Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG

Abstract

Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes)1,2,3. They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia1,4,5. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF6,7. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric phenylalanine, asparagine and tyrosine rings that may guide the extended polypeptide substrate through the secretion pore. The specificity factor CsgE forms a nonameric adaptor that binds and closes off the periplasmic face of the secretion channel, creating a 24,000 Å3 pre-constriction chamber. Our structural, functional and electrophysiological analyses imply that CsgG is an ungated, non-selective protein secretion channel that is expected to employ a diffusion-based, entropy-driven transport mechanism.

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Figure 1: X-ray structure of CsgGC1S in pre-pore conformation.
Figure 2: Structure of CsgG in its channel conformation.
Figure 3: CsgG channel constriction.
Figure 4: Model of CsgG transport mechanism.

Accession codes

Accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

Coordinates and structure factors for CsgGC1S and CsgG have been deposited in the Protein Data Bank under accession codes 4uv2 and 4uv3, respectively. The cryo-EM map for CsgG–CsgE has been deposited in the EMDataBank under accession code EMDB-2750.

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Acknowledgements

This research was supported by VIB through project grant PRJ9 (P.G., N.V.G. and H.R.), by Hercules Foundation through equipment grant UABR/09/005, by National Institutes of Health RO1 grants AI099099 and AI048689 (J.P. and S.J.H.) and A1073847 (M.R.C.), and by Institut Pasteur and Centre national de la recherche scientifique (F.G., G.P.A. and R.F.). S.H. is funded by the Engineering and Physical Sciences Research Council (Institutional Sponsorship Award), the National Physical Laboratory and University College London Chemistry. F.G. is the recipient of a ‘Bourse Roux’ from Institut Pasteur. P.V.K. was supported by the European Research Council (ERC). We acknowledge Diamond Light Source for time on beamlines I02, I03, I04 and I24 under proposal mx7351, and the Soleil synchrotron for access to Proxima-1 and Proxima-2a under proposals 20100734, 20110924 and 20121253.

Author information

P.G. produced, purified and crystallized CsgG and CsgGC1S, and determined their X-ray structures. Single-particle EM was performed by P.V.K., F.G. and G.P.H. and supervised by R.F. P.V.K., F.G. and W.J. performed the in vitro characterization of the CsgG–CsgE complex. N.V.G. and W.J. performed mutagenesis and phenotyping experiments. I.V.d.B. conducted the single-channel recordings, and S.H. supervised the acquisition and analysis of the recordings. A.T.T. and P.G. recorded and analysed FTIR spectra. J.S.P., M.R.C. and S.J.H. conceived the study and contributed expression constructs and protein. H.R. conceived and supervised the study, analysed data and wrote the paper with contributions from all authors.

Correspondence to Rémi Fronzes or Han Remaut.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Curli biosynthesis pathway in E. coli.

The major curli subunit CsgA (light green) is secreted from the cell as a soluble monomeric protein. The minor curli subunit CsgB (dark green) is associated with the outer membrane (OM) and acts as a nucleator for the conversion of CsgA from a soluble protein to amyloid deposit. CsgG (orange) assembles into an oligomeric curli-specific translocation channel in the outer membrane. CsgE (purple) and CsgF (light blue) form soluble accessory proteins required for productive CsgA and CsgB transport and deposition. CsgC forms a putative oxidoreductase of unknown function. All curli proteins have putative Sec signal sequences for transport across the cytoplasmic (inner) membrane (IM).

Extended Data Figure 2 In-solution oligomerization states of CsgG and CsgGC1S analysed by size-exclusion chromatography and negative-stain electron microscopy.

