Jang Hyunbum, Zhang Mingzhen, Nussinov Ruth
Computational Structural Biology Section, Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.
Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
Comput Struct Biotechnol J. 2020 Mar 25;18:737-748. doi: 10.1016/j.csbj.2020.03.018. eCollection 2020.
Proximally located in the membrane, oncogenic Ras dimers (or nanoclusters) can recruit and promote Raf dimerization and MAPK (Raf/MEK/ERK) signaling. Among Ras isoforms, KRas4B is the most frequently mutated. Recent data on the binary KRas4B-Raf-1 complex suggested that Raf-1 CRD not only executes membrane anchorage, but also supports the high-affinity interaction of Raf-1 RBD with KRas4B catalytic domain. For a detailed mechanistic picture of Raf activation at the membrane, we employ explicit MD simulations of the quaternary KRas4B-Raf-1 complex. The complex contains two active GTP-bound KRas4B proteins forming a dimer through the allosteric lobe interface and two tandem RBD-CRD segments of Raf-1 interacting with the effector lobes at both ends of the KRas4B dimer. We show that Raf-1 RBD-CRD supports stable KRas4B dimer at preferred interface and orientation at the membrane, thereby cooperatively enhancing the affinity of the KRas4B-Raf-1 interaction. We propose that a Ras dimer at the membrane can increase the population of proximal Raf kinase domains, promoting kinase domain dimerization in the cytoplasm. Collectively, the dynamic Ras-Raf assembly promotes Raf activation not by allostery; instead, Ras activates Raf by shifting its ensemble toward kinase domain-accessible states through enhanced affinity at the membrane.
致癌性Ras二聚体（或纳米簇）位于膜的近端，可招募并促进Raf二聚化以及MAPK（Raf/MEK/ERK）信号传导。在Ras亚型中，KRas4B是最常发生突变的。关于二元KRas4B-Raf-1复合物的最新数据表明，Raf-1 CRD不仅执行膜锚定作用，还支持Raf-1 RBD与KRas4B催化结构域的高亲和力相互作用。为了详细了解膜上Raf激活的机制，我们对四元KRas4B-Raf-1复合物进行了显式分子动力学模拟。该复合物包含两个结合有活性GTP的KRas4B蛋白，它们通过变构叶界面形成二聚体，以及Raf-1的两个串联RBD-CRD片段，与KRas4B二聚体两端的效应叶相互作用。我们表明，Raf-1 RBD-CRD在膜上的首选界面和取向下支持稳定的KRas4B二聚体，从而协同增强KRas4B-Raf-1相互作用的亲和力。我们提出，膜上的Ras二聚体可以增加近端Raf激酶结构域的数量，促进细胞质中的激酶结构域二聚化。总的来说，动态的Ras-Raf组装促进Raf激活并非通过变构作用；相反，Ras通过在膜上增强亲和力，将其整体状态转变为激酶结构域可及状态来激活Raf。
