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,Tohru Ohyama

doi:10.1074/jbc.m703446200

Abstract

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.

Highlights

The number of HA-331⁄7HA-17 molecules observed in all TC species consisted of one molecule less than the number predicted by our initial schematic model (21), which was derived from titration-based experiments
Our present results indicate that the L-TC species may have flexible characteristics, for instance the relative positions of the arms of the 650-kDa L-TC varied, and sometimes fewer arms were observed, probably because some HA-331⁄7HA-17 molecules could adhere to the carbon-coated membrane and be hidden beneath the TC molecule itself
The HA-331⁄7HA-17 subcompo- molecules are related by 2-fold axial symmetry, showing both nent complex, which could not be separated as a pure compo- upswept C-terminal domains with putative carbohydrate-bindnent during the isolation procedure, was isolated from L-TC ing sites separated by a small angle (Fig. 3A)

Summary

Introduction

The number of HA-331⁄7HA-17 molecules observed in all TC species consisted of one molecule less than the number predicted by our initial schematic model (21), which was derived from titration-based experiments. The HA-70 molecules seemed to acquire a dramatically more rigid folded configuration upon binding of HA-331⁄7HA-17 to form complexes.

Results

Conclusion