title: Engineering domain-swapped binding interfaces by mutually exclusive folding.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA.
authors.forename: Jeung-Hoi
authors.initials: JH
authors.lastname: Ha
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Karchin
authors.forename: Nancy
authors.initials: N
authors.lastname: Walker-Kopp
authors.forename: Li-Shar
authors.initials: LS
authors.lastname: Huang
authors.forename: Edward A
authors.initials: EA
authors.lastname: Berry
authors.forename: Stewart N
authors.initials: SN
authors.lastname: Loh
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012260:Ribonucleases / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D025801:Ubiquitin; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease