title: Effects of introducing a single charged residue into the phenylalanine clamp of multimeric anthrax protective antigen.
abstract: Multimeric pores formed in the endosomal membrane by the Protective Antigen moiety of anthrax toxin translocate the enzymatic moieties of the toxin to the cytosolic compartment of mammalian cells. There is evidence that the side chains of the Phe(427) residues come into close proximity with one another in the lumen of the pore and form a structure, termed the Phe clamp, that catalyzes the translocation process. In this report we describe the effects of replacing Phe(427) in a single subunit of the predominantly heptameric pore with a basic or an acidic amino acid. Incorporating any charged residue at this position inhibited cytotoxicity >or=1,000-fold in our standard assay and caused strong inhibition of translocation in a planar phospholipid bilayer system. His and Glu were the most strongly inhibitory residues, ablating both cytotoxicity and translocation. Basic residues at position 427 prevented the Phe clamp from interacting with a translocation substrate to form a seal against the passage of ions and accelerated dissociation of the substrate from the pore. Acidic residues, in contrast, allowed the seal to form and the substrate to remain firmly bound, but blocked its passage, perhaps via electrostatic interactions with the positively charged N-terminal segment. Our findings are discussed in relation to the role of the Phe clamp in a Brownian ratchet model of translocation.
authors.affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Blythe E
authors.initials: BE
authors.lastname: Janowiak
authors.forename: Audrey
authors.initials: A
authors.lastname: Fischer
authors.forename: R John
authors.initials: RJ
authors.lastname: Collier
mesh_terms: D019943:Amino Acid Substitution; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D011992:Endosomes / Q000378:metabolism*; D005973:Glutamine / Q000737:chemistry; D006632:Histamine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D008564:Membrane Potentials; D016296:Mutagenesis; D010649:Phenylalanine / Q000235:genetics*; D020836:Protein Structure, Quaternary; D021381:Protein Transport
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D008051:Lipid Bilayers; C030325:anthrax toxin; D005973:Glutamine; D010649:Phenylalanine; D006632:Histamine