Structure-based mutagenesis reveals distinct functions for Ras switch 1 and switch 2 in Sos-catalyzed guanine nucleotide exchange
Structure-based mutagenesis reveals distinct functions for Ras switch 1 and switch 2 in Sos-catalyzed guanine nucleotide exchange
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DOI：
10.1074/jbc.m101727200
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发表时间：
2001-07-20
影响因子：
4.8
通讯作者：
Bar-Sagi, D
中科院分区：
文献类型：
--
作者：
Hall, BE;Yang, SS;Bar-Sagi, D
RAS GTP酶在控制细胞生长和分化的信号通路中起着二元开关的作用。鸟嘌呤核苷酸交换因子SOS介导RAS的激活，以响应细胞外信号。我们以前已经解决了无核苷酸RAS的晶体结构与SOS催化结构域的络合物(Boriack-Sjodin，P.A.，Margarit，S.M.，Bar-Sagi，D.和Kuriyan，J.(1998)Natural 394,337-343)，其结构表明SOS诱导RAS的两个环区的构象变化，称为开关1和开关2，在本研究中，我们采用定点突变的方法来研究构象变化对SOS催化功能的功能意义。RAS的开关2被SOS紧紧地抱在怀里，几乎每个外部侧链都由SOS协调。对Switch 2-SOS界面接触残基的突变表明，只有少数侧链影响结合，其中最重要的接触是由酪氨酸介导的，它被埋在RAS SOS复合体中的SOS疏水口袋中。插入开关2(Ras Ala(59)和SOS Leu(938)和Glu(942))的镁离子和核苷酸磷酸结合部位的RAS和SOS侧链的取代对SOS的催化功能没有影响。这些结果表明，SOS与开关2的相互作用是紧密结合所必需的，但不是GDP位移的关键驱动力。SOS诱导的Switch 1的结构扭曲是由RAS和SOS上高度保守的残基之间的少量特异性接触所介导的。这些残基的一个子集(Ras Tyr(32)和Tyr(40))的突变导致核苷酸从RAS解离的固有速率增加，并损害RAS与SOS的结合。基于这一分析，我认为SOS与RAS的Snitch 1和Switch 2区域的相互作用具有不同的功能后果：与Switch 2的相互作用介导RAS与SOS的锚定，而与Switch 1的相互作用导致核苷酸结合位点的破坏和GDP的解离。
Ras GTPases function as binary switches in signaling pathways controlling cell growth and differentiation. The guanine nucleotide exchange factor Sos mediates the activation of Ras in response to extracellular signals. We have previously solved the crystal structure of nucleotide-free Ras in complex with the catalytic domain of Sos (Boriack-Sjodin, P.A., Margarit, S.M., Bar-Sagi, D., and Kuriyan, J. (1998) Nature 394, 337-343), The structure demonstrates that Sos induces conformational changes in two loop regions of Ras known as switch 1 and switch 2, In this study, we have employed site-directed mutagenesis to investigate the functional significance of the conformational changes for the catalytic function of Sos. Switch 2 of Ras is held in a very tight embrace by Sos, with almost every external side chain coordinated by Sos. Mutagenesis of contact residues at the switch 2-Sos interface shows that only a small set of side chains affect binding, with the most important contact being mediated by tyrosine 64, which is buried in a hydrophobic pocket of Sos in the Ras Sos complex. Substitutions of Ras and Sos side chains that are inserted into the Mg2+- and nucleotide phosphate-binding site of switch 2 (Ras Ala(59) and Sos Leu(938) and Glu(942)) have no effect on the catalytic function of Sos. These results indicate that the interaction of Sos with switch 2 is necessary for tight binding, but is not the critical driving force for GDP displacement. The structural distortion of switch 1 induced by Sos is mediated by a small number of specific contacts between highly conserved residues on both Ras and Sos. Mutations of a subset of these residues (Ras Tyr(32) and Tyr(40)) result in an increase in the intrinsic rate of nucleotide dissociation from Ras and impair the binding of Ras to Sos. Based on this analysis, me propose that the interactions of Sos with the snitch 1 and switch 2 regions of Ras have distinct functional consequences: the interaction with switch 2 mediates the anchoring of Ras to Sos, whereas the interaction with switch 1 leads to disruption of the nucleotide-binding site and GDP dissociation.