Domain-swapped cytochrome dimer and its nanocage encapsulating a Zn-SO 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 -type cytochromes and myoglobin form oligomers by domain swapping. Herein, we show that a four-helix bundle protein cyt , with the cyt heme attached to the protein moiety by two Cys residues insertion, forms a domain-swapped dimer. Dimeric cyt did not dissociate to monomers at 4 °C, whereas dimeric cyt 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 , 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 dimers formed a unique cage structure with a Zn-SO cluster inside the cavity. The Zn-SO cluster consisted of fifteen Zn and seven SO ions, whereas six additional Zn ions were detected inside the cavity. The cage structure was stabilized by coordination of the amino acid side chains of the dimers to the Zn 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.