title: Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: Insights from X-ray crystallography.
abstract: The seven antigenically distinct serotypes (A-G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins consisting of a ∼ 100 kDa heavy chain and a ∼ 50 kDa light chain; the former is responsible for neurospecific binding, internalization and translocation, and the latter for cleavage of neuronal SNARE proteins. Because of their extreme toxicity and history of weaponization, the BoNTs are regarded as potential biowarfare/bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than intensive care; therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was evaluated in a FRET assay for its ability to inhibit BoNT/A light chain (Balc). CPI was found to be highly potent, exhibiting a Ki of 12.3 nM with full-length Balc448 and 39.2 nM using a truncated crystallizable form of the light chain (Balc424). Cocrystallization studies revealed that in the Balc424-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic manner with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn(2+) binding region involved in catalysis. This differs from linear peptide inhibitors described to date which block only the latter.
authors.affiliation: Biological, Environmental & Climate Sciences Department, Brookhaven National Laboratory, Upton, NY 11973, United States.
authors.forename: Desigan
authors.initials: D
authors.lastname: Kumaran
authors.affiliation: Analytical Toxicology Division, U.S. Army Medical Research Institute of Chemical Defense (USAMRICD), Aberdeen Proving Ground, MD 21010, United States. Electronic address: michael.adler2.civ@mail.mil.
authors.forename: Michael
authors.initials: M
authors.lastname: Adler
authors.affiliation: Analytical Toxicology Division, U.S. Army Medical Research Institute of Chemical Defense (USAMRICD), Aberdeen Proving Ground, MD 21010, United States.
authors.forename: Matthew
authors.initials: M
authors.lastname: Levit
authors.affiliation: Analytical Toxicology Division, U.S. Army Medical Research Institute of Chemical Defense (USAMRICD), Aberdeen Proving Ground, MD 21010, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Krebs
authors.affiliation: Research Division, USAMRICD, Aberdeen Proving Ground, MD 21010, United States.
authors.forename: Richard
authors.initials: R
authors.lastname: Sweeney
authors.affiliation: Biological, Environmental & Climate Sciences Department, Brookhaven National Laboratory, Upton, NY 11973, United States.
authors.forename: Subramanyam
authors.initials: S
authors.lastname: Swaminathan
mesh_terms: D001665:Binding Sites; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006801:Humans; D056004:Molecular Dynamics Simulation; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism
chemical_list: D010456:Peptides, Cyclic; D050825:Synaptosomal-Associated Protein 25; D019274:Botulinum Toxins, Type A
keywords: Botulinum neurotoxin; Botulinum toxin; Cyclic peptide; Inhibition mechanism; Light chain; SNAP-25; SNARE protein; X-ray crystallography