5'-d( Tp Tp Tp Tp Gp Cp Cp Ap Tp Gp Tp Ap Ap Tp C Gp Gp A)-3'. Chain: c. Engineered: yes. 5'-d( Ap Tp Cp Cp Gp Gp Gp Gp Ap Tp Tp Ap Cp Ap T Ap Ap A)-3'. Chain: d. Engineered: yes. Segmentation polarity homeobox protein engrailed.
Synthetic: yes. Other_details: solid-phase DNA synthesis. Other_details: commercial solid-phase DNA synthesis. Drosophila melanogaster. Fruit fly. Organism_taxid: 7227. Gene: en. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.
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