1. Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution
- Author
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Rebekah P. Dyer, Hariny M. Isoda, Gabriela S. Salcedo, Gaetano Speciale, Madison H. Fletcher, Linh Q. Le, Yi Liu, Karen Brami-Cherrier, Shiazah Z. Malik, Edwin J. Vazquez-Cintron, Andrew C. Chu, David C. Rupp, Birgitte P. S. Jacky, Thu T. M. Nguyen, Benjamin B. Katz, Lance E. Steward, Sudipta Majumdar, Amy D. Brideau-Andersen, and Gregory A. Weiss
- Subjects
Multidisciplinary ,Botulinum Toxins ,Prevention ,Protein Engineering ,Foodborne Illness ,Catalysis ,Substrate Specificity ,Type A ,Emerging Infectious Diseases ,Infectious Diseases ,Catalytic Domain ,Clostridium botulinum ,Botulinum Toxins, Type A ,Peptide Hydrolases - Abstract
The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A’s protease domain (LC/A) could expand its therapeutic applications; however, LC/A’s extended substrate recognition (≈ 60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A retains its ability to form full-length holotoxin, infiltrate neurons, and cleave SNAP23. The identification of substrate control loops outside BoNT/A’s active site could guide the design of improved BoNT proteases and inhibitors.
- Published
- 2022