Structures of BRAF-MEK1-14-3-3 sheds light on drug discovery
Structures of BRAF-MEK1-14-3-3 sheds light on drug discovery
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BRAF-MEK1-14-3-3 的结构揭示了药物发现
DOI：
10.1038/s41392-019-0096-z
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发表时间：
2019
影响因子：
39.3
通讯作者：
Wang Wenjing
中科院分区：
文献类型：
--
作者：
Sun Qiu;Wang Wenjing
在最近发表在《自然》杂志上的一篇论文中，Eunyoung Park等人报道了自抑制和活性BRAF-MEK1-14-3-3复合物的结构。这一发现揭示了RAF调控的详细机制，有助于开发新的治疗策略来克服RAF相关的癌症。在所有真核细胞中，RAF/MEK/ERK信号转导通路通过磷酸化级联反应调控广泛的细胞事件，包括细胞增殖、分化和存活。先前的研究已经部分证实了Raf/MEK/ERK循环的分子机制。2最初，gtp结合的RAS将Raf招募到质膜，促使Raf从n端释放抑制14-3-3蛋白并形成二聚体，从而从自抑制状态切换到激活状态。然后，活化的Raf二聚体使MEK募集和磷酸化，从而将信号传递给ERK。最后，ERK信号传导实现了一个负反馈回路，其中ERK磷酸化活化RAF不同区域的几个抑制位点，导致活化RAS的释放和RAF二聚体的破坏。一些研究表明，BRAF和MEK在静息状态下是预相关的。除了在生理过程中发挥作用外，关键通路中异常的RAF激活有助于致癌；因此，RAF激酶被认为是抗癌治疗的靶点。几种Raf激酶抑制剂已被开发用于临床应用，并取得了显著的临床效果。然而，由于获得性耐药，大多数患者在治疗一年内复发。一些RAF抑制剂出乎意料地促进RAF二聚化，诱导ERK信号的上调，而一些抑制剂通过破坏BRAF激酶与其n端区域的相互作用来打破自身抑制状态。在过去的几十年里，针对这一途径的激酶的分离结构域或片段的结构已经被表征，以阐明RAF/MEK/ERK的机制4-6；然而，缺乏具有完整RAF调控结构信息的更深层次的结构表征阻碍了新型RAF/MEK/ERK激酶抑制剂的开发。为了了解raf的正常调控，Park等人最近报道了具有MEK1和14-3-3蛋白的全长BRAF在自抑制和活性状态下的冷冻电镜复合物结构。1 RAF激酶的哺乳动物同源物（ARAF、BRAF和CRAF）有三个保守区域，称为CR1、CR2和CR3。位于n末端的CR1包含RBD （RAS结合域）和CRD（半胱氨酸富结构域）结构域，CR2区域包含14-3-3蛋白的结合位点，而位于c末端的CR3具有另一个14-3-3蛋白的结合位点、磷酸化基元和丝氨酸/苏氨酸激酶结构域。自抑制BRAF/MEK1AA/14-3-3复合体（MEK1AA：激活段磷酸化位点有丙氨酸突变）的冷冻电镜结构揭示了14-3-3二聚体（PDB: 6NYB）的抑制机制。在自抑制状态下，BRAF激酶结构域的活性位点远离14-3-3结构域，而面向MEK1，两种激酶的c叶之间有广泛的接触。ATP-γS结合在BRAF活性位点间隙，而ADP似乎在MEK活性位点。GDC-0623和ATP类似物amppnp结合的BRAF/MEK1AA复合物的2.6 Å晶体结构与cryo-EM结构高度相似（PDB: 6PP9）。螺旋状的旋转，称为抑制旋转，与富含甘氨酸的环和β3链中的疏水残基堆积在一起……
In a recent paper published in Nature, Eunyoung Park et al. reported structures of autoinhibited and active BRAF-MEK1-14-3-3 complexes. This finding reveals the detailed mechanism underlying RAF regulation, which could facilitate the development of novel therapeutic strategies to overcome RAF-related cancer. 1 The RAF/MEK/ERK signal transduction pathway regulates a wide set of cellular events, including cell proliferation, differentiation, and survival, via phosphorylation cascades in all eukaryotic cells. 2 Previous studies have partially demonstrated the molecular mechanisms of the Raf/MEK/ERK cycle. 2 Initially, GTP-bound RAS recruits Raf to the plasma membrane, which drives RAF to release inhibitory 14-3-3 protein from the N-terminus and form a dimer, thereby switching from an autoinhibited state to an activated state. Then, the activated Raf dimers enable MEK recruitment and phosphorylation, thereby transmitting the signal down to ERK. Finally, ERK signaling implements a negative feedback loop in which ERK phosphorylates several inhibitory sites in distinct regions of activated RAF, resulting in a release from the activated RAS and the disruption of RAF dimers. Some studies have revealed that BRAF and MEK are pre-associated in the quiescent state. In addition to its role in physiological processes, aberrant RAF activation in the key pathway contributes to causing cancer; thus, RAF kinase has been considered a target for anticancer treatment. Several Raf kinase inhibitors have been developed for clinical use and have achieved remarkable clinical outcomes. Nevertheless, most patients relapse within a year of treatment due to acquired resistance. Some RAF inhibitors unexpectedly promote RAF dimerization to induce the upregulation of ERK signaling, while some break the autoinhibitory status by disrupting the interactions of BRAF kinase with its N-terminal region. 3 In the past decades, structures of isolated domains or fragments of the kinases targeting this pathway have been characterized to elucidate the mechanism of RAF/MEK/ERK 4-6; however, the lack of a deeper structural characterization with intact structural information of RAF regulation has hindered the development of novel RAF/MEK/ERK kinase inhibitors. To understand the normal regulation of RAFs, Park et al. recently reported the cryo-EM complex structures of full-length BRAF with MEK1 and 14-3-3 proteins in both the autoinhibited and active status. 1 The mammalian homologs of RAF kinases (ARAF, BRAF, and CRAF) have three conserved regions called CR1, CR2, and CR3. CR1 at the N-terminal site contains the RBD (RAS binding domain) and the CRD (cysteine-rich domain) domain, the CR2 region consists of a binding site for the 14-3-3 protein, and CR3 at the C-terminal site has another binding site for the 14-3-3 protein, a phosphorylated motif and a serine/threonine kinase domain. The cryo-EM structure of an autoinhibited BRAF/MEK1AA/14-3-3 complex (MEK1AA: with alanine mutations at the phosphorylation sites of the activation segment) reveals the inhibitory mechanisms of the 14-3-3 dimer (PDB: 6NYB). In an autoinhibited state, the active site of the BRAF kinase domain faces away from the 14-3-3 domain but faces MEK1 with extensive contact between the C-lobes of both kinases. ATP-γS is bound in the BRAF active site cleft, while ADP seems to be in a MEK active site. A 2.6 Å crystal structure of GDC-0623 and the ATP analog AMPPNP-bound BRAF/MEK1AA complex reveals high similarity with the cryo-EM structure (PDB: 6PP9). A helix-like turn, called an inhibitory turn, stacks with hydrophobic residues in the glycine-rich loop and the β3 strand and …