title: Structure and properties of a re-engineered homeodomain protein-DNA interface.
abstract: The homeodomain (HD)-DNA interface has been conserved over 500 million years of evolution. Despite this conservation, we have successfully re-engineered the engrailed HD to specifically recognize an unnatural nucleotide using a phage display selection. Here we report the synthesis of novel nucleosides and the selection of mutant HDs that bind these nucleotides using phage display. The high-resolution crystal structure of one mutant in complex with modified and unmodified DNA demonstrates that, even with the substantial perturbation to the interface, this selected mutant retains a canonical HD structure. Dissection of the contributions due to each of the selected mutations reveals that the majority of the modification-specific binding is accomplished by a single mutation (I47G) but that the remaining mutations retune the stability of the HD. These results afford a detailed look at a re-engineered protein-DNA interaction and provide insight into the opportunities for re-engineering highly conserved interfaces.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Simon
authors.forename: Morris E
authors.initials: ME
authors.lastname: Feldman
authors.forename: Daniel
authors.initials: D
authors.lastname: Rauh
authors.forename: Ann E
authors.initials: AE
authors.lastname: Maris
authors.forename: David E
authors.initials: DE
authors.lastname: Wemmer
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000235:genetics; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics
chemical_list: D018398:Homeodomain Proteins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C106185:engrailed homeobox proteins; D004247:DNA