Metal ion induced folding of a de novo designed coiled-coil peptide

A disulfide-bridged two-stranded alpha-helical coiled-coil peptide has been designed which undergoes a random coil to alpha-helical transition at pH 7 and 20 degrees C upon metal bindine to two sites engineered into the molecule. The metal binding coiled-coil, dubbed Gla(2)Nx, is composed of identical 35 residue polypeptide chains based on the "Native" heptad sequence repeat Q(g)V(a)G(b)A(c)L(d)Q(e)K(f) and contains a Cys substitution for Val at position 2 to allow formation of an interchain disulfide bridge. The coiled-coil structure is destabilized in the apo form by two interhelical i to i' + 5 ionic repulsions between negatively charged gamma-carboxyglutamic acid (Gla) side chains at position 15 of one strand and position 20 of the other strand. In contrast, the folded form is stabilized by metal binding because the metal is chelated by the Gla side chains which otherwise (in the absence of metal) repel one another. One such metal binding interaction was predicted to occur between each pair of repelling Gla side chains. Metal titration monitored indirectly by circular dichroism spectroscopy showed that folding (increased helicity) is directly correlated with addition of 2 equiv of metal ion, supporting the presence of two specific metal-binding sites. The estimated metal dissociation constant, K-d, is 0.6 +/- 0.3 mu M for La3+ and 0.4 +/- 0.2 mu M for Yb3+. In addition, Zn2+ bound with a K-d of 1.7 +/- 0.3 mu M but Ca2+ had a very weak affinity with a K-d of 18 +/- 2 mM. Because folding was associated with a 2:1 ratio of metal to peptide monomer (disulfide-bridged two-stranded coiled-coil), the data suggests cooperativity occurs, so that binding of the second metal is significantly tighter than that of the first, and the measured affinity corresponds to that of the first bound metal. Metal titration monitored by nuclear magnetic resonance spectroscopy also supported the prediction that the peptide binds two metal ions specifically and the conclusion that binding is cooperative because it showed no evidence for the existence of a singly bound intermediate. In addition. Gla(2)Nx displays a sharp folding transition over the range from pH 6.2 to 5.2 apparently as a result of Gla side chain carboxylate protonation.