Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.
Bioinformatics Institute (BII), Agency for Science Technology and Research (ASTAR), Singapore 138671, Singapore.
Sci Adv. 2019 Aug 14;5(8):eaav8463. doi: 10.1126/sciadv.aav8463. eCollection 2019 Aug.
Oncogenic BRAF mutations initiate tumor formation by unleashing the autoinhibited kinase conformation and promoting RAS-decoupled proliferative RAF-MEK-ERK signaling. We have engineered luciferase-based biosensors to systematically track full-length BRAF conformations and interactions affected by tumorigenic kinase mutations and GTP loading of RAS. Binding of structurally diverse αC-helix-OUT BRAF inhibitors (BRAFi) showed differences in specificity and efficacy by shifting patient mutation-containing BRAF reporters from the definitive opened to more closed conformations. Unexpectedly, BRAFi engagement with the catalytic pocket of V600E-mutated BRAF stabilized an intermediate and inactive kinase conformation that enhanced binary RAS:RAF interactions, also independently of RAF dimerization in melanoma cells. We present evidence that the interference with RAS interactions and nanoclustering antagonizes the sequential formation of drug-induced RAS:RAF tetramers. This suggests a previously unappreciated allosteric effect of anticancer drug-driven intramolecular communication between the kinase and RAS-binding domains of mutated BRAF, which may further promote paradoxical kinase activation and drug resistance mechanisms.
致癌 BRAF 突变通过释放自动抑制激酶构象并促进 RAS 去耦的促有丝分裂 RAF-MEK-ERK 信号转导来引发肿瘤形成。我们已经设计了基于荧光素酶的生物传感器，以系统地跟踪全长 BRAF 构象和受致癌激酶突变和 RAS GTP 加载影响的相互作用。结构不同的 αC-helix-OUT BRAF 抑制剂（BRAFi）的结合显示出特异性和功效的差异，通过将含有患者突变的 BRAF 报告基因从明确的开放构象转变为更封闭的构象。出乎意料的是，BRAFi 与 V600E 突变 BRAF 的催化口袋结合稳定了一种中间和非活性的激酶构象，这种构象增强了二元 RAS:RAF 相互作用，这也独立于黑色素瘤细胞中 RAF 二聚化。我们提出的证据表明，与 RAS 相互作用的干扰和纳米簇抑制了药物诱导的 RAS:RAF 四聚体的顺序形成。这表明抗癌药物驱动的突变 BRAF 的激酶和 RAS 结合域之间的分子内通信的先前未被认识的变构效应，这可能进一步促进反常的激酶激活和药物耐药机制。
