A Protective Antigen Mutation Increases the pH Threshold of Anthrax Toxin Receptor 2-Mediated Pore Formation

Abstract

Anthrax toxin protective antigen (PA) binds cellular receptors and self-assembles into oligomeric prepores. A prepore converts to a protein translocating pore after it has been transported to an endosome where the low pH triggers formation of a membrane-spanning β-barrel channel. Formation of this channel occurs after some PA–receptor contacts are broken to allow pore formation, while others are retained to preserve receptor association. The interaction between PA and anthrax toxin receptor 1 (ANTXR1) is weaker than its interaction with ANTXR2 such that the pH threshold of ANTXR1-mediated pore formation is higher by 1 pH unit. Here we examine receptor-specific differences in toxin binding and pore formation by mutating PA residue G342 that selectively abuts ANTXR2. Mutation of G342 to valine, leucine, isoleucine, or tryptophan increased the amount of PA bound to ANTXR1-expressing cells and decreased the amount of PA bound to ANTXR2-expressing cells. The more conservative G342A mutation did not affect the level of binding to ANTXR2, but ANTXR2-bound PA-G342A prepores exhibited a pH threshold higher than that of wild-type prepores. Mixtures of wild-type PA and PA-G342A were functional in toxicity assays, and the pH threshold of ANTXR2-mediated pore formation was dictated by the relative amounts of the two proteins in the hetero-oligomers. These results suggest that PA subunits within an oligomer do not have to be triggered simultaneously for a productive membrane insertion event to occur.

Roles of Anthrax Toxin Receptor 2 in Anthrax Toxin Membrane Insertion and Pore Formation

“…Moreover, the residue G342 of PA was found to be involved in receptor-specific binding and pore formation. Replacement of G342 with a hydrophobic residue, such as valine, leucine, isoleucine or tryptophan, increased the binding of PA to ANTXR1-expressing cells and decreased the binding of PA to ANTXR2-expressing cells [ 43 ].…”

The Disulfide Bond Cys255-Cys279 in the Immunoglobulin-Like Domain of Anthrax Toxin Receptor 2 Is Required for Membrane Insertion of Anthrax Protective Antigen Pore

“…Most recently, a study has shown that binding of the VWA domain largely increases the stability of PA and the effect reaches up to 70 Å from the receptor binding interface [ 20 ]. While the role of VWA domain in receptor-PA interaction and in PA pore formation has been well studied [ 21 – 25 ], little is known about the structure and function of the stalk region (219–318). Recently, we have found that the stalk region is an immunoglobulin-like (Ig) domain and the disulfide bonds in this domain are essential for functioning of the PA pore [ 26 ].…”