Transformation efficiency of RasQ61 mutants linked to structural features of the switch regions in the presence of Raf
Transformation efficiency of RasQ61 mutants linked to structural features of the switch regions in the presence of Raf
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DOI：
10.1016/j.str.2007.10.011
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发表时间：
2007-12-01
期刊：
影响因子：
5.7
通讯作者：
Mattos, Carla
中科院分区：
文献类型：
--
作者：
Buhrman, Greg;Wink, Glenna;Mattos, Carla
Ras突变体在残基61处的转化效率在三个数量级以上，但所有突变体的体外GTP酶活性都下降了10倍。我们发现Raf损害了RasQ61L的GTP酶活性，表明Ras/Raf复合体对转化起差异调控作用。我们的晶体结构表明，在转化突变体的过程中，Switch 11参与了掩埋核苷酸和前催化水分子的疏水相互作用网络。我们的结果表明，在没有GAP的情况下，Y32和连接它与野生型结构中的伽马-磷酸的水分子代替Arg指在GTP的水解中发挥作用。在转化突变体中没有桥接水分子，这有助于核苷酸的掩埋。我们提出了Raf结合的RAS的内在水解机制，并阐明了Q61突变体中与其转化细胞能力相关的结构特征。
Transformation efficiencies of Ras mutants at residue 61 range over three orders of magnitude, but the in vitro GTPase activity decreases 10-fold for all mutants. We show that Raf impairs the GTPase activity of RasQ61L, suggesting that the Ras/Raf complex differentially modulates transformation. Our crystal structures show that, in transforming mutants, switch 11 takes part in a network of hydrophobic interactions burying the nucleotide and precatalytic water molecule. Our results suggest that Y32 and a water molecule bridging it to the gamma-phosphate in the wild-type structure play a role in GTP hydrolysis in lieu of the Arg finger in the absence of GAP. The bridging water molecule is absent in the transforming mutants, contributing to the burying of the nucleotide. We propose a mechanism for intrinsic hydrolysis in Raf-bound Ras and elucidate structural features in the Q61 mutants that correlate with their potency to transform cells.