Class 3 mutations in B-Raf proto-oncogene, Ser/Thr kinase (BRAF), that result in kinase-impaired or kinase-dead BRAF have the highest mutation frequency in BRAF gene in lung adenocarcinoma. Several studies have reported that kinase-dead BRAF variants amplify mitogen-activated protein kinase (MAPK) signaling by dimerizing with and activating WT C-Raf proto-oncogene, Ser/Thr kinase (CRAF). However, the structural and functional principles underlying their activation remain elusive. Herein, using cell biology and various biochemical approaches, we established that variant BRAFD594G, a kinase-dead representative of class 3 mutation-derived BRAF variants, has a higher dimerization potential as compared with WT BRAF. Molecular dynamics simulations uncovered that the D594G substitution orients the αC-helix toward the IN position and extends the activation loop within the kinase domain, shifting the equilibrium toward the active, dimeric conformation, thus priming BRAFD594G as an effective allosteric activator of CRAF. We found that B/CRAF heterodimers are the most thermodynamically stable RAF dimers, suggesting that RAF heterodimers, and not homodimers, are the major players in determining the amplitude of MAPK signaling in cells. Additionally, we show that BRAFD594G:CRAF heterodimers bypass autoinhibitory P-loop phosphorylation, which might contribute to longer duration of MAPK pathway signaling in cancer cells. Last, we propose that the dimer interface of the BRAFD594G:CRAF heterodimer may represent a promising target in the design of novel anticancer therapeutics.

中文翻译：

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致癌性BRAFD594G变体的分析显示，BRAF在激活MAPK信号传导中具有激酶独立功能。

B-Raf原癌基因Ser / Thr激酶（BRAF）中的3类突变导致激酶受损或激酶死亡的BRAF在肺腺癌中的BRAF基因突变频率最高。几项研究报道，死于激酶的BRAF变体通过与WT C-Raf原癌基因Ser / Thr激酶（CRAF）二聚并激活，从而放大了促分裂原活化蛋白激酶（MAPK）信号。但是，激活它们的结构和功能原理仍然难以捉摸。本文中，我们利用细胞生物学和各种生化方法，确定了BRAFD594G变体（一种3级突变衍生的BRAF变体的激酶死亡代表），与WT BRAF相比，具有更高的二聚化潜力。分子动力学模拟发现，D594G取代可将αC螺旋定向至IN位置，并在激酶结构域内延伸活化环，使平衡朝着有效的二聚体构象移动，从而引发BRAFD594G作为CRAF的有效变构活化剂。我们发现B / CRAF异二聚体是最热力学稳定的RAF二聚体，表明RAF异二聚体而不是同二聚体是决定细胞中MAPK信号传导幅度的主要因素。此外，我们显示BRAFD594G：CRAF异二聚体绕过了自抑制性P环磷酸化作用，这可能有助于延长MAPK信号通路在癌细胞中的持续时间。最后，我们建议BRAFD594G的二聚体接口：