Identification and Characterization of Botulinum Neurotoxin A Substrate Binding Pockets and Their Re-Engineering for XXXX @entity24835 .

@entity1 @entity3534 @entity24835 @entity8816 @entity375 @entity12252
UNASSIGNED: Botulinum neurotoxins (BoNTs) are highly potent bacterial proteins that block neurotransmitter release at the neuromuscular junction by cleaving SNAREs (soluble @entity8816 sensitive factor attachment protein receptors). However, their serotype A (BoNT/A) that cleaves @entity12252 ( @entity12252 ) has also been an established pharmaceutical for treatment of medical conditions that rely on hyperactivity of cholinergic nerve terminals for 25years. The expansion of its use to a variety of further medical conditions associated with hypersecretion components is prevented partly because the involved SNARE isoforms are not cleaved. Therefore, we examined by mutational analyses the reason for the resistance of @entity1 @entity24835 , an isoform of @entity12252 . We show that replacement of 10 @entity24835 residues with their @entity12252 counterparts effects @entity12252 -like cleavability. Conversely, transfer of each of the replaced @entity24835 residues to @entity12252 drastically decreased the cleavability of @entity12252 . By means of the existing @entity12252 -toxin co-crystal structure, molecular dynamics simulations, and corroborative mutagenesis studies, the appropriate binding pockets for these residues in BoNT/A were characterized. Systematic mutagenesis of two major BoNT/A binding pockets was conducted in order to adapt these pockets to corresponding @entity3534 of @entity1 @entity24835 . @entity1 @entity24835 cleaving mutants were isolated using a newly established @entity375 -based screening system. This method may be useful for engineering novel BoNT/A pharmaceuticals for the treatment of diseases that rely on @entity24835 -mediated hypersecretion.
