Strand-Swapped SH3 Domain Dimer with Superoxide Dismutase Activity

Abstract

The design of metalloproteins allows us to better understand metal complexation in proteins and the resulting function. In this study, we incorporated a Cu²⁺-binding site into a natural protein domain, the 58 amino acid c-Crk-SH3, to create a miniaturized superoxide dismutase model, termed SO1. The resulting low complexity metalloprotein was characterized for structure and function by circular dichroism and UV spectroscopy as well as EPR spectroscopy and X-ray crystallography. The miniprotein was found to be a strand-swapped dimer with an unusual coupled binuclear Type 2-like copper center in each protomer. SO1-Cu was found to be SOD-active with an activity only 1 order of magnitude lower than that of natural SOD enzymes and 1 to 2 orders of magnitude higher than that of other low-complexity SOD protein models of similar size. This serendipitous design provides us with a new structural template for future designs of binuclear metalloproteins with different metal ions and functions.

The catalytic copper site of human Cu,Zn Superoxide Dismutase 1 was grafted onto the c-Crk SH3 domain, resulting in a strand-swapped SH3 domain dimer with remarkable superoxide dismutase activity.