Experimental and computational analyses of the energetic basis for dual recognition of immunity proteins by colicin endonucleases

被引:37
作者
Keeble, Anthony H. [1 ]
Joachimiak, Lukasz A. [2 ]
Mate, Maria Jesus [1 ]
Meenan, Nicola [1 ]
Kirkpatrick, Nadine [1 ]
Baker, David [2 ]
Kleanthous, Cohn [1 ]
机构
[1] Univ York, Dept Biol, York YO10 5YW, N Yorkshire, England
[2] Univ Washington, Howard Hughes Med Inst, Dept Biochem, Seattle, WA 98195 USA
基金
英国生物技术与生命科学研究理事会;
关键词
protein-protein interactions; specificity; alanine scanning; double-mutant cycles; crystallography;
D O I
10.1016/j.jmb.2008.03.055
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:745 / 759
页数:15
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