title: Identification and Characterization of Botulinum Neurotoxin A Substrate Binding Pockets and Their Re-Engineering for Human SNAP-23.
abstract: Botulinum neurotoxins (BoNTs) are highly potent bacterial proteins that block neurotransmitter release at the neuromuscular junction by cleaving SNAREs (soluble N-ethyl maleimide sensitive factor attachment protein receptors). However, their serotype A (BoNT/A) that cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa) has also been an established pharmaceutical for treatment of medical conditions that rely on hyperactivity of cholinergic nerve terminals for 25 years. 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 human SNAP-23, an isoform of SNAP-25. We show that replacement of 10 SNAP-23 residues with their SNAP-25 counterparts effects SNAP-25-like cleavability. Conversely, transfer of each of the replaced SNAP-23 residues to SNAP-25 drastically decreased the cleavability of SNAP-25. By means of the existing SNAP-25-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 amino acids of human SNAP-23. Human SNAP-23 cleaving mutants were isolated using a newly established yeast-based screening system. This method may be useful for engineering novel BoNT/A pharmaceuticals for the treatment of diseases that rely on SNAP-23-mediated hypersecretion.
authors.affiliation: Institut für Physiologische Chemie, OE 4310, Medizinische Hochschule Hannover, D-30623 Hannover, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Sikorra
authors.affiliation: Institut für Physiologische Chemie, OE 4310, Medizinische Hochschule Hannover, D-30623 Hannover, Germany.
authors.forename: Christa
authors.initials: C
authors.lastname: Litschko
authors.affiliation: Institut für Physiologische Chemie, OE 4310, Medizinische Hochschule Hannover, D-30623 Hannover, Germany.
authors.forename: Carina
authors.initials: C
authors.lastname: Müller
authors.affiliation: Institut für Physiologische Chemie, OE 4310, Medizinische Hochschule Hannover, D-30623 Hannover, Germany.
authors.forename: Nadine
authors.initials: N
authors.lastname: Thiel
authors.affiliation: Sorbonne Paris Cité, Université Paris Diderot, Institut Jacques Monod, CNRS UMR 7592, INSERM ERL U950, F-75013 Paris, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Galli
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, MA 02454, USA.
authors.forename: Timo
authors.initials: T
authors.lastname: Eichner
authors.affiliation: Institut für Physiologische Chemie, OE 4310, Medizinische Hochschule Hannover, D-30623 Hannover, Germany. Electronic address: binz.thomas@mh-hannover.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Binz
mesh_terms: D001665:Binding Sites; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D005820:Genetic Testing; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D059748:Proteolysis; D050766:Qb-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D050767:Qc-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; C100493:SNAP23 protein, human; D019274:Botulinum Toxins, Type A
keywords: SNAP-25; molecular dynamics simulation; screening method; substrate specificity; zinc protease