Abstract
The bacterial flagellar motor is a unique supramolecular complex which converts ion flow into rotational force. Many biological devices mainly use two types of ions, proton and sodium ion. This is probably because of the fact that life originated in seawater, which is rich in protons and sodium ions. The polar flagellar motor in Vibrio is coupled with sodium ion and the energy converting unit of the motor is composed of two membrane proteins, PomA and PomB. It has been shown that the ion binding residue essential for ion transduction is the conserved aspartic acid residue (PomB-D24) in the PomB transmembrane region. To reveal the mechanism of ion selectivity, we identified essential residues, PomA-T158 and PomA-T186, other than PomB-D24, in the Na+-driven flagellar motor. It has been shown that the side chain of threonine contacts Na+ in Na+-coupled transporters. We monitored the Na+-binding specific structural changes using ATR-FTIR spectroscopy. The signals were abolished in PomA-T158A and -T186A, as well as in PomB-D24N. Molecular dynamics simulations further confirmed the strong binding of Na+ to D24 and showed that T158A and T186A hindered the Na+ binding and transportation. The data indicate that two threonine residues (PomA-T158 and PomA-T186), together with PomB-D24, are important for Na+ conduction in the Vibrio flagellar motor. The results contribute to clarify the mechanism of ion recognition and conversion of ion flow into mechanical force.
Highlights
A fundamental part of the development of life on Earth was the evolution of cells possessing a membrane between the interior and exterior environments
Escherichia coli and Salmonella have H+-driven motors, Vibrio alginolyticus and alkalophilic Bacillus have Na+-driven motors, the alkaliphilic species B. alcalophilus has a motor coupled with K+ and rubidium ion (Rb+), and Paenibacillus has a motor driven by divalent cations (Ca2+ and Mg2+)
On soft-agar plates, PomA-T5A and PomB-T21A retained motility (Fig. 2A). These mutants swam in the liquid, the swimming speeds were less than the wild type (WT) (Fig. 2B,C)
Summary
The PomA-T158A and PomA-T186A mutants were completely incapable of motility and ion transport activity, but could form the stable complex even in detergent micelle, as observed with the PomB-D24N mutant We hypothesized that these residues are involved in Na+-binding. For T158A-50, once the D24-A158 distance started to shrink, the Na+ ion near D24 totally escaped from the vicinity of the protein and did not rebind to D24 for a long period of time (it returned only at the end of the 200 ns simulation, when the D24-A158 distance started to slightly grow; see Fig. S4C,D) This behavior suggested that the configuration in which D24 and residue 158 are in close proximity might be unstable for Na+ binding when residue 158 is mutated to Ala. No compensating chain sliding in channel 2 was observed for the mutant in the simulations (Fig. S4B). In the T186 site, only one water molecule is lost, resulting in a smaller negative peak for T186A
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