Structural analysis of SecA-SecYE complex from Thermus thermophilus

进化上保守的膜蛋白复合体Sec易位作为蛋白质传导通道，促进分泌蛋白输出和膜蛋白整合。在这个项目中，我们从含有SecA（细菌易位马达）的生物Thermus thermophilus中确定了SecYE易位子的第一个原子分辨率晶体结构，该结构与抗secy Fab片段相结合。该结构与先前确定的古细菌SecYEβ结构有显著的构象变化，其中几个SecY跨膜螺旋被转移，形成一个向细胞质开放的疏水裂缝。二硫化物定位表明，细菌SecYE复合体的这种“预打开”状态是由Fab片段和SecA诱导的，它们通常与细胞质结构域尖端的SecY残基结合。我们的二硫交联实验表明，这些SecY残基中的一些与SecA的特定残基接触，否则这些残基在分离的SecA分子中是隐藏的和不可用的。这些结果表明，Sec机制的运动组分在相互作用时发生了协同构象变化，这可能是前蛋白-SecA复合物进入和SecA atp酶活性增强的重要步骤。

Evolutionarily conserved membrane protein complex, the Sec translocon, functions as a protein-conducting channel to facilitate secretory protein export and membrane protein integration. In this project, we determined the first atomic resolution crystal structure of the SecYE translocon from a SecA (bacterial translocation motor)-containing organism, Thermus thermophilus, in complex with an anti-SecY Fab fragment. The structure has revealed a significant conformational change from the previously determined archaeal SecYEβ structure, in that several SecY transmembrane helices are shifted to create a hydrophobic crevasse open to the cytoplasm. Disulfide mapping indicate that this "pre-open" state of the bacterial SecYE complex was induced by the Fab fragment and SecA, which in common bind to SecY residues at the tip of the cytoplasmic domain. Our disulfide crosslinking experiments revealed that some of these SecY residues contact specific residues of SecA that are otherwise buried and unavailable in the isolated SecA molecule. These results suggest that the motor components of the Sec machinery undergo cooperative conformational changes upon their interaction, presumably as important steps for the entrance of the preprotein-SecA complex and enhancement of the ATPase activity of SecA.