The protective antigen (PA) moiety of anthrax toxin forms oligomeric pores that translocate the enzymatic moieties of the toxin -lethal factor (LF) and edema factor (EF)-across the endosomal membrane of mammalian cells. Here we describe site-directed spin-labeling studies that identify interactions of LF with the prepore and pore conformations of PA. Our results reveal a direct interaction between the extreme N terminus of LF (residues 2-5) and the Φ-clamp, a structure within the lumen of the pore that catalyzes translocation. Also, consistent with a recent crystallographic model, we find that, upon binding of the translocation substrate to PA, LF helix α1 separates from helices α2 and α3 and binds in the α-clamp of PA. These interactions, together with the binding of the globular part of the N-terminal domain of LF to domain 1′ of PA, indicate that LF interacts with the PA pore at three distinct sites. Our findings elucidate the state from which translocation of LF and EF proceeds through the PA pore.A nthrax toxin, in addition to its importance in regard to the pathogenesis of Bacillus anthracis, is one of the simplest and most tractable systems for studying protein translocation across membranes. The toxin is composed of two intracellularly acting enzymes-edema factor (EF) (1) and lethal factor (LF) (2)-and a receptor-binding (3, 4), pore-forming (5) protein, termed protective antigen (PA). These three proteins are secreted from the Bacillus anthracis as monomeric units. Monomeric PA 83 is activated by furin-family proteases (6) on the host cell surface and spontaneously assembles into homoheptamers, ½PA 63 7 (7), and homooctamers, ½PA 63 8 (8) (collectively termed prepores). ½PA 63 7 binds up to three, and ½PA 63 8 binds up to four, molecules of LF and/or EF (8, 9), forming noncovalent receptor-bound complexes at the cell surface. These complexes are taken into the cell through endocytosis (10) and trafficked to the endosome, where, upon exposure to low pH, the prepores insert into the membrane (5, 11, 12), forming pores through which LF and/or EF translocate to the cytosol (13).LF and EF bind to the surface of PA (on domain 1′) (14) through their homologous, ∼250-residue N-terminal domains (LF N and EF N , Fig. 1E) (15). Each has a positively charged, unstructured N terminus (16, 17) (∼20-30 residues), which is positioned at or near the entry of the lumen when the proteins bind to the prepore (18). PA pores have a negatively charged lumen (7) and form cation-selective channels in planar lipid bilayers (19). LF or LF N blocks current flow through these channels (20), whereas N-terminally truncated LF N mutants do not (21). These observations have led to the hypothesis that upon binding of LF N to domain 1′ of a pore, the N terminus is drawn into the lumen by electrostatic forces, initiating N-to C-terminal translocation. However, data have been lacking to indicate precisely how the unstructured LF or EF N terminus interacts with the lumen of the pore.The Phe427 residues of the PA prepore, located in the lume...