title: Studies of the mechanistic details of the pH-dependent association of botulinum neurotoxin with membranes.
abstract: Botulinum neurotoxin (BoNT) belongs to a large class of toxic proteins that act by enzymatically modifying cytosolic substrates within eukaryotic cells. The process by which a catalytic moiety is transferred across a membrane to enter the cytosol is not understood for any such toxin. BoNT is known to form pH-dependent pores important for the translocation of the catalytic domain into the cytosol. As a first step toward understanding this process, we investigated the mechanism by which the translocation domain of BoNT associates with a model liposome membrane. We report conditions that allow pH-dependent proteoliposome formation and identify a sequence at the translocation domain C terminus that is protected from proteolytic degradation in the context of the proteoliposome. Fluorescence quenching experiments suggest that residues within this sequence move to a hydrophobic environment upon association with liposomes. EPR analyses of spin-labeled mutants reveal major conformational changes in a distinct region of the structure upon association and indicate the formation of an oligomeric membrane-associated intermediate. Together, these data support a model of how BoNT orients with membranes in response to low pH.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Mushrush
authors.forename: Hanane A
authors.initials: HA
authors.lastname: Koteiche
authors.forename: Morgan A
authors.initials: MA
authors.lastname: Sammons
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Link
authors.forename: Hassane S
authors.initials: HS
authors.lastname: McHaourab
authors.forename: D Borden
authors.initials: DB
authors.lastname: Lacy
mesh_terms: D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008567:Membranes, Artificial; D008956:Models, Chemical; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology
chemical_list: D008567:Membranes, Artificial; D019274:Botulinum Toxins, Type A; C545476:incobotulinumtoxinA