Structural characterization of components of the flagellar export apparatus from the H. pylori

Abstract

H. pylori is a human gastric pathogen responsible for serious health conditions such as gastritis and peptic ulcers. It requires polar sheathed flagella for initial colonization and manifestation of infection in the human stomach. The export of flagellum components requires a specialized protein complex called the export apparatus. FliI ATPase, FliH, and FliJ are major soluble components of the flagellar export apparatus. FliI is thought to provide energy for export of flagellum components by converting the energy of ATP hydrolysis into energy for export of proteins. In vitro, FliH is a negative regulator of FliI ATPase activity. FliJ is a general export co-chaperone. In the case of H. pylori, there is no strong evidence for the existence of FliJ. There are other important proteins involved in flagellum assembly such as flagellin, FliS and FlhA. The project involves protein characterization and crystallization studies of proteins FliH, FliI, FliS, flagellin and the identification of FliJ in H. pylori. The in-vitro characterization studies of two constructs of H. pylori FliH (FliH (57-258) and FliH (73-258)) suggests that FliH likely forms a homodimer in solution and it forms an elongated structure. The crystallization studies of both these proteins yielded beautiful crystals but getting structural information was challenging due to diffraction quality of the crystals. The site-directed mutagenesis of FliI was successfully performed to produce FliIE193Q mutant. The protein characterization studies showed that FliIE193Q mutant precipitates at every stage of purification. The gel filtration suggests that it forms a monomer in solution. The crystallization studies of FliIE193Q mutant did not produce any crystals possibly due to solubility issues. The interaction studies of FliH and the FliIE193Q mutant suggests that it most likely forms FliH2:FliI complex. Bioinformatics studies suggests HP0256 as a potential homolog of FliJ (Douillard et al., 2010). The protein characterization studies showed that HP0256 forms a monomer in solution. Unfortunately, no crystals were observed for HP0256. The structural studies of FliS showed that it forms an antiparallel four-helix bundle. The binding studies (GST-pulldown) of FliS and flagellin showed that the flagellin forms a complex with the FliS. Overall, we have characterized some of the key proteins from the H. pylori flagellar export system.