De novo design of cysteine proteases

Despite advances in de novo enzyme design, success has been largely limited to low-energy-barrier model reactions. Amide bonds such as those linking amino acids along the peptide backbone are stable for hundreds of years in neutral aqueous solution because of the high energy barrier to hydrolysis¹. Here we describe the de novo design of enzymes that use an activated cysteine nucleophile to hydrolyse the polypeptide backbone in a sequence-dependent manner, with a success rate of 13/69 = 19% and rate enhancements over the background reaction (k_cat/k_uncat) of up to 3 × 10⁷. The designed proteases have folds that are very different from proteases in nature (TM score < 0.50), and six crystal structures are very close to the design models (Cα root mean squared deviation of <1.2 Å), highlighting the capacity for generalization and the accuracy of the design methodology. Experimental and computational analyses suggest that the remaining gap in activity to the most active native cysteine proteases arises from imperfections in active-site preorganization and substrate positioning. The designed proteases efficiently cleave their targets in mammalian cells, opening the door to a wide range of synthetic biology applications.