Domain‐Swapping Design by Polyproline Rod Insertion

Abstract

During domain swapping, proteins mutually interconvert structural elements to form a di‐/oligomer. Engineering this process by design is important for creating a higher order protein assembly with minimal modification. Herein, a simple design strategy is shown for domain‐swapping formation by loop deletion and insertion of a polyproline rod. Crystal structures revealed the formation of the domain‐swapped dimers and polyproline portion formed a polyproline II (PPII) structure. Small‐angle X‐ray scattering demonstrated that an extended orientation of domain‐swapped dimer was retained in solution. It is found that a multiple of three of inserting proline residue is favored for domain swapping because of the helical nature of PPII. The rigid nature of the polyproline rod enables precise control of the interdomain distance and orientation.

Use of 3D domain swapping in constructing supramolecular metalloproteins

“…For example, 3D-DS formation by loop deletion and insertion of a polyproline rod to the outer-surface protein A (OspA) from Borrelia, which is composed of sequential 21 antiparallel b-strands, has been reported. 104 The rigid nature of the polyproline rod enabled precise control of the interdomain distance and orientation. It was shown that 3D-DS propensity may depend on the amino acid composition of the hinge loop by investigating the 3D-DS of loop-deletion mutants of a non-3D-DS b-sheet protein monellin.…”

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.…”

Experimental and theoretical study on converting myoglobin into a stable domain-swapped dimer by utilizing a tight hydrogen bond network at the hinge region

“… 34 On the other hand, the 3D-DS dimer structure has been stabilized by forming an anti-parallel β-sheet at the hinge region between protomers with insertion of a QVVAG motif, 35 whereas loop deletion and insertion of a polyproline rod to the outer-surface protein A from Borrelia stabilized the 3D-DS structure by formation of the polyproline II structure at the hinge region. 36 In this study, to investigate the effect of the H-bond network constructed at the hinge loop containing amino acid residues and water molecules on 3D-DS, we introduced two Ala residues at G80 and H81 but retained H82 to interact with K79 and D141 through water molecules at the hinge region (G80A/H81A (K 3 A 2 H) Mb). Additionally, L137 is located relatively close to the H-bond network in the WT Mb 3D-DS dimer; thus, L137 was additionally mutated to a hydrophilic amino acid: Glu and Asp (G80A/H81A/L137E (K 3 A 2 H-L137E) Mb and G80A/H81A/L137D (K 3 A 2 H-L137D) Mb).…”