Arrangement of core membrane segments in the MotA/MotB proton-channel complex of Escherichia coli
Arrangement of core membrane segments in the MotA/MotB proton-channel complex of Escherichia coli
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DOI：
10.1021/bi035406d
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发表时间：
2004-01-13
期刊：
影响因子：
2.9
通讯作者：
Blair, DF
中科院分区：
文献类型：
--
作者：
Braun, TF;Al-Mawsawi, LQ;Blair, DF
细菌鞭毛马达的定子由膜蛋白MotA和MotB形成，它们以化学计量比为MotA(4)MotB(2)的复合物形式结合（小岛，S.，和布莱尔，D.F.，本期前文）。MotA/MotB复合物使离子跨膜传导，并通过一种似乎涉及复合物内构象变化的机制将离子流与鞭毛旋转耦合。MotA有四个跨膜区段，称为A1 - A4，MotB有一个，称为B。我们正在通过靶向二硫键交联研究MotA(4)MotB(2)复合物中18个膜区段的组织。先前的一项交联研究表明，复合物中的两个B区段（每个MotB亚基各一个）排列成α - 螺旋的对称二聚体。在此，我们将交联研究扩展到A3和A4区段。在A3和A4区段的几个连续位置通过突变引入单个半胱氨酸残基，并通过A3、A4和B区段中半胱氨酸替换子集的成对组合构建双突变体。在全细胞、膜和去污剂溶液中研究了单半胱氨酸和双半胱氨酸蛋白质的二硫键交联。A3和B区段中半胱氨酸残基的几种组合在碘氧化时产生了高产率的二硫键连接的MotA/MotB异二聚体。有效交联的位置确定了A3区段上与B区段邻近的一个螺旋面。A4和B区段中半胱氨酸残基的一些组合也产生了显著量的二硫键连接的异二聚体，表明A4区段也靠近B区段。A3和A4区段中半胱氨酸残基的某些组合在氧化时交联形成高产率的MotA四聚体。高产率位置确定了A3和A4上位于MotA亚基之间界面的面。结合突变研究和氨基酸保守模式，交联结果描绘了MotA/MotB复合物中10个膜区段的整体排列，并确定了可能排列在质子通道内的螺旋面。
The stator of the bacterial flagellar motor is formed from the membrane proteins MotA and MotB, which associate in complexes with stoichiometry MotA(4)MotB(2) (Kojima, S., and Blair, D. F., preceding paper in this issue). The MotA/MotB complexes conduct ions across the membrane, and couple ion flow to flagellar rotation by a mechanism that appears to involve conformational changes within the complex. MotA has four membrane-crossing segments, termed A1-A4, and MotB has one, termed B. We are studying the organization of the 18 membrane segments in the MotA(4)MotB(2) complex by using targeted disulfide cross-linking. A previous cross-linking study showed that the two B segments in the complex (one from each MotB subunit) are arranged as a symmetrical dimer of alpha-helices. Here, we extend the cross-linking study to segments A3 and A4. Single Cys residues were introduced by mutation in several consecutive positions in segments A3 and A4, and double mutants were made by pairwise combination of subsets of the Cys replacements in segments A3, A4, and B. Disulfide cross-linking of the single- and double-Cys proteins was studied in whole cells, in membranes, and in detergent solution. Several combinations of Cys residues in segments A3 and B gave a high yield of disulfide-linked MotA/MotB heterodimer upon oxidation with iodine. Positions of efficient cross-linking identify a helix face on segment A3 that is in proximity to segment(s) B. Some combinations of Cys residues in segments A4 and B also gave a significant yield of disulfide-linked heterodimer, indicating that segment A4 is also near segment(s) B. Certain combinations of Cys residues in segments A3 and A4 cross-linked to form MotA tetramers in high yield upon oxidation. The high-yield positions identify faces on A3 and A4 that are at an interface between MotA subunits. Taken together with mutational studies and patterns of amino acid conservation, the cross-linking results delineate the overall arrangement of 10 membrane segments in the MotA/MotB complex, and identify helix faces likely to line the proton channels.