Multivalent assembly of KRAS with the RAS-binding and cysteine-rich domains of CRAF on the membrane
Multivalent assembly of KRAS with the RAS-binding and cysteine-rich domains of CRAF on the membrane
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DOI：
10.1073/pnas.1914076117
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发表时间：
2020-06-02
影响因子：
11.1
通讯作者：
Marshall, Christopher B.
中科院分区：
文献类型：
--
作者：
Fang, Zhenhao;Lee, Ki-Young;Marshall, Christopher B.
法尼基化 KRAS 的膜锚定对于 RAF 激酶的激活至关重要，但我们对这些蛋白质如何在膜上相互作用的理解仅限于孤立的域。 RAF 的 RAS 结合结构域 (RBD) 和富含半胱氨酸结构域 (CRD) 与 KRAS 和质膜结合，从而释放激酶结构域的自动抑制。由于实验的挑战，对这种三部分 KRAS:RBD-CRD:膜复合物的结构洞察依赖于分子动力学模拟。在这里，我们报告了 KRAS:CRAF RBD-CRD 复合物的 NMR 研究。我们发现核苷酸依赖性 KRAS-RBD 相互作用会导致 KRAS 和 CRD 之间的瞬时静电相互作用，并且我们绘制了 CRD、RBD-CRD 和 KRAS:RBD-CRD 复合物的膜界面。 RBD-CRD 通过典型的 CRD 脂质结合位点（CRD beta 7-8）以及包含 beta 6 和 CRD 的 C 末端以及 RBD 的 beta 2 的替代界面表现出与膜的动态相互作用。在与 KRAS 形成复合物后，通过 NMR 观察到两种不同的状态：状态 A 通过 CRD beta 7-8 和 KRAS alpha 4-alpha 5 的膜结合而稳定，而状态 B 涉及 CRD 的 C 末端、RBD 的 beta 3-5 和 KRAS alpha 5 的一部分。值得注意的是，已被提议介导 KRAS 二聚化的 alpha 4-alpha 5 仅在状态 B 中才可接近。状态 B 膜上的癌症相关突变CRAF RBD (E125K) 的界面稳定 B 状态并增强激酶活性和细胞 MAPK 信号传导。这些研究通过 KRAS、CRAF RBD-CRD 和膜之间的多价动态相互作用揭示了 KRAS-CRAF 复合物组装的动态图景。
Membrane anchoring of farnesylated KRAS is critical for activation of RAF kinases, yet our understanding of how these proteins interact on the membrane is limited to isolated domains. The RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF engage KRAS and the plasma membrane, unleashing the kinase domain from autoinhibition. Due to experimental challenges, structural insight into this tripartite KRAS:RBD-CRD:membrane complex has relied on molecular dynamics simulations. Here, we report NMR studies of the KRAS:CRAF RBD-CRD complex. We found that the nucleotide-dependent KRAS-RBD interaction results in transient electrostatic interactions between KRAS and CRD, and we mapped the membrane interfaces of the CRD, RBD-CRD, and the KRAS:RBD-CRD complex. RBD-CRD exhibits dynamic interactions with the membrane through the canonical CRD lipid-binding site (CRD beta 7-8), as well as an alternative interface comprising beta 6 and the C terminus of CRD and beta 2 of RBD. Upon complex formation with KRAS, two distinct states were observed by NMR: State A was stabilized by membrane association of CRD beta 7-8 and KRAS alpha 4-alpha 5 while state B involved the C terminus of CRD, beta 3-5 of RBD, and part of KRAS alpha 5. Notably, alpha 4-alpha 5, which has been proposed to mediate KRAS dimerization, is accessible only in state B. A cancer-associated mutation on the state B membrane interface of CRAF RBD (E125K) stabilized state B and enhanced kinase activity and cellular MAPK signaling. These studies revealed a dynamic picture of the assembly of the KRAS-CRAF complex via multivalent and dynamic interactions between KRAS, CRAF RBD-CRD, and the membrane.