a, Raw negative-stain EM image of C8E4/LDAO-solubilized CsgG. Arrows indicate the different particle populations as labelled in the size exclusion profile shown in g, being (I) aggregates of CsgG nonamers, (II) CsgG octadecamers and (III) CsgG nonamers. Scale bar, 20 nm. b, Representative class average for top and side views of the indicated oligomeric states. c, Rotational autocorrelation function graph of LDAO-solubilized CsgG in top view, showing nine-fold symmetry. d, Raw negative-stain EM image of CsgGC1S. Arrows indicate the hexadecameric (IV) and octameric (V) particles observed by size-exclusion chromatography in g. e, Representative class average for side views of CsgGC1S oligomers. No top views were observed for this construct. f, Table of elution volumes (EV) of CsgGC1S and CsgG particles observed by size-exclusion chromatography shown in g, calculated molecular mass (MWcalc), expected molecular mass (MWCsgG) corresponding CsgG oligomerization state (CsgGn) and the particles’ symmetry as observed by negative-stain EM and X-ray crystallography. g, Size-exclusion chromatogram of CsgGC1S (black) and C8E4/LDAO-solubilized CsgG (grey) run on Superdex 200 10/300 GL (GE Healthcare). h, i, Ribbon representation of crystallized oligomers in top and side view, showing the D8 hexadecamers for CsgGC1S (h) and D9 octadecamers for membrane-extracted CsgG (i). One protomer is coloured in rainbow from N terminus (blue) to C terminus (red). The two C8 octamers (CsgGC1S) or C9 nonamers (CsgG) that form the tail-to-tail dimers captured in the crystals are coloured blue and tan. r and θ give radius and interprotomer rotation, respectively.

Extended Data Figure 3 Comparison of CsgG with structural homologues and interprotomer contacts in CsgG.

a, b, Ribbon diagram for the CsgGC1S monomer (for example CsgG in pre-pore conformation) (a) and the nucleotide-binding-domain-like domain of TolB (b) (PDB 2hqs), both coloured in rainbow from N terminus (blue) to C terminus (red). Common secondary structure elements are labelled equivalently. c, CsgGC1S (grey) in superimposition with, from left to right, Xanthomonas campestris rare lipoprotein B (PDB 2r76, coloured pink), Shewanella oneidensis hypothetical lipoprotein DUF330 (PDB 2iqi, coloured pink) and Escherichia coli TolB (PDB 2hqs, coloured pink and yellow for the N-terminal and β-propeller domains, respectively). CsgG-specific structural elements are labelled and coloured as in the upper left panel. d, e, Ribbon diagram of two adjacent protomers as found in the CsgG structure, viewed along the plane of the bilayer, either from outside (c) or inside (d) the oligomer. One protomer is shown in rainbow (dark blue to red) from N terminus to C terminus; a second protomer is shown in light blue (core domain), blue (helix 2) and tan (TM domain). Four main oligomerization interfaces are apparent: β6–β3′ main-chain interactions inside the β-barrel, the constriction loop (CL), side-chain packing of helix 1 (α1) against β1–β3–β4–β5, and helix–helix packing of helix 2 (α2). The 18-residue N-terminal loop connecting the lipid anchor (a magenta sphere shows the Cα position of Leu 2) and N-terminal helix (αN) is also seen to wrap over the adjacent two protomers. The projected position of the lipid anchor is expected to lie against the TM1 and TM2 hairpins of the +2 protomer (not shown for clarity).

Extended Data Figure 4 Cys accessibility assays for selected surface residues in the CsgG oligomers.

ac, Ribbon representation of CsgG nonamers shown in periplasmic (a), side (b) and extracellular (c) views. One protomer is coloured in rainbow from N terminus (blue) to C terminus (red). Cysteine substitutions are labelled and the equivalent locations of the S atoms are shown as spheres, coloured according to accessibility to MAL-PEG (5,000 Da) labelling in E. coli outer membranes. d, Western blot of MAL-PEG reacted samples analysed on SDS–PAGE, showing 5 kDa increase on MAL-PEG binding of the introduced cysteine. Accessible (++ and +++), moderately accessible (+) and inaccessible (−) sites are coloured green, orange and red, respectively, in ae. For Arg 97 and Arg 110 a second species at 44 kDa is present, corresponding to a fraction of protein in which both the introduced and native cysteine became labelled. Data are representative of four independent experiments from biological replicates. e, Side view of the dimerization interface in the D9 octadecamer as present in the X-ray structure. Introduced cysteines in the dimerization interface or inside the lumen of the D9 particle are labelled. In membrane-bound CsgG, these residues are accessible to MAL-PEG, demonstrating that the D9 particles are an artefact of concentrated solutions of membrane-extracted CsgG and that the C9 complex forms the physiologically relevant species. Residues in the C-terminal helix (αC; Lys 242, Asp 248 and His 255) are found to be inaccessible to poorly accessible, indicating that αC may form additional contacts with the E. coli cell envelope, possibly the peptidoglycan layer.

