Mutational analysis of the engrailed homeodomain recognition helix by phage display.

The homeodomain (HD) is a ubiquitous protein fold that confers DNA binding function on a superfamily of eukaryotic gene regulatory proteins. Here, the DNA binding of recognition helix variants of the HD from the engrailed gene of Drosophila melanogaster was investigated by phage display. Nineteen different combinations of pairwise mutations at positions 50 and 54 were screened against a panel of four DNA sequences consisting of the engrailed consensus, a non-specific DNA control based on the lambda repressor operator OR1 and two model sequence targets con-taining imperfect versions of the 5'-TAAT-3' consensus. The resulting mutant proteins could be divided into four groups that varied with respect to their affinity for DNA and specificity for the engrailed consensus. The altered specificity phenotypes of several mutant proteins were confirmed by DNA mobility shift analysis. Lys50/Ala54 was the only mutant protein that exhibited preferential binding to a sequence other than the engrailed consensus. Arginine was also demonstrated to be a functional replacement for Ala54. The functional combinations at 50 and 54 identified by these experiments recapitulate the distribution of naturally occurring HD sequences and illustrate how the engrailed HD can be used as a framework to explore covariation among DNA binding residues.同源结构域（homeodomain, HD）是一类广泛存在的蛋白质折叠结构，可赋予真核基因调控蛋白超家族DNA结合功能。本研究通过噬菌体展示技术，探究了黑腹果蝇engrailed基因编码的同源结构域识别螺旋变体的DNA结合特性。研究针对4组DNA序列共筛选了19种位于第50和54位的成对突变组合，这4组DNA序列分别为engrailed共识序列、基于λ阻遏蛋白操纵基因OR1构建的非特异性DNA对照，以及两组带有不完全匹配5'-TAAT-3'共识序列的模型靶序列。所得突变蛋白可根据其对DNA的亲和力以及对engrailed共识序列的结合特异性分为4个组别。通过DNA迁移率变动分析法，验证了数种突变蛋白的特异性改变表型。Lys50/Ala54是唯一一款可优先结合engrailed共识序列以外序列的突变蛋白。研究同时证实，精氨酸可有效替代Ala54发挥功能。本实验鉴定出的第50和54位功能突变组合，重现了天然同源结构域序列的分布特征，并阐明了以engrailed同源结构域为框架，探究DNA结合残基间共变异关系的可行路径。