Back to Search Start Over

Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

Authors :
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
Gregory A. Weiss
Source :
Scientific Reports, Vol 12, Iss 1, Pp 1-11 (2022)
Publication Year :
2022
Publisher :
Nature Portfolio, 2022.

Abstract

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

Subjects :
Medicine
Science

Details

Language :
English
ISSN :
20452322
Volume :
12
Issue :
1
Database :
Directory of Open Access Journals
Journal :
Scientific Reports
Publication Type :
Academic Journal
Accession number :
edsdoj.1faa403f9c0b49ad85a29291d7a72970
Document Type :
article
Full Text :
https://doi.org/10.1038/s41598-022-13617-z