3-DIMENSIONAL STRUCTURES AND PROPERTIES OF A TRANSFORMING AND A NONTRANSFORMING GLYCINE-12 MUTANT OF P21(H-RAS)
3-DIMENSIONAL STRUCTURES AND PROPERTIES OF A TRANSFORMING AND A NONTRANSFORMING GLYCINE-12 MUTANT OF P21(H-RAS)
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DOI：
10.1021/bi00084a005
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发表时间：
1993-08-24
期刊：
影响因子：
2.9
通讯作者：
WITTINGHOFER, A
中科院分区：
文献类型：
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作者：
FRANKEN, SM;SCHEIDIG, AJ;WITTINGHOFER, A
用鸟苷5‘-(β，γ-亚胺)三磷酸(GppNHp)测定了p21H-ras G区(残基1-166)的两个突变体p21(G12D)和p21(G12P)在三磷酸结合形式下的三维结构和生化性质。它们分别对应于Gly-12最常见的致癌突变和唯一的非致癌突变。G12D突变是迄今为止分析的唯一一个在不同于野生型的空间群中结晶的突变体，与野生型p21相比，该蛋白质的原子模型显示出活性部位周围结构的最大变化。这是由于天冬氨酸侧链与Tyr-32、Gln-61和伽马-磷酸的相互作用，导致这些结构元素的流动性降低。Asp-12的羧酸基与γ-磷酸之间的相互作用是由一个共享的质子介导的，P-31核磁共振测量表明，这个质子也存在于溶液中。P21(G12P)在活性部位的结构与野生型p21非常相似，包括亲核水的位置。Pro-12的吡咯烷环向外指向，这可能是导致GAP(GTP酶激活蛋白)亲和力较弱以及GAP不能刺激GTP水解的原因。
The three-dimensional structures and biochemical properties of two mutants of the G-domain (residues 1-166) of p21H-ras, p21 (G12D) and p21 (G12P), have been determined in the triphosphate-bound form using guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp). They correspond to the most frequent oncogenic and the only nononcogenic mutation of Gly-12, respectively. The G12D mutation is the only mutant analyzed so far that crystallizes in a space group different from wild type, and the atomic model of the protein shows the most drastic changes of structure around the active site as compared to wild-type p21. This is due to the interactions of the aspartic acid side chain with Tyr-32, Gln-61, and the gamma-phosphate, which result in reduced mobility of these structural elements. The interaction between the carboxylate group of Asp-12 and the gamma-phosphate is mediated by a shared proton, which we show by P-31 NMR measurements to exist in solution as well. The structure of p21 (G12P) is remarkably similar to that of wild-type p21 in the active site, including the position of the nucleophilic water. The pyrrolidine ring of Pro-12 points outward and seems to be responsible for the weaker affinity toward GAP (GTPase-activating protein) and the failure of GAP to stimulate GTP hydrolysis.