3bwi | pdb_00003bwi
Crystal structure of the catalytic domain of botulinum neurotoxin serotype A with an acetate ion bound at the active site
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. Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedThe seven antigenically distinct serotypes of Clostridium botulinum neurotoxins cleave specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex proteins and block the release of neurotransmitters that cause flaccid paralysis and are considered potential bioweapons. Botulinum neurotoxin type A is the most potent among the clostridial neurotoxins, and to date there is no post-exposure therapeutic intervention available. To develop inhibitors leading to drug design, it is imperative that critical interactions between the enzyme and the substrate near the active site are known. Although enzyme-substrate interactions at exosites away from the active site are mapped in detail for botulinum neurotoxin type A, information about the active site interactions is lacking. Here, we present the crystal structures of botulinum neurotoxin type A catalytic domain in complex with four inhibitory substrate analog tetrapeptides, viz. RRGC, RRGL, RRGI, and RRGM at resolutions of 1.6-1.8 A. These structures show for the first time the interactions between the substrate and enzyme at the active site and delineate residues important for substrate stabilization and catalytic activity. We show that OH of Tyr(366) and NH(2) of Arg(363) are hydrogen-bonded to carbonyl oxygens of P1 and P1' of the substrate analog and position it for catalytic activity. Most importantly, the nucleophilic water is replaced by the amino group of the N-terminal residue of the tetrapeptide. Furthermore, the S1' site is formed by Phe(194), Thr(215), Thr(220), Asp(370), and Arg(363). The K(i) of the best inhibitory tetrapeptide is 157 nm. Structure- and substrate-based inhibitor design for Clostridium botulinum neurotoxin serotype A.,Kumaran D, Rawat R, Ludivico ML, Ahmed SA, Swaminathan S J Biol Chem. 2008 Jul 4;283(27):18883-91. Epub 2008 Apr 22. PMID:18434312[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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