Binding stoichiometry and kinetics of the interaction of a human anthrax toxin receptor, CMG2, with protective antigen.
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.
Wigelsworth
Darran J
DJ
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Krantz
Bryan A
BA
Christensen
Kenneth A
KA
Lacy
D Borden
DB
Juris
Stephen J
SJ
Collier
R John
RJ
D000942:Antigens, Bacterial / Q000737:chemistry* / Q000378:metabolism; D001408:Bacillus anthracis / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018000:Receptors, Peptide / Q000737:chemistry* / Q000378:metabolism
D000942:Antigens, Bacterial; D018000:Receptors, Peptide; C070309:anthrax toxin receptors