Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity Switch Protein in the Yersinia Type III Secretion System

Abstract

Edited by Thomas Sö llnerMany pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia, the switch to secretion of effector proteins is induced first after intimate contact between the bacterium and its eukaryotic target cell has been established, and the T3SS proteins YscP and YscU play a central role in this process. Here we identify the molecular details of the YscP binding site on YscU by means of nuclear magnetic resonance (NMR) spectroscopy. The binding interface is centered on the C-terminal domain of YscU. Disrupting the YscU-YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the binding of YscP to the slowly selfcleaving YscU variant P264A conferred significant protection against autoproteolysis. The YscP-mediated inhibition of YscU autoproteolysis suggests that the cleavage event may act as a timing switch in the regulation of early versus late T3SS substrates. We also show that YscU C binds to the inner rod protein YscI with a dissociation constant (K d ) of 3.8 M and with 1:1 stoichiometry. The significant similarity among different members of the YscU, YscP, and YscI families suggests that the protein-protein interactions discussed in this study are also relevant for other T3SS-containing Gram-negative bacteria.

The flexible linker of the secreted FliK ruler is required for export switching of the flagellar protein export apparatus

“…Recent photo-crosslinking experiments have demonstrated that the conserved core domain of FliK C directly binds to FlhB C 45 . Similar protein-protein interactions have been observed in the injectisome 55 . These suggest that length control and substrate specificity switching mechanisms are conserved in both flagellar and injectisome systems.…”

The role of intrinsically disordered C‐terminal region of FliK in substrate specificity switching of the bacterial flagellar type III export apparatus

“…It has been shown that the C-terminal domain of YscP (YscP C ), which is a FliK homologue of the Yersinia type III secretion system, directly binds to the C-terminal domain of YscU (YscU C ), which is a FlhB homologue. Ala-355 of YscU C , which corresponds to Ala-343 of FlhB CC , is critical for the binding to YscP C , and Leu-280 of YscU C , which corresponds to Ala-286 of FlhB CC , contributes to this binding (Ho et al, 2017). Ala-343 is located in helix a4, and Ala-286 is located in a loop between the b2 and b3 strands.…”

Mutational analysis of the C‐terminal cytoplasmic domain of FlhB, a transmembrane component of the flagellar type III protein export apparatus in Salmonella

“…FlhB C is involved in the substrate specificity switching along with the molecular ruler protein FliK, which is also secreted via the flagellar type III protein export apparatus during hook assembly (Erhardt, Singer, Wee, Keener, & Hughes, ; Kutsukake, Minamino, & Yokoseki, ; Minamino, González‐Pedrajo, Yamaguchi, Aizawa, & Macnab, ; Minamino, Moriya, Hirano, Hughes, & Namba, ; Moriya, Minamino, Hughes, Macnab, & Namba, ; Shibata et al, ; Uchida & Aizawa, ; Williams et al, ). The C‐terminal domain of a FliK homologue, YscP, of the Yersinia injectisome binds to the C‐terminal cytoplasmic domain of a FlhB homologue, YscU, to promote substrate specificity switching of the virulence‐associated type III protein export apparatus (Ho et al, ). Consistently, photo‐cross‐linking experiments have showed a direct interaction between FlhB C and the C‐terminal domain of FliK (FliK C ; Kinoshita, Aizawa, Inoue, Namba, & Minamino, ).…”