Nat. Chem. https://doi.org/10.1038/s41557-019-0218-9 (2019)

The design of proteins with novel metal centers generally requires the pre-existence of a suitable binding site in the host scaffold or the use of a consensus sequence derived from natural scaffolds. With the aim of efficiently diversifying the design process, Rittle et al. developed MASCoT (metal active sites by covalent tethering), in which two copies of a stable protein are linked via a disulfide bond to form an interface where a new metal-binding site can be engineered. Using cytochrome cb562 as a proof of concept, the authors created a disulfide-linked dimeric protein that binds divalent metal ions in the newly created interface. Further engineering created a small hydrophobic pocket and a high-affinity manganese-binding site. Removal of the heme cofactors from the cytochrome scaffold followed by iron binding in the MASCoT-generated interface demonstrated that the dimeric scaffold can bind ions in multiple oxidation states, and the resulting nonheme iron protein could also bind nitric oxide. MASCoT provides rapid access to potential novel metal-binding sites not found in nature and could help to understand how such sites evolved in biological metalloproteins.