Abstract
Botulinum Neurotoxins (BoNTs) are the causative agents of botulism, which act by potently inhibiting the neurotransmitter release in motor neurons. Seven serotypes of BoNTs designated as BoNT/A-G have been identified. Recently, two novel types of Botulinum neurotoxins, which cleave a novel scissile bond, L54-E55, of VAMP-2 have been reported including BoNT/F subtype F5 and serotype H. However, little has been known on how these BoNTs recognize their substrates. The present study addressed for the first time the unique substrate recognition mechanism of LC/F5. Our data indicated that the optimal peptide required for efficient LC/F5 substrate cleavage is VAMP-2 (20–65). Interestingly, the overall mode of substrate recognition adopted by LC/F5 was similar to LC/F1, except that its recognition sites were shifted one helix toward the N-terminus of VAMP-2 when compared to that of LC/F1. The composition of LC/F5 pockets were found to have changed accordingly to facilitate specific recognition of these new sites of VAMP-2, including the P2′, P1′, P2, P3, B3, B2 and B1 sites. The study provides direct evidence of the evolutionary adaption of BoNT to recognize its substrate which is useful for effective antitoxin and inhibitor development.
Highlights
Botulism, named after the Latin word “botulus” for sausage, was first described by Justinus Kerner after a food poisoning outbreak associated with ingestion of blood sausages[1]
Saturation mutagenesis analysis on substrate VAMP-2 revealed that the minimal peptide region required for efficient substrate cleavage by LC/F5 was VAMP-2 (20–65), in which both the P1′ (E55) and P2′ (R56) site residues played a very significant role in substrate recognition by LC/F5 (Fig. 1), which is different from other serotypes of Botulinum Neurotoxins (BoNTs), for which the P1′ residue played the most important role on substrate hydrolysis[25,26,28]
LC/F5 recognition of substrate VAMP-2 was initiated through interaction between the B1 site of VAMP-2 (R31) and the B1 pocket of LC/F5, which is composed of the residue E147
Summary
Botulinum Neurotoxins (BoNTs) are the causative agents of botulism, which act by potently inhibiting the neurotransmitter release in motor neurons. Two novel types of Botulinum neurotoxins, which cleave a novel scissile bond, L54-E55, of VAMP-2 have been reported including BoNT/F subtype F5 and serotype H. Two novel BoNTs, BoNT/F subtype F5 and serotype H, which cleave a novel scissile bond of VAMP-2 (Vesicle-associated membrane protein 2), namely L54-E55, have been identified. BoNT/F5 exhibits highest homology to serotype F with 46–49% identity in amino acid sequence to the other six subtypes within the BoNT/F serotype[21,22] It cleaves the substrate VAMP-2 at a different scissile bond. We investigated the substrate recognition and cleavage mechanism of one of this novel toxin, LC/F5.
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