Acid-induced unfolding of the amino-terminal domains of the lethal and edema factors of anthrax toxin

J Mol Biol. 2004 Nov 26;344(3):739-56. doi: 10.1016/j.jmb.2004.09.067.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acids / chemistry*
  • Antigens, Bacterial / chemistry*
  • Bacterial Toxins / chemistry*
  • Circular Dichroism
  • Fluorescence Resonance Energy Transfer
  • Guanidine
  • Hydrogen-Ion Concentration
  • Protein Denaturation*
  • Protein Structure, Secondary
  • Spectrophotometry, Ultraviolet


  • Acids
  • Antigens, Bacterial
  • Bacterial Toxins
  • anthrax toxin
  • Guanidine