Experimental and computational analyses of the energetic basis for dual recognition of immunity proteins by colicin endonucleases
Experimental and computational analyses of the energetic basis for dual recognition of immunity proteins by colicin endonucleases
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DOI：
10.1016/j.jmb.2008.03.055
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发表时间：
2008-06-13
影响因子：
5.6
通讯作者：
Kleanthous, Cohn
中科院分区：
文献类型：
--
作者：
Keeble, Anthony H.;Joachimiak, Lukasz A.;Kleanthous, Cohn
大肠杆菌素核酸内切酶（DNA酶）被免疫（Im）蛋白结合并失活。Im蛋白是广泛的交叉反应性但特异性的抑制剂，其结合同源和非同源DNA酶，K-d值在10（-4）和10（-14）M之间变化，其特征由“双重识别”机制解释。在这项工作中，我们首次解决了大肠杆菌素DNA酶通过E9 DNA酶丙氨酸扫描和双突变体循环（DMC）的组合，再加上动力学和量热分析的同源Im 9和非同源Im 2结合，以及丙氨酸扫描和DMC数据的计算分析的Im蛋白识别的能量学。我们发现，四个E9 DNA酶残基观察到的差异Δ Δ G累积区分同源Im 9协会从非同源Im 2协会。E9 DNA酶Phe 86是界面中心的主要特异性热点残基，其由同源Im蛋白的保守和可变热点残基协调。实验DMC分析显示，仅观察到与Im 9残基的适度偶联能，与使用程序ROSETTA计算的DMC一致，并且与E9 DNA酶-Im 9特异性接触的大部分疏水性质一致。12个E9 DNA酶丙氨酸突变体的计算值与实验Δ Δ G数据显示出合理的一致性，特别是对于界面水分子不介导的相互作用。使用溶剂化旋转异构体模型计算的接触掩埋水分子的残留物的Δ Δ G预测取得了混合的成功，然而，我们能够以高度的准确度预测一个这样的接触的位置和能量贡献。我们的研究强调了大肠杆菌素DNA酶如何能够利用保守和可变氨基酸来区分同源与非同源的Im蛋白，其中保守残基的能量贡献由相邻的特异性位点调节。(C)2008爱思唯尔有限公司保留所有权利。
Colicin endonucleases (DNases) are bound and inactivated by immunity (Im) proteins. Im proteins are broadly cross-reactive yet specific inhibitors binding cognate and non-cognate DNases with K-d values that vary between 10(-4) and 10(-14) M, characteristics that are explained by a 'dual-recognition' mechanism. In this work, we addressed for the first time the energetics of Im protein recognition by colicin DNases through a combination of E9 DNase alanine scanning and double-mutant cycles (DMCs) coupled with kinetic and calorimetric analyses of cognate Im9 and non-cognate Im2 binding, as well as computational analysis of alanine scanning and DMC data. We show that differential Delta Delta Gs observed for four E9 DNase residues cumulatively distinguish cognate Im9 association from non-cognate Im2 association. E9 DNase Phe86 is the primary specificity hotspot residue in the centre of the interface, which is coordinated by conserved and variable hotspot residues of the cognate Im protein. Experimental DMC analysis reveals that only modest coupling energies to Im9 residues are observed, in agreement with calculated DMCs using the program ROSETTA and consistent with the largely hydrophobic nature of E9 DNase-Im9 specificity contacts. Computed values for the 12 E9 DNase alanine mutants showed reasonable agreement with experimental Delta Delta G data, particularly for interactions not mediated by interfacial water molecules. Delta Delta G predictions for residues that contact buried water molecules calculated using solvated rotamer models met with mixed success; however, we were able to predict with a high degree of accuracy the location and energetic contribution of one such contact. Our study highlights how colicin DNases are able to utilise both conserved and variable amino acids to distinguish cognate from non-cognate Im proteins, with the energetic contributions of the conserved residues modulated by neighbouring specificity sites. (C) 2008 Elsevier Ltd. All rights reserved.