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Mechanism of type‐ III protein secretion: Regulation of F lh A conformation by a functionally critical charged‐residue cluster
Author(s) -
Erhardt Marc,
Wheatley Paige,
Kim Eun A,
Hirano Takanori,
Zhang Yang,
Sarkar Mayukh K.,
Hughes Kelly T.,
Blair David F.
Publication year - 2017
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.13623
Subject(s) - biology , transmembrane domain , secretion , cytoplasm , flagellum , inner membrane , microbiology and biotechnology , transport protein , transmembrane protein , protonation , biophysics , protein structure , biochemistry , chemiosmosis , membrane , gene , atp synthase , receptor , ion , physics , quantum mechanics
Summary 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.