title: The C terminus of the catalytic domain of type A botulinum neurotoxin may facilitate product release from the active site.
abstract: Botulinum neurotoxins are the most toxic of all compounds. The toxicity is related to a poor zinc endopeptidase activity located in a 50-kDa domain known as light chain (Lc) of the toxin. The C-terminal tail of Lc is not visible in any of the currently available x-ray structures, and it has no known function but undergoes autocatalytic truncations during purification and storage. By synthesizing C-terminal peptides of various lengths, in this study, we have shown that these peptides competitively inhibit the normal catalytic activity of Lc of serotype A (LcA) and have defined the length of the mature LcA to consist of the first 444 residues. Two catalytically inactive mutants also inhibited LcA activity. Our results suggested that the C terminus of LcA might interact at or near its own active site. By using synthetic C-terminal peptides from LcB, LcC1, LcD, LcE, and LcF and their respective substrate peptides, we have shown that the inhibition of activity is specific only for LcA. Although a potent inhibitor with a Ki of 4.5 μm, the largest of our LcA C-terminal peptides stimulated LcA activity when added at near-stoichiometric concentration to three versions of LcA differing in their C-terminal lengths. The result suggested a product removal role of the LcA C terminus. This suggestion is supported by a weak but specific interaction determined by isothermal titration calorimetry between an LcA C-terminal peptide and N-terminal product from a peptide substrate of LcA. Our results also underscore the importance of using a mature LcA as an inhibitor screening target.
authors.affiliation: Integrated Toxicology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702, USA.
authors.forename: Rahman M
authors.initials: RM
authors.lastname: Mizanur
authors.forename: Verna
authors.initials: V
authors.lastname: Frasca
authors.forename: Subramanyam
authors.initials: S
authors.lastname: Swaminathan
authors.forename: Sina
authors.initials: S
authors.lastname: Bavari
authors.forename: Robert
authors.initials: R
authors.lastname: Webb
authors.forename: Leonard A
authors.initials: LA
authors.lastname: Smith
authors.forename: S Ashraf
authors.initials: SA
authors.lastname: Ahmed
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis / Q000187:drug effects; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D002151:Calorimetry; D020134:Catalytic Domain; D003014:Clostridium botulinum / Q000145:classification / Q000378:metabolism*; D004795:Enzyme Stability / Q000187:drug effects; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009498:Neurotoxins / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D058767:Protein Unfolding / Q000187:drug effects; D012703:Serotyping; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D013696:Temperature
chemical_list: D050505:Mutant Proteins; D009498:Neurotoxins; D010455:Peptides; D050825:Synaptosomal-Associated Protein 25; D019274:Botulinum Toxins, Type A
keywords: Botulinum Toxin; C Terminus; Enzyme Mechanisms; Enzyme Structure; Metalloprotease; Neurotoxin; Peptide Interactions; Protein Chemistry; Zinc Endopeptidase