title: Insertion of anthrax protective antigen into liposomal membranes: effects of a receptor.
abstract: Protective antigen (PA), the receptor-binding component of anthrax toxin, heptamerizes and inserts into the endosomal membrane at acidic pH, forming a pore that mediates translocation of the enzymic components of the toxin to the cytosol. When the heptameric pre-insertion form of PA (the prepore) is acidified in solution, it rapidly loses the ability to insert into membranes. To maximize insertion into model membranes, we examined two ways to bind the protein to large unilamellar vesicles (LUV). One involved attaching a His tag to the von Willebrand factor A domain of one of the PA receptors, ANTXR2, and using this protein as a bridge to bind PA to LUV containing a nickel-chelating lipid. The other involved using a His tag fused to the C terminus of PA to bind the protein directly to LUV containing the same lipid. Both ways enhanced pore formation at pH 5.0 strongly and about equally, as measured by the release of K+. Controls showed that pore formation in this system faithfully reproduced that in vivo. We also showed that binding unmodified ANTXR2 von Willebrand factor A to the prepore in solution enhanced its pore forming activity by slowing its inactivation at acidic pH. These findings indicate that an important role of PA receptors is to promote partitioning of PA into the bilayer by maintaining the prepore close to the target membrane and presumably in the optimal orientation as it undergoes the acidic pH-dependent conformational transition to the pore.
authors.affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Sun
authors.forename: Gregory
authors.initials: G
authors.lastname: Vernier
authors.forename: Darran J
authors.initials: DJ
authors.lastname: Wigelsworth
authors.forename: R John
authors.initials: RJ
authors.lastname: Collier
mesh_terms: D000143:Acids / Q000378:metabolism; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives; D008565:Membrane Proteins / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism; D009829:Oleic Acids; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D018000:Receptors, Peptide; D013386:Succinates; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism
chemical_list: C447150:ANTXR2 protein, human; D000143:Acids; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; C476561:N(alpha),N(alpha)-bis(carboxymethyl)-N(epsilon)-(1,2-dioleyl-sn-glycero-3-succinyl)-L-lysine; D009829:Oleic Acids; D018000:Receptors, Peptide; D013386:Succinates; C030325:anthrax toxin; D014841:von Willebrand Factor; D006639:Histidine; D009532:Nickel; D008239:Lysine