De novo design of cysteine proteases

Key Points:

• SDS–PAGE and size-exclusion chromatography analyses of 69 enzyme designs identified several variants exhibiting cleavage activity, with results consistent across three independent experiments.
• A pLDDT-guided redesign workflow applied to the Dokki enzyme yielded 24 variants outperforming the parent design in catalytic rate, with Dokki-15 showing the highest improvement up to 31-fold.
• Detailed kinetic and biochemical assays, including Michaelis–Menten analysis and pH/temperature profiling, characterized the enhanced catalytic efficiencies and stability of Dokki-15 and other variants.
• Mutational screening combined with in silico predictions and wet-lab validation identified beneficial single and combinatorial mutations that further improved enzymatic activity, culminating in the Dokki-v2 variant with superior catalytic parameters and turnover numbers.
• Structural analyses using PLACER ensembles and molecular dynamics simulations revealed stepwise improvements in active-site geometry and productive conformations correlating with increased enzyme activity, supported by cellular fluorescence microscopy demonstrating functional protease activity in HeLa cells.