Rational Design of Domain‐Swapping‐Based c‐Type Cytochrome Heterodimers by Using Chimeric Proteins

Abstract

The design of protein oligomers with multiple active sites has been gaining interest, owing to their potential use for biomaterials, which has encouraged researchers to develop a new design method. Three-dimensional domain swapping is the unique phenomenon in which protein molecules exchange the same structural region between each other. Herein, to construct oligomeric heme proteins with different active sites by utilizing domain swapping, two c-type cytochrome-based chimeric proteins have been constructed and the domains swapped. According to X-ray crystallographic analysis, the two chimeric proteins formed a domain-swapped dimer with two His/Met coordinated hemes. By mutating the heme coordination structure of one of the two chimeric proteins, a domainswapped heterodimer with His/Met and His/H O coordinated hemes was formed. Binding of an oxygen molecule to the His/H O site of the heterodimer was confirmed by resonance Raman spectroscopy, in which the Fe-O stretching band was observed at 580 cm for the reduced/oxygenated heterodimer (at 554 cm under an O atmosphere). These results show that domain swapping is a useful method to design multiheme proteins.

Protein surface charge effect on 3D domain swapping in cells for c-type cytochromes

“…C-type cytochromes (cyts) are heme proteins that act as electron carriers, functioning in a variety of cellular processes [1,2]. Many c-type cyts can form oligomers by 3D domain swapping (herein, domain swapping) [3][4][5][6][7][8][9][10][11]. In domain swapping, two or more identical protein monomers exchange the same domain or secondary structural elements and fold into dimers or higher oligomers whose units are structurally similar to the original monomer [12][13][14][15].…”

Human Cellular Retinol Binding Protein II Forms a Domain‐Swapped Trimer Representing a Novel Fold and a New Template for Protein Engineering

“…Providing larger binding surfaces, enhancing the number of binding sites for specific ligands, generating a higher local concentration of active sites, and creating new opportunities for allosteric regulation are some examples of functional diversification through domain‐swapping [6,11–16] . Domain swapping also is a useful protein engineering tool for creating new protein conformational switches, optogenetic tools, artificial enzymes, biosensors and so on as shown by many research groups including us [17–31] . Now our work in engineering the DS trimer as a novel protein engineering template can also be added to this literature.…”

“…[6,[11][12][13][14][15][16] Domain swapping also is a useful protein engineering tool for creating new protein conformational switches, optogenetic tools, artificial enzymes, biosensors and so on as shown by many research groups including us. [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] Now our work in engineering the DS trimer as a novel protein engineering template can also be added to this literature.…”

Oligomerization of cytochrome c, myoglobin, and related heme proteins by 3D domain swapping

“…PA cyt c551 and HT cyt c552 exhibit similar three-dimensional monomeric structures and their domain-swapped dimers exchange the N-terminal regions containing the N-terminal α-helix and heme between protomers, where the hinge loop locations are essentially the same (Figures 2A and 2B). To construct a c-type cyt heterodimer similar to what was performed with Mb, we constructed two chimeric proteins (PA cyt c551−HT cyt c552 and HT cyt c552−PA cyt c551), in which the sequences of N-terminal domain swapping regions were exchanged between the original proteins (Figure 5C) [85]. The two chimeric proteins formed a domain-swapped heterodimer with two His/Met coordinate hemes (PDB ID: 5XEC).…”