1H, 15N backbone assignment and comparative analysis of the wild type and G12C, G12D, G12V mutants of K-Ras bound to GDP at physiological pH
1H, 15N backbone assignment and comparative analysis of the wild type and G12C, G12D, G12V mutants of K-Ras bound to GDP at physiological pH
复制标题
DOI：
10.1007/s12104-019-09909-7
复制
发表时间：
2020-04-01
影响因子：
0.9
通讯作者：
Perczel, Andras
中科院分区：
文献类型：
--
作者：
Palfy, Gyula;Vida, Istvan;Perczel, Andras
K-Ras蛋白是一种膜结合的小G蛋白，起着分子开关的作用。它在调节细胞增殖、分化、存活等的许多信号转导途径中起关键作用。它在受鸟嘌呤核苷酸交换因子和GTP酶激活蛋白调节的GTP结合活性构象和GDP结合非活性构象之间交替。其最常见的致癌突变体是G12 C、G12 D和G12 V，这些突变体具有受损的GT3活性，从而诱导恶性肿瘤。本文报道了K-Ras野生型的骨架H-1和N-15核，G12 C，G12 D和G12 V蛋白的催化G结构域（1-169个残基）在GDP结合状态下的共振归属，以及G12 C突变体在生理pH 7.4下的骨架和侧链的C-13。三重共振数据用于获得K-Ras突变体中最知名的药物靶标G12 C的二级结构信息和主链动力学。同时研究G12 C、G12 D和G12 V突变体，沿着与野生型形式在非常相同的条件下允许我们基于组合化学位移进行综合分析，以揭示G12位置突变对结构的影响。有趣的是，发现G12 C和G12 V突变体在K-Ras的三个最重要的区域（P-环、Switch-I、Switch-II）处结构非常相似，而G12 D突变体在P-环和Switch-II处与野生型以及G12 C和G12 V突变体显著不同。然而，在Switch-I中，它几乎不偏离野生型蛋白。
K-Ras protein is a membrane-bound small GTPase acting as a molecular switch. It plays a key role in many signal transduction pathways regulating cell proliferation, differentiation, survival, etc. It alternates between its GTP-bound active and the GDP-bound inactive conformers regulated by guanine nucleotide exchange factors and GTPase activating proteins. Its most frequent oncogenic mutants are G12C, G12D, and G12V that have impaired GTPase activity, thus induce malignant tumors. Here we report the resonance assignment of the backbone H-1 and N-15 nuclei of K-Ras wildtype, G12C, G12D and G12V proteins' catalytic G domain (1-169 residues) in GDP-bound state, and C-13 of backbone and side chains of G12C mutant at physiological pH 7.4. Triple resonance data were used to get secondary structure information and backbone dynamics of G12C, the best-known drug target among K-Ras mutants. Simultaneous investigation of G12C, G12D and G12V mutants, along with the wild type form at the very same conditions allowed us to perform a comprehensive analysis based on the combined chemical shifts to reveal the effect of mutation at G12 position on structure. Intriguingly, the G12C and G12V mutants found to be structurally very similar at the three most important regions of K-Ras (P-loop, Switch-I, Switch-II), while the G12D mutant significantly differs at P-loop and Switch-II from the wildtype as well as G12C and G12V mutants. However, in Switch-I it hardly deviates from the wildtype protein.