Conformational basis for SH2-Tyr(P)527 binding in Src inactivation
Conformational basis for SH2-Tyr(P)527 binding in Src inactivation
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DOI：
10.1074/jbc.m604219200
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发表时间：
2006-08-18
影响因子：
4.8
通讯作者：
Sun, Gongqin
中科院分区：
文献类型：
--
作者：
Ayrapetov, Marina K.;Wang, Yue-Hao;Sun, Gongqin
Src蛋白酪氨酸激酶包含豆蔻酰化基序、独特区域、Src同源（SH）3结构域、SH 2结构域、催化结构域和C末端尾。C-末端尾部含有Tyr残基Tyr（527）。Tyr（527）的磷酸化触发Src失活，这是由Tyr（P）（527）与SH 2结构域结合引起的。在这项研究中，我们证明了构象贡献，而不是亲和力，是分子内SH 2-Tyr（P）（527）结合的主要力量，我们表征了这种构象贡献的结构基础。首先，模拟C-末端尾部的磷酸肽是比最佳磷酸肽pYEEI弱80倍的配体，并且类似于在结合Src SH 2结构域时在Tyr（P）之后含有三个Ala残基的磷酸肽。其次，SH 2-Tyr（P）（527）结合在很大程度上不依赖于Tyr（P）（527）周围的氨基酸序列，并且仅因SH 2结构域中的失活突变而略微降低。此外，甚至具有YEEI序列的未磷酸化的C-末端尾部也通过与SH 2结构域结合来抑制Src活性。这些实验证明，在Src失活中，非常弱的亲和力足以用于SH 2-Tyr（P）（527）结合。第三，有效的分子内SH 2-Tyr（P）（527）结合归因于需要残基Trp（260）和Leu（255）的构象贡献。尽管SH 3结构域对于Tyr（P）的Src失活是必需的（527），但它不促进SH 2-Tyr（P）（527）结合。这些研究结果表明，构象为基础的Src失活模型，它提供了一个统一的框架，了解Src激活的各种机制。
Src protein-tyrosine kinase contains a myristoylation motif, a unique region, an Src homology (SH) 3 domain, an SH2 domain, a catalytic domain, and a C-terminal tail. The C-terminal tail contains a Tyr residue, Tyr(527). Phosphorylation of Tyr(527) triggers Src inactivation, caused by Tyr(P)(527) binding to the SH2 domain. In this study, we demonstrated that a conformational contribution, not affinity, is the predominant force for the intramolecular SH2-Tyr(P)(527) binding, and we characterized the structural basis for this conformational contribution. First, a phosphopeptide mimicking the C-terminal tail is an 80-fold weaker ligand than the optimal phosphopeptide, pYEEI, and similar to a phosphopeptide containing three Ala residues following Tyr( P) in binding to the Src SH2 domain. Second, the SH2-Tyr(P)(527) binding is largely independent of the amino acid sequence surrounding Tyr(P)(527), and only slightly decreased by an inactivating mutation in the SH2 domain. Furthermore, even the unphosphorylated C-terminal tail with the sequence of YEEI suppresses Src activity by binding to the SH2 domain. These experiments demonstrate that very weak affinity is sufficient for the SH2-Tyr(P)(527) binding in Src inactivation. Third, the effective intramolecular SH2-Tyr(P)(527) binding is attributed to a conformational contribution that requires residues Trp(260) and Leu(255). Although the SH3 domain is essential for Src inactivation by Tyr(P)(527), it does not contribute to the SH2-Tyr(P)(527) binding. These findings suggest a conformation-based Src inactivation model, which provides a unifying framework for understanding Src activation by a variety of mechanisms.