Anthrax toxin complexes: heptameric protective antigen can bind lethal factor and edema factor simultaneously Ruth-Anne L. Pimental 1 , Kenneth A. Christensen 1 , Bryan A. Krantz, R. John Collier * Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA Received 14 July 2004 Available online 7 August 2004 Abstract The 83 kDa protective antigen (PA 83 ) component of anthrax toxin, after proteolytic activation, self-associates to form ring- shaped heptamers ([PA 63 ] 7 ) that bind and aid delivery of the Edema Factor (EF) and Lethal Factor (LF) components to the cytosol. Here we show using fluorescence (Fo ¨ rster) resonance energy transfer that a molecule of [PA 63 ] 7 can bind EF and LF simultaneously. We labeled EF and LF with an appropriate donor/acceptor pair and found quenching of the donor and an increase in sensitized emission of the acceptor when, and only when, a mixture of the labeled proteins was combined with [PA 63 ] 7 . Addition of unlabeled PA 63 -binding domain of LF to the mixture competitively displaced labeled EF and LF, causing a loss of energy transfer. In view of the known maximum occupancy of 3 ligand molecules per [PA 63 ] 7 , these findings indicate that PA, EF, and LF can form mixtures of liganded toxin complexes containing both EF and LF. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Anthrax toxin; Edema factor; Lethal factor; Protective antigen; Stoichiometry; Ligand binding; Macromolecular interactions Anthrax toxin consists of three non-toxic proteins that interact at the mammalian cell surface to form toxic non-covalent complexes [1,2]. The three proteins are: edema factor (EF), a calmodulin dependent adenylate cyclase; lethal factor (LF), a Zn 2+ -metalloprotease; and protective antigen (PA 83 , 83 kDa), a protein that binds the enzymatic components and delivers them to the cytosol of mammalian cells. Once within the cytosol, EF and LF contact their substrates and catalyze reac- tions that result in toxicity [3–5]. The current, generally accepted, model of anthrax toxin assembly and action is as follows. PA 83 binds to either of two known cell surface receptors, anthrax toxin receptor/tumor endothelial marker-8 (ATR/TEM8) and capillary morphogenesis protein 2 (CMG2) [6,7]. A cel- lular protease of the furin family then cleaves PA 83 into two fragments: PA 20 (20 kDa), corresponding to the N-terminus, and PA 63 (63 kDa), corresponding to the C-terminus [8,9]. PA 20 dissociates into the medium and plays no known further role in toxin action. Removal of PA 20 eliminates a steric barrier to self-association, allowing PA 63 to oligomerize and form a heptameric ring-shaped structure ([PA 63 ] 7 ), termed the prepore [10]. This structure can bind up to three molecules of LF or EF competitively and with nanomolar affinity [11,12]. The resulting complexes are localized to deter- gent-resistant lipid microdomains, where they undergo receptor-mediated endocytosis [13]. Acidification of the toxin-containing compartment then causes a structural rearrangement of the prepore that enables it to insert into the membrane, forming a pore [14,15]. Pore forma- tion is linked to translocation of the bound enzymatic cargo (LF or EF) to the cytosol. LF has been shown to cleave members of the mitogen-activated protein ki- nase kinase family [4,5], but the events leading to death are poorly defined. Elevation of cAMP concentration by 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.07.105 * Corresponding author. Fax: +1 617 432 0115. E-mail address: jcollier@hms.harvard.edu (R.J. Collier). 1 These authors contributed equally to this work. www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 322 (2004) 258–262 BBRC