class of functional amyloids termed curli, which are critical for biofilm formation, host cell adhesion, and colonization of inert surfaces. Curli are secreted via the type VIII bacterial secretion system, and they share biochemical and structural characteristics with amyloid fibers that have been implicated in deleterious disease in humans. Here, we report the crystal structure of Escherichia coli CsgG, which is an essential lipoprotein component of the type VIII secretion system and which forms a secretion channel in the bacterial outer membrane for transporting curli subunits. CsgG forms a crown-shaped, symmetric nonameric channel that spans the outer membrane via a 36-strand β-barrel, with each subunit contributing four β-strands. This nonameric complex contains a central channel with a pore located at the middle. The eyelet of the pore is ∼12 Å in diameter and is lined with three stacked nine-residue rings consisting of Tyr-66, Asn-70, or Phe-71. Our structure-based functional studies suggest that Tyr-66 and Phe-71 residues function as gatekeepers for the selective secretion of curli subunits. Our study describes in detail, to our knowledge, the first core structure of the type VIII bacterial secretion machinery. Importantly, our structural analysis suggests that the curli subunits are secreted via CsgG across the bacterial outer membrane in an unfolded form.

Keywords: CsgG; amyloids; biofilm; curli; outer membrane protein.

Fig. 1.

General architecture of the nonameric CsgG channel. (A) Overall structure of the nonameric CsgG channel shown in ribbon representation from different perspectives: the side view (Left), downward view from the extracellular space (Center), and upward view from the periplasmic space (Right). The CsgG channel resembles a crown and contains nine protomers, all of which are highlighted in individual colors as well as numbered 1–9. (Left) The TM domain consists of a 36-strand β-barrel fully open to the extracellular environment, with each protomer contributing four consecutive β-strands. The periplasmic region of each protomer consists of three α-helices and a three-strand β-sheet. (Center and Right) The pore eyelet of the nonamer is defined by nine loops arranged in a ring. (B) Ribbon representation of a CsgG protomer colored in rainbow showing three structural modules: the TM β-sheet, the eyelet loop and the periplasmic module. The TM β-sheet consists of four antiparallel β-strands (β2, β3, β4, and β5); the eyelet loop includes residues 55−80; and the periplasmic module primarily consists of three α-helices (α1, α2, and α3) and a three-strand β-sheet (β1 and the periplasmic portions of β3 and β4). The N and C termini of CsgG are in blue and red, respectively. (C) Amino acid sequence and secondary structures of the CsgG promoter. Residues not included in the final model are indicated by dashed lines. Chymotrypsin removed the first 34 residues at the N terminus and 12 residues at the C terminus of the mature CsgG protein. Residues Tyr-61, Asn-70, and Phe-71 that line the pore eyelet are marked by an asterisk.

Fig. 2.

Dimensions and surface attributes of the nonameric CsgG channel. (A) Hydrophobicity of the outer surfaces of the nonameric CsgG channel. The outer surface exhibits a clear subdivision into an upper nonpolar surface (the TM region) and a lower polar surface (the periplasmic region). (B) The pore radius of the CsgG channel along the channel axis from the periplasmic space to the extracellular space, calculated by using EDPDB (23). The eyelet of the pore, which is formed by the nine eyelet loops and lies at the middle of the channel, is only 12 Å in diameter and 20 Å in length. Because of the ninefold rotational symmetry, the entire channel is nearly circular. (C) A slab view from the plane of the OM showing the electrostatic properties inside of the channel. Positive and negative charges are in blue and red, respectively. The interior surface of the channel is predominantly negatively charged (contoured from +10 to −10 kT/e). (D) Hydrophobicity of the channel constriction region that is formed by nine eyelet loops viewed from the extracellular (Left) and the periplasmic (Right) space. In contrast to the hydrophilic nature of the channel wall, the surface of the eyelet loops is highly hydrophobic (gold, W/Y/F/M/L/I/V/P/A/G; turquoise, K/R/H/E/D/Q/N/T/S/C).

Fig. 3.

Detailed view of the eyelet of the nonameric CsgG channel. (A) Ribbon representation of a CsgG protomer with the side chains of residues Phe-63 (magenta), Tyr-66 (orange), Asn-70 (cyan), and Phe-71 (magenta) represented as sticks (side view from the plane of the OM). Residues Phe-71, Asn-70, and Tyr-66 of CsgG in the structure lie sequentially along the channel axis. (B) A detailed view of the eyelet. The side chains of Tyr-66, Asn-70, and Phe-71 point at the channel axis. The pore eyelet is lined with three stacked nine-residue rings, and each ring is formed by a single identical residue from each CsgG protomer. Nine Phe-63 residues form an external ring (indicated by a dashed circle) outside of the inner ring formed by nine Phe-71 residues.

Fig. 4.

Mutational analysis of the nonameric CsgG channel. (A) Negative-stain EM micrographs of E. coli K-12 strain BW25113 WT (Left), ΔcsgG mutant (Center), and BW25113 ΔcsgG mutant transformed with pBAD22-CsgG plasmid (Right). Hair-like surface appendages are curli. (Scale bars: 500 nm.) (B) CR–yeast extract casamino acids (YESCA) plate with E. coli K-12 strain BW25113ΔcsgG mutant transformed with vector control (pBAD22), plasmids encoding WT CsgG or CsgG mutants at position of Tyr-66. Eleven of 19 point mutants (Y66G, Y66V, Y66I, Y66T, Y66C, Y66P, Y66N, Y66K, Y66R, Y66D, and Y66E) have noticeable decreased curli production compared with the WT. (C) CR-YESCA plate with E. coli K-12 strain BW25113ΔcsgG mutant transformed with vector control (pBAD22), plasmids encoding WT CsgG or CsgG mutants at position of Phe-71. Nine of 19 point mutants (F71A, F71G, F71V, F71S, F71T, F71P, F71D, and F71E) interfere with curli production. Reduced curli production of CsgG mutants was quantified by using a Thioflavin T (ThT) fluorescence assay as shown in Fig. S5G. All CR-YESCA plates contained 0.01 mM arabinose. Experiments were repeated at least three times. Representative data are shown.