We previously reported the IZ-3adH peptide, which formed a triple-stranded coiled-coil after binding Ni(II), Cu(II), or Zn(II). In this paper, we report the peptide, IZ-3aH, having a new metal binding specificity. The IZ-3aH peptide was found to bind Cu(II) and Zn(II) and form a triple-stranded coiled-coil. However, it did not bind Ni(II). Metal ion titrations monitored by circular dichroism revealed that the dissociation constants, K(d) were 9 microm for Zn(II) and 10 microm for Cu(II). The bound Cu(II) ion has a planar tetragonal geometry, where the coordination positions are three nitrogens of the His residues and one H(2)O.

Metalloproteins are an attractive target for de novo design. Usually, natural proteins incorporate two or more (hetero- or homo-) metal ions into their frameworks to perform their functions, but the design of multiple metal-binding sites is usually difficult to achieve. Here, we undertook the de novo engineering of heterometal-binding sites, Ni(II) and Cu(II), into a designed coiled coil structure based on an isoleucine zipper (IZ) peptide. Previously, we described two peptides, IZ-3adH and IZ-3aH. The former has two His residues and forms a triple-stranded coiled coil after binding Ni(II), Zn(II), or Cu(II). The latter has one His residue, which allowed binding with Cu(II) and Zn(II), but not with Ni(II). On the basis of these properties, we newly designed IZ(5)-2a3adH as a heterometal-binding peptide. This peptide can bind Cu(II) and Ni(II) simultaneously in the hydrophobic core of the triple-stranded coiled coil. The first metal ion binding induced the folding of the peptide into the triple-stranded coiled coil, thereby promoting the second metal ion binding. This is the first example of a peptide that can bind two different metal ions. This construction should provide valuable insights for the de novo design of metalloproteins.