Weak Interactions between Salmonella enterica FlhB and Other Flagellar Export Apparatus Proteins Govern Type III Secretion Dynamics

• Published in: PloS one 2015-08, Vol.10 (8), p.e0134884-e0134884
• Main Authors: McMurry, Jonathan L, Minamino, Tohru, Furukawa, Yukio, Francis, Joshua W, Hill, Stephanie A, Helms, Katy A, Namba, Keiichi
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Adenosine Triphosphate - pharmacology; Affinity; Algorithms; ATP; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding; Cellular biology; Computer Simulation; Exports; Flagella; Flagella - metabolism; Immunoblotting; Kinetics; Membrane proteins; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mutation; Oligomerization; Physiological aspects; Plasmids; Protein Binding; Protein interaction; Protein Multimerization - drug effects; Protein Transport; Protein-protein interactions; Proteins; Proton-Translocating ATPases - chemistry; Proton-Translocating ATPases - genetics; Proton-Translocating ATPases - metabolism; Salmonella; Salmonella enterica - genetics; Salmonella enterica - metabolism; Secretion; Substrate specificity; Substrates
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0134884

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.