Domain swapping occurs when identical proteins exchange segments in reciprocal fashion. Natural swapping mechanisms remain poorly understood and engineered swapping has the potential for creating self-assembling biomaterials that encode for emergent functions. We demonstrate that induced swapping can be used to regulate function of a target protein. Swapping is triggered by inserting a ‘lever’ protein (ubiquitin) into one of four loops of the ribose binding protein (RBP) target. The lever splits the target, forcing RBP to refold in trans to generate swapped oligomers. Identical RBP-ubiquitin fusions form homo-swapped complexes with the ubiquitin domain acting as the hinge. Surprisingly, some pairs of non-identical fusions swap more efficiently with each other than they do with themselves. NMR experiments reveal that the hinge of these hetero-swapped complexes maps to a region of RBP distant from both ubiquitins. This design is expected to be applicable to other proteins to convert them into functional switches.

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 is inserted into one of six surface loops of barnase. The 38 Å amino-to-carboxy terminal distance of ubiquitin stresses the barnase 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 barnase domain, resulting in a domain-swapped polymer. All six constructs oligomerize suggesting that inserting ubiquitin into each surface loop of barnase 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.

Domain swapping is the process by which identical proteins exchange reciprocal segments to generate dimers. Here we introduce induced domain swapping (INDOS) as a mechanism for regulating protein function. INDOS employs a modular design consisting of the fusion of two proteins: a recognition protein that binds a triggering molecule, and a target protein that undergoes a domain swap in response to binding of the triggering ligand. The recognition protein (FK506 binding protein) is inserted into functionally-inactivated point mutants of two target proteins (staphylococcal nuclease and ribose binding protein). Binding of FK506 to the FKBP domain causes the target domain to first unfold, then refold via domain swap. The inactivating mutations become ‘swapped out’ in the dimer, increasing nuclease and ribose binding activities by 100-fold and 15-fold, respectively, restoring them to near wild-type values. INDOS is intended to convert an arbitrary protein into a functional switch, and is the first example of rational design in which a small molecule is used to trigger protein domain swapping and subsequent activation of biological function.

We describe the results of a systematic search for a class of hitherto-overlooked chemical-genetic interactions in the Saccharomyces cerevisiae genome, which exists between a detrimental genetic mutation and a chemical/drug that can ameliorate, rather than exacerbate, that detriment. We refer to this type of interaction as “chemical suppression.” Our work was driven by the hypothesis that genome instability in a certain class of mutants could be alleviated by mild replication inhibition using chemicals/drugs. We queried a collection of conditionally lethal, i.e., temperature-sensitive, alleles representing 40% of the yeast essential genes for those mutants whose growth defect can be suppressed by hydroxyurea (HU), known as a potent DNA replication inhibitor, at the restrictive temperature. Unexpectedly, we identified a number of mutants defective in diverse cellular pathways other than DNA replication. Here we report that HU suppresses selected mutants defective in the kinetochore-microtubule attachment pathway during mitotic chromosome segregation. HU also suppresses an ero1-1 mutant defective for a thiol oxidase of the endoplasmic reticulum by providing oxidation equivalents. Finally, we report that HU suppresses an erg26-1 mutant defective for a C-3 sterol dehydrogenase through regulating iron homeostasis and in turn impacting ergosterol biosynthesis. We further demonstrate that cells carrying the erg26-1 mutation show an increased rate of mitochondrial DNA loss and delayed G1 to S phase transition. We conclude that systematic gathering of a compendium of “chemical suppression” of yeast mutants by genotoxic drugs will not only enable the identification of novel functions of both chemicals and genes, but also have profound implications in cautionary measures of anticancer intervention in humans.

performed to confirm the stability of complexes. The MM/PBSA binding free energies of these peptide-cytokine complexes were also calculated for comparison. The results showed that de novo peptides have better binding affinities than wild type peptides. Surface plasmon resonance (SPR) detection were performed to validate the docking and binding free energy calculations. The results indicated that the de novo peptides are able to capture the cytokines consistent with our predictions. The outcome of the research may be applied to manufacture a cytokines adsorber for treating patients with severe sepsis in the future.