Computational design of metalloproteases

De novo design of enzymes that cleave amide bonds represents a major challenge^1,2. Here we use RFdiffusion methods^3,4 to de novo design proteins with water-activating zinc-based active sites adjacent to a peptide substrate binding pocket. Of 135 designs tested, 36% catalyse cleavage of their cognate peptide substrates at the targeted scissile bond. The best design accelerates peptide-bond hydrolysis by more than 10⁸-fold relative to the uncatalysed reaction; four substitutions increase this acceleration to more than 10¹⁰-fold by improving active-site preorganization⁵. We use this approach to design metalloproteases that selectively cleave the human disease-associated proteins TDP-43, amyloid-β and serum amyloid A, with rate enhancements of up to 10⁹-fold. We further design caged cytokines and receptor antagonists that are selectively activated by the designed proteases, enabling bioorthogonal control of cell state. Thus, de novo design can yield highly active, programmable proteases with applications in therapeutics, biotechnology and bioremediation.