The structure of an injectisome export gate demonstrates conservation of architecture in the core export gate between flagellar and virulence type three secretion systems

Abstract

Export of proteins through type three secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported though a genetically defined export gate complex consisting of three proteins. We have recently shown at 4.2 Å that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assemble into an extra-membrane helical assembly that likely seeds correct assembly of the rod above. Here we present the structure of an equivalent complex from the more fragile Shigella virulence system at 3.5 Å by cryo-electron microscopy. This higher resolution structure reveals further detail and confirms the prediction of 30 structural conservation in this core complex. Analysis of particle heterogeneity also reveals details of how the SctS/FliQ subunits sequentially assemble in the complex.Introduction:

The inner rod of virulence‐associated type III secretion systems constitutes a needle adapter of one helical turn that is deeply integrated into the system's export apparatus

“…In general, crosslinks to SctT were weaker than to SctR, which may imply a less optimal fit of the two interaction partners. In fact, SctR and SctT show the biggest structural difference in their distal, inner rod facing part (Johnson et al , ). Very prominent crosslinks between SctI and SctR were observed with p Bpa at positions 52, 83 and 95 of SctI (Fig.…”

“…The SctRST complex structure from Shigella flexneri (PDB 6R6B) (Johnson et al , ) was used as template to model the St 1 SctRST complex, due to the high similarity (Table ). The template structure has missing residues that encompass the St 1 SctR N‐terminal region, until the residue 8, and the loops between Val73–Leu96 and Phe115–Pro141, as well as the St 1 SctT N‐ and C‐terminal regions until Ile9 and from Tyr254 respectively.…”

“…The inner rod itself is anchored to the core export apparatus components SctR and SctT, that, together with SctS, also form a helical assembly with nearly identical helical parameters as the needle filament ( Fig. 1B,C) (Dietsche et al, 2016;Kuhlen et al, 2018;Johnson et al, 2019).…”

Structural dynamics of the functional nonameric Type III translocase export gate

“…Chaperone/exported protein complexes also interact with the hexameric ATPase of the system in NMR-identified complexes (20) and with cytoplasmic platform components in pull-down experiments (17, 21, 22). Although the order of chaperone/exported protein interactions with machinery components during secretion remains debatable, such interactions presumably attract exported substrates to SctV (9, 19), dissociate them from chaperones, let them diffuse to the SctV vestibule and, finally, to the inner membrane SctV/RSTU channel for export, possibly via a putative channel running longitudinally through SctRST (23, 24). Although the internal SctRST pathway is closed in all available structures (24, 25), it may dilate like a diaphragm.…”

“…Although the order of chaperone/exported protein interactions with machinery components during secretion remains debatable, such interactions presumably attract exported substrates to SctV (9, 19), dissociate them from chaperones, let them diffuse to the SctV vestibule and, finally, to the inner membrane SctV/RSTU channel for export, possibly via a putative channel running longitudinally through SctRST (23, 24). Although the internal SctRST pathway is closed in all available structures (24, 25), it may dilate like a diaphragm. These steps are poorly understood, have not been reconstituted in vitro , and the precise order and roles of the translocase components are unknown.…”