JST, ICORP* Graduate School of Frontier Biosciences, Osaka University, JAPAN and ICORP, JST** Department of Molecular Biophysics and Biochemistry, Yale Univ*** Institute of Molecular and Cellular Biosciences, Univ. of Tokyo and CREST, JST****
â—‹Yumiko Saijo-Hamano* Katsumi Imada** Tohru Minamino** May Kihara*** Akio Kitao**** Keiichi Namba**
The bacteria flagellum has the type III protein export apparatus as one part of the basal body. It selectively translocates flagellar axial proteins into the central channel of the flagellum. The export apparatus consists of six integral membrane components (FlhA, FlhB, FliO, FliP, FliQ and FliR) and three cytoplasmic components (FliH, FliI and FliJ). Genetic and biochemical information are available for some of these proteins. However, the export mechanism is not clear except that the energy released by ATP hydrolysis by FliI is used for the export process. FlhA from Salmonella is an integral membrane component of the flagellar specific type III protein export apparatus. It consists of an N-terminal transmembrane domain (FlhATM) and a C-terminal cytoplasmic domain (FlhAC). We solved a 2.8 Å crystal structure of FlhAC, which is composed of one elongated linker region continued from FlhATM and four subdomains (ACD1, ACD2, ACD3 and ACD4). We thought that FlhAC could conformational change from the structure, therefore we curried out molecular dynamics simulation of FlhAC. The simulation showed that FlhAC periodically repeated conformational change to open and then closed state using two hinges between ACD1 and ACD3, and ACD3 and ACD4. And also substitution mutations of key residues interacting between the subdomains failed to complement to motility of flhA-null mutant. These results suggest that the fluctuation of conformational change is important to function of FlhAC. The crystal structure, together with the result of multicopy effects of N-terminal half of FlhAC to motility of wild-type cell and previous studies, suggest that linker region and ACD1 act as holding soluble export apparatus components, probably FliI, which is a ATP-ase.