Zhang Mingzhen, Maloney Ryan, Jang Hyunbum, Nussinov Ruth
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 Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University Tel Aviv 69978 Israel.
Chem Sci. 2021 Nov 18;12(47):15609-15619. doi: 10.1039/d1sc03444h. eCollection 2021 Dec 8.
Raf, a threonine/serine kinase in the Raf/MEK/ERK pathway, regulates cell proliferation. Raf's full activation requires dimerization. Aberrant activation through dimerization is an important therapeutic target. Despite its clinical importance, fundamental questions, such as how the side-to-side dimerization promotes the OFF-to-ON transition of Raf's kinase domain and how the fully activated ON-state kinase domain is stabilized in the dimer for Raf signaling, remain unanswered. Herein, we decipher an atomic-level mechanism of Raf activation through dimerization, clarifying this enigma. The mechanism reveals that the replacement of intramolecular π-π stacking by intermolecular π-π stacking at the dimer interface releases the structural constraint of the αC-helix, promoting the OFF-to-ON transition. During the transition, the inhibitory hydrophobic interactions were disrupted, making the phosphorylation sites in A-loop approach the HRD motif for -autophosphorylation. Once fully activated, the ON-state kinase domain can be stabilized by a newly identified functional N-terminal basic (NtB) motif in the dimer for Raf signaling. This work provides atomic level insight into critical steps in Raf activation and outlines a new venue for drug discovery against Raf dimerization.
Raf是Raf/MEK/ERK信号通路中的一种苏氨酸/丝氨酸激酶，可调节细胞增殖。Raf的完全激活需要二聚化。通过二聚化的异常激活是一个重要的治疗靶点。尽管其具有临床重要性，但一些基本问题仍未得到解答，比如侧向二聚化如何促进Raf激酶结构域从关闭状态转变为开启状态，以及完全激活的开启状态激酶结构域如何在二聚体中稳定以进行Raf信号传导。在此，我们解析了Raf通过二聚化激活的原子水平机制，揭开了这个谜团。该机制表明，在二聚体界面处分子间的π-π堆积取代分子内的π-π堆积，释放了αC螺旋的结构限制，促进了从关闭状态到开启状态的转变。在转变过程中，抑制性疏水相互作用被破坏，使得A环中的磷酸化位点接近HRD模体进行自磷酸化。一旦完全激活，开启状态的激酶结构域可通过二聚体中新发现的功能性N端碱性（NtB）模体稳定下来以进行Raf信号传导。这项工作为Raf激活的关键步骤提供了原子水平的见解，并为针对Raf二聚化进行药物研发开辟了新途径。
