Abstract
The protective antigen (PA) moiety of anthrax toxin binds to cellular receptors and mediates entry of the two enzymatic moieties of the toxin into the cytosol. Two PA receptors, anthrax toxin receptor (ATR)/tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2), have been identified. We expressed and purified the von Willebrand A (VWA) domain of CMG2 and examined its interactions with monomeric and heptameric forms of PA. Monomeric PA bound a stoichiometric equivalent of CMG2, whereas the heptameric prepore form bound 7 eq. The Kd of the VWA domain-PA interaction is 170 pm when liganded by Mg2+, reflecting a 1000-fold tighter interaction than most VWA domains with their endogenous ligands. The dissociation rate constant is extremely slow, indicating a 30-h lifetime for the CMG2.PA monomer complex. CMG2 metal ion-dependent adhesion site (MIDAS) was studied kinetically and thermodynamically. The association rate constant (approximately 10(5) m(-1) s(-1)) is virtually identical in the presence or absence of Mg2+ or Ca2+ , but the dissociation rate of metal ion liganded complex is up to 4 orders of magnitude slower than metal ion free complex. Residual affinity (Kd approximately 960 nm) in the absence of divalent metal ions allowed the free energy for the contribution of the metal ion to be calculated as 5 kcal mol(-1), demonstrating that the metal ion-dependent adhesion site is directly coordinated by CMG2 and PA in the binding interface. The high affinity of the VWA domain for PA supports its potency in neutralizing anthrax toxin, demonstrating its potential utility as a novel therapeutic for anthrax.
Highlights
The initial step in the action of the toxin is the binding of protective antigen (PA) to a cell surface receptor
PA83 Binds 1 Stoichiometric Equivalent of capillary morphogenesis protein 2 (CMG2) von Willebrand A (VWA) Domain—We measured the stoichiometry of CMG2 VWA domain binding to PA83 by three independent methods, isothermal titration calorimetry, fluorescence resonance energy transfer (FRET)-based titration, and size-exclusion chromatography coupled with multiangle laser light scattering
For isothermal titration calorimetry measurements, PA83 was loaded into the measurement cell, and CMG235–225 was added in small increments under continuous stirring
Summary
The initial step in the action of the toxin is the binding of PA to a cell surface receptor. Each is a single peptide chain consisting of an extracellular domain, a membrane-spanning region, and a cytoplasmic tail In their extracellular domains, there is an ϳ200-amino acid von Willebrand type A (VWA) domain that shows 60% amino acid identity between the two proteins [11]. The pathology of the anthrax bacillus, Bacillus anthracis, is due in part to the production of anthrax toxin, an ensemble of three nontoxic monomeric proteins that combine at the surface of host cells to form toxic noncovalent complexes (see Fig. 1A). Two of these proteins are enzymes that modify cytosolic substrates.
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