Structure of the dominant negative S17N mutant of Ras.
Structure of the dominant negative S17N mutant of Ras.
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DOI：
10.1021/bi9020742
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发表时间：
2010-03-09
期刊：
影响因子：
2.9
通讯作者：
Garcia-Diaz, Miguel
中科院分区：
文献类型：
--
作者：
Nassar, Nicolas;Singh, Kavita;Garcia-Diaz, Miguel
Ras显性负突变体的使用在阐明Ras响应于各种膜结合受体的激活的细胞信号传导中是至关重要的。虽然Ras的几个点突变体表现出显性负效应，但在位置17（S17 N）处的天冬酰胺至丝氨酸突变仍然是最流行的，并且在抑制内源性Ras的活化方面最有效。现在广泛接受的是，显性负效应是由于突变体的能力，以螯合上游激活剂和其无法激活下游效应。在这里，我们提出了在GDP结合形式的RasS 17 N的晶体结构。在占据不对称单元的三个分子中，由于Asn 17侧链的空间位阻，通常与β-磷酸盐配位的Mg 2+离子不存在。相反，Ca 2+离子与α-磷酸盐配位。一个分子中还缺少Phe 28的电子密度，Phe 28是一个保守的残基，通常稳定核苷酸的鸟嘌呤碱基。除Phe 28外，该核苷酸与Ras的相互作用是保守的。结合，Phe 28不能稳定鸟嘌呤碱基和缺乏Mg 2+离子来中和磷酸盐上的负电荷解释了GDP对Ras的较弱亲和力。我们的数据表明，Mg 2+的情况下，也应该显着影响GTP结合Ras和适当的定位Thr 35必要的开关1的激活和下游效应器的结合，触发信号通路的先决条件。
The use of the dominant negative mutant of Ras has been crucial in elucidating the cellular signaling of Ras in response to the activation of various membrane-bound receptors. Although several point mutants of Ras exhibit a dominant negative effect, the asparagine to serine mutation at position 17 (S17N) remains the most popular and the most effective at inhibiting the activation of endogenous Ras. It is now widely accepted that the dominant negative effect is due to the ability of the mutant to sequester upstream activators and its inability to activate downstream effectors. Here, we present the crystal structure of RasS17N in the GDP-bound form. In the three molecules that populate the asymmetric unit, the Mg2+ ion that normally coordinates the β-phosphate is absent because of steric hindrance from the Asn17 side chain. Instead, a Ca2+ ion is coordinating the α-phosphate. Also absent from one molecule is electron density for Phe28, a conserved residue that normally stabilizes the nucleotide’s guanine base. Except for Phe28, the nucleotide makes conserved interactions with Ras. Combined, the inability of Phe28 to stabilize the guanine base and the absence of a Mg2+ ion to neutralize the negative charges on the phosphates explain the weaker affinity of GDP for Ras. Our data suggest that the absence of the Mg2+ should also dramatically affect GTP binding to Ras and the proper positioning of Thr35 necessary for the activation of switch 1 and the binding to downstream effectors, a prerequisite for the triggering of signaling pathways.
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