Site-directed mutational analysis of human tumor necrosis factor-alpha receptor binding site and structure-functional relationship.
In order to define the receptor binding site and the structure-functional relationship of tumor necrosis factor (TNF), single amino acid substitutions were made by site-directed mutagenesis at selected residues of human tumor necrosis factor, using a phagemid mutagenesis/expression vector. The recombinant TNF mutants were compared to the wild type TNF in assays using crude bacterial lysates, for protein yield, solubility, subunit trimerization, receptor binding inhibition activity, and in vitro cytotoxic activity. All mutants which did not form cross-linkable trimer also showed little cytotoxic activity or receptor binding inhibition activity, indicating that trimer formation is obligatory for TNF-alpha activity. Most mutations of internal residues yielded no cross-linkable trimer, while most mutations of surface residues yielded cross-linkable trimer. Mutations at surface residues Leu29, Arg31, and Ala35 yielded cross-linkable trimers with good activities, except proline substitutions which may cause conformational changes in the polypeptide chain. This suggested that these residues are near the receptor binding site. Mutations at other strictly conserved internal residues such as Ser60, His78, and Tyr119 form cross-linkable trimer with little activity. These mutations may indirectly affect the receptor binding site by forming trimers with undetectable abnormalities. