Interactions of anthrax lethal factor with protective antigen defined by site-directed spin labeling.

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.