The structural characterization of chimeric EF-hands based on the helix-turn-helix.

摘要

By exploiting a near super-imposable turn of two similar yet unrelated protein folds, we have designed a new class of DNA nucleases. The combination of the recognition helices of the engrailed homeodomain with the metal binding loop of calmodulin has generated chimeric peptides that possess both affinity for DNA and hydrolytically active metal ions.{09}Studies by isothermal titration calorimetry and fluorescence of a tryptophan residue document the affinity for lanthanide or calcium similar to traditional EF-hand peptides (Kd ≈ 1--10 muM). Upon binding of one equivalent of lanthanide or calcium, one such chimeric peptide, P3W folds into an extensively alpha helical structure which possesses 40% alpha helicity. This chimera, anchored by key hydrophobic residues flanking the metal binding loop, successfully integrates structural elements from both parent folds, creating a true helix-loop-helix fold. A solution structure model based on Nuclear Overhauser Effect constraints coupled with an analysis using the chemical shift index confirms this observation. Europium (III) 7F0 → 5D 0 excitation spectroscopy demonstrates the presents of two distinct isomers about the metal center, in good agreement with the behavior of native EF-hands. Additionally, a third pH dependent peak is observed in each spectrum corresponding to the deprotonation of one ligand in the metal's first coordination sphere. However, unlike previous reports, this deprotonation likely corresponds to a coordinated water molecule, and indeed the pKa regime observed for a series of the chimera correlates nicely to the observed nuclease activity. Moreover, binding of DNA does not compromise the integrity of the metal binding loop, rather for one poorly folded design P5B, it even serves to organize the fold, illustrated by the decrease in the number of coordinated solvent molecules from 3 to 2. Circular dichroism experiments of titrations of DNA with GdP3W document the binding of GdP3W to a 14-mer sequence of DNA and provide an estimate of the affinity at 15 muM. The changes are even incremental, and suggest a 1:1 ratio between DNA and the metallopeptide adduct.