Single-amino acid substitutions alter the specificity and affinity of PDZ domains for their ligands
Single-amino acid substitutions alter the specificity and affinity of PDZ domains for their ligands
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DOI：
10.1021/bi001633t
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发表时间：
2000-11-28
期刊：
影响因子：
2.9
通讯作者：
Chabot, J
中科院分区：
文献类型：
--
作者：
Gee, SH;Quenneville, S;Chabot, J
PDZ结构域是模块化蛋白质-蛋白质相互作用结构域，其结合至膜蛋白的特异性C-末端序列和/或结合至其他PDZ结构域。PSD-95和促突触蛋白中的某些PDZ结构域与C-末端肽配体相互作用，并与延伸的nNOS PDZ结构域异二聚化。与nNOS相互作用的能力与这些PDZ结构域的羧酸结合环中的Lys残基的存在相关。在这里，我们报告说，在PSD-95 PDZ 2的Lys-165的精氨酸取代破坏其与nNOS的相互作用，但不与C端的摇床型K+通道Kv1.4。相同的突变影响nNOS结合α 1和β 1-syntrophin PDZ结构域在较小程度上，部分原因是三级相互作用与典型的nNOS PDZ结构域的稳定作用。在羧酸结合环中具有Arg的PDZ结构域不结合nNOS;然而，用Lys或Ala取代能够赋予nNOS结合。我们的研究结果表明，羧酸结合环赖氨酸或精氨酸是nNOS结合的一个关键决定因素，这个残基的身份可以深刻地改变一种模式的PDZ识别，而不影响另一个。我们还分析了突变Asp-143的影响，Asp-143是alpha 1-syntrophin的alphaB螺旋中的一个残基，与nNOS PDZ结构域形成三级接触。该残基对于nNOS和C-末端肽结合都是重要的，并且赋予在位置-4处具有带正电荷残基的肽的优先性。在此基础上，我们已经确定了Kir2.1通道的C末端作为可能的结合伴侣的syntrophin PDZ结构域。总之，我们的研究结果表明，单氨基酸取代改变了PDZ结构域对其配体的特异性和亲和力。
PDZ domains are modular protein-protein interaction domains that bind to specific C-terminal sequences of membrane proteins and/or to other PDZ domains. Certain PDZ domains in PSD-95 and syntrophins interact with C-terminal peptide ligands and heterodimerize with the extended nNOS PDZ domain. The capacity to interact with nNOS correlates with the presence of a Lys residue in the carboxylate-binding loop of these PDZ domains. Here, we report that substitution of an Arg for Lys-165 in PSD-95 PDZ2 disrupted its interaction with nNOS, but not with the C terminus of the Shaker-type K+ channel Kv1.4. The same mutation affected nNOS binding to alpha1- and beta1-syntrophin PDZ domains to a lesser extent, due in part to the stabilizing effect of tertiary interactions with the canonical nNOS PDZ domain. PDZ domains with an Arg in the carboxylate-binding loop do not bind nNOS; however, substitution with Lys or Ala was able to confer nNOS binding. Our results indicate that the carboxylate-binding loop Lys or Arg is a critical determinant of nNOS binding and that the identity of this residue can profoundly alter one mode of PDZ recognition without affecting another. We also analyzed the effects of mutating Asp-143, a residue in the alphaB helix of alpha1-syntrophin that forms a tertiary contact with the nNOS PDZ domain. This residue is important for both nNOS and C-terminal peptide binding and confers a preference for peptides with a positively charged residue at position -4. On this basis, we have identified the C terminus of the Kir2.1 channel as a possible binding partner for syntrophin PDZ domains. Together, our results demonstrate that single-amino acid substitutions alter the specificity and affinity of PDZ domains for their ligands.