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- Title
Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution.
- Authors
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 P. S.; Nguyen, Thu T. M.; Katz, Benjamin B.; Steward, Lance E.; Majumdar, Sudipta; Brideau-Andersen, Amy D.; Weiss, Gregory A.
- 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.
- Subjects
CLOSTRIDIUM botulinum; BOTULINUM toxin; PROTEOLYTIC enzymes; PEPTIDE bonds; NEURONS
- Publication
Scientific Reports, 2022, Vol 12, Issue 1, p1
- ISSN
2045-2322
- Publication type
Article
- DOI
10.1038/s41598-022-13617-z