Metal ion induced self-assembly of a designed peptide into a triple-stranded alpha-helical bundle: A novel metal binding site in the hydrophobic core

Coiled coils, which mediate the associations and regulate the functions of various proteins, have a representative amino acid sequence of (defgabc)(n) heptad repeats and usually have hydrophobic residues at the a and d positi ons. We have designed a triple-stranded parallel or-helical coiled coil, in which the amino acid sequence is YGG(IEKKIEA)(4). To construct a peptide t hat undergoes metal ion induced self-assembly into a triple-stranded coiled coil, we engineered a metal binding site in the hydrophobic core of the co iled coil. We replaced two lie residues of the third heptad with His residu es. The peptide had a random structure in aqueous solution. In contrast, in the presence of a transition metal ion, the peptide exhibited an alpha-hel ical conformation The metal-complexed peptide was triple stranded and had a parallel orientation, as determined by sedimentation equilibrium and fluor escence quenching analyses. Metal ion titrations monitored by circular dich roism revealed that the dissociation constants, K-d, were 35 +/- 1 mu M for Co(II), 5.0 +/- 0.3 mu M for Ni(II), 17 +/- 1 mu M for Cu(II), and 23 +/- 2 mu M for Zn(II). The Ni(II) binds to the His residues, as judged by both pH titration monitored by circular dichroism and metal ion titration monito red by nuclear magnetic resonance. The highest affinity for NL(II) suggests that the metal binding site has six-coordinated octahedral geometry. Thus, the peptide is a useful tool to control the associations of functional dom ains attached to the peptide.