Highly discriminating protein-protein interaction specificities in the context of a conserved binding energy hotspot
Highly discriminating protein-protein interaction specificities in the context of a conserved binding energy hotspot
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DOI：
10.1016/j.jmb.2004.02.005
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发表时间：
2004-03-26
影响因子：
5.6
通讯作者：
Kleanthous, C
中科院分区：
文献类型：
--
作者：
Li, W;Keeble, AH;Kleanthous, C
我们探讨了高亲和力结合和特异性的保守蛋白质复合物的热力学基础，大肠杆菌素核酸内切酶免疫蛋白复合物作为我们的模型系统。我们研究了每种大肠杆菌素特异性免疫蛋白（Im 2，Im 7，Im.8和Im 9）在体外结合大肠杆菌素E2，E7和E8的核酸内切酶（DNase）结构域的能力，并将其与先前研究的大肠杆菌素E9进行了比较。我们发现（~（14）M）是同源蛋白的共同特征，高亲和力结合（Kd 10大肠杆菌素DNase-Im蛋白复合物）比非同源蛋白-蛋白18弱10（6）-10（8）倍。比较丙氨酸协会，这通常是10个扫描的Im 2和Im 9残基参与结合E2 DNA酶揭示了类似的行为，这两种蛋白质结合E9 DNA酶;螺旋III形成一个保守的结合能热点与特异性残基从螺旋II只有助于有利的同源相互作用，我们称之为“双重识别”的组合。然而，在有助于同源结合的特异性位点的数量和侧链化学方面存在显著差异。在Im 2中，Asp 33从螺旋II占主导地位大肠杆菌素E2特异性，而在Im 9几个疏水残基，包括位置33（亮氨酸），帮助定义其大肠杆菌素特异性。使用噬菌体展示观察到特异性位点的类似分布，其中以Im 2为模板，在螺旋II中产生随机序列的文库，并且文库针对E2或E9 DNA酶进行淘选。位置33是在所有E2 DNA酶选择的克隆中回收的显性特异性位点，而在E9 DNA酶选择的克隆中回收了许多Im 9特异性位点，包括位置33。为了探索生物特异性和体外结合亲和力之间的关系，我们比较了一组免疫蛋白对大肠杆菌素E9毒性的保护程度，这些免疫蛋白对E9 DNA酶的亲和力相差高达10个数量级。该分析表明，完全生物保护所需的Kd < 10（-10）M，并且新免疫蛋白特异性的选择可能演变的“亲和窗口”为10（-6）-10（-10）M。大肠杆菌素DNA酶免疫蛋白复合物的这一全面调查说明了如何高亲和力的蛋白质-蛋白质相互作用可以是非常有区别的，即使结合是由一个保守的热点为主，与单个或多个特异性位点调节整体结合自由能。我们讨论了这些结果的背景下，其他保守的蛋白质复合物，并建议他们指出一个通用的特异性机制，在分化进化的蛋白质-蛋白质相互作用。(C)2004爱思唯尔有限公司保留所有权利。
We explore the thermodynamic basis for high affinity binding and specificity in conserved protein complexes using colicin endonuclease-immunity protein complexes as our model system. We investigated the ability of each colicin-specific immunity protein (Im2, Im7, Im.8 and Im9) to bind the endonuclease (DNase) domains of colicins E2, E7 and E8 in vitro and compared these to the previously studied colicin E9. We find (-14) M) is a common feature of cognate that high affinity binding (Kd 10 colicin DNase-Im protein complexes as are non-cognate protein-protein 1 8 10(6)-10(8) -fold weaker. Comparative alanine associations, which are generally 10 scanning of Im2 and Im9 residues involved in binding the E2 DNase revealed similar behaviour to that of the two proteins binding the E9 DNase; helix III forms a conserved binding energy hotspot with specificity residues from helix II only contributing favourably in a cognate interaction, a combination we have termed as "dual recognition". Significant differences are seen, however, in the number and side-chain chemistries of specificity sites that contribute to cognate binding. In Im2, Asp33 from helix II dominates colicin E2 specificity, whereas in Im9 several hydrophobic residues, including position 33 (leucine), help define its colicin specificity. A similar distribution of specificity sites was seen using phage display where, with Im2 as the template, a library of randomised sequences was generated in helix II and the library panned against either the E2 or E9 DNase. Position 33 was the dominant specificity site recovered in all E2 DNase-selected clones, whereas a number of Im9 specificity sites were recovered in E9 DNase-selected clones, including position 33. In order to probe the relationship between biological specificity and in vitro binding affinity we compared the degree of protection afforded to bacteria against colicin E9 toxicity by a set of immunity proteins whose affinities for the E9 DNase differed by up to ten orders of magnitude. This analysis indicated that the Kd required for complete biological protection is < 10(-10) M and that the "affinity window" over which the selection of novel immunity protein specificities likely evolves is 10(-6)-10(-10) M. This comprehensive survey of colicin DNase-immunity protein complexes illustrates how high affinity protein-protein interactions can be very discriminating even though binding is dominated by a conserved hotspot, with single or multiple specificity sites modulating the overall binding free energy. We discuss these results in the context of other conserved protein complexes and suggest that they point to a generic specificity mechanism in divergently evolved protein-protein interactions. (C) 2004 Elsevier Ltd. All rights reserved.