Intermolecular cross-linking between the periplasmic Loop3-4 regions of PomA, a component of the Na+-driven flagellar motor of Vibrio alginolyticus

PomA and PomB form a complex that conducts sodium ions and generates the torque for the Na+-driven polar flagellar motor of Vibrio alginolyticus, PomA has four transmembrane segments. One periplasmic loop (loop,,) connects segments 1 and 2, and another (loop(3-4)), in which cysteine-scanning mutagenesis had been carried out, connects segments 3 and 4, When PomA with an introduced Cys residue (Cys-PomA) in the C-terminal periplasmic loop (loop(3-4)) was examined without exposure to a reducing reagent, a 43-kDa band was observed, whereas only a 25-kDa band, which corresponds to monomeric PomA, was observed under reducing conditions, The intensity of the 43-kDa band was enhanced in most mutants by the oxidizing reagent CuCl2. The 43-kDa band was strongest in the P172C mutant. The motility of the P172C mutant was severely reduced, and P172C showed a dominant-negative effect, whereas substitution of Pro with Ala, Ile, or Ser at this position did not affect motility, In the presence of DTT, the ability to swim was partially restored; and the amount of 43-kDa protein was reduced. These results suggest that the disulfide cross-link disturbs the Reaction of PomA, When the mutated Cys residue was modified with N-ethyhmaleimide, only the 25-kDa PomA band was labeled, demonstrating that the 43-kDa form is a cross-linked homodimer and suggesting that the loops,4 of adjacent subunits of PomA are close to each other in the assembled motor. We propose that this loop region is important for dimer formation and motor function.