title: Acid-induced unfolding of the amino-terminal domains of the lethal and edema factors of anthrax toxin.
abstract: The two enzymatic components of anthrax toxin, lethal factor (LF) and edema factor (EF), are transported to the cytosol of mammalian cells by the third component, protective antigen (PA). A heptameric form of PA binds LF and/or EF and, under the acidic conditions encountered in endosomes, generates a membrane-spanning pore that is thought to serve as a passageway for these enzymes to enter the cytosol. The pore contains a 14-stranded transmembrane beta-barrel that is too narrow to accommodate a fully folded protein, necessitating that LF and EF unfold, at least partly, in order to pass. Here, we describe the pH-dependence of the unfolding of LF(N) and EF(N), the 30kDa N-terminal PA-binding domains, and minimal translocatable units, of LF and EF. Equilibrium chemical denaturation studies using fluorescence and circular dichroism spectroscopy show that each protein unfolds via a four-state mechanism: N<-->I<-->J<-->U. The acid-induced N-->I transition occurs within the pH range of the endosome (pH 5-6). The I state predominates at lower pH values, and the J and U states are populated significantly only in the presence of denaturant. The I state is compact and has characteristics of a molten globule, as shown by its retention of significant secondary structure and its ability to bind an apolar fluorophore. The N-->I transition leads to an overall 60% increase in buried surface area exposure. The J state is expanded significantly and has diminished secondary structure content. We analyze the different protonation states of LF(N) and EF(N) in terms of a linked equilibrium proton binding model and discuss the implications of our findings for the mechanism of acidic pH-induced translocation of anthrax toxin. Finally, analysis of the structure of the transmembrane beta-barrel of PA shows that it can accommodate alpha-helix, and we suggest that the steric constraints and composition of the lumen may promote alpha-helix formation.
authors.affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115, USA.
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Krantz
authors.forename: Amar D
authors.initials: AD
authors.lastname: Trivedi
authors.forename: Kristina
authors.initials: K
authors.lastname: Cunningham
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Christensen
authors.forename: R John
authors.initials: RJ
authors.lastname: Collier
mesh_terms: D000143:Acids / Q000737:chemistry*; D000942:Antigens, Bacterial / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry*; D002942:Circular Dichroism; D031541:Fluorescence Resonance Energy Transfer; D019791:Guanidine; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000143:Acids; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin; D019791:Guanidine