Mechanistic target of rapamycin complex 2 (mTORC2) is a multi-subunit kinase complex, central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1, distinguish it from the related mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously; however, important questions remain, particularly regarding the structural determinants mediating substrate specificity and context-dependent activity. Here, we used cryo-EM to obtain high-resolution structures of the human mTORC2 apo-complex in the presence of substrates Akt and SGK1. Using functional assays, we then tested predictions suggested by substrate-induced structural changes in mTORC2. For the first time, we visualized in the apo-state the side chain interactions between Rictor and mTOR that sterically occlude recruitment of mTORC1 substrates and confer resistance to the mTORC1 inhibitor rapamycin. Also in the apo-state, we observed that mSin1 formed extensive contacts with Rictor via a pair of short α-helices nestled between two Rictor helical repeat clusters, as well as by an extended strand that makes multiple weak contacts with Rictor helical cluster 1. In co-complex structures, we found that SGK1, but not Akt, markedly altered the conformation of the mSin1 N-terminal extended strand, disrupting multiple weak interactions while inducing a large rotation of mSin1 residue Arg-83, which then interacts with a patch of negatively charged residues within Rictor. Finally, we demonstrate mutation of Arg-83 to Ala selectively disrupts mTORC2-dependent phosphorylation of SGK1, but not of Akt, supporting context-dependent substrate selection. These findings provide new structural and functional insights into mTORC2 specificity and context-dependent activity.

雷帕霉素复合物 2 (mTORC2) 的机制靶点是一种多亚基激酶复合物，是多个重要信号通路的核心。两个核心亚基 Rictor 和 mSin1 将其与相关的 mTORC1 区分开来，并支持其底物的上下文依赖性磷酸化。mTORC2 结构先前已确定；然而，重要的问题仍然存在，特别是关于介导底物特异性和上下文相关活动的结构决定因素。在这里，我们使用冷冻电镜在底物 Akt 和 SGK1 存在下获得人类 mTORC2 载脂蛋白复合物的高分辨率结构。然后使用功能分析，我们测试了由底物诱导的 mTORC2 结构变化所暗示的预测。首次，我们在载脂蛋白状态下观察到 Rictor 和 mTOR 之间的侧链相互作用，它们在空间上阻断了 mTORC1 底物的募集并赋予对 mTORC1 抑制剂雷帕霉素的抗性。同样在 apo 状态下，我们观察到 mSin1 与 Rictor 形成了广泛的接触通过位于两个 Rictor 螺旋重复簇之间的一对短 α-螺旋，以及通过与 Rictor 螺旋簇 1 进行多次弱接触的延伸链。在共复合结构中，我们发现 SGK1，但不是 Akt，显着改变了 mSin1 N 末端延伸链的构象，破坏了多个弱相互作用，同时诱导 mSin1 残基 Arg-83 的大旋转，然后与 Rictor 内的一小块带负电荷的残基相互作用。最后，我们证明了 Arg-83 向 Ala 的突变选择性地破坏了 mTORC2 依赖性 SGK1 的磷酸化，但不破坏 Akt 的磷酸化，从而支持上下文依赖性底物选择。这些发现为 mTORC2 特异性和上下文相关活动提供了新的结构和功能见解。