Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA 92697-4560, United States.
Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA 92697-4560, United States.
J Mol Biol. 2022 Jun 30;434(12):167607. doi: 10.1016/j.jmb.2022.167607. Epub 2022 Apr 27.
The soluble cytoplasmic ATPase motor protein SecA powers protein transport across the Escherichia coli inner membrane via the SecYEG translocon. Although dimeric in solution, SecA associates monomerically with SecYEG during secretion according to several crystallographic and cryo-EM structural studies. The steps SecA follows from its dimeric cytoplasmic state to its active SecYEG monomeric state are largely unknown. We have previously shown that dimeric SecA in solution dissociates into monomers upon electrostatic binding to negatively charged lipid vesicles formed from E. coli lipids. Here we address the question of the disposition of SecA on the membrane prior to binding to membrane embedded SecYEG. We mutated to cysteine, one at a time, 25 surface-exposed residues of a Cys-free SecA. To each of these we covalently linked the polarity-sensitive fluorophore NBD whose intensity and fluorescence wavelength-shift change upon vesicle binding report on the the local membrane polarity. We established from these measurements the disposition of SecA bound to the membrane in the absence of SecYEG. Our results confirmed that SecA is anchored in the membrane interface primarily by the positive charges of the N terminus domain. But we found that a region of the nucleotide binding domain II is also important for binding. Both domains are rich in positively charged residues, consistent with electrostatic interactions playing the major role in membrane binding. Selective replacement of positively charged residues in these domains with alanine resulted in weaker binding to the membrane, which allowed us to quantitate the relative importance of the domains in stabilizing SecA on membranes. Fluorescence quenchers inside the vesicles had little effect on NBD fluorescence, indicating that SecA does not penetrate significantly across the membrane. Overall, the topology of SecA on the membrane is consistent with the conformation of SecA observed in crystallographic and cryo-EM structures of SecA-SecYEG complexes, suggesting that SecA can switch between the membrane-associated and the translocon-associated states without significant changes in conformation.
可溶性细胞质 ATP 酶马达蛋白 SecA 通过 SecYEG 转运蛋白在大肠杆菌内膜上推动蛋白质运输。尽管在溶液中是二聚体，但根据几项晶体学和 cryo-EM 结构研究，SecA 在分泌过程中与 SecYEG 单体结合。SecA 从其二聚体细胞质状态到其活性 SecYEG 单体状态的步骤在很大程度上是未知的。我们之前已经表明，溶液中的二聚体 SecA 在静电结合到由大肠杆菌脂质形成的带负电荷的脂质小泡时会解离成单体。在这里，我们研究了 SecA 在与嵌入膜中的 SecYEG 结合之前在膜上的位置问题。我们逐个突变了 25 个表面暴露的残基中的一个半胱氨酸，这些残基位于无半胱氨酸 SecA 的一个区域中。我们用极性敏感荧光染料 NBD 共价连接到每个突变体上，NBD 的强度和荧光波长的变化反映了小泡结合时的局部膜极性。我们从这些测量结果中确定了在没有 SecYEG 的情况下 SecA 与膜的结合方式。我们的结果证实，SecA 主要通过 N 端结构域的正电荷锚定在膜界面上。但我们发现核苷酸结合结构域 II 的一个区域对于结合也很重要。这两个结构域富含带正电荷的残基，这与静电相互作用在膜结合中起主要作用是一致的。用丙氨酸选择性替换这些结构域中的带正电荷的残基会导致与膜的结合减弱，这使我们能够定量评估这些结构域在稳定 SecA 与膜结合中的相对重要性。小泡内的荧光猝灭剂对 NBD 荧光的影响很小，表明 SecA 没有显著穿透膜。总的来说，SecA 在膜上的拓扑结构与 SecA-SecYEG 复合物的晶体学和 cryo-EM 结构中观察到的构象一致，这表明 SecA 可以在不发生构象显著变化的情况下在膜相关状态和转运蛋白相关状态之间切换。
