Abstract
Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their hollow needle into a host, requiring co-ordination. In the Salmonella enterica serovar Typhimurium SPI-2 injectisome, this switch is triggered by sensing the neutral pH of the host cytoplasm. Central to specificity switching is a nonameric SctV protein with an N-terminal transmembrane domain and a toroidal C-terminal cytoplasmic domain. A ‘gatekeeper’ complex interacts with the SctV cytoplasmic domain in a pH dependent manner, facilitating translocon secretion while repressing effector secretion through a poorly understood mechanism. To better understand the role of SctV in SPI-2 translocon-effector specificity switching, we purified full-length SctV and determined its toroidal cytoplasmic region’s structure using cryo-EM. Structural comparisons and molecular dynamics simulations revealed that the cytoplasmic torus is stabilized by its core subdomain 3, about which subdomains 2 and 4 hinge, varying the flexible outside cleft implicated in gatekeeper and substrate binding. In light of patterns of surface conservation, deprotonation, and structural motion, the location of previously identified critical residues suggest that gatekeeper binds a cleft buried between neighboring subdomain 4s. Simulations suggest that a local pH change from 5 to 7.2 stabilizes the subdomain 3 hinge and narrows the central aperture of the nonameric torus. Our results are consistent with a model of local pH sensing at SctV, where pH-dependent dynamics of SctV cytoplasmic domain affect binding of gatekeeper complex.
Highlights
Bacterial type III secretion systems (T3SSs) are required for virulence by many bacteria
How does SctV detect changes in pH? Through direct deprotona tion of SctV, communicated by other proteins of the injectisome, or through a more indirect process? What is the role of the unique Salmonella Pathogenicity Island 2 (SPI-2) Cterminal extension? What conformation or dynamic changes in SctV arise, and how do they cause changes in export specificity? Here we describe our work to determine the structure of SctVSPI-2 and simulate its dynamics at the different pH values that determine the substrate switch
SctV is composed of an N-terminal transmembrane domain (TMD) SctVN joined by a linker to a C-terminal cytosolic domain SctVC
Summary
Bacterial type III secretion systems (T3SSs) are required for virulence by many bacteria. T3SSs are multiprotein inner membrane complexes that export virulence proteins and self-assembling components of one of two types of trans-periplasmic molecular machines through hollow axial structures. Upon the hook reaching a predetermined length, the T3SS switches export specificity to export subunits of the multi-micron long flagellum, which forms a helical propellor for motility. The second type of T3SSbased molecular machines, injectisomes (sometimes referred to as “type III secretion systems”, or non-flagellar T3SSs), are used by a wide variety of bacterial pathogens of animals and plants but differ functionally to flagella as virulence protein delivery machines that evoke molecular hypodermic syringes (Fig. 1A). Upon the needle reaching a predetermined length, the T3SS switches specificity to export translocon proteins that cap the needle and form a pore in host cell membranes (Journet et al, 2003). The T3SS switches specificity again to export virulence proteins (effectors) through the translocon pore into the host cell where they hijack host cell physiology to benefit the pathogen (Figueira and Holden, 2012)
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