Aspartic acid 50 and tyrosine 108 are essential for receptor binding and cytotoxic activity of tumour necrosis factor beta (lymphotoxin).
Single amino acid substitutions were generated in predicted hydrophilic loop regions of the human tumour necrosis factor beta (TNF-beta) molecule, and the mutant proteins were expressed in Escherichia coli and purified. Mutants with single amino acid changes at either of two distinct loop regions, at positions aspartic acid 50 or tyrosine 108, were found to have greatly reduced receptor binding and cytotoxic activity. These two regions in TNF-beta correspond to known loop regions where mutations also result in loss of biological activity of TNF-alpha, a related cytokine which shares the same cellular receptors with TNF-beta. The two distinct loops at positions 31-34 and 84-89 in the known three-dimensional structure of TNF-alpha (equivalent to positions 46-50 and 105-110 respectively in TNF-beta), lie on opposite sides of the TNF-alpha monomer. When the TNF-alpha monomer forms a trimer, the two loops, each from a different subunit of the trimer, come together and lie in a cleft between adjacent subunits. Together, these findings suggest that a TNF receptor binds to a cleft between subunits via surface loops at amino acid residues 31-34 and 84-89 in TNF-alpha, and similarly via surface loops including amino acids aspartic acid 50 and tyrosine 108 in TNF-beta.
Goh
C R
CR
Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge Crescent.
Loh
C S
CS
Porter
A G
AG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003602:Cytotoxicity, Immunologic; D015870:Gene Expression; D006801:Humans; D008233:Lymphotoxin-alpha / Q000235:genetics*; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D008233:Lymphotoxin-alpha; D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins