Characterizing the binding interactions between anthrax toxin receptor 1 and anthrax protective antigen

Bacillus anthrach is the etiological agent of the anthrax disease. A primary virulence factor encoded by B. anthracis is anthrax toxin (AT). For AT to function within the host, the cell-binding subunit protective antigen (PA) must bind to one of two proteinaceous cell-surface anthrax toxin receptors (ANTXR1 or -2). Thus, characterization of the PA/ANTXR1 interaction provides insight into a critical intoxication step and further sheds light on the development of therapeutics directed against ANTXR1 expressing tumor vasculature.;Work presented here tests the hypothesis that ANTXR1 exists as a population of active/inactive receptors on the cell surface where inside-out signaling regulates PA binding by an activation dependent mechanism through changes in the extracellular toxin-binding inserted (I) domain. Conserved ANTXR1 I domain residues were mutated to explore putative conformational regulation of activation status. An inactivating mutant of ANTXR1 (T118A) results in a locked inactive state whereby PA binding is repressed. Conversely, an activating mutation of a conserved I domain phenylalanine (F205W) increases the proportion of cell-surface ANTXR1 that binds PA without affecting affinity. Interestingly, the affinity and total amount of PA bound by isolated ANTXR1 I domain (sANTXR1) is not affected by the activating mutation (F205 W), indicating that ANTXR1 I domain is preferentially found in the active state in the absence of inside-out signaling.