Extended Data Figure 5 Molecular dynamics simulation of CsgG constriction with model polyalanine chain.

a, b, Top (a) and side (b) views of the CsgG constriction modelled with a polyalanine chain threaded through the channel in an extended conformation, here shown in a C-terminal to N-terminal direction. Substrate passage through the CsgG transporter is itself not sequence specific16,23. For clarity, a polyalanine chain was used for modelling the putative interactions of a passing polypeptide chain. The modelled area is composed of nine concentric CsgG C-loops, each comprising residues 47–58. Side chains lining the constriction are shown in stick representation, with Phe 51 coloured slate blue, Asn 55 (amide-clamp) cyan, and Phe 48 and Phe 56 (ϕ-clamp) in light and dark orange, respectively. N, O and H atoms (only hydroxyl or side-chain amide H atoms are shown) are coloured blue, red and white, respectively. The polyalanine chain is coloured green, blue, red and white for C, N, O and H atoms, respectively. Solvent molecules (water) within 10 Å of the polyalanine residues inside the constriction (residues labelled +1 to +5) are shown as red dots. c, Modelled solvation of the polyalanine chain, position as in b and with C-loops removed for clarity (shown solvent molecules are those within 10 Å of the full polyalanine chain). At the height of the amide-clamp and ϕ-clamp, the solvation of the polyalanine chain is reduced to a single water shell that bridges the peptide backbone and amide-clamp side chains. Most side chains in the Tyr 51 ring have rotated towards the solvent in comparison with their inward, centre-pointing position observed in the CsgG (and the CsgGC1S) X-ray structure. The model is the result of a 40 ns all-atom explicit solvent molecular dynamics simulation with GROMACS54 using the AMBER99SB-ILDN55 force field and with the Cα atoms of the residues at the extremity of the C-loop (Gln 47 and Thr 58) positionally restricted.

Extended Data Figure 6 Sequence conservation in CsgG homologues.

a, Surface representation of the CsgG nonamer coloured according to sequence similarity (coloured yellow to blue from low to high conservation score)56 and viewed from the periplasm (far left), the side (middle left), the extracellular milieu (middle right) or as a cross-sectional side view (far right). The figures show that the regions of highest sequence conservation map to the entry of the periplasmic vestibule, the vestibular side of the constriction loop and the luminal surface of the TM domain. b, Multiple sequence alignment of CsgG-like lipoproteins. The selected sequences were chosen from monophyletic clades across the phylogenetic three of CsgG-like sequences (not shown), to give a representative view of sequence diversity. Secondary structure elements are shown as arrows or bars for β-strands and α-helices, respectively, and are based on the E. coli CsgG crystal structure. c, d, CsgG protomer in secondary structure representation (c) and a cross-sectional side view (d) of the CsgG nonamer in surface representation, both coloured grey and with three continuous blocks of high sequence conservation coloured red (HCR1), blue (HCR2) and yellow (HCR3). HCR1 and HCR2 shape the vestibular side of the constriction loop; HCR3 corresponds to helix 2, lying at the entry of the periplasmic vestibule. Inside the constriction, Phe 56 is 100% conserved, whereas Asn 55 can be conservatively replaced by Ser or Thr, for example by a small polar side chain that can act as hydrogen-bond donor/acceptor. The concentric side-chain ring at the exit of the constriction (Tyr 51) is not conserved. The presence of the Phe-ring at the entrance of the constriction is topologically similar to the Phe 427-ring (referred to as the ϕ-clamp) in the anthrax protective antigen PA63, in which it was shown to catalyse polypeptide capture and passage20. MST of toxB superfamily proteins reveals a conserved motif D(D/Q)(F)(S/N)S at the height of the Phe-ring. This is similar to the S(Q/N/T)(F)ST motif seen in curli-like transporters. Although an atomic resolution structure of PA63 in pore conformation is not yet available, available structures suggest the Phe-ring may similarly be followed by a conserved hydrogen-bond donor/acceptor (Ser/Asn 428) as a subsequent concentric ring in the translocation channel (note that the orientation of the element is inverted in both transporters).

