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J. Biol. Chem., Vol. 281, Issue 43, 32335-32343, October 27, 2006

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Anthrax Edema Factor, Voltage-dependent Binding to the Protective Antigen Ion Channel and Comparison to LF Binding^*

Tobias Neumeyer, Fiorella Tonello, Federica Dal Molin^¶, Bettina Schiffler, and Roland Benz¹

From the Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany, Istituto di Neuroscienze del Consiglio Nazionale delle Ricerche, and ^¶Dipartimento di Scienze Biomediche, Università degli Studi di Padova, I-35121 Padova, Italy

Anthrax toxin complex consists of three different molecules, the binding component protective antigen (PA, 83 kDa), and the enzymatic components lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). The 63-kDa N-terminal part of PA, PA₆₃, forms a heptameric channel that inserts at low pH in endosomal membranes and that is necessary to translocate EF and LF in the cytosol of the target cells. EF is an intracellular active enzyme, which is a calmodulin-dependent adenylate cyclase (89 kDa) that causes a dramatic increase of intracellular cAMP level. Here, the binding of full-length EF on heptameric PA₆₃ channels was studied in experiments with artificial lipid bilayer membranes. Full-length EF blocks the PA₆₃ channels in a dose, temperature, voltage, and ionic strength-dependent way with half-saturation constants in the nanomolar concentration range. EF only blocked the PA₆₃ channels when PA₆₃ and EF were added to the same side of the membrane, the cis side. Decreasing ionic strength and increasing transmembrane voltage at the cis side of the membranes resulted in a strong decrease of the half-saturation constant for EF binding. This result suggests that ion-ion interactions are involved in EF binding to the PA heptamer. Increasing temperature resulted in increasing half-saturation constants for EF binding to the PA₆₃ channels. The binding characteristics of EF to the PA₆₃ channels are compared with those of LF binding. The comparison exhibits similarities but also remarkable differences between the bindings of both toxins to the PA₆₃ channel.

Received for publication, July 10, 2006 , and in revised form, August 23, 2006.

^* This work was supported by the Deutsche Forschungsgemeinschaft (SFB 487, project A5), by the Fonds der Chemischen Industrie, by the Anthrax-Euronet Coordination Action (FP6, contract no. 503616) and by the Consorzio Interuniversitario Biotecnologie (CIB). 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 To whom correspondence should be addressed: Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany. Tel.: 49-931-8884501; Fax: 49-931-8884509; E-mail: roland.benz{at}mail.uni-wuerzburg.de.

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