Structure Determination of SH2-Phosphopeptide Complexes by X-Ray Crystallography: The Example of p120RasGAP.
Structure Determination of SH2-Phosphopeptide Complexes by X-Ray Crystallography: The Example of p120RasGAP.
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通过 X 射线晶体学测定 SH2-磷酸肽复合物的结构：以 p120RasGAP 为例。
DOI：
10.1007/978-1-0716-3393-9_5
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Boggon,TitusJ
中科院分区：
文献类型：
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作者：
Stiegler,AmyL;Boggon,TitusJ
p120RasGAP 蛋白包含两个 Src 同源 2 (SH2) 结构域，每个结构域都具有磷酸酪氨酸结合活性。我们描述了分离和纯化的 p120RasGAP SH2 结构域与源自结合伴侣蛋白 p190RhoGAP 的磷酸肽的结晶。将纯化的重组 SH2 结构域蛋白与合成磷酸肽以化学计量比混合，在体外形成复合物。然后通过悬滴蒸汽扩散法在特定的储存溶液上实现结晶，产生含有 SH2 结构域蛋白和磷酸肽的单一大分子共晶。该方案产生了适合 X 射线衍射研究的晶体，我们最近对 p120RasGAP 的两个 SH2 结构域的 X 射线晶体学研究表明，N 端 SH2 结构域以规范相互作用结合磷酸肽。相比之下，C 端 SH2 结构域通过独特的非典型结合模式结合磷酸肽。 p120RasGAP 的晶体学研究表明，尽管 SH2 结构域的三维结构及其与磷酸酪氨酸肽结合的分子细节已经明确，但仔细的结构分析可以继续产生新的分子水平见解。
The p120RasGAP protein contains two Src homology 2 (SH2) domains, each with phosphotyrosine-binding activity. We describe the crystallization of the isolated and purified p120RasGAP SH2 domains with phosphopeptides derived from a binding partner protein, p190RhoGAP. Purified recombinant SH2 domain protein is mixed with synthetic phosphopeptide at a stoichiometric ratio to form the complex in vitro. Crystallization is then achieved by the hanging drop vapor diffusion method over specific reservoir solutions that yield single macromolecular co-crystals containing SH2 domain protein and phosphopeptide. This protocol yields suitable crystals for X-ray diffraction studies, and our recent X-ray crystallography studies of the two SH2 domains of p120RasGAP demonstrate that the N-terminal SH2 domain binds phosphopeptide in a canonical interaction. In contrast, the C-terminal SH2 domain binds phosphopeptide via a unique atypical binding mode. The crystallographic studies for p120RasGAP illustrate that although the three-dimensional structure of SH2 domains and the molecular details of their binding to phosphotyrosine peptides are well defined, careful structural analysis can continue to yield new molecular-level insights.