UC San Diego

The de novo design of protein-protein interactions (PPIs) has proven to be an immense challenge due to the difficulty in controlling and predicting the weak forces that govern them. In order to circumvent this challenge, we set out to induce new PPIs between monomeric proteins through the coordination of metal ions. In our strategy, the strength of the metal bonding interactions should be sufficient to drive the formation of PPIs without initial consideration of the weak non-covalent interactions along the newly formed interfaces. Additionally, the distinct geometric preferences of metal ions should only allow for a limited number of organizations of proteins around a given metal center, thus permitting a degree of foresight into the oligomeric architectures that will be formed. As a model system, we chose a stable, four-helix bundle protein, cytochrome cb₅₆₂, onto which two i and i+4 bis- histidine "clamp" coordination motifs were installed. The resulting metal-binding construct, MBPC-1, is shown to be capable of forming discrete oligomeric species whose supramolecular architectures depend on the stereochemical preferences of the added metal. Further studies to exert control over the morphology of a Zn-mediated tetrameric assembly of MBPC-1 (Zn₄:MBPC-1₄) highlighted the fact that , although we initially ignored secondary, non-covalent interactions along our newly induced interfaces, these interactions do play a role in determining the overall structure of the complex. This idea led us to computationally "evolve" our protein by re-designing residues along interfaces of the Zn₄:MBPC-1₄ tetramer to include more favorable hydrophobic packing interactions. The resulting constructs were shown to not only form a significantly stabilized tetramer, but also to have the ability to form oligomers in the absence of metals. Further stabilization of this tetrameric architecture by way of chemical crosslinking has allowed for the formation of a potentially metal-selective complex that can serve as a stable platform onto which functionalized metal centers, and in turn enzymatic activity, may be engineered in the future