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 gen e of Drosophila melanogaster was investigated by phage display. Nineteen di fferent combinations of pairwise mutations at positions 50 and 54 were scre ened against a panel of four DNA sequences consisting of the engrailed cons ensus, a non-specific DNA control based on the lambda repressor operator O( R)1 and two model sequence targets containing 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 sequen ce 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 natural ly occurring HD sequences and illustrate how the engrailed HD can be used a s a framework to explore covariation among DNA binding residues.