title: Structural framework for covalent inhibition of Clostridium botulinum neurotoxin A by targeting Cys165.
abstract: 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 Cys(165), a residue located in the enzyme active site just above the catalytic zinc ion. However, no direct proof of Cys(165) 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 Cys(165) was first assessed through a combination of site-directed mutagenesis and structural studies. These data suggested that Cys(165) 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 Cys(165). 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.
authors.affiliation: Commissariat à l'Energie Atomique, iBiTec-S, Service d'Ingénierie Moléculaire des Protéines, CE-Saclay, 91191 Gif-sur-Yvette, Cedex, France.
authors.forename: Enrico A
authors.initials: EA
authors.lastname: Stura
authors.forename: Laura
authors.initials: L
authors.lastname: Le Roux
authors.forename: Karine
authors.initials: K
authors.lastname: Guitot
authors.forename: Sandra
authors.initials: S
authors.lastname: Garcia
authors.forename: Sarah
authors.initials: S
authors.lastname: Bregant
authors.forename: Fabrice
authors.initials: F
authors.lastname: Beau
authors.forename: Laura
authors.initials: L
authors.lastname: Vera
authors.forename: Guillaume
authors.initials: G
authors.lastname: Collet
authors.forename: Denis
authors.initials: D
authors.lastname: Ptchelkine
authors.forename: Huseyin
authors.initials: H
authors.lastname: Bakirci
authors.forename: Vincent
authors.initials: V
authors.lastname: Dive
mesh_terms: D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors*; D020134:Catalytic Domain; D002626:Chemistry, Pharmaceutical / Q000379:methods; D003014:Clostridium botulinum / Q000378:metabolism*; D018360:Crystallography, X-Ray / Q000379:methods; D003545:Cysteine / Q000737:chemistry*; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D010455:Peptides; D011994:Recombinant Proteins; D050825:Synaptosomal-Associated Protein 25; D010447:Peptide Hydrolases; D019274:Botulinum Toxins, Type A; D015032:Zinc; D003545:Cysteine