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Cross-Linked Forms of the Isolated N-Terminal Domain of the Lethal Factor Are Potent Inhibitors of Anthrax Toxin

Stephen J. Juris,^1,, Roman A. Melnyk,^1, Robert E. Bolcome III,² Joanne Chan,² and R. John Collier^1*

Harvard Medical School, Department of Microbiology and Molecular Genetics, 200 Longwood Avenue, Boston, Massachusetts 02115,¹ Harvard Medical School, Children's Hospital, Vascular Biology Program, 300 Longwood Avenue, Boston, Massachusetts 02115²

Received 5 April 2007/ Returned for modification 21 May 2007/ Accepted 3 July 2007

The proteins that comprise anthrax toxin self-assemble at the mammalian cell surface into a series of toxic complexes, each containing a heptameric form of protective antigen (PA) plus up to a total of three molecules of the enzymatic moieties of the toxin (lethal factor [LF] and edema factor [EF]). These complexes are trafficked to the endosome, where the PA heptamer forms a pore in the membrane under the influence of low pH, and bound LF and EF unfold and translocate through the pore to the cytosol. To explore the hypothesis that the PA pore can translocate multiple, cross-linked polypeptides simultaneously, we cross-linked LF_N, the N-terminal domain of LF, via an introduced cysteine at its N or C terminus and characterized the products. Both dimers and trimers of LF_N retained the ability to bind to PA pores and block ion conductance, but they were unable to translocate across the membrane, even at high voltages or with a transmembrane pH gradient. The multimers were remarkably potent inhibitors of toxin action in mammalian cells (20- to 50-fold more potent than monomeric LF_N) and in a zebrafish model system. These findings show that the PA pore cannot translocate multimeric, cross-linked polypeptides and demonstrate a new approach to generating potent inhibitors of anthrax toxin.

Published ahead of print on 16 July 2007.

Editor: V. J. DiRita

These authors contributed equally to this work.

Present address: Central Michigan University, 268 Dow Science, Mount Pleasant, MI 48859.

This article has been cited by other articles:

• Bolcome, R. E. III, Sullivan, S. E., Zeller, R., Barker, A. P., Collier, R. J., Chan, J. (2008). Anthrax lethal toxin induces cell death-independent permeability in zebrafish vasculature. Proc. Natl. Acad. Sci. USA 105: 2439-2444 [Abstract] [Full Text]