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

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, k_off, of 3.3 × 10^-2 s^-1 (t_1/2 of 21 s). A two-step association process was observed using stopped-flow: a fast bimolecular step (k_on = 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, K_iso, 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.