A phage display selection of engrailed homeodomain mutants and the importance of residue Q50

Abstract

Mutants of engrailed homeodomain (HD) that retain DNA-binding activity were isolated using a phage display selection. This selection was used to enrich for active DNA-binding clones from a complex library consisting of over a billion members. A more focused library of mutant homeodomains consisting of all possible amino acid combinations at two DNA-contacting residues (I47 and Q50) was constructed and screened for members capable of binding tightly and specifically to the engrailed consensus sequence, TAATTA. The isolated mutants largely recapitulated the distribution of amino acids found at these positions in natural homeodomains thus validating the in vitro selection conditions. In particular, the unequivocal advantage enjoyed by glutamine at residue 50 is surprising in light of reports that minimize the importance of this residue. Here, the subtle contributions of residue Q50 are demonstrated to play a functionally important role in specific recognition of DNA. These results highlight the complex subtlety of protein-DNA interactions, underscoring the value of the first reported in vitro selection of a homeodomain.

Figure 1

Ribbon representation of EnHD bound to TAATTA based on the reported crystal structure (11). Residues labeled are those examined by phage display in this study (I47 and Q50). Residue N51 is also shown.

Figure 2

Development of an EnHD phage display construct with specific DNA-binding activity. (A) The DNA-binding activity of EnHD phage constructs as examined by phage capture titering experiments measured by the number of colony forming units recovered after elution. (B) Binding of H₆-EnHD-P8 to TAATTA₂₀ (closed circles) and TATATA₂₀ (open triangles).

Figure 3

Examination of the dynamic range of a phage pull-down titering experiment. The input phage consisted of H6-EnHD-P8 diluted at various concentrations into helper phage. Yields are reported relative to undiluted H6-EnHD-P8.

Figure 4

Amino-acid distribution in 129 human HDs (33) found (A) at residue 47 and (B) at residue 50, compared with the distribution of 45 clones recovered from a phage selection of EnHD (C) at residue 47 and (D) at residue 50.

Figure 5

Examination of the affinity (A and B) and specificity (C) of various EnHD mutants. (A) EMSA comparing the affinity between the EnHD I47V;Q50R and EnHD WT. The asterisk indicates addition of excess competitor salmon-sperm DNA to demonstrate the specificity of binding. (B) EMSA comparing the affinities of EnHD WT, EnHD I47T;Q50A and EnHD Q50A binding to TAATTA₂₀. (C) EMSA competition experiment with increasing concentrations of salmon sperm DNA comparing the specificities of binding. Experiments in (B) and (C) were performed under rapid loading conditions.