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.