Structural Determinants of Affinity Enhancement between GoLoco Motifs and G-Protein α Subunit Mutants
Structural Determinants of Affinity Enhancement between GoLoco Motifs and G-Protein α Subunit Mutants
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DOI：
10.1074/jbc.m110.190496
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发表时间：
2011-02-04
影响因子：
4.8
通讯作者：
Siderovski, David P.
中科院分区：
文献类型：
--
作者：
Bosch, Dustin E.;Kimple, Adam J.;Siderovski, David P.
GoLoco基序蛋白结合G蛋白α亚基的抑制G(i)亚类并减缓结合的GDP的释放；这种相互作用被认为是不对称细胞分裂和神经上皮和上皮祖细胞分化的关键。为了提供蛋白质工具来询问GoLoco基序/G α (i)复合物的精确细胞作用，我们采用了基于结构的蛋白质设计策略来预测增加GoLoco基序结合亲和力的功能获得突变。在这里，我们描述了荧光偏振和等温滴定量热测量显示三个预测的G α (i1)点突变，E116L， Q147L和E245L；每个都增加了对多个GoLoco基序的亲和力。这种亲和力增强的一个组成部分是由于G α突变体和GoLoco基序之间的解离率降低。对于G α (Q147L)(i1)，亲和增强被认为是由结合焓的有利变化驱动的，尽管结合熵的贡献减少了。结合RGS14 GoLoco基序的G(α i1)（Q147L）的晶体结构揭示了围绕明确的Leu-147侧链的三个肽残基之间的无序性。蒙特卡罗模拟的肽在这个区域显示了多个骨干构象的采样，而不是野生型复合物。我们得出的结论是，将Glu-147突变为亮氨酸产生了一个有利于GoLoco肽结合的疏水表面，但疏水的Glu-147也促进了RGS14 GoLoco肽残基511-513之间的柔韧性。
GoLoco motif proteins bind to the inhibitory G(i) subclass of G-protein alpha subunits and slow the release of bound GDP; this interaction is considered critical to asymmetric cell division and neuro-epithelium and epithelial progenitor differentiation. To provide protein tools for interrogating the precise cellular role(s) of GoLoco motif/G alpha(i) complexes, we have employed structure-based protein design strategies to predict gain-of-function mutations that increase GoLoco motif binding affinity. Here, we describe fluorescence polarization and isothermal titration calorimetry measurements showing three predicted G alpha(i1) point mutations, E116L, Q147L, and E245L; each increases affinity for multiple GoLoco motifs. A component of this affinity enhancement results from a decreased rate of dissociation between the G alpha mutants and GoLoco motifs. For G alpha(Q147L)(i1), affinity enhancement was seen to be driven by favorable changes in binding enthalpy, despite reduced contributions from binding entropy. The crystal structure of G(alpha i1)(Q147L) bound to the RGS14 GoLoco motif revealed disorder among three peptide residues surrounding a well defined Leu-147 side chain. Monte Carlo simulations of the peptide in this region showed a sampling of multiple backbone conformations in contrast to the wild-type complex. We conclude that mutation of Glu-147 to leucine creates a hydrophobic surface favorably buried upon GoLoco peptide binding, yet the hydrophobic Leu-147 also promotes flexibility among residues 511-513 of the RGS14 GoLoco peptide.