Backbone dynamics of an oncogenic mutant of Cdc42Hs shows increased flexibility at the nucleotide-binding site
Backbone dynamics of an oncogenic mutant of Cdc42Hs shows increased flexibility at the nucleotide-binding site
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DOI：
10.1021/bi0490901
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发表时间：
2004-08-10
期刊：
影响因子：
2.9
通讯作者：
Oswald, RE
中科院分区：
文献类型：
--
作者：
Adams, PD;Loh, AP;Oswald, RE
Cdc42Hs是gtp结合信号转导蛋白Ras表面的成员，与鸟嘌呤核苷酸结合，在多种信号转导途径中起分子定时开关的作用。野生型蛋白的结构已经确定（Feltham et al. (1997) Biochemistry 36, 8755-8766），骨架动力学已通过NMR波谱表征（Loh et al. (1999) Biochemistry 38, 12547-12557）。Cdc42Hs的F28L突变的特点是GTP和gdp结合形式之间的循环速率增加，导致细胞转化(Lin et al. (1997) Curr。生物学报，7,794 -797)。在这里，我们用H-1-N-15核磁共振测量了两种磁场强度下的T-1、T-1p和稳态NOE来描述Cdc42Hs(F28L)-GDP的骨架动力学。利用Lipari-Szabo形式得到了广义阶参数S-s(2)和S-f(2)、局部相关时间tau(e)和汇率R-ex的残差特异值。化学位移微扰分析表明，核苷酸结合位点外的结构变化很小。然而，在Cdc42Hs（F28L）中，与野生型相比，包含核苷酸结合位点的残基以及核苷酸本身在大范围的时间尺度上表现出增加的动态。除了弛豫法测量的动力学变化外，氢-氘交换表明核苷酸结合位点内的氢键网络发生了实质性的破坏。因此，由单点突变引入的局部动态变化可以影响信号传导过程的重要方面，而不会破坏整个蛋白质的构象。
Cdc42Hs, a member of the Ras superfantily of GTP-binding signal transduction proteins, binds guanine nucleotides, and acts as a molecular-timing switch in multiple signal transduction pathways. The structure of the wild-type protein has been solved (Feltham et al. (1997) Biochemistry 36, 8755-8766), and the backbone dynamics have been characterized by NMR spectroscopy (Loh et al. (1999) Biochemistry 38, 12547-12557). The F28L mutation of Cdc42Hs is characterized by an increased rate of cycling between the GTP and GDP-bound forms leading to cell transformation (Lin et al. (1997) Curr. Biol. 7, 794-797). Here, we describe the backbone dynamics of Cdc42Hs(F28L)-GDP using H-1-N-15 NMR measurements of T-1, T-1p, and steady-state NOE at two magnetic field strengths. Residue-specific values of the generalized order parameters (S-s(2) and S-f(2)), local correlation time (tau(e)), and exchange rate (R-ex) were obtained using the Lipari-Szabo formalism. Chemical-shift perturbation analysis suggested that very little structural change was evident outside of the nucleotide-binding site. However, residues comprising the nucleotide-binding site, as well as the nucleotide itself, exhibit increased dynamics over a wide range of time scales in Cdc42Hs(F28L) relative to the wild type. In addition to changes in dynamics measured by relaxation methods, hydrogen-deuterium exchange indicated a substantial disruption of the hydrogen-bonding network within the nucleotide-binding site. Thus, local dynamic changes introduced by a single-point mutation can affect important aspects of signaling processes without disrupting the conformation of the whole protein.