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BIOLOGICAL SCIENCES / NEUROSCIENCE
Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept
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*Institut für Biochemie, OE 4310, and Institut für Toxikologie, OE 5340, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany; Merz Pharmaceuticals GmbH, Eckenheimer Landstrasse 100, 60318 Frankfurt am Main, Germany; ¶Kekule-Institut für Organische Chemie und Biochemie der Universität Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany; ||Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892; and **Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom
Communicated by Axel T. Brunger, Stanford University, Stanford, CA, November 1, 2006 (received for review August 24, 2006)
Botulinum neurotoxins (BoNTs) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery within motoneurons. Complex gangliosides initially bind into a pocket that is conserved among the seven BoNTs and tetanus neurotoxin. Productive neurotoxin uptake also requires protein receptors. The interaction site of the protein receptor within the neurotoxin is currently unknown. We report the identification and characterization of the protein receptor binding site of BoNT/B and BoNT/G. Their protein receptors, synaptotagmins I and II, bind to a pocket at the tip of their HCC (C-terminal domain of the C-terminal fragment of the heavy chain) that corresponds to the unique second carbohydrate binding site of tetanus neurotoxin, the sialic acid binding site. Substitution of amino acids in this region impaired binding to synaptotagmins and drastically decreased toxicity at mouse phrenic nerve preparations; CD-spectroscopic analyses evidenced that the secondary structure of the mutated neurotoxins was unaltered. Deactivation of the synaptotagmin binding site by single mutations led to virtually inactive BoNT/B and BoNT/G when assayed at phrenic nerve preparations of complex-ganglioside-deficient mice. Analogously, a BoNT B mutant with deactivated ganglioside and synaptotagmin binding sites lacked appreciable activity at wild-type mouse phrenic nerve preparations. Thus, these data exclude relevant contributions of any cell surface molecule other than one ganglioside and one protein receptor to the entry process of BoNTs, which substantiates the double-receptor concept. The molecular characterization of the synaptotagmin binding site provides the basis for designing a novel class of potent binding inhibitors.
synaptotagmin | tetanus
Author contributions: A.R. and T.B. designed research; A.R., T.E., T.W., T.K., A.G., S.M., and T.B. performed research; K.S., R.L.P., and H.B. contributed new reagents/analytic tools; A.R., T.E., T.W., A.G., K.R.A., H.B., and T.B. analyzed data; and A.R. and T.B. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0609713104/DC1.
To whom correspondence may be addressed. E-mail: rummel.andreas{at}mh-hannover.de or binz.thomas{at}mh-hannover.de
© 2007 by The National Academy of Sciences of the USA
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  M. Dong, W. H. Tepp, H. Liu, E. A. Johnson, and E. R. Chapman Mechanism of botulinum neurotoxin B and G entry into hippocampal neurons J. Cell Biol., December 31, 2007; 179(7): 1511 - 1522. [Abstract] [Full Text] [PDF]
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