Franken S M, Scheidig A J, Krengel U, Rensland H, Lautwein A, Geyer M, Scheffzek K, Goody R S, Kalbitzer H R, Pai E F
Abteilung Biophysik, Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany.
Biochemistry. 1993 Aug 24;32(33):8411-20. doi: 10.1021/bi00084a005.
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 31P 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.
已使用鸟苷5'-（β，γ-亚氨基）三磷酸（GppNHp）以三磷酸结合形式测定了p21H-ras的G结构域（第1至166位氨基酸残基）的两个突变体p21（G12D）和p21（G12P）的三维结构及生化特性。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活性位点周围的结构变化最为剧烈。这是由于天冬氨酸侧链与Tyr-32、Gln-61以及γ-磷酸之间的相互作用，导致这些结构元件的流动性降低。Asp-12的羧基与γ-磷酸之间的相互作用由一个共享质子介导，我们通过31P NMR测量表明其在溶液中也存在。p21（G12P）在活性位点的结构与野生型p21非常相似，包括亲核水的位置。Pro-12的吡咯烷环向外突出，似乎是其对GAP（GTP酶激活蛋白）亲和力较弱以及GAP无法刺激GTP水解的原因。
