title: Mechanism of type-III protein secretion: Regulation of FlhA conformation by a functionally critical charged-residue cluster.
abstract: The bacterial flagellum contains a specialized secretion apparatus in its base that pumps certain protein subunits through the growing structure to their sites of installation beyond the membrane. A related apparatus functions in the injectisomes of gram-negative pathogens to export virulence factors into host cells. This mode of protein export is termed type-III secretion (T3S). Details of the T3S mechanism are unclear. It is energized by the proton gradient; here, a mutational approach was used to identify proton-binding groups that might function in transport. Conserved proton-binding residues in all the membrane components were tested. The results identify residues R147, R154 and D158 of FlhA as most critical. These lie in a small, well-conserved cytoplasmic domain of FlhA, located between transmembrane segments 4 and 5. Two-hybrid experiments demonstrate self-interaction of the domain, and targeted cross-linking indicates that it forms a multimeric array. A mutation that mimics protonation of the key acidic residue (D158N) was shown to trigger a global conformational change that affects the other, larger cytoplasmic domain that interacts with the export cargo. The results are discussed in the framework of a transport model based on proton-actuated movements in the cytoplasmic domains of FlhA.
authors.affiliation: Department of Biology, University of Utah, Salt Lake City, UT, 84112, USA.|Junior Research Group Infection Biology of Salmonella, Helmholtz Centre for Infection Research, Inhoffenstraβe 7, Braunschweig, 38124, Germany.
authors.forename: Marc
authors.identifier: 0000-0001-6292-619X
authors.initials: M
authors.lastname: Erhardt
authors.affiliation: Department of Biology, University of Utah, Salt Lake City, UT, 84112, USA.
authors.forename: Paige
authors.identifier: 0000-0002-5324-0089
authors.initials: P
authors.lastname: Wheatley
authors.affiliation: Department of Biology, University of Utah, Salt Lake City, UT, 84112, USA.
authors.forename: Eun A
authors.initials: EA
authors.lastname: Kim
authors.affiliation: Department of Biology, University of Utah, Salt Lake City, UT, 84112, USA.|Toshiba Medical Service Corporation, 1385 Shimoichigami, Otawara-shi, Tochigi, 324-8550, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Hirano
authors.affiliation: Department of Biology, University of Utah, Salt Lake City, UT, 84112, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Mayukh K
authors.initials: MK
authors.lastname: Sarkar
authors.affiliation: Department of Biology, University of Utah, Salt Lake City, UT, 84112, USA.
authors.forename: Kelly T
authors.initials: KT
authors.lastname: Hughes
authors.affiliation: Department of Biology, University of Utah, Salt Lake City, UT, 84112, USA.
authors.forename: David F
authors.initials: DF
authors.lastname: Blair
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D005407:Flagella / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D013329:Structure-Activity Relationship; D000069296:Type III Secretion Systems / Q000378:metabolism* / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; C080688:FlhA protein, Bacteria; D008565:Membrane Proteins; D000069296:Type III Secretion Systems