Identification of the interaction region between hemagglutinin components of the botulinum toxin complex.
The large toxin complex (L-TC) produced by Clostridium botulinum is formed from the M-TC (BoNT/NTNHA complex) by conjugation of M-TC with HA-33/HA-17 trimer consists of two HA-33 proteins and a single HA-17 protein. This association is mediated by HA-70, which interacts with HA-17. The current study aims to identify the regions of the HA-70 molecule that adhere to the HA-33/HA-17 complex. Products from limited proteolysis of HA-70 were resolved by SDS-PAGE and transferred onto PVDF membranes, where they were probed with HA-33/HA-17 in a far-western blot. Among the HA-70 fragments, HA-33/HA-17 bound to those containing at least the C-terminal half of the HA-70 molecule, but not those carrying the N-terminal half. Additional docking simulation analysis indicated that the HA-70 region Gln420-Tyr575 is responsible for binding to HA-17, which is consistent with the far-western blot data. The findings here reveal additional details concerning the three-dimensional structure of the functional HA sub-complex in the botulinum toxin complex.
Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan. Electronic address: suzuk-t2@cc.okayama-u.ac.jp.
Tomonori
T
Suzuki
Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
Shin-Ichiro
S
Miyashita
Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
Shintaro
S
Hayashi
Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
Keita
K
Miyata
Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
Ken
K
Inui
Department of Medicinal and Life Science, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan.
Yosuke
Y
Kondo
Department of Medicinal and Life Science, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan.
Satoru
S
Miyazaki
Department of Health and Nutrition, Faculty of Human Science, Hokkaido Bunkyo University, 5-196-1 Kogane-chuo, Eniwa 061-1449, Japan.
Tohru
T
Ohyama
Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
Koichi
K
Niwa
Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
Toshihiro
T
Watanabe
Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
Yoshimasa
Y
Sagane
D001905:Botulinum Toxins / Q000737:chemistry* / Q000378:metabolism*; D003014:Clostridium botulinum; D006388:Hemagglutinins / Q000737:chemistry* / Q000378:metabolism*; D008567:Membranes, Artificial; D062105:Molecular Docking Simulation; D011145:Polyvinyls / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D005465:Fluorocarbon Polymers
D001905:Botulinum Toxins; D006388:Hemagglutinins; D008567:Membranes, Artificial; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers
Clostridium botulinum; Far-western blot; Hemagglutinin