Alignment of the protein substrate hairpin along the SecA two-helix finger primes protein transport in Escherichia coli.
Alignment of the protein substrate hairpin along the SecA two-helix finger primes protein transport in Escherichia coli.
复制标题
蛋白质底物发夹沿着 SecA 双螺旋指的排列启动了大肠杆菌中的蛋白质运输。
DOI：
10.1073/pnas.1702201114
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发表时间：
2017
影响因子：
11.1
通讯作者：
Mukerji,Ishita
中科院分区：
文献类型：
--
作者：
Zhang,Qi;Lahiri,Sudipta;Banerjee,Tithi;Sun,Zhongmou;Oliver,Donald;Mukerji,Ishita
由信号肽和早期成熟区域组成的保守发夹样结构分别启动细菌或古细菌和真核生物中穿过 SecY 或 Sec61α 通道的蛋白质转运。这种引发剂底物发夹何时以及如何形成仍然是个谜。在这里，我们使用细菌 SecA ATPase 运动蛋白和 SecYEG 通道复合物来解决这个问题。将功能性小蛋白底物工程化到 SecA 末端，使我们能够与 SecYEG 有效形成三元复合物，用于光谱研究。该三元复合物内关键残基的 Förster 共振能量转移图谱表明，蛋白质底物在进入通道之前采用紧邻 SecA 双螺旋指亚结构域的发夹状结构。 ADP 和 ATP-γS 结合状态的比较表明，信号肽在后一种状态下部分插入 SecY 通道。我们的研究定义了一种独特的插入前中间状态，其中 SecA 双螺旋指似乎在通道入口处的底物发夹模板化和促进其随后的 ATP 依赖性插入中发挥作用。
A conserved hairpin-like structure comprised of a signal peptide and early mature region initiates protein transport across the SecY or Sec61α channel in Bacteria or Archaea and Eukarya, respectively. When and how this initiator substrate hairpin forms remains a mystery. Here, we have used the bacterial SecA ATPase motor protein and SecYEG channel complex to address this question. Engineering of a functional miniprotein substrate onto the end of SecA allowed us to efficiently form ternary complexes with SecYEG for spectroscopic studies. Förster resonance energy transfer mapping of key residues within this ternary complex demonstrates that the protein substrate adopts a hairpin-like structure immediately adjacent to the SecA two-helix finger subdomain before channel entry. Comparison of ADP and ATP-γS–bound states shows that the signal peptide partially inserts into the SecY channel in the latter state. Our study defines a unique preinsertion intermediate state where the SecA two-helix finger appears to play a role in both templating the substrate hairpin at the channel entrance and promoting its subsequent ATP-dependent insertion.