Studies on the Interaction between Subunits of the Botulinum Toxin Complex and X-Ray Crystallography

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Botulinum toxin is produced as a 650 kDa toxin complex(TC) with subunit structure that non-covalently associated with five different protein components, 150 kDa neurotoxin(NT), nontoxic non-hemagglutinin(NTNHA) and three hemagglutinin subcomponents, in culture supernatant. In this study, we have clarified molecular ration of subunit components in the TC. Type D strain 4947(D-4947) produces a large amount of TC species (650 kDa L-TC,610 kDa and 540 kDa TC). They revealed same banding patterns on SDS-PAGE, but less number of HA-33/17 complexes in the TC. Based on titration experiments with HA-33/17 complex, we have concluded that complete subunit composition of the TC is a dodecamer composed of a single NT, a single NTNHA, two HA-70,four HA-33 and four HA-17 molecules. Accordingly, we have preliminary information on the analysis of the three-dimensional structure of botulinum TC by X-ray crystallography. Of the TC species, recombination of the components was found in 610 and 540 kDa TCs depending upon storage condition such as temperature, and was thus depressed by the addition of glycerol leading supply of stable TC for the experiments. Additionally, we have established new method to separate considerable amount of the NT from the D-4947 TC, which has been technically difficult to separate. Although we have attempted to crystallize TCs (M and L-TC) that were examined using dynamic light scattering from serotype C and D strains by 130 combinations of experimental condition, it has not been yet obtain large crystals for X-ray analysis.