RAF 蛋白激酶是 MAPK 通路中的重要效应器，也是重要的癌症药物靶点。迄今为止，对 RAF 激活的结构理解基于冷冻电子显微镜 (cryo-EM) 和 BRAF 在不同构象状态下的 X 射线结构，即与 KRAS、14-3-3 和 MEK1 的非活性或活性复合物。在这项研究中，我们使用表达为组成型活性 Y340D/Y341D 的 CRAF 激酶结构域解析了第一个 3.4 Å 分辨率的 CRAF2/14-3-32 和 4.2 Å 分辨率的 CRAF2/14-3-32/MEK12 的冷冻电镜结构。昆虫细胞中的突变体。我们的 CRAF2/14-3-32 和 CRAF2/14-3-32/MEK12 冷冻电镜结构的整体架构与与 14-3-3 或 14-3-3/MEK1 复合的相应 BRAF 结构高度相似，代表激活的二聚体 RAF 构象。我们的 CRAF 冷冻电镜结构为了解激活的 CRAF2/14-3-32/MEK12 复合体的结构提供了更多见解。版权所有 © 2024 Elsevier Ltd。保留所有权利。

RAF protein kinases are essential effectors in the MAPK pathway and are important cancer drug targets. Structural understanding of RAF activation is so far based on cryo-electron microscopy (cryo-EM) and X-ray structures of BRAF in different conformational states as inactive or active complexes with KRAS, 14-3-3 and MEK1. In this study, we have solved the first cryo-EM structures of CRAF2/14-3-32 at 3.4 Å resolution and CRAF2/14-3-32/MEK12 at 4.2 Å resolution using CRAF kinase domain expressed as constitutively active Y340D/Y341D mutant in insect cells. The overall architecture of our CRAF2/14-3-32 and CRAF2/14-3-32/MEK12 cryo-EM structures is highly similar to corresponding BRAF structures in complex with 14-3-3 or 14-3-3/MEK1 and represent the activated dimeric RAF conformation. Our CRAF cryo-EM structures provide additional insights into structural understanding of the activated CRAF2/14-3-32/MEK12 complex.Copyright © 2024 Elsevier Ltd. All rights reserved.