Structural dynamics of the functional nonameric Type III translocase export gate

Abstract

The Type III protein secretion (T3S) pathway is widespread in bacterial Gram-negative pathogens. It comprises the injectisome with a cytoplasm-facing inner membrane translocase and a surface-exposed needle. The translocase comprises a conical SctR5S4T1 export channel, decorated by SctU, and enveloped by SctV. The large cytoplasmic domain (C-domain) of SctV binds T3S chaperone/exported protein and forms a putative ante-chamber leading to the membrane translocase. Here we probed the mechanism of assembly and function of SctV. Using live cell imaging, SctV was shown to assemble in peripheral oligomeric clusters in both EPEC and a non-T3SS harbouring E.coli strain. Non-ionic detergents extracted SctV homo-nonamers from membranes of both strains. His-SctV9 reconstituted in peptidiscs revealed an elongated, tripartite particle of ~22nm with a membrane domain and a narrower linker connecting to a C-domain. The C-domain assembles in a hollow asymmetric ring with a 5-6 nm-wide inner opening. SctV9 is necessary and sufficient to act as a receptor for two different chaperone/exported protein pairs by binding them at distinct C-domain sites identified by immobilized peptide arrays. Binding sites are not only important for binding but also essential for secretion suggesting a close mechanistic link between the receptor and secretion activities. These findings advance structural understanding of injectisome assembly and reveal that chaperone/exported protein targeting is mechanistically uncoupled from the succeeding translocation step.Author summaryThe export apparatus of the Type III secretion pathway is conserved in flagellar and virulence injectisomes. Its major component SctV, is essential for T3S substrate targeting and translocation. Here, we analysed SctV assembly and function as a receptor for targeting T3S exported proteins. SctV was shown to self-nonamerize, in a structure that is sufficient for functional binding of chaperone/exported protein complexes. Nonameric SctV reconstituted in peptidiscs and its nonameric ring-forming cytoplasmic domain reveal structural features and lay the foundation for high-resolution cryoEM. These tools set the stage for mechanistic dissection of the structural interactions of the export apparatus with the exported proteins, independently of the transmembrane crossing reaction.

Crystals of SctV from different species reveal variable symmetry for the cytosolic domain of the type III secretion system export gate

“…T3SS export gates have been shown to form cyclic nonamers via their cytoplasmic domains (Abrusci et al, 2013;Majewski et al, 2020;Jensen et al, 2020;Matthews-Palmer et al, 2021;Xu et al, 2021;Kuhlen et al, 2021;Yuan et al, 2021;Gilzer et al, 2022). We purified the cytosolic domain of P. luminescens LscV (LscV C ) and successfully crystallized it using 0.1 M Tris-HCl pH 8.0, 1.3 M ammonium sulfate at 293 K. Unfortunately, the purification of LscV C could not be reproduced.…”

TypeIIIsecretion byYersinia pseudotuberculosisis reliant upon an authentic N‐terminalYscXsecretor domain

“…Thus, it is utterly intriguing that the Ysc‐T3SS clade has evolved a unique system in YscX–YscY‐SctV for coordinating early substrate secretion. Several structural studies have proposed a conserved region in the cytoplasmic domain of SctV that is thought to recognize a chaperone‐substrate complex and prepare substrates for secretion (Erhardt et al, 2017; Kuhlen et al, 2021; Majewski et al, 2020; Matthews‐Palmer et al, 2021; Xing et al, 2018; Yuan et al, 2021). Hence, SctV has a conserved universal role in all T3SSs, although bacteria producing the Ysc‐T3SS clade seems to have customized this function in a way that relies on the YscX–YscY complex.…”

Recent structural advances towards understanding of the bacterial type III secretion injectisome

“…However, many of the structures obtained using these techniques represent individual protein monomers or truncated subdomains, and therefore provide limited insights into the dynamic interactions and transient nature of the larger oligomeric multiprotein complexes that make up the injectisome. Our molecular understanding of the injectisome has accelerated in the past 5 years, and the structures of many of its components have been determined, including those of the central needle bound complex [22][23][24], basal body [25,26], export gate [27][28][29][30], ATPase [31], needle filament [23,32], and tip [33,34]. The progression made has been greatly facilitated by technological improvements in the field of cryoelectron microscopy (cryoEM): namely single-particle analysis (SPA) and cryoelectron tomography (cryoET).…”

“…Structural characterisation efforts have been performed in several homologues, using a combination of XRC, SPA (Figure 2E) and cryoET approaches [15,21,[28][29][30]73,74]. SctV forms a multimeric assembly; however, to date, atomic resolution structures defining only the soluble cytosolic domain are available from both XRC [15] and SPA [27][28][29][30], revealing a homo-nonomeric ring. The soluble cytosolic domain is thought to be involved in substrate binding, selection, and switching [75,76].…”

“…Despite successful efforts to express and purify the full-length export gate, deriving a high-resolution structure of the transmembrane domain has proved challenging [27,28,30]. Kuhlen et al expressed and purified full-length YscV FL and flagellar orthologue FlhA FL for structural determination by SPA (Figure 2Eiii) [28].…”