Domain-swapped cytochrome cb₅₆₂ dimer and its nanocage encapsulating a Zn-SO₄ cluster in the internal cavity

Protein nanostructures have been gaining in interest, along with developments in new methods for construction of novel nanostructures. We have previously shown that c-type cytochromes and myoglobin form oligomers by domain swapping. Herein, we show that a four-helix bundle protein cyt cb₅₆₂, with the cyt b₅₆₂ heme attached to the protein moiety by two Cys residues insertion, forms a domain-swapped dimer. Dimeric cyt cb₅₆₂ did not dissociate to monomers at 4 °C, whereas dimeric cyt b₅₆₂ dissociated under the same conditions, showing that heme attachment to the protein moiety stabilizes the domain-swapped structure. According to X-ray crystallographic analysis of dimeric cyt cb₅₆₂, the two helices in the N-terminal region of one protomer interacted with the other two helices in the C-terminal region of the other protomer, where Lys51-Asp54 served as a hinge loop. The heme coordination structure of the dimer was similar to that of the monomer. In the crystal, three domain-swapped cyt cb₅₆₂ dimers formed a unique cage structure with a Zn-SO₄ cluster inside the cavity. The Zn-SO₄ cluster consisted of fifteen Zn²⁺ and seven SO₄^2- ions, whereas six additional Zn²⁺ ions were detected inside the cavity. The cage structure was stabilized by coordination of the amino acid side chains of the dimers to the Zn²⁺ ions and connection of two four-helix bundle units through the conformation-adjustable hinge loop. These results show that domain swapping can be applied in the construction of unique protein nanostructures.