Incorporating Metal–Ligand and Salt-Bridge Interactions in the Design of Protein Heterodimers

The design of chemically controlled asymmetric protein-protein interfaces will further enhance the building of precise protein-based biomaterials. Driving protein-protein interactions through engineered metal-ligand coordination and salt-bridge formation enables the reversible association of two unique binding partners. Creation of precise biomaterial is enhanced through the temporal and chemical control afforded by metal-controlled heterodimeric proteins. In addition, heterodimers enable the specific association of different passenger proteins expressed as fusions to the heterodimeric binding partners. To increase the versatility of protein-based tools, we converted a previously engineered metal-controlled homodimer into a metal-controlled heterodimer. To promote specificity of the heterodimer complex and prevent self-association, it was necessary to incorporate elements of positive and negative design, which was achieved through the incorporation of a cross-interface electrostatic interaction, as well as modifications to hydrophobic contacts at the protein-protein interface. The resulting metal-controlled heterodimer binds with low micromolar affinity, and the crystal structures indicate the presence of the designed dual-interaction motifs at the protein-protein interface.