Structure of the core of the type III secretion system export apparatus

Abstract

Export of proteins through type III secretion systems is critical for motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP, FliQ, FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. Here, we present the cryoelectron microscopy structure of the Salmonella Typhimurium FliP-FliQ-FliR complex at 4.2 Å. None of the subunits adopt canonical integral membrane protein topologies, and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems, combined with cross-linking, localize the export gate as a core component of the periplasmic portion of the machinery. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.

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

“…The core export apparatus components SctR, SctS and SctT form a helical assembly with nearly the same helical parameters as the needle filament: about 5.5 subunits per turn and a helical rise of about 4.0 Å per subunit (Kuhlen et al , ). These data suggest that the core export apparatus templates assembly of the helical filament and thus, also the inner rod should possess these helical properties.…”

“…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).…”

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

“…The high level of sequence conservation within all T3SS implied that this complex would be similarly assembled in virulence T3SS. Native mass spectrometry (nMS) of purified virulence system complexes supported this, revealing a core SctR5T1 complex equivalent to FliP5R1 (Kuhlen, Abrusci et al 2018). However, the number of SctS subunits was highly variable in samples and seemed to reflect lower stability of these complexes c.f.…”

“…We have recently demonstrated that a subset of these proteins assembles into a core exportgate complex, and reported the structure of a flagellar FliP5Q4R1 (hereafter termed FliPQR) complex at 4.2 Å structure by cryo-electron microscopy (Kuhlen, Abrusci et al 2018). Strikingly placing the FliPQR structure into lower resolution basal body structures revealed that this complex, built from three putative membrane proteins and purified from membranes when expressed in isolation of the rest of the T3SS, physiologically exists in an 20 extra-membrane location at the core of the basal body (Worrall, Hong et al 2016, Kuhlen, Abrusci et al 2018}. This positioning of the complex, in conjunction with the observation that it exhibits helical symmetry, suggested that it seeds assembly of the axial helical components that culminate in the flagellum or needle.…”

“…However, the number of SctS subunits was highly variable in samples and seemed to reflect lower stability of these complexes c.f. the flagellar ones (Kuhlen, Abrusci et al 2018). This decreased stability meant that determination of a structure from a non-flagellar export gate complex was not previously 30 possible.…”