Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, M5G 1L7, Canada.
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, and Perlmutter Cancer Center, New York University Langone Health, New York, NY, 10016, USA.
Angew Chem Int Ed Engl. 2020 Jun 26;59(27):11037-11045. doi: 10.1002/anie.202001758. Epub 2020 Apr 30.
KRAS homo-dimerization has been implicated in the activation of RAF kinases, however, the mechanism and structural basis remain elusive. We developed a system to study KRAS dimerization on nanodiscs using paramagnetic relaxation enhancement (PRE) NMR spectroscopy, and determined distinct structures of membrane-anchored KRAS dimers in the active GTP- and inactive GDP-loaded states. Both dimerize through an α4-α5 interface, but the relative orientation of the protomers and their contacts differ substantially. Dimerization of KRAS-GTP, stabilized by electrostatic interactions between R135 and E168, favors an orientation on the membrane that promotes accessibility of the effector-binding site. Remarkably, "cross"-dimerization between GTP- and GDP-bound KRAS molecules is unfavorable. These models provide a platform to elucidate the structural basis of RAF activation by RAS and to develop inhibitors that can disrupt the KRAS dimerization. The methodology is applicable to many other farnesylated small GTPases.
KRAS 同源二聚化已被牵涉到 RAF 激酶的激活中，然而，其机制和结构基础仍然难以捉摸。我们开发了一种使用顺磁弛豫增强 (PRE) NMR 光谱研究纳米盘上 KRAS 二聚化的系统，并确定了膜锚定的 KRAS 二聚体在活性 GTP 和非活性 GDP 加载状态下的不同结构。两者都通过 α4-α5 界面二聚化，但构象异构体的相对取向和它们的接触有很大的不同。KRAS-GTP 的二聚化通过 R135 和 E168 之间的静电相互作用稳定，有利于促进效应物结合位点可及性的膜定位。值得注意的是，GTP 和 GDP 结合的 KRAS 分子之间的“交叉”二聚化是不利的。这些模型为阐明 RAF 激活的结构基础提供了一个平台，并开发了可以破坏 KRAS 二聚化的抑制剂。该方法适用于许多其他法尼基化的小 GTP 酶。
