Photocontrol of DNA Binding Specificity of a Miniature Engrailed Homeodomain

Control of DNA binding of HDH-3, a 18-residue polypeptide based on the recognition helix of
the Q50K engrailed homeodomain, has been achieved. HDH-3 was linked to an azobenzene cross-linker
through two cysteine residues in an i, i + 11 spacing. For the thermodynamically stable trans configuration
of the cross-linker, the dark-adapted peptide (dad-HDH-3) adopted a mainly α-helical structure as judged
by circular dichroism (CD) spectroscopy. After irradiation with light of 360 nm, the helical content of the
peptide (irrad-HDH-3) was reduced significantly and the CD spectrum of the irradiated peptide resembled
that of the largely unstructured, unalkylated peptide. Despite lacking helices-1 and -2 and the N-terminal
arm of Q50K engrailed, dad-HDH-3 bound to its natural DNA target sequence TAATCC (QRE) with high
affinity (KD = 7.5 ± 1.3 nM). The binding affinity for the mutant DNA sequence, TAATTA (ERE), was reduced
significantly (KD = 140 ± 11 nM). Unlike irrad-HDH-3, which like the unalkylated parent peptide displayed
only marginal DNA binding specificity, dad-HDH-3 specified base pairs 5 and 6 of QRE with an accuracy
rivaling that of the intact wild-type Q50K engrailed homeodomain, making dad-HDH-3 the most specific
designed DNA binding miniature homeodomain reported to date. Moreover, DNA binding affinity and
specificity of HDH-3 could be controlled externally by irradiation with light.