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J. Biol. Chem., Vol. 283, Issue 7, 3904-3914, February 15, 2008

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Clostridium botulinum C2 Toxin

IDENTIFICATION OF THE BINDING SITE FOR CHLOROQUINE AND RELATED COMPOUNDS AND INFLUENCE OF THE BINDING SITE ON PROPERTIES OF THE C2II CHANNEL^*

Tobias Neumeyer, Bettina Schiffler, Elke Maier, Alexander E. Lang, Klaus Aktories, and Roland Benz¹

From the Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany and Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany

Clostridium botulinum C2 toxin belongs to the family of binary AB type toxins that are structurally organized into distinct enzyme (A, C2I) and binding (B, C2II) components. The proteolytically activated 60-kDa C2II binding component is essential for C2I transport into target cells. It oligomerizes into heptamers and forms channels in lipid bilayer membranes. The C2II channel is cation-selective and can be blocked by chloroquine and related compounds. Residues 303–330 of C2II contain a conserved pattern of alternating hydrophobic and hydrophilic residues, which has been implicated in the formation of two amphipathic β-strands involved in membrane insertion and channel formation. In the present study, C2II mutants created by substitution of different negatively charged amino acids by alanine-scanning mutagenesis were analyzed in artificial lipid bilayer membranes. The results suggested that most of the C2II mutants formed SDS-resistant oligomers (heptamers) similar to wild type. The mutated negatively charged amino acids did not influence channel properties with the exception of Glu³⁹⁹ and Asp⁴²⁶, which are probably localized in the vestibule near the channel entrance. These mutants show a dramatic decrease in their affinity for binding of chloroquine and its analogues. Similarly, F428A, which represents the -clamp in anthrax protective antigen, was mutated in C2II in several other amino acids. The C2II mutants F428A, F428D, F428Y, and F428W not only showed altered chloroquine binding but also had drastically changed single channel properties. The results suggest that amino acids Glu³⁹⁹, Asp⁴²⁶, and Phe⁴²⁸ have a major impact on the function of C2II as a binding protein for C2I delivery into target cells.

Received for publication, November 30, 2007

^* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 487, project A5, and SFB 388, project A13, and by the Fonds der Chemischen Industrie. 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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