Interaction of the 20 kDa and 63 kDa Fragments of Anthrax Protective Antigen: Kinetics and Thermodynamics

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Abstract
The action of anthrax toxin begins when the protective antigen (PA83, 83 kDa) moiety binds to a mammalian cell-surface receptor and is cleaved by a furin-family protease into two fragments: PA20 (20 kDa) and PA63 (63 kDa). After PA20 dissociates, receptor-bound PA63 spontaneously oligomerizes to form a heptameric species, which is able to bind the two enzymatic components of the toxin and transport them to the cytosol. Treatment of PA83 with trypsin yielded PA63 and a form of PA20 lacking unstructured regions at the N- and C-termini. We labeled these fragments with dyes capable of fluorescence resonance energy transfer to quantify their association in solution. We kinetically determined that the equilibrium dissociation constant is 190 nM with a dissociation rate constant, koff, of 3.3 × 10-2 s-1 (t1/2 of 21 s). A two-step association process was observed using stopped-flow: a fast bimolecular step (kon = 1.4 × 105 M-1 s-1) was followed by a slower unimolecular step (k = 3.5 × 10-3 s-1) with an equilibrium isomerization constant, Kiso, of 2.1. The two-step mechanism most consistent with the data is one in which the dissociation of the PA20·PA63 complex is followed by an isomerization in the PA63 moiety. Our results indicate that, following the cleavage of PA on the cell surface, PA20 is largely dissociated within a minute. A slow isomerization step in PA63 may then potentiate it for oligomerization and subsequent steps in toxin action.