Computational Structural Biology Section, Frederick National Laboratory for Cancer Research in the Laboratory of Cancer ImmunoMetabolism, National Cancer Institute, Frederick, MD 21702, USA.
Department of Laboratory Medicine, National Institutes of Health, Bethesda, MD 20892, USA.
Structure. 2021 Jul 1;29(7):768-777.e2. doi: 10.1016/j.str.2021.02.005. Epub 2021 Mar 11.
Raf-activating mutations are frequent in cancer. In the basal state, B-Raf is autoinhibited by its upstream Ras-binding domain (RBD) and cysteine-rich domain (RBD-CRD) interacting with its kinase domain (KD) and the 14-3-3 dimer. Our comprehensive molecular dynamics simulations explore two autoinhibition scenarios in the presence and absence of the 14-3-3 dimer. When present, the 14-3-3 interaction with B-Raf stabilizes the RBD-CRD-KD interaction, interfering with the KD dimerization. Raf's pSer365 removal fails to induce large disruption. RBD-CRD release promotes KD fluctuations and reorientation for dimerization, consistent with experimental data. In the absence of 14-3-3, our sampled B-Raf conformations suggest that RBD-CRD can block the KD dimerization surface. Our results suggest a B-Raf activation mechanism, whereby one KD monomer is donated by 14-3-3-free B-Raf KD and the other by 14-3-3-bound KD. This mechanism can lead to homo- and heterodimers. These autoinhibition scenarios can transform autoinhibited B-Raf monomers into active B-Raf dimers.
Raf 激活突变在癌症中很常见。在基础状态下，B-Raf 被其上游 Ras 结合结构域（RBD）和富含半胱氨酸的结构域（RBD-CRD）自动抑制，该结构域与激酶结构域（KD）和 14-3-3 二聚体相互作用。我们的综合分子动力学模拟在存在和不存在 14-3-3 二聚体的情况下探索了两种自动抑制情况。当 14-3-3 存在时，与 B-Raf 的相互作用稳定了 RBD-CRD-KD 相互作用，干扰了 KD 二聚化。Raf 的 pSer365 去除未能诱导大的破坏。RBD-CRD 的释放促进 KD 的波动和重定向以进行二聚化，与实验数据一致。在没有 14-3-3 的情况下，我们采样的 B-Raf 构象表明 RBD-CRD 可以阻止 KD 二聚化表面。我们的结果表明了一种 B-Raf 激活机制，其中一个 KD 单体由无 14-3-3 的 B-Raf KD 提供，另一个由 14-3-3 结合的 KD 提供。这种机制可以导致同型和异型二聚体。这些自动抑制情况可以将自动抑制的 B-Raf 单体转化为活性 B-Raf 二聚体。
