Construction of a Cyclic Regular-Triangle Trimer of Cytochrome c₅₅₅ with a Central Hole Using Sortase A

Protein-based supramolecules require precise arrangement of building blocks. A regular-triangle trimer (cp-c₅₅₅)₃ has been constructed using an α-helix-inserted-circular permutant (cp-c₅₅₅) of Aquifex aeolicus cytochrome (cyt) c₅₅₅, where the trimers may dissociate to monomers. In this study, we stabilized the regular-triangle structure by constructing a cyclic regular-triangle of three α-helix-linked cyt c₅₅₅ molecules using sortase-mediated ligation (SML). Comparing SML using sortase A for six cp-c₅₅₅ variant trimers, the variant with GGG at the N-terminus and LPETG at the C-terminus reacted most efficiently. OP-(c₅₅₅)₃ was designed, in which two cyt c₅₅₅ molecules were fused using an α-helix, generating a dimer. The cyt c₅₅₅ C-terminal region was attached to the N-terminus of the dimer, and the cyt c₅₅₅ N-terminal region was attached to the C-terminus of the dimer using the same α-helix. OP-(c₅₅₅)₃ was expressed in Escherichia coli cells, and the termini were connected by SML, forming a cyclic regular-triangle, CL-(c₅₅₅)₃. CL-(c₅₅₅)₃ showed higher thermostability than (cp-c₅₅₅)₃ and OP-(c₅₅₅)₃. CL-(c₅₅₅)₃ structural stability was confirmed using high-speed atomic force microscopy. The X-ray crystal structure of CL-(c₅₅₅)₃ showed a cyclic structure and a nanoporous supramolecular assembly. These results demonstrate that a nanoporous supramolecular assembly can be constructed by designing a cyclic molecule with a central hole using SML.