1. Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution
- Author
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Dyer, Rebekah P, Isoda, Hariny M, Salcedo, Gabriela S, Speciale, Gaetano, Fletcher, Madison H, Le, Linh Q, Liu, Yi, Brami-Cherrier, Karen, Malik, Shiazah Z, Vazquez-Cintron, Edwin J, Chu, Andrew C, Rupp, David C, Jacky, Birgitte PS, Nguyen, Thu TM, Katz, Benjamin B, Steward, Lance E, Majumdar, Sudipta, Brideau-Andersen, Amy D, and Weiss, Gregory A
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,Emerging Infectious Diseases ,Foodborne Illness ,Infectious Diseases ,Prevention ,Botulinum Toxins ,Type A ,Catalysis ,Catalytic Domain ,Clostridium botulinum ,Peptide Hydrolases ,Protein Engineering ,Substrate Specificity - 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