Dual recognition and the role of specificity-determining residues in colicin E9 DNase-immunity protein interactions
Dual recognition and the role of specificity-determining residues in colicin E9 DNase-immunity protein interactions
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DOI：
10.1021/bi9808621
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发表时间：
1998-08-25
期刊：
影响因子：
2.9
通讯作者：
Kleanthous, C
中科院分区：
文献类型：
--
作者：
Li, W;Hamill, SJ;Kleanthous, C
免疫蛋白Im 2可以结合并抑制细菌毒素大肠杆菌素E9的非同源核酸内切酶结构域，其Kd为19 nM，比同源免疫蛋白Im 9的Kd弱6个数量级，与Im 9具有68%的序列同一性。我们实验室以前的工作已经表明，这些四螺旋免疫蛋白的特异性差异几乎完全是由于螺旋II，其在免疫蛋白家族中的序列在很大程度上是可变的。从Im 9的丙氨酸扫描诱变结合高场NMR数据，已经提出了大肠杆菌素-免疫蛋白特异性的双重识别模型，其中免疫蛋白的螺旋III的保守残基作为核酸内切酶结合位点的锚，而螺旋II的可变残基控制蛋白质-蛋白质相互作用的特异性。在这项工作中，我们确定了三个残基（在位置33，34和38）在螺旋II定义的特异性差异的Im 2和Im 9大肠杆菌素E9，并使用丙氨酸诱变的推定的核酸内切酶结合表面的Im 2，比较保守和非保守位点的结合能分布在两个免疫蛋白。这种比较突出了Im 2和Im 9的保守残基作为E9 DNA酶结合能的主要决定因素。相反，非保守的，特异性决定残基只有助于E9 DNA酶的结合能在同源的Im 9蛋白，而在非同源的免疫蛋白Im 2，他们要么不稳定的复合物或不有助于结合能。因此，两种免疫蛋白的比较丙氨酸扫描支持大肠杆菌素-免疫蛋白相互作用中选择性的双重识别机制，并为理解涉及结构保守蛋白质家族的其他蛋白质-蛋白质相互作用系统中的特异性提供了基础。
The immunity protein Im2 can bind and inhibit the noncognate endonuclease domain of the bacterial toxin colicin E9 with a K-d Of 19 nM, 6 orders of magnitude weaker than that of the cognate immunity protein Im9 with which it shares 68% sequence identity. Previous work from our laboratory has shown that the specificity differences of these four-helix immunity proteins is due almost entirely to helix II which is largely variable in sequence in the immunity protein family. From alanine scanning mutagenesis of Im9 in conjunction with high-field NMR data, a dual recognition model for colicin-immunity protein specificity has been proposed whereby the conserved residues of helix III of the immunity protein act as the anchor of the endonuclease binding site while the variable residues of helix II control the specificity of the protein-protein interaction. In this work, we identify three residues (at positions 33, 34, and 38) in helix II which define the specificity differences of Im2 and Im9 for colicin E9 and, using alanine mutagenesis of the putative endonuclease binding surface of Im2, compare the distribution of binding energies for conserved and nonconserved sites in both immunity proteins. This comparison highlights the conserved residues of both Im2 and Im9 as the major determinants of E9 DNase binding energy. Conversely, the nonconserved, specificity-determining residues only contribute to the E9 DNase binding energy in the cognate Im9 protein, while in the noncognate immunity protein Im2, they either destabilize the complex or do not contribute to the binding energy. This comparative alanine scan of two immunity proteins therefore supports the dual recognition mechanism of selectivity in colicin-immunity protein interactions and provides a basis for understanding specificity in other protein-protein interaction systems involving structurally conserved protein families.