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

Abstract

Bacillus anthracis 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 (PA(63)). PA(63) 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 PA(63) by means of surface plasmon resonance (SPR) measurements. Heptameric PA(63) was covalently bound by amine coupling to an SPR chip, or noncovalently bound via a C-terminal hexahistidine tag on the protein to Ni(2+)nitrilotriacetate groups on the chip. Values of k(on) and k(off) for EF at 23 degrees C were approximately 3 x 10(5) M(-)(1) s(-)(1) and (3-5) x 10(-)(4) s(-)(1), respectively, giving a calculated K(d) of (1-2) x 10(-)(9) M. A similar value of K(d) (7 x 10(-)(10) M) was obtained when we measured the binding of radiolabeled EF to receptor-bound PA(63) on the surface of L6 cells (at 4 degrees 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 PA(63) and EF/LF.

The lethal and edema factors of anthrax toxin bind only to oligomeric forms of the protective antigen

“…The kinetics of these pathways depend on the relative stabilities of the PA 63 -PA 63 and PA 63 -ligand intermediates, which are unknown. Values of K d and the on-and off-rate constants of the interactions of the enzymatic moieties for oligomeric PA 63 have been measured (12), but other parameters affecting the assembly process have not. Regardless of the pathway by which it formed, the liganded dimer of the mixed oligomerization-deficient mutant PAs is sufficiently stable in solution to be identified as a peak from a size-exclusion column and a band on PAGE.…”

Sensitizing Anthrax Lethal Toxin-resistant Macrophages to Lethal Toxin-induced Killing by Tumor Necrosis Factor-α

“…PA binds to a cell-surface receptor (7), where it is proteolytically processed on the cell surface (8,9). The processed PA heptamerizes and binds 3 molecules of either EF or LF, resulting in either a PA⅐EF or a PA⅐LF complex, which is internalized through receptor-mediated endocytosis (10). PA then forms a hole in the acidified endocytic vesicle through which EF or LF is delivered to the cytosol (11).…”

Anthrax toxin-induced rupture of artificial lipid bilayer membranes

“…The process begins when the B-component (83 kDa protective antigen PA 83 protein) binds to receptors on cell membranes [1][2][3] and is cleaved by a furin-type protease on the cell surface. The 63 kg/mol fragment, PA 63 , remains receptor bound 4,5 and forms a nanometer-scale heptameric pre-pore, 6 (PA 63 ) 7 , that binds up to three A-components, the 90 kDa lethal factor (LF), or the 89 kDa edema factor (EF) molecules. 7 PA 63 then forms nanometer-scale pores in membranes.…”