Substrate recognition strategy for botulinum neurotoxin serotype A

Abstract

Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus. CNTs impair neuronal exocytosis through specific proteolysis of essential proteins called SNAREs. SNARE assembly into a low-energy ternary complex is believed to catalyse membrane fusion, precipitating neurotransmitter release; this process is attenuated in response to SNARE proteolysis. Site-specific SNARE hydrolysis is catalysed by the CNT light chains, a unique group of zinc-dependent endopeptidases. The means by which a CNT properly identifies and cleaves its target SNARE has been a subject of much speculation; it is thought to use one or more regions of enzyme-substrate interaction remote from the active site (exosites). Here we report the first structure of a CNT endopeptidase in complex with its target SNARE at a resolution of 2.1 A: botulinum neurotoxin serotype A (BoNT/A) protease bound to human SNAP-25. The structure, together with enzyme kinetic data, reveals an array of exosites that determine substrate specificity. Substrate orientation is similar to that of the general zinc-dependent metalloprotease thermolysin. We observe significant structural changes near the toxin's catalytic pocket upon substrate binding, probably serving to render the protease competent for catalysis. The novel structures of the substrate-recognition exosites could be used for designing inhibitors specific to BoNT/A.

An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists

“…The catalytic LC domain of BoNT/A is a compact globule consisting of a mixture of ␣-helices, ␤-sheets, and ␤-strands with a zinc-containing metalloprotease active site bound deeply inside a large open cavity (2). The remarkable substrate selectivity of BoNT/A for SNAP-25 has been explained to be a consequence of extensive interactions with two exosite domains distinct from the active site (19). A model for substrate recognition has been proposed in which ␣-exosite binding occurs first via helix formation in the appropriate region of SNAP-25, followed by ␤-exosite recognition and subsequent conformational changes in the enzyme to facilitate efficient substrate cleavage (19).…”

“…The remarkable substrate selectivity of BoNT/A for SNAP-25 has been explained to be a consequence of extensive interactions with two exosite domains distinct from the active site (19). A model for substrate recognition has been proposed in which ␣-exosite binding occurs first via helix formation in the appropriate region of SNAP-25, followed by ␤-exosite recognition and subsequent conformational changes in the enzyme to facilitate efficient substrate cleavage (19). This model argues that, without exosite binding, BoNT/A is a significantly less efficient enzyme, and thus these regions could be targeted for lead development.…”

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

“…Finally, inside the cytosol, the LC acts as zinc-dependent metallo¬protease and cleaves proteins of the SNARE complex, which are the part of exocytosis apparatus, effectively destroying this apparatus and leading to inhibition of neurotransmitter release [3]. In this last step of SNARE complex protein cleavage, each of the seven different BoNT serotypes cleaves a unique peptide bond located on one of the SNARE proteins [16,17,18]. BoNT/A, /C and /E cleave synaptosomal associated protein of 25 kDa (SNAP-25), at positions 197-198, 198-199 and 180-181, respectively.…”

A yeast assay probes the interaction between botulinum neurotoxin serotype B and its 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). The structure reveals multiple substrate exosites, which position the cleavage site at the catalytic Zn 2ϩ -binding motif in the LC.…”

“…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). Whereas the structure reveals numerous interactions distal to the site of substrate cleavage, SNARE motif residues appear to be excluded from the substrateendoprotease interface.…”