Rational design of metal-binding sites in domain-swapped myoglobin dimers

The metal active site is precisely designed in metalloproteins. Here we applied 3D domain swapping, a phenomenon in which a partial protein structure is exchanged between molecules, to introduce metal sites in proteins. We designed multiple metal-binding sites specific to domain-swapped myoglobin (Mb) with His mutation. Stable dimeric Mbs with metal-binding sites were obtained by shifting the His position and introducing two Ala residues in the hinge region (K78H/G80A/H82A and K79H/G80A/H81A Mbs). The absorption and circular dichroism spectra of the monomer and dimer of K78H/G80A/H82A and K79H/G80A/H81A Mbs were similar to the corresponding spectra, respectively, of wild-type Mb. No negative peak due to dimer-to-monomer dissociation was observed below the denaturation temperature in the differential scanning calorimetry thermograms of K78H/G80A/H82A and K79H/G80A/H81A Mbs, whereas the dimer dissociates into monomers at 68 °C for wild-type Mb. These results show that the two mutants were stable in the dimer state. Metal ions bound to the metal-binding sites containing the introduced His in the domain-swapped Mb dimers. Co²⁺-bound and Ni²⁺-bound K78H/G80A/H82A Mb exhibited octahedral metal-coordination structures, where His78, His81, Glu85, and three H₂O/OH^- molecules coordinated to the metal ion. On the other hand, Co²⁺-bound and Zn²⁺-bound K79H/G80A/H81A Mb exhibited tetrahedral metal-coordination structures, where His79, His82, Asp141, and a H₂O/OH^- molecule coordinated to the metal ion. The Co²⁺-bound site exists deep inside the protein in the K79H/G80A/H81A Mb dimer, which may allow the unique tetrahedral coordination for the Co²⁺ ion. These results show that we can utilize domain swapping to construct artificial metalloproteins.