Topology of the SecA ATPase Bound to Large Unilamellar Vesicles.
Topology of the SecA ATPase Bound to Large Unilamellar Vesicles.
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与大单层囊泡结合的SecA ATP酶的拓扑结构。
DOI：
10.1016/j.jmb.2022.167607
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发表时间：
2022-06-30
影响因子：
5.6
通讯作者：
White, Stephen H.
中科院分区：
文献类型：
--
作者：
Roussel, Guillaume;Lindner, Eric;White, Stephen H.
可溶性细胞质 ATP 酶马达蛋白 SecA 通过 SecYEG 易位子为蛋白质运输穿过大肠杆菌内膜提供动力。根据多项晶体学和冷冻电镜结构研究，虽然在溶液中为二聚体，但 SecA 在分泌过程中与 SecYEG 单体结合。 SecA 从二聚体细胞质状态到活性 SecYEG 单体状态的步骤在很大程度上是未知的。我们之前已经证明，溶液中的二聚体 SecA 在与由大肠杆菌脂质形成的带负电荷的脂质囊泡静电结合后解离成单体。在这里，我们解决了在与膜嵌入的 SecYEG 结合之前 SecA 在膜上的处理问题。我们突变为半胱氨酸，一次一个，25 个无 Cys 的 SecA 的表面暴露残基。我们将极性敏感的荧光团 NBD 与其中每一个共价连接，其强度和荧光波长偏移在囊泡结合报告局部膜极性时发生变化。我们根据这些测量结果确定了在没有 SecYEG 的情况下 SecA 与膜结合的情况。我们的结果证实 SecA 主要通过 N 末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。这两个结构域都富含带正电荷的残基，这与在膜结合中起主要作用的静电相互作用一致。用丙氨酸选择性替换这些结构域中的带正电残基会导致与膜的结合较弱，这使我们能够定量这些结构域在稳定膜上 SecA 方面的相对重要性。囊泡内的荧光猝灭剂对 NBD 荧光几乎没有影响，表明 SecA 没有显着穿透膜。总体而言，膜上 SecA 的拓扑结构与 SecA-SecYEG 复合物的晶体学和冷冻电镜结构中观察到的 SecA 构象一致，表明 SecA 可以在膜相关状态和易位子相关状态之间切换，而构象没有显着变化。
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.
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影响因子：
3.5
作者：
BREUKINK, E;KELLER, RCA;DEKRUIJFF, B
通讯作者：
DEKRUIJFF, B
影响因子：
8.3
作者：
Keller RC
通讯作者：
Keller RC
通过冷冻电子显微镜测定全长大肠杆菌 SecA 在溶液中以闭合构象二聚化
影响因子：
4.8
作者：
Chen, Yong;Pan, Xijiang;Sui, Sen-Fang
通讯作者：
Sui, Sen-Fang
DOI：
10.1006/bbrc.1994.2185
发表时间：
1994-08-30
影响因子：
3.1
作者：
AHN, T;KIM, H
通讯作者：
KIM, H
影响因子：
6.4
作者：
Knuepffer, Lara;Fehrenbach, Clara;Koch, Hans-Georg
通讯作者：
Koch, Hans-Georg