Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin

Insight into the ganglioside binding mechanism

Nipawan Nuemket, Yoshikazu Tanaka, Kentaro Tsukamoto, Takao Tsuji, Keiji Nakamura, Shunji Kozaki, Min Yao, Isao Tanaka

Research output: Contribution to journalArticle

16 Citations (Scopus)

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 3.0 Å. 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.

Original languageEnglish
Pages (from-to)433-439
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume411
Issue number2
DOIs
Publication statusPublished - 29-07-2011

Fingerprint

Dilatation and Curettage
Gangliosides
Neurotoxins
Clostridium botulinum type D
Binding Sites
Botulism
Clostridium
Mutagenesis
Poisons
Site-Directed Mutagenesis
Protein Binding
Alanine
Disease Outbreaks
Carrier concentration
Japan
Crystal structure
Electrons
Proteins

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Nuemket, Nipawan ; Tanaka, Yoshikazu ; Tsukamoto, Kentaro ; Tsuji, Takao ; Nakamura, Keiji ; Kozaki, Shunji ; Yao, Min ; Tanaka, Isao. / Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin : Insight into the ganglioside binding mechanism. In: Biochemical and Biophysical Research Communications. 2011 ; Vol. 411, No. 2. pp. 433-439.
@article{a189d03be67e44929e8c823b35aee47f,
title = "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 3.0 {\AA}. 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.",
author = "Nipawan Nuemket and Yoshikazu Tanaka and Kentaro Tsukamoto and Takao Tsuji and Keiji Nakamura and Shunji Kozaki and Min Yao and Isao Tanaka",
year = "2011",
month = "7",
day = "29",
doi = "10.1016/j.bbrc.2011.06.173",
language = "English",
volume = "411",
pages = "433--439",
journal = "Biochemical and Biophysical Research Communications",
issn = "0006-291X",
publisher = "Academic Press Inc.",
number = "2",

}

Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin : Insight into the ganglioside binding mechanism. / Nuemket, Nipawan; Tanaka, Yoshikazu; Tsukamoto, Kentaro; Tsuji, Takao; Nakamura, Keiji; Kozaki, Shunji; Yao, Min; Tanaka, Isao.

In: Biochemical and Biophysical Research Communications, Vol. 411, No. 2, 29.07.2011, p. 433-439.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin

T2 - Insight into the ganglioside binding mechanism

AU - Nuemket, Nipawan

AU - Tanaka, Yoshikazu

AU - Tsukamoto, Kentaro

AU - Tsuji, Takao

AU - Nakamura, Keiji

AU - Kozaki, Shunji

AU - Yao, Min

AU - Tanaka, Isao

PY - 2011/7/29

Y1 - 2011/7/29

N2 - 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 3.0 Å. 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.

AB - 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 3.0 Å. 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.

UR - http://www.scopus.com/inward/record.url?scp=79960844485&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79960844485&partnerID=8YFLogxK

U2 - 10.1016/j.bbrc.2011.06.173

DO - 10.1016/j.bbrc.2011.06.173

M3 - Article

VL - 411

SP - 433

EP - 439

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 2

ER -