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J. Biol. Chem., Vol. 282, Issue 2, 1059-1065, January 12, 2007

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Insertion of Anthrax Protective Antigen into Liposomal Membranes

EFFECTS OF A RECEPTOR^*

From the Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

Protective antigen (PA), the receptor-binding component of anthrax toxin, heptamerizes and inserts into the endosomal membrane at acidic pH, forming a pore that mediates translocation of the enzymic components of the toxin to the cytosol. When the heptameric pre-insertion form of PA (the prepore) is acidified in solution, it rapidly loses the ability to insert into membranes. To maximize insertion into model membranes, we examined two ways to bind the protein to large unilamellar vesicles (LUV). One involved attaching a His tag to the von Willebrand factor A domain of one of the PA receptors, ANTXR2, and using this protein as a bridge to bind PA to LUV containing a nickel-chelating lipid. The other involved using a His tag fused to the C terminus of PA to bind the protein directly to LUV containing the same lipid. Both ways enhanced pore formation at pH 5.0 strongly and about equally, as measured by the release of K⁺. Controls showed that pore formation in this system faithfully reproduced that in vivo. We also showed that binding unmodified ANTXR2 von Willebrand factor A to the prepore in solution enhanced its pore forming activity by slowing its inactivation at acidic pH. These findings indicate that an important role of PA receptors is to promote partitioning of PA into the bilayer by maintaining the prepore close to the target membrane and presumably in the optimal orientation as it undergoes the acidic pH-dependent conformational transition to the pore.

Received for publication, October 20, 2006 , and in revised form, November 14, 2006.

^* This work was supported by National Institutes of Health Grants RO1 AI 22021, AI 48489, and PO1 AI 56013. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ Holds equity in PharmAthene, Inc. and is a consultant for CombinatoRx, Inc. To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1930; Fax: 617-432-0115; E-mail: jcollier{at}hms.harvard.edu.

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