Botulinum Neurotoxin Serotype F:  Identification of Substrate Recognition Requirements and Development of Inhibitors with Low Nanomolar Affinity

Abstract

Botulinum neurotoxins (BoNTs A-G) are zinc metalloendoproteases that exhibit extraordinary specificities for proteins involved in neurotransmitter release. In view of the extreme toxicities of these molecules, their applications in human medicine, and potential for misuse, it is of considerable importance to elucidate the mechanisms underlying substrate recognition and to develop inhibitors, with the ultimate goal of obtaining anti-botulinum drugs. We synthesized peptides based on vesicle-associated membrane protein (VAMP) to investigate the substrate requirements of BoNT F, which cleaves VAMP between residues Q58 and K59. The minimum substrate was a peptide containing VAMP residues 32-65, which includes only one of the two VAMP structural motifs thought to be required for botulinum substrate recognition. BoNT F exhibited a strict requirement for residues D57 (P 2 ), K59 (P 1 ′), and L60 (P 2 ′), but peptides containing substitutions for R56 (P 3 ), Q58 (P 1 ), and S61 (P 3 ′) were cleaved. Therefore, the P 2 , P 1 ′, and P 2 ′ residues of VAMP are of paramount importance for BoNT F substrate recognition near the scissile bond. K i values of uncleavable analogues were similar to K m values of the substrate, suggesting that substrate discrimination occurs at the cleavage step, not at the initial binding step. We then synthesized inhibitors of BoNT F that incorporated D-cysteine in place of glutamine 58, exhibited K i values of 1-2 nM, and required binding groups on the N-terminal but not the C-terminal side of the zinc ligand. The latter characteristic distinguishes BoNT F from other zinc metalloendoproteases, including BoNTs A and B.The clostridial neurotoxins consist of tetanus toxin, secreted by Clostridium tetani, and the seven serologically distinct botulinum toxins, designated types A-G, produced by various strains of Clostridium botulinum, Clostridium baratii, and Clostridium butyricum (1-3). They are the most toxic substances known (4). Each neurotoxin is synthesized as a single-chain protein with an M r of ∼150000. Endogenous proteases then act to produce the dichain structure, consisting of an M r ∼ 100 000 heavy chain and an M r ∼ 50 000 light chain, covalently linked by a disulfide bond. The dichain is thought to be the active form of the toxin (1). In the United States, there are ∼200 cases of human botulism each year, due to ingestion of toxin-contaminated food, wound infections, or colonization of the intestinal tract by C. botulinum (5-7). However, much of the current interest in BoNTs 1 stems from their use as tools in research on the mechanisms of neurotransmission (1,8,9), an ever-expanding number of applications in the treatment of human muscle dysfunctions (10-13), and their potential for use as bioterrorist weapons (14).Intoxication resulting in the flaccid paralysis of botulism or the spastic paralysis of tetanus has been described as a four-step process, consisting of (1) toxin binding to specific receptors on neurons, (2) receptor-mediated toxin endocytosis, (3) translocation th...

A yeast assay probes the interaction between botulinum neurotoxin serotype B and its SNARE substrate

“…2a). Despite these differences, our data show that mutations at the P2 and PЈ2 positions inhibit the B-LC͞Sb interaction and cleavage, an intriguing result in light of the recently reported role for P2 and PЈ2 residues in the serotype F-LC͞Sb interaction (5).…”

“…SNARE proteins are thought to be the primary and perhaps only intracellular target for the BoNT͞LCs, and each serotype cleaves a different peptide bond within its corresponding SNARE substrate. This remarkable specificity results from the LCs' recognition of unusually long SNARE sequences (Ϸ30-50 residues, depending on the serotype), suggesting that substrate residues distal to the cleavage site are recognized (3)(4)(5). This hypothesis is supported by the recently elucidated crystal structure of serotype A-LC in complex with a fragment of SNAP-25 (6,7).…”

“…To probe LC substrate specificity, cell-free assays have been developed, employing purified endoprotease and soluble SNARE protein fragments to probe cleavage of mutated SNARE sequences. The model emerging from these studies is one where cleavage site residues and residues positioned in the SNARE motif, a conserved 9-to 10-residue element, are important for LC substrate recognition (4,5,(8)(9)(10)). An alternative approach to elucidate substrate specificity comes from the threedimensional structure of serotype A-LC in complex with a SNAP-25 protein fragment (6,7).…”

Transmembrane-domain determinants for SNARE-mediated membrane fusion

“…The expression of botF/LC-resistant VAMP2 (K59R) (Schmidt and Stafford, 2005) restored secretion (102% of 2475 SNARE transmembrane domain and fusion control) in the presence of toxin (Fig. 4B).…”

Structural Studies on Intact Clostridium botulinum Neurotoxins Complexed with Inhibitors Leading to Drug Design

“…They require an extended substrate segment for optimal catalytic activity as indicated by studies employing truncated substrates. [9][10][11][12][13][14] Interestingly, despite sharing the same scissile bond in VAMPs, even TeNT and BoNT/B appear to interact with different substrate sites.…”