3Q3F

Engineering Domain-Swapped Binding Interfaces by Mutually Exclusive Folding: Insertion of Ubiquitin into position 103 of Barnase

Summary for 3Q3F

• Entry DOI: 10.2210/pdb3q3f/pdb
• Descriptor: Ribonuclease/Ubiquitin chimeric protein, SULFATE ION (3 entities in total)
• Functional Keywords: domain swap, oligomerization, ubiquitin insertion, hydrolase, protein binding
• Biological source: Bacillus amyloliquefaciens; More
• Cellular location: Secreted: P00648
• Total number of polymer chains: 1
• Total formula weight: 22665.72

Authors

Ha, J.-H.,Karchin, J.M.,Walker-Kopp, N.,Huang, L.-S.,Berry, E.A.,Loh, S.N. (deposition date: 2010-12-21, release date: 2012-01-25, Last modification date: 2023-09-13)

Primary citation

Ha, J.H.,Karchin, J.M.,Walker-Kopp, N.,Huang, L.S.,Berry, E.A.,Loh, S.N.
Engineering domain-swapped binding interfaces by mutually exclusive folding.
J.Mol.Biol., 416:495-502, 2012

PubMed Abstract: Domain swapping is a mechanism for forming protein dimers and oligomers with high specificity. It is distinct from other forms of oligomerization in that the binding interface is formed by reciprocal exchange of polypeptide segments. Swapping plays a physiological role in protein-protein recognition, and it can also potentially be exploited as a mechanism for controlled self-assembly. Here, we demonstrate that domain-swapped interfaces can be engineered by inserting one protein into a surface loop of another protein. The key to facilitating a domain swap is to destabilize the protein when it is monomeric but not when it is oligomeric. We achieve this condition by employing the "mutually exclusive folding" design to apply conformational stress to the monomeric state. Ubiquitin (Ub) is inserted into one of six surface loops of barnase (Bn). The 38-Å amino-to-carboxy-terminal distance of Ub stresses the Bn monomer, causing it to split at the point of insertion. The 2.2-Å X-ray structure of one insertion variant reveals that strain is relieved by intermolecular folding with an identically unfolded Bn domain, resulting in a domain-swapped polymer. All six constructs oligomerize, suggesting that inserting Ub into each surface loop of Bn results in a similar domain-swapping event. Binding affinity can be tuned by varying the length of the peptide linkers used to join the two proteins, which modulates the extent of stress. Engineered, swapped proteins have the potential to be used to fabricate "smart" biomaterials, or as binding modules from which to assemble heterologous, multi-subunit protein complexes.

PubMed: 22245575
DOI: 10.1016/j.jmb.2011.12.050

Experimental method

X-RAY DIFFRACTION (2.169 Å)