4el4
Crystal structure of the catalytic domain of botulinum neurotoxin BoNT/A C134S/C165S double mutant
Structural highlights
FunctionBXA1_CLOBH Inhibits acetylcholine release. The botulinum toxin binds with high affinity to peripheral neuronal presynaptic membrane to the secretory vesicle protein SV2. It binds directly to the largest luminal loop of SV2A, SV2B and SV2C. It is then internalized by receptor-mediated endocytosis. The C-terminus of the heavy chain (H) is responsible for the adherence of the toxin to the cell surface while the N-terminus mediates transport of the light chain from the endocytic vesicle to the cytosol. After translocation, the light chain (L) hydrolyzes the 197-Gln-|-Arg-198 bond in SNAP-25, thereby blocking neurotransmitter release. Inhibition of acetylcholine release results in flaccid paralysis, with frequent heart or respiratory failure. Publication Abstract from PubMedClostridium botulinum neurotoxin type A (BoNT/A) is one of the most potent toxin for humans and a major biothreat agent. Despite intense chemical efforts over the ten past 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 data base with a cysteine at a similar location, some expressed by major human pathogens, thus this work should find broader applications for developing covalent inhibitors. Structural framework for covalent inhibition of Clostridium botulinum neurotoxin A by targeting Cys165.,Stura EA, Le Roux L, Guitot K, Garcia S, Bregant S, Beau F, Vera L, Collet G, Ptchelkine D, Bakirci H, Dive V J Biol Chem. 2012 Aug 6. PMID:22869371[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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