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J. Biol. Chem., Vol. 281, Issue 3, 1630-1635, January 20, 2006

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Structural Determinants for the Binding of Anthrax Lethal Factor to Oligomeric Protective Antigen^*

Roman A. Melnyk1, Krissi M. Hewitt1, D. Borden Lacy, Henry C. Lin, Chris R. Gessner, Sheng Li, Virgil L. Woods, Jr.2, and R. John Collier3

From the Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115 and the Department of Medicine and Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, California 92093

Anthrax lethal toxin assembles at the surface of mammalian cells when the lethal factor (LF) binds via its amino-terminal domain, LF_N, to oligomeric forms of activated protective antigen (PA). LF·PA complexes are then trafficked to acidified endosomes, where PA forms heptameric pores in the bounding membrane and LF translocates through these pores to the cytosol. We used enhanced peptide amide hydrogen/deuterium exchange mass spectrometry and directed mutagenesis to define the surface on LF_N that interacts with PA. A continuous surface encompassing one face of LF_N became protected from deuterium exchange when LF_N was bound to a PA dimer. Directed mutational analysis demonstrated that residues within this surface on LF_N interact with Lys-197 on two PA subunits simultaneously, thereby showing that LF_N spans the PA subunit:subunit interface and explaining why heptameric PA binds a maximum of three LF_N molecules. Our results elucidate the structural basis for anthrax lethal toxin assembly and may be useful in developing drugs to block toxin action.

Received for publication, October 13, 2005 , and in revised form, November 9, 2005.

^* This work was supported by NCI, National Institutes of Health Grants CA105347 and CA099835 (to V. L. W.) and AI22021 (to R. J. C.). 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.

¹ Both authors contributed equally to this work.

² To whom correspondence may be addressed. E-mail: vwoods{at}ucsd.edu. ³ To whom correspondence may be addressed. E-mail: jcollier{at}hms.harvard.edu.

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