title: Weak Interactions between Salmonella enterica FlhB and Other Flagellar Export Apparatus Proteins Govern Type III Secretion Dynamics.
abstract: The bacterial flagellum contains its own type III secretion apparatus that coordinates protein export with assembly at the distal end. While many interactions among export apparatus proteins have been reported, few have been examined with respect to the differential affinities and dynamic relationships that must govern the mechanism of export. FlhB, an integral membrane protein, plays critical roles in both export and the substrate specificity switching that occurs upon hook completion. Reported herein is the quantitative characterization of interactions between the cytoplasmic domain of FlhB (FlhBC) and other export apparatus proteins including FliK, FlhAC and FliI. FliK and FlhAC bound with micromolar affinity. KD for FliI binding in the absence of ATP was 84 nM. ATP-induced oligomerization of FliI induced kinetic changes, stimulating fast-on, fast-off binding and lowering affinity. Full length FlhB purified under solubilizing, nondenaturing conditions formed a stable dimer via its transmembrane domain and stably bound FliH. Together, the present results support the previously hypothesized central role of FlhB and elucidate the dynamics of protein-protein interactions in type III secretion.
authors.affiliation: Department of Molecular & Cellular Biology, Kennesaw State University, Kennesaw, Georgia, United States of America.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: McMurry
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Minamino
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Furukawa
authors.affiliation: Department of Molecular & Cellular Biology, Kennesaw State University, Kennesaw, Georgia, United States of America.
authors.forename: Joshua W
authors.initials: JW
authors.lastname: Francis
authors.affiliation: Department of Molecular & Cellular Biology, Kennesaw State University, Kennesaw, Georgia, United States of America.
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Hill
authors.affiliation: Department of Molecular & Cellular Biology, Kennesaw State University, Kennesaw, Georgia, United States of America.
authors.forename: Katy A
authors.initials: KA
authors.lastname: Helms
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Namba
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000465:Algorithms; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003198:Computer Simulation; D005407:Flagella / Q000378:metabolism*; D015151:Immunoblotting; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization / Q000187:drug effects; D021381:Protein Transport; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019779:Salmonella enterica / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C080688:FlhA protein, Bacteria; C085668:FlhB protein, Bacteria; C099486:FliK protein, Bacteria; D008565:Membrane Proteins; C069250:fliH protein, Bacteria; C069251:fliI protein, bacteria; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases