A five-residue motif for the design of domain swapping in proteins

Neha Nandwani,Ranabir Das,Jayant B Udgaonkar,Parag Surana,Hitendra Negi,Shachi Gosavi,Nahren M Mascarenhas

doi:10.1038/s41467-019-08295-x

Abstract

Domain swapping is the process by which identical monomeric proteins exchange structural elements to generate dimers/oligomers. Although engineered domain swapping is a compelling strategy for protein assembly, its application has been limited due to the lack of simple and reliable design approaches. Here, we demonstrate that the hydrophobic five-residue ‘cystatin motif’ (QVVAG) from the domain-swapping protein Stefin B, when engineered into a solvent-exposed, tight surface loop between two β-strands prevents the loop from folding back upon itself, and drives domain swapping in non-domain-swapping proteins. High-resolution structural studies demonstrate that engineering the QVVAG stretch independently into various surface loops of four structurally distinct non-domain-swapping proteins enabled the design of different modes of domain swapping in these proteins, including single, double and open-ended domain swapping. These results suggest that the introduction of the QVVAG motif can be used as a mutational approach for engineering domain swapping in diverse β-hairpin proteins.

Highlights

Domain swapping is the process by which identical monomeric proteins exchange structural elements to generate dimers/oligomers
The major conformational difference between the stefin B dimer and the MNEI monomer occurs in loop[1], the loop connecting the β2 and β3 strands
Here, we have shown that engineering the QVVAG sequence from stefin B into any of the three β-turn-β loops of MNEI induces dimerization through domain swapping

Summary

Introduction

Domain swapping is the process by which identical monomeric proteins exchange structural elements to generate dimers/oligomers. The underlying driving force for oligomerization is the change in the conformation of the modified and strained hinge loop to an energetically favorable extended conformation in the domainswapped structure[6,7,12] These studies have not led to a specific mutational strategy for the introduction of domain swapping into diverse monomeric proteins. We noticed that placing a bulky, hydrophobic residue at the apex of a solvent-exposed, strained β-turn results in domain swapping[34] Based on this result, we asked if engineering the largely hydrophobic pentapeptide motif present in the domain-swapping cystatin (β1-α1-β2-β3-β4-β5 topology) proteins[35] into the β-turns of proteins could be a general strategy for designing domain swapping. In the domain-swapping cystatins, the loop connecting β2–β3 has a QXVXG consensus motif, which has been implicated in both protease inhibition and domain swapping[35,36,37,38,39] (Fig. 1b)

Methods

Results

Conclusion