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)1–3. 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 trans-membrane 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.

Solution NMR structure of CsgE: Structural insights into a chaperone and regulator protein important for functional amyloid formation

“…The approximate ratio of the SEC molecular weight for the main (red*) and minor (red arrow) peaks was about 1:9 ( Fig. S1), suggesting the minor peak (red arrow) of WT on SEC is a nonamer, consistent with the previous report (37). On SDS-and native-PAGE (Inset), lane # was the same sample as loaded in SEC analysis; lane * was the peak marked * in SEC for WT CsgE and W48A/F79A CsgE.…”

“…S7). Interestingly, CsgG is also structurally homologous with TolB, as reported previously (37). The topology of W48A/F79A CsgE is partially similar to CsgG and its homologs (Fig.…”

“…Recently, cryo-EM data have shown that CsgE binds to the periplasmic domain of CsgG with 9:9 (CsgE:CsgG subunit) stoichiometry and forms a capping adaptor that closes off the periplasmic face of the CsgG secretion channel (37). The CsgG-CsgE complex has a preconstriction chamber (∼24,000 Å 3 ), probably entrapping substrates such as CsgA and facilitating the entropy-driven diffusion across the outer membrane (37). Hence, CsgE appears to interact with CsgA as a periplasmic chaperone that delivers the structural subunits to the periplasmic vestibule of the CsgG pore.…”

“…CsgD controls expression of the csgBAC operon as well as the biosynthesis of cellulose, the primary polysaccharide component of the biofilm extracellular matrix (33)(34)(35). CsgE and CsgF are also encoded by the operon and have long been considered accessory proteins that function in concert with CsgG, the outer membrane pore responsible for the translocation of CsgA and CsgB to the outer membrane space (1,2,(36)(37)(38). CsgG forms a symmetric, nonameric, ungated, and nonselective protein secretion channel, as revealed recently (37,39).…”

“…CsgE and CsgF are also encoded by the operon and have long been considered accessory proteins that function in concert with CsgG, the outer membrane pore responsible for the translocation of CsgA and CsgB to the outer membrane space (1,2,(36)(37)(38). CsgG forms a symmetric, nonameric, ungated, and nonselective protein secretion channel, as revealed recently (37,39). CsgF is secreted to the cell surface, binds to CsgG, and appears to be critical for the specific localization of CsgB (38).…”