Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: Insights from X-ray crystallography. Issue 22 (15th November 2015)

• Record Type: Journal Article
• Authors: Kumaran, Desigan; Adler, Michael; Levit, Matthew; Krebs, Michael; Sweeney, Richard; Swaminathan, Subramanyam
• Is Part Of: Bioorganic & medicinal chemistry. Volume 23:Issue 22(2015)
• Page Start: 7264
• Page End: 7273
• Publication Date: 2015-11-15
• Subjects: BoNT botulinum neurotoxin -- Balc botulinum neurotoxin A light chain -- Balc424 Balc truncated at residue 424 -- Balc448 full-length Balc -- CPI-1 cyclic peptide inhibitor-1 -- CPI-2 cyclic peptide inhibitor-2 -- DAB 2, 4-diaminobutanoic acid -- FRET Förster resonance energy transfer -- HBAT hepatavalent botulism antitoxin -- LC light chain -- PDB protein data bank -- PLM peptide-like molecule -- SNARE soluble N-ethylmaleimide-sensitive factor attachment protein receptor -- SNAP-25 synaptosome-associated protein of 25 kDa -- TCEP tris(2-carboxyethyl)phosphine -- TPI tetra peptide inhibitor -- VAMP1/2 vesicle-associated membrane protein isoforms 1 or 2; Botulinum toxin -- Botulinum neurotoxin -- Cyclic peptide -- Inhibition mechanism -- Light chain -- SNAP-25 -- SNARE protein -- X-ray crystallography; Bioorganic chemistry -- Periodicals; Pharmaceutical chemistry -- Periodicals; Biochemistry -- Periodicals; Chemistry, Clinical -- Periodicals; Chemistry, Organic -- Periodicals; Chimie bio-organique -- Périodiques; Chimie pharmaceutique -- Périodiques; 615.19
• DOI: 10.1016/j.bmc.2015.10.024
• Languages: English
• ISSNs: 0968-0896

Abstract

Graphical abstract: 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 K i 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.