A Tripartite Efflux System Affects Flagellum Stability in Helicobacter pylori.

Helicobacter pylori uses a cluster of polar, sheathed flagella for swimming motility. A search for homologs of H. pylori proteins that were conserved in Helicobacter species that possess flagellar sheaths but were underrepresented in Helicobacter species with unsheathed flagella identified several candidate proteins. Four of the identified proteins are predicted to form part of a tripartite efflux system that includes two transmembrane domains of an ABC transporter (HP1487 and HP1486), a periplasmic membrane fusion protein (HP1488), and a TolC-like outer membrane efflux protein (HP1489). Deleting hp1486/hp1487 and hp1489 homologs in H. pylori B128 resulted in reductions in motility and the number of flagella per cell. Cryo-electron tomography studies of intact motors of the Δhp1489 and Δhp1486/hp1487 mutants revealed many of the cells contained a potential flagellum disassembly product consisting of decorated L and P rings, which has been reported in other bacteria. Aberrant motors lacking specific components, including a cage-like structure that surrounds the motor, were also observed in the Δhp1489 mutant. These findings suggest a role for the H. pylori HP1486-HP1489 tripartite efflux system in flagellum stability. Three independent variants of the Δhp1486/hp1487 mutant with enhanced motility were isolated. All three motile variants had the same frameshift mutation in fliL, suggesting a role for FliL in flagellum disassembly.

Irregular structure of the HIV fusion peptide in membranes demonstrated by solid-state NMR and MD simulations.

To better understand peptide-induced membrane fusion at a molecular level, we set out to determine the structure of the fusogenic peptide FP23 from the HIV-1 protein gp41 when bound to a lipid bilayer. An established solid-state (19)F nuclear magnetic resonance (NMR) approach was used to collect local orientational constraints from a series of CF(3)-phenylglycine-labeled peptide analogues in macroscopically aligned membranes. Fusion assays showed that these (19)F-labels did not significantly affect peptide function. The NMR spectra were characteristic of well-behaved samples, without any signs of heterogeneity or peptide aggregation at 1:300 in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC). We can conclude from these NMR data that FP23 has a well-defined (time-averaged) conformation and undergoes lateral diffusion in the bilayer plane, presumably as a monomer or small oligomer. Attempts to evaluate its conformation in terms of various secondary structures, however, showed that FP23 does not form any type of regular helix or ß-strand. Therefore, all-atom molecular dynamics (MD) simulations were carried out using the orientational NMR constraints as pseudo-forces to drive the peptide into a stable alignment and structure. The resulting picture suggests that FP23 can adopt multiple ß-turns and insert obliquely into the membrane. Such irregular conformation explains why the structure of the fusion peptide could not be reliably determined by any biophysical method so far.