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

Abstract

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.journal articl