Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya, Japan.
J Mol Biol. 2010 Apr 2;397(3):689-96. doi: 10.1016/j.jmb.2010.01.050. Epub 2010 Feb 1.
Rotation of the bacterial flagellar motor exploits the electrochemical potential of the coupling ion (H(+) or Na(+)) as its energy source. In the marine bacterium Vibrio alginolyticus, the stator complex is composed of PomA and PomB, and conducts Na(+) across the cytoplasmic membrane to generate rotation. The transmembrane (TM) region of PomB, which forms the Na(+)-conduction pathway together with TM3 and TM4 of PomA, has a highly conserved aspartate residue (Asp24) that is essential for flagellar rotation. This residue contributes to the Na(+)-binding site. However, it is not clear whether residues other than Asp24 are involved in binding the coupling ion. We examined the possibility that loss of the negative charge of Asp24 can be suppressed by introduction of negatively charged residues in TM3 or TM4 of PomA. The motility defect associated with the D24N substitution in PomB could be rescued only by a N194D substitution in PomA. This result suggests that there must be a negatively charged ion-binding pocket in the stator complex but that the presence of a negatively charged residue at position 24 of PomB is not essential. A tandemly fused PomA dimer containing the N194D mutation either in its N-terminal or C-terminal half with PomB-D24N was functional, suggesting that PomB-D24N can form an ion-binding pocket with either subunit of PomA dimer. The findings obtained in this study provide important clues to the mechanism of ion binding in the stator complex.
细菌鞭毛马达的旋转利用耦合离子（H(+)或 Na(+)）的电化学势作为其能量来源。在海洋细菌 AlginoLyticus 中，定子复合物由 PomA 和 PomB 组成，并通过质膜传导 Na(+)以产生旋转。PomB 的跨膜（TM）区域与 PomA 的 TM3 和 TM4 一起形成 Na(+)传导途径，其中含有一个高度保守的天冬氨酸残基（Asp24），对于鞭毛旋转至关重要。该残基有助于 Na(+)结合位点。然而，目前尚不清楚除 Asp24 以外的残基是否参与结合耦合离子。我们研究了这种可能性，即 Asp24 的负电荷丧失可以通过在 PomA 的 TM3 或 TM4 中引入带负电荷的残基来抑制。只有在 PomA 中进行 N194D 取代，才能挽救 PomB 中 D24N 取代所导致的运动缺陷。这一结果表明，定子复合物中必须存在一个带负电荷的离子结合口袋，但 PomB 中第 24 位带负电荷的残基的存在并非必需。一个串联融合的 PomA 二聚体，在其 N 端或 C 端的一半与 PomB-D24N 中含有 N194D 突变，是有功能的，这表明 PomB-D24N 可以与 PomA 二聚体的任何一个亚基形成一个离子结合口袋。本研究的结果为定子复合物中离子结合的机制提供了重要线索。
