Figure 7.

Mapping the binding interface between FliH and FliY-FliN. A, excerpted T-COFFEE alignment of H. pylori (HPY) FliH with its homologues from Helicobacter hepaticus (HHE), C. jejuni (CJJ), Wolinella succinogenes (WSU), S. enterica Typhimurium (STY), Shigella flexneri (SFL), Yersinia pestis (YPE), and E. coli (ECO). The conserved hydrophobic residues are colored orange (H. pylori numbering). B, effect of FliH truncation on the binding to FliY-FliN. GST, GST-FliH, or GST-FliH^Δ27 was immobilized, and the beads were incubated with purified FliY-FliN. C, model of H. pylori FliH_N (residues 18–29) docked onto FliY_C–FliN_C. The initial model of FliH_N was generated by Modeler using FliH_N of S. enterica Typhimurium (PDB code 4YXC) as a template (44). The peptide was docked onto the FliY_C–FliN_C complex using the flexible docking protocol Rosetta FlexPepDock (55). The model with the highest score was chosen. The binding site for FliH_N is shown as electrostatic surface in the boxed-in region. The molecular surface was calculated by APBS (Adaptive Poisson-Boltzmann Solver) and contoured at keT = ±3. The conserved hydrophobic residues of FliH_N and FliY_C–FliN_C that participated in the interaction are represented as stick and ball-and-stick models, respectively. D, effect of FliY–FliN mutations on FliH interaction. The GST-FliY–FliN mutants were immobilized on resin followed by incubation with purified FliH.