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

Many 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 that intimate contact between the bacterium and its eukaryotic target cell has been established and the T3SS proteins YscP and YscU are playing 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 self-cleaving YscU variant P264A conferred significant protection against auto-proteolysis. The YscP mediated inhibition of YscU auto-proteolysis suggest that the cleavage event may act as a timing switch in the regulation of early vs. late T3SS substrates. We also show that YscUC binds to the inner-rod protein YscI with a Kd of 3.8 μM and with one-to-one stoichiometry. The significant similarity between different members of the YscU, YscP, YscI families suggests that the protein-protein interactions discussed in this study are also relevant for other T3SS-containing Gram-negative bacteria.

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