<i>De Novo</i> Design of a Metalloprotein with a Synthetically Inspired Dinuclear Paddlewheel Coordination Motif

A metal-centered design approach was used to design a tetrameric protein assembly, PW1, with a new-to-nature metal center inspired by the structures of transition metal paddlewheel complexes such as di-Cu^II tetraacetate. The structure of PW1 matches the design prediction with atomic accuracy, preorganizing four Glu residues to form the desired dinuclear Cu^II center. Notably, Cu-PW1 has a reduction potential of -0.085 V at pH 7.0, which is more negative than any natural or engineered Cu redox protein. In addition to Cu^II, PW1 can also scaffold Rh^II ions in the dinuclear paddlewheel geometry <i>in crystallo</i> as well as trivalent lanthanide ions in a mononuclear fashion. These results demonstrate the feasibility of designing new protein architectures around synthetically inspired, nonbiological metal centers, thus expanding the scope of artificial metalloproteins beyond those constructed around pre-existing protein scaffolds and opening new avenues to discover structure-function relationships outside the constraints of natural evolution.