RT loop flexibility enhances the specificity of Src family SH3 domains for HIV-1 Nef
RT loop flexibility enhances the specificity of Src family SH3 domains for HIV-1 Nef
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DOI：
10.1021/bi980989q
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发表时间：
1998-10-20
期刊：
影响因子：
2.9
通讯作者：
Ladbury, JE
中科院分区：
文献类型：
--
作者：
Arold, S;O'Brien, R;Ladbury, JE
对SH3结构域相互作用的特异性问题的理解在很大程度上是在研究这些结构域潜在配体衍生的富含脯氨酸的氨基酸序列相互作用的研究中解决的。尽管与该基序的相互作用形成了SH3结构域结合的重要平台，但在许多情况下，几乎没有观察到特异性，所谓的特异和非特异性富含脯氨酸序列的亲和力差异不大。此外，SH3结构域与蛋白质配体之间的结合界面似乎包含了比富含Pro基序的结合界面更多的相互作用。在这里，我们从相反的角度研究特异性问题；即，配体如何识别不同的SH3结构域？我们给出了来自造血细胞激酶(HCK)的未结合的SH3结构域的晶体结构，该蛋白是酪氨酸激酶Src家族的成员之一。这种结构表明，与其他Src激酶SH3结构域的结构不同，RT环区具有高度的流动性，并且缺乏其他地方明显的氢键网络。RT环已被证明构成了SH3结构域与HIV-1Nef之间结合界面的主要部分。等温滴定量热法得到的HCK SH3与HIV-1 Nef结合的热力学数据表明，HCK(K-D=1.5muM)的结合比所研究的其他Src家族激酶(Fyn、Lck和Src)的结合紧密约一个数量级。这种亲和力的增加尤其归因于RT环中固有的灵活性，它不需要破坏氢键网络来采用结合所需的构象。
Understanding the issue of specificity imposed in the interactions of SH3 domains has largely been addressed in studies investigating the interaction of proline-rich amino acid sequences derived from potential ligands for these domains. Although the interaction with this motif forms an essential platform in the binding of SH3 domains, in many cases little specificity is observed and the difference in affinity for so-called specific and nonspecific proline-rich sequences is not great. Furthermore, the binding interface between an SH3 domain and a protein ligand appears to encompass more interactions than are represented by that involving the proline-rich motif. Here we investigate the issue of specificity from the opposite point of view; namely, how does a ligand recognize different SH3 domains? We present the crystal structure of the unbound SH3 domain from hemopoietic cell kinase (Hck) which isa member of the Src family of tyrosine kinases. This structure reveals that, unlike the structures of other Src kinase SH3 domains, the RT loop region is highly mobile and lacks a network of hydrogen bonds that is elsewhere apparent. The RT loop has been shown to form a major part of the binding interface between SH3 domains and HIV-1 Nef. Thermodynamic data, derived from isothermal titration calorimetry, for the binding of Hck SH3 to HIV-1 Nef show that the binding of Hck (K-D = 1.5 mu M) is approximately an order of magnitude tighter than those of other Src family kinases that were investigated (Fyn, Lck, and Src). This increase in affinity is attributed to, among other effects, the inherent flexibility in the RT loop which does not require breaking the network of hydrogen bonds to adopt the conformation required for binding.