Structure and properties of a re-engineered homeodomain protein-DNA interface

ACS Chem Biol. 2006 Dec 15;1(12):755-60. doi: 10.1021/cb6003756.


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.

Publication types

  • Letter
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • Binding Sites
  • Crystallography, X-Ray
  • DNA* / chemistry
  • DNA* / genetics
  • Homeodomain Proteins* / chemistry
  • Homeodomain Proteins* / genetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Nucleic Acid Conformation
  • Peptide Library
  • Protein Binding
  • Protein Engineering*
  • Recombinant Fusion Proteins / chemistry*
  • Transcription Factors* / chemistry
  • Transcription Factors* / genetics


  • Homeodomain Proteins
  • Peptide Library
  • Recombinant Fusion Proteins
  • Transcription Factors
  • engrailed homeobox proteins
  • DNA

Associated data

  • PDB/2HOS
  • PDB/2HOT