Extended Data Figure 7 Single-channel current analysis of CsgG and CsgG + CsgE pores.

a, Under negative field potential, CsgG pores show two conductance states. The upper left and right panels show a representative single-channel current trace of, respectively, the normal (measured at +50, 0 and −50 mV) and the low-conductance forms (measured at 0, +50 and −50 mV). No conversions between both states were observed during the total observation time (n = 22), indicating that the conductance states have long lifetimes (second to minute timescale). The lower left panel shows a current histogram for the normal and low-conductance forms of CsgG pores acquired at +50 and −50 mV (n = 33). IV curves for CsgG pores with regular and low conductance are shown in the lower right panel. These data represent averages and standard deviations from at least four independent recordings. The nature or physiological existence of the low-conductance form is unknown. b, Electrophysiology of CsgG channels titrated with the periplasmic factor CsgE. The plots display the normalized occurrence, that is, the fractions of open, closed and intermediate-state channels, as a function of CsgE concentration. Open and closed states of CsgG are illustrated in Fig. 4f. Increasing the concentration of CsgE to more than 10 nM leads to the closure of CsgG pores. The effect occurs at +50 mV (left) and −50 mV (right), ruling out the possibility that the pore blockade is caused by electroosmosis or electrophoresis of CsgE (calculated pI 4.7) into the CsgG pore. An infrequent (<5%) intermediate state has roughly half the conductance of the open channel. It may represent CsgE-induced incomplete closures of the CsgG channel. Alternatively, it could represent the temporary formation of a CsgG dimer caused by the binding of residual CsgG monomer from the electrolyte solution to the membrane-embedded pore. The fractions for the three states were obtained from all-point histogram analysis of single-channel current traces. The histograms yielded peak areas for up to three states, and the fraction for a given state was obtained by dividing the corresponding peak area by the sum of all other states in the recording. Under negative field potential, two open conductance states are discerned, similar to the observations for CsgG (see a). Because both open channel variations were blocked by higher CsgE concentrations, the ‘open’ traces in b combine both conductance forms. The data in the plot represent averages and standard deviations from three independent recordings. c, The crystal structure, size-exclusion chromatography and EM show that detergent extracted CsgG pores form non-native tail-to-tail stacked dimers (for example, two nonamers as D9 particle; Extended Data Fig. 2) at higher protein concentration. These dimers can also be observed in single-channel recordings. The upper panel shows the single-channel current trace of stacked CsgG pores at +50, 0 and −50 mV (left to right). The lower left panel shows a current histogram of dimeric CsgG pores recorded at +50 and −50 mV. The experimental conductances of +16.2 ± 1.8 and –16.0 ± 3.0 pA (n = 15) at +50 and −50 mV, respectively, are near the theoretically calculated value of 23 pA. The lower right panel shows an IV curve for the stacked CsgG pores. The data represent averages and standard deviations from six independent recordings. d, The ability of CsgE to bind and block stacked CsgG pores was tested by electrophysiology. Shown are single-channel current traces of stacked CsgG pore in the presence of 10 or 100 nM CsgE at +50 mV (upper) and −50 mV (lower). The current traces indicate that otherwise saturating concentrations of CsgE do not lead to pore closure for stacked CsgG dimers. These observations are in good agreement with the mapping of the CsgG–CsgE contact zone to helix 2 and the mouth of the CsgG periplasmic cavity as discerned by EM and site-directed mutagenesis (Fig. 4 and Extended Data Fig. 7).

