Ke Haoliang, Matsumoto Shigeyuki, Murashima Yosuke, Taniguchi-Tamura Haruka, Miyamoto Ryo, Yoshikawa Yoko, Tsuda Chiemi, Kumasaka Takashi, Mizohata Eiichi, Edamatsu Hironori, Kataoka Tohru
Division of Molecular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Chuo-ku, Kobe, Japan.
Japan Synchrotron Radiation Research Institute (JASRI), Sayo-gun, Hyogo, Japan.
FEBS Lett. 2017 Aug;591(16):2470-2481. doi: 10.1002/1873-3468.12759. Epub 2017 Aug 2.
Ras undergoes post-translational modifications including farnesylation, proteolysis, and carboxymethylation at the C terminus, which are necessary for membrane recruitment and effector recognition. Full activation of c-Raf-1 requires cooperative interaction of the farnesylated C terminus and the activator region of Ras with its cysteine-rich domain (CRD). However, the molecular basis for this interaction remains unclear because of difficulties in preparing modified Ras in amounts sufficient for structural studies. Here, we use Sortase A-catalyzed protein ligation to prepare modified Ras in sufficient amounts for NMR and X-ray crystallographic analyses. The results show that the farnesylated C terminus establishes an intramolecular interaction with the catalytic domain and brings the farnesyl moiety to the proximity of the activator region, which may be responsible for their cooperative recognition of c-Raf-1-CRD.
Ras会经历翻译后修饰，包括法尼基化、蛋白水解以及C端的羧甲基化，这些修饰对于膜招募和效应器识别是必需的。c-Raf-1的完全激活需要法尼基化的C端与Ras的激活区域及其富含半胱氨酸的结构域（CRD）协同相互作用。然而，由于难以制备出足够量用于结构研究的修饰Ras，这种相互作用的分子基础仍不清楚。在此，我们使用分选酶A催化的蛋白质连接反应制备出足够量的修饰Ras，用于核磁共振（NMR）和X射线晶体学分析。结果表明，法尼基化的C端与催化结构域建立了分子内相互作用，并将法尼基部分带到激活区域附近，这可能是它们协同识别c-Raf-1-CRD的原因。
