title: Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity Switch Protein in theYersiniaType III Secretion System.
abstract: Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. InYersinia, 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 self-cleaving 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 earlyversuslate T3SS substrates. We also show that YscUCbinds to the inner rod protein YscI with a dissociation constant (Kd) of 3.8 μm 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.
authors.affiliation: Department of Chemistry, Chemical Biological Centre.
authors.forename: Oanh
authors.initials: O
authors.lastname: Ho
authors.affiliation: Department of Chemistry, Chemical Biological Centre.
authors.forename: Per
authors.initials: P
authors.lastname: Rogne
authors.affiliation: Department of Molecular Biology and The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, S-901 87 Umeå, Sweden.
authors.forename: Tomas
authors.initials: T
authors.lastname: Edgren
authors.affiliation: Department of Molecular Biology and The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, S-901 87 Umeå, Sweden.
authors.forename: Hans
authors.initials: H
authors.lastname: Wolf-Watz
authors.affiliation: Department of Molecular Biology and The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, S-901 87 Umeå, Sweden. Electronic address: frederic.login@clin.au.dk.
authors.forename: Frédéric H
authors.initials: FH
authors.lastname: Login
authors.affiliation: Department of Chemistry, Chemical Biological Centre. Electronic address: magnus.wolf-watz@umu.se.
authors.forename: Magnus
authors.initials: M
authors.lastname: Wolf-Watz
mesh_terms: D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D060066:Protein Interaction Maps; D013379:Substrate Specificity; D000069296:Type III Secretion Systems / Q000737:chemistry / Q000378:metabolism*; D015011:Yersinia pseudotuberculosis / Q000737:chemistry / Q000378:metabolism*; D015012:Yersinia pseudotuberculosis Infections / Q000378:metabolism* / Q000382:microbiology
chemical_list: D000069296:Type III Secretion Systems
keywords: Gram-negative bacteria; YscI; YscP; YscU; nuclear magnetic resonance (NMR); protein conformation; secretion; type III secretion system (T3SS)