A Quantitative Study of the Interactions of Bacillus anthracis Edema Factor and Lethal Factor with Activated Protective Antigen

Abstract

Bacillusanthracis secretes three proteins, which associate in binary combinations to form toxic complexes at the surface of mammalian cells. Receptor-bound protective antigen (PA) is proteolytically activated, yielding a 63 kDa fragment (PA₆₃). PA₆₃ oligomerizes into heptamers, which bind edema factor (EF) or lethal factor (LF) to form the toxic complexes. We undertook a quantitative analysis of the interactions of EF with PA₆₃ by means of surface plasmon resonance (SPR) measurements. Heptameric PA₆₃ was covalently bound by amine coupling to an SPR chip, or noncovalently bound via a C-terminal hexahistidine tag on the protein to Ni²⁺nitrilotriacetate groups on the chip. Values of k_on and k_off for EF at 23 °C were ∼3 × 10⁵ M^-1 s^-1 and (3−5) × 10^-4 s^-1, respectively, giving a calculated K_d of (1−2) × 10^-9 M. A similar value of K_d (7 × 10^-10 M) was obtained when we measured the binding of radiolabeled EF to receptor-bound PA₆₃ on the surface of L6 cells (at 4 °C). Each of these analyses was also performed with LF and LF_N (the N-terminal 255 residues of LF), and values obtained were comparable to those for EF. The similarity in the dissociation constants determined by SPR and by measurements on the cell surface suggests that the presence of the receptor does not play a large role in the interaction between PA₆₃ and EF/LF.