Identification of a binding site for ganglioside on the receptor binding domain of tetanus toxin.
The carboxyl-terminal region of the tetanus toxin heavy chain (H(C) fragment) binds to di- and trisialylgangliosides on neuronal cell membranes. To determine which amino acids in tetanus toxin are involved in ganglioside binding, homology modeling was performed using recently resolved X-ray crystallographic structures of the tetanus toxin H(C) fragment. On the basis of these analyses, two regions in tetanus toxin that are structurally homologous with the binding domains of other sialic acid and galactose-binding proteins were targeted for mutagenesis. Specific amino acids within these regions were altered using site-directed mutagenesis. The amino acid residue tryptophan 1288 was found to be critical for binding of the H(C) fragment to ganglioside GT1b. Docking of GD1b within this region of the toxin suggested that histidine 1270 and aspartate 1221 were within hydrogen bonding distance of the ganglioside. These two residues were mutagenized and found also to be important for the binding of the tetanus toxin H(C) fragment to ganglioside GT1b. In addition, the H(C) fragments mutagenized at these residues have reduced levels of binding to neurites of differentiated PC-12 cells. These studies indicate that the amino acids tryptophan 1288, histidine 1270, and aspartate 1221 are components of the GT1b binding site on the tetanus toxin H(C) fragment.
Louch
Heather A
HA
Laboratory of Bacterial Toxins, Division of Bacterial, Parasitic, and Allergenic Products, Office of Vaccine Research and Review, Center for Biologics Evaluation and Research, Federal Drug Administration, 8800 Rockville Pike, Bethesda, Maryland 20892, USA.
Buczko
Ellen S
ES
Woody
Mary A
MA
Venable
Richard M
RM
Vann
Willie F
WF
D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D017931:DNA Primers / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D005732:Gangliosides / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008081:Liposomes; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells / Q000166:cytology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013744:Tetanus Toxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D005732:Gangliosides; D008024:Ligands; D008081:Liposomes; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C; C033851:ganglioside, GD1b; D006639:Histidine