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J. Biol. Chem., Vol. 282, Issue 34, 24777-24783, August 24, 2007

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A Novel Subunit Structure of Clostridium botulinum Serotype D Toxin Complex with Three Extended Arms^*

Kimiko Hasegawa, Toshihiro Watanabe, Tomonori Suzuki, Akihito Yamano, Tetsuo Oikawa^¶, Yasuhiko Sato^¶, Hirokazu Kouguchi^||, Tohru Yoneyama, Koichi Niwa, Toshihiko Ikeda^**, and Tohru Ohyama¹

From the Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan, PharmAxess, Inc., 3-9-12 Matsubara-Cho, Akishima, Tokyo 196-8666, Japan, ^¶JEOL Ltd., 3-1-2 Musashino, Akishima, Tokyo 196-8558, Japan, ^||Hokkaido Institute of Public Health, N19, W12, Sapporo 060-0819, Japan, and ^**Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa, Tokyo 140-8710, Japan

The botulinum neurotoxins (BoNTs) are the most potent toxins known in nature, causing the lethal disease known as botulism in humans and animals. The BoNTs act by inhibiting neurotransmitter release from cholinergic synapses. Clostridium botulinum strains produce large BoNTs toxin complexes, which include auxiliary non-toxic proteins that appear not only to protect BoNTs from the hostile environment of the digestive tract but also to assist BoNT translocation across the intestinal mucosal layer. In this study, we visualize for the first time a series of botulinum serotype D toxin complexes using negative stain transmission electron microscopy (TEM). The complexes consist of the 150-kDa BoNT, 130-kDa non-toxic non-hemagglutinin (NTNHA), and three kinds of hemagglutinin (HA) subcomponents: 70-kDa HA-70, 33-kDa HA-33, and 17-kDa HA-17. These components assemble sequentially to form the complex. A novel TEM image of the mature L-TC revealed an ellipsoidal-shaped structure with "three arms" attached. The "body" section was comprised of a single BoNT, a single NTNHA and three HA-70 molecules. The arm section consisted of a complex of HA-33 and HA-17 molecules. We determined the x-ray crystal structure of the complex formed by two HA-33 plus one HA-17. On the basis of the TEM image and biochemical results, we propose a novel 14-mer subunit model for the botulinum toxin complex. This unique model suggests how non-toxic components make up a "delivery vehicle" for BoNT.

Received for publication, April 25, 2007

^* This work was supported by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research 17300161. Part of this work was supported by a Sasagawa Scientific Research Grant from The Japan Science Society. 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.

The atomic coordinates and structure factors (code 2E4M) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¹ To whom correspondence should be addressed: Dept. of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan. Tel.: 81-152-48-3838; Fax: 81-152-48-2940; E-mail: t-oyama{at}bioindustry.nodai.ac.jp.

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