Assessing the Influence of Mutation on GTPase Transition States by using X-ray Crystallography, 19F NMR, and DFT Approaches,Angewandte Chemie International Edition

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Assessing the Influence of Mutation on GTPase Transition States by using X-ray Crystallography, 19F NMR, and DFT Approaches
Angewandte Chemie International Edition ( IF 16.1 ) Pub Date : 2017-05-24 08:50:50 , DOI: 10.1002/anie.201703074
Yi Jin _{1,

2} , Robert W. Molt _{2,

3,

4} , Erika Pellegrini ₅ , Matthew J. Cliff ₆ , Matthew W. Bowler _{5,

7} , Nigel G. J. Richards ₂ , G. Michael Blackburn ₁ , Jonathan P. Waltho _{1,

6}

Affiliation

We report X-ray crystallographic and ¹⁹F NMR studies of the G-protein RhoA complexed with MgF₃⁻, GDP, and RhoGAP, which has the mutation Arg85′Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X-ray data show how Tyr34 maintains solvent exclusion and the core H-bond network in the active site by relocating to replace the missing Arg85′ sidechain. The ¹⁹F NMR data show deshielding effects that indicate the main function of Arg85′ is electronic polarization of the transferring phosphoryl group, primarily mediated by H-bonding to O^3G and thence to P^G. DFT calculations identify electron-density redistribution and pinpoint why the TS for guanosine 5′-triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAP_Arg85′Ala relative to wild-type (WT) RhoGAP. This study demonstrates that ¹⁹F NMR measurements, in combination with X-ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site-specific modifications.

中文翻译：

使用X射线晶体学，19F NMR和DFT方法评估突变对GTPase过渡态的影响

我们报告的X射线晶体学和^19个G蛋白RhoA的络合氟化镁的F NMR研究₃^-，GDP和RhoGAP，它具有突变Arg85'Ala。与DFT计算结合使用时，这些数据可以识别过渡状态（TS）属性的变化。X射线数据显示Tyr34如何通过重新定位来替换缺失的Arg85'侧链，从而在活性位点保持溶剂排除和核心H键网络。的^19个F NMR数据显示去屏蔽指示Arg85的主要功能效应“是所述转印磷酰基的电子极化，主要由氢键介导至O ^3G并从那里至P ^ģ。DFT计算确定了电子密度的重新分布，并指出了当RhoA与RhoGAP _Arg85'Ala相比于野生型（WT）RhoGAP时，鸟苷5'-三磷酸（GTP）水解的TS在能量上更高的原因。这项研究表明¹⁹ F NMR测量与X射线晶体学和DFT计算相结合，可以可靠地剖析小GTP酶对位点特异性修饰的响应。

更新日期：2017-05-25

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