title: 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.
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720, USA.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Simon
authors.forename: Ken
authors.initials: K
authors.lastname: Sato
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Weiss
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
mesh_terms: D001665:Binding Sites; D016384:Consensus Sequence; D005973:Glutamine / Q000235:genetics / Q000502:physiology*; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics*; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D019151:Peptide Library; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics
chemical_list: D018398:Homeodomain Proteins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D005973:Glutamine