Studies of the mechanistic details of the pH-dependent association of botulinum neurotoxin with membranes.

Mushrush DJ, Koteiche HA, Sammons MA, Link AJ, McHaourab HS, Lacy DB
J Biol Chem. 2011 286 (30): 27011-8

PMID: 21652698 · PMCID: PMC3143659 · DOI:10.1074/jbc.M111.256982

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.

MeSH Terms (8)

Botulinum Toxins, Type A Cytosol Hydrogen-Ion Concentration Membranes, Artificial Models, Chemical Protein Multimerization Protein Structure, Tertiary Protein Transport

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