Surface change of Ras enabling effector binding monitored in real time at atomic resolution
Surface change of Ras enabling effector binding monitored in real time at atomic resolution
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DOI：
10.1002/cbic.200600552
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发表时间：
2007-05-07
期刊：
影响因子：
3.2
通讯作者：
Gerwert, Klaus
中科院分区：
文献类型：
--
作者：
Koetting, Carsten;Kallenbach, Angela;Gerwert, Klaus
Ras是Ras超家族的原型，充当细胞生长的分子开关。外部增长信号诱导GDP到GTP的交换。这修饰Ras表面（Ras（on）GTP）并使效应物结合成为可能，然后激活信号转导途径。由Ras和GAP催化的GTP水解将信号返回到"关闭“（Ras（关闭）GDP）。Ras的致癌突变阻止了这种水解，从而导致细胞生长失控。在Ras（关闭）到RaSon的转变中，Ras表面通过控制效应物结合的开关I环的移动而改变。我们监测原子分辨率的表面变化，在真实的时间的时间分辨傅里叶变换红外光谱（trFTIR）光谱。在从Ras（off）到RaSon的转变中，现在鉴定出一种GTP结合的中间体，其中效应物结合仍然被阻止（Ras（off）GTP）。通过Thr 35的C=O振动直接监测从Ras（off）GTP到Ras（on）GTP的环运动。这种结构变化产生了一个结合位点，在260 K下的速率常数为5 s（-1）。一个小分子将平衡从RaS（on）GTP状态转移到Ras（off）GTP状态，将阻止效应物结合，即使水解被致癌突变阻断。我们提出了一个光谱指纹的两种状态，可以用作药物筛选这种小分子的测定。
Ras, the prototype of the Ras superfamily, acts as a molecular switch for cell growth. External growth signals induce a GDP-to-GTP exchange. This modifies the Ras surface (Ras(on)GTP) and enables effector binding, which then activates signal-transduction pathways. GTP hydrolysis, catalysed by Ras and GAP returns the signal to '' off '' (Ras(off)GDP). Oncogenic mutations in Ras prevent this hydrolysis, and thereby cause uncontrolled cell growth. In the Ras(off)to-RaSon transition, the Ras surface is changed by a movement of the switch I loop that controls effector binding. We monitored this surface change at atomic resolution in real time by time-resolved FTIR (trFTIR) spectroscopy. In the transition from Ras(off) to RaSon a GTP-bound intermediate is now identified in which effector binding is still prevented (Ras(off)GTP). The loop movement from Ras(off)GTP to Ras(on)GTP was directly monitored by the C=O vibration of Thr35. The structural change creates a binding site with a rate constant of 5 s(-1) at 260 K A small molecule that shifted the equilibrium from the RaS(on)GTP state towards the Ras(off)GTP state would prevent effector binding, even if hydrolysis were blocked by oncogenic mutations. We present a spectroscopic fingerprint of both states that can be used as an assay in drug screening for such small molecules.