Mechanism of type‐III protein secretion: Regulation of FlhA conformation by a functionally critical charged‐residue cluster

• Published in: Molecular microbiology Vol. 104; no. 2; pp. 234 - 249
• Main Authors: Erhardt, Marc, Wheatley, Paige, Kim, Eun A, Hirano, Takanori, Zhang, Yang, Sarkar, Mayukh K., Hughes, Kelly T., Blair, David F.
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01-04-2017
• Subjects: Amino Acid Sequence; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Crystal structure; Flagella - metabolism; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mutation; Protein Conformation; Protein Structure, Tertiary; Protein Transport - physiology; Proteins; Structure-Activity Relationship; Type III Secretion Systems - metabolism; Type III Secretion Systems - physiology
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/mmi.13623

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.
A key proton‐interacting site is identified in the flagellar protein FlhA and is shown to modulate conformation of the cargo‐engaging parts of the protein. These findings give clues to the mechanism of the remarkably rapid protein export process known as type‐III secretion.