Structural Framework for Covalent Inhibition of Clostridium botulinum Neurotoxin A by Targeting Cys165

• Published in: The Journal of biological chemistry Vol. 287; no. 40; pp. 33607 - 33614
• Main Authors: Stura, Enrico A., Le Roux, Laura, Guitot, Karine, Garcia, Sandra, Bregant, Sarah, Beau, Fabrice, Vera, Laura, Collet, Guillaume, Ptchelkine, Denis, Bakirci, Huseyin, Dive, Vincent
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.09.2012; American Society for Biochemistry and Molecular Biology
• Subjects: Active Site Cysteine; Biochemistry, Molecular Biology; Bioinformatics; Botulinum Neurotoxin; Botulinum Toxin; Botulinum Toxins, Type A - antagonists & inhibitors; Catalytic Domain; Chemistry, Pharmaceutical - methods; Clostridium botulinum - metabolism; Computer Science; Crystallography, X-Ray - methods; Cysteine - chemistry; Drug Design; Enzyme Inhibitors; Enzyme Inhibitors - pharmacology; Enzymology; Humans; Inhibitor Design; Kinetics; Life Sciences; Mutagenesis, Site-Directed; Peptide Hydrolases - chemistry; Peptides - chemistry; Protein Structure, Tertiary; Proteolytic Enzymes; Quantitative Methods; Recombinant Proteins - chemistry; Structural Biology; Synaptosomal-Associated Protein 25 - chemistry; Targeted Covalent Inhibitors; Zinc - chemistry; Zinc Protease; Botulinum Toxin; Inhibitor Design; Enzyme Inhibitors; Botulinum Neurotoxin; Structural Biology; Drug Design; Zinc Protease; Active Site Cysteine; Targeted Covalent Inhibitors; Proteolytic Enzymes
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.396697

Clostridium botulinum neurotoxin type A (BoNT/A) is one of the most potent toxins for humans and a major biothreat agent. Despite intense chemical efforts over the past 10 years to develop inhibitors of its catalytic domain (catBoNT/A), highly potent and selective inhibitors are still lacking. Recently, small inhibitors were reported to covalently modify catBoNT/A by targeting Cys165, a residue located in the enzyme active site just above the catalytic zinc ion. However, no direct proof of Cys165 modification was reported, and the poor accessibility of this residue in the x-ray structure of catBoNT/A raises concerns about this proposal. To clarify this issue, the functional role of Cys165 was first assessed through a combination of site-directed mutagenesis and structural studies. These data suggested that Cys165 is more involved in enzyme catalysis rather than in structural property. Then by peptide mass fingerprinting and x-ray crystallography, we demonstrated that a small compound containing a sulfonyl group acts as inhibitor of catBoNT/A through covalent modification of Cys165. The crystal structure of this covalent complex offers a structural framework for developing more potent covalent inhibitors catBoNT/A. Other zinc metalloproteases can be founded in the protein database with a cysteine at a similar location, some expressed by major human pathogens; thus this work should find broader applications for developing covalent inhibitors. Background: Development of small potent synthetic inhibitors of Clostridium botulinum neurotoxin A remains an unresolved challenge. Results: Small compounds incorporating electrophile moiety can block enzyme activity by covalent modification of Cys165. Conclusion: A structural framework for developing potent covalent inhibitors of Clostridium botulinum neurotoxin A is provided. Significance: This study uncovers a subfamily of zinc proteases containing a conserved cysteine in their active site.