Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA.
Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
EMBO Rep. 2020 Nov 5;21(11):e50905. doi: 10.15252/embr.202050905. Epub 2020 Sep 24.
Bacterial secretory proteins are translocated post-translationally by the SecA ATPase through the protein-conducting SecY channel in the plasma membrane. During the ATP hydrolysis cycle, SecA undergoes large conformational changes of its two-helix finger and clamp domains, but how these changes result in polypeptide movement is unclear. Here, we use a reconstituted purified system and protease protection assays to show that ATP binding to SecA results in a segment of the translocation substrate being pushed into the channel. This motion is prevented by mutation of conserved residues at the finger's tip. Mutation of SecA's clamp causes backsliding of the substrate in the ATP-bound state. Together, these data support a power stroke model of translocation in which, upon ATP binding, the two-helix finger pushes the substrate into the channel, where it is held by the clamp until nucleotide hydrolysis has occurred.
细菌分泌蛋白通过 SecA ATP 酶在后翻译阶段穿过质膜中的蛋白传导 SecY 通道进行易位。在 ATP 水解循环中，SecA 的双螺旋指和夹子结构域发生了很大的构象变化，但这些变化如何导致多肽运动尚不清楚。在这里，我们使用重组的纯化系统和蛋白酶保护实验证明，ATP 与 SecA 的结合导致易位底物的一段被推入通道。这种运动被位于指状物顶端的保守残基的突变所阻止。SecA 夹子的突变导致在 ATP 结合状态下底物的回溯。这些数据共同支持一种易位的动力冲程模型，即在 ATP 结合后，双螺旋指将底物推入通道，在核苷酸水解发生之前，夹子将其固定在通道中。
