Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: insight into the ganglioside binding mechanism.

Abstract

Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0Å. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.

• Authors: Nipawan Nuemket, Yoshikazu Tanaka, Kentaro Tsukamoto, Takao Tsuji, Keiji Nakamura, Shunji Kozaki, Min Yao, Isao Tanaka
• Journal: Biochemical and biophysical research communications (Biochem Biophys Res Commun) Vol. 411 Issue 2 Pg. 433-9 (Jul 29 2011) ISSN: 1090-2104 [Electronic] United States
• PMID: 21749855 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2011 Elsevier Inc. All rights reserved.

Chemical References

• 3'-sialyllactose
• Gangliosides
• Oligosaccharides
• botulinum toxin type D
• Botulinum Toxins

Topics

• Animals
• Botulinum Toxins (chemistry, genetics)
• Cell Line, Tumor
• Clostridium botulinum type D
• Crystallography, X-Ray
• DNA Mutational Analysis
• Gangliosides (chemistry)
• Mice
• Mosaicism
• Oligosaccharides (chemistry)
• Protein Structure, Tertiary (genetics)