Conformational change in the stator of the bacterial flagellar motor
Conformational change in the stator of the bacterial flagellar motor
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DOI：
10.1021/bi011263o
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发表时间：
2001-10-30
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：
Kojima, S;Blair, DF
MOTA和MOTB是构成质子燃料鞭毛旋转马达定子的大肠杆菌的完整膜蛋白。马达包含几个MOTA/MOTB复合体，它们独立地发挥作用，引导质子穿过细胞膜，并将质子流动耦合到旋转。MOTB含有一个保守的天冬氨酸残基，Asp32，对旋转至关重要。我们提出，激励电机的质子与MOTB的Asp32相互作用，诱导定子的构象变化，从而驱动转子的运动。为了测试构象变化，我们检测了MOTA在与野生型MOTB或在第32位突变的MOTB的膜结合复合体中的蛋白酶敏感性。MOTB中Asp32的小而不带电荷的替代(D32N、D32A、D32G、D32S或D32C)引起了MOTA构象的显著变化，蛋白质降解片段的模式发生了变化。这种构象变化除了MOTA和MOTB外不需要任何鞭毛蛋白，因为它仍然在一株不表达其他鞭毛基因的菌株中看到。它影响MOTA的一个细胞质结构域，该结构域包含已知与转子相互作用的残基，与产生扭矩的作用一致。同时考察了MOTA关键残基对构象的影响。MoTA的Pro173对旋转是重要的，是构象的重要决定因素：显性的Pro173突变，而不是隐性的突变，改变了MoTA的蛋白分解模式，也阻止了Asp32替换引起的构象变化。Arg90和Glu98是MoTA的残基，与转子发生静电相互作用，似乎不是膜中MoTA/MOTB络合物构象的强烈决定因素。我们注意到MoTA和ExbB的序列相似，ExbB是一种细胞质膜蛋白，它为革兰氏阴性细菌的外膜运输提供能量。ExbB和相关蛋白质也可能使用一种涉及质子驱动的构象变化的机制。
MotA and MotB are integral membrane proteins of Escherichia coli that form the stator of the proton-fueled flagellar rotary motor. The motor contains several MotA/MotB complexes, which function independently to conduct protons across the cytoplasmic membrane and couple proton flow to rotation. MotB contains a conserved aspartic acid residue, Asp32, that is critical for rotation. We have proposed that the protons energizing the motor interact with Asp32 of MotB to induce conformational changes in the stator that drive movement of the rotor. To test for conformational changes, we examined the protease susceptibility of MotA in membrane-bound complexes with either wild-type MotB or MotB mutated at residue 32. Small, uncharged replacements of Asp32 in MotB (D32N, D32A, D32G, D32S, or D32C) caused a significant change in the conformation of MotA, as evidenced by a change in the pattern of proteolytic fragments. The conformational change does not require any flagellar proteins besides MotA and MotB, as it was still seen in a strain that expresses no other flagellar genes. It affects a cytoplasmic domain of MotA that contains residues known to interact with the rotor, consistent with a role in the generation of torque. Influences of key residues of MotA on conformation were also examined. Pro173 of MotA, known to be important for rotation, is a significant determinant of conformation: Dominant Pro173 mutations, but not recessive ones, altered the proteolysis pattern of MotA and also prevented the conformational change induced by Asp32 replacements. Arg90 and Glu98, residues of MotA that engage in electrostatic interactions with the rotor, appear not to be strong determinants of conformation of the MotA/MotB complex in membranes. We note sequence similarity between MotA and ExbB, a cytoplasmic-membrane protein that energizes outer-membrane transport in Gram-negative bacteria. ExbB and associated proteins might also employ a mechanism involving proton-driven conformational change.