Extended Data Figure 8 CsgE oligomer and CsgG–CsgE complex.

a, Size-exclusion chromatography of CsgE (Superose 6, 16/600; running buffer 20 mM Tris-HCl pH 8, 100 mM NaCl, 2.5% glycerol) shows an equilibrium of two oligomeric states, 1 and 2, with an apparent molecular mass ratio of 9.16:1. Negative-stain EM inspection of peak 1 shows discrete CsgE particles (five representative class averages are shown in the inset, ordered by increasing tilt angles) compatible in size with nine CsgE copies. b, Selected class average of CsgE oligomer observed in top view by cryo-EM and its rotational autocorrelation show the presence of C9 symmetry. c, FSC analysis of CsgG–CsgE cryo-EM model. Three-dimensional reconstruction achieved a resolution of 24 Å as determined by FSC at a threshold of 0.5 correlation using 125 classes corresponding to 1,221 particles. d, Overlay of CsgG–CsgE cryo-EM density and the CsgG nonamer observed in the X-ray structure. The overlays are shown viewed from the side as semi-transparent density (left) or as a cross-sectional view. e, Congo red binding of E. coli BW25141DcsgG complemented with wild-type csgG (WT), empty vector (DcsgG) or csgG helix 2 mutants (single amino acid replacements labelled in single-letter code). Data are representative of four biological replicates. f, Effect of bile salt toxicity on E. coli LSR12 complemented with csgG (WT) or on csgG carrying different helix 2 mutations, complemented with (+) or without (−) csgE. Tenfold serial dilution starting from 107 bacteria were spotted on McConkey agar plates. Expression of the CsgG pore in the outer membrane leads to an increased bile salt sensitivity that can be blocked by co-expression of CsgE (n = 6, three biological replicates, with two repetitions each). g, Cross-sectional view of CsgG X-ray structure in molecular surface representation. CsgG mutants without an effect on Congo red binding or toxicity are shown in blue; mutants that interfere with CsgE-mediated rescue of bile salt sensitivity are indicated in red.

Extended Data Figure 9 Assembly and substrate recruitment of the CsgG secretion complex.

The curli transporter CsgG and the soluble secretion cofactor CsgE form a secretion complex with 9:9 stoichiometry that encloses a 24,000 Å3 chamber that is proposed to entrap the CsgA substrate and facilitate its entropy-driven diffusion across the outer membrane (OM; see the text and Fig. 4). On theoretical grounds, three putative pathways (ac) for substrate recruitment and assembly of the secretion complex can be envisaged. a, A ‘catch-and-cap’ mechanism entails the binding of CsgA to the apo CsgG translocation channel (1), leading to a conformational change in the latter that exposes a high-affinity binding platform for CsgE binding (2). CsgE binding leads to capping of the substrate cage. On secretion of CsgA, CsgG would fall back into its low-affinity conformation, leading to CsgE dissociation and liberation of the secretion channel for a new secretion cycle. b, In a ‘dock-and-trap’ mechanism, periplasmic CsgA is first captured by CsgE (1), causing the latter to adopt a high-affinity complex that docks onto the CsgG translocation pore (2), enclosing CsgA in the secretion complex. CsgA binding could be directly to CsgE oligomers or to CsgE monomers, the latter leading to subsequent oligomerization and CsgG binding. Secretion of CsgA leads CsgE to fall back into its low-affinity conformation and to dissociate from the secretion channel. c, CsgG and CsgE form a constitutive complex, in which CsgE conformational dynamics cycle between open and closed forms in the course of CsgA recruitment and secretion. Currently published or available data do not allow us to discriminate between these the putative recruitment modes or derivatives thereof, or to put forward one of them.

Extended Data Figure 10 Data collection statistics and electron density maps of CsgGC1S and CsgG.

a, Data collection statistics for CsgGC1S and CsgG X-ray structures. b, Electron density map at 2.8 Å for CsgGC1S calculated using NCS-averaged and density-modified experimental SAD phases, and contoured at 1.5σ. The map shows the region of the channel construction (CL; a single protomer is labelled) and is overlaid on the final refined model. c, Electron density map (resolutions 3.6, 3.7 and 3.8 Å along reciprocal vectors a*, b* and c*, respectively) in the CsgG TM domain region, calculated from NCS-averaged and density-modified molecular replacement phases (TM loops were absent from the input model); B-factor sharpened by −20 Å2 and contoured at 1.0σ. The figure shows the TM1 (Lys 135–Leu 154) and TM2 (Leu 182–Asn 209) region of a single CsgG protomer, overlaid on the final refined model.

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Goyal, P., Krasteva, P., Van Gerven, N. et al. Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG. Nature 516, 250–253 (2014) doi:10.1038/nature13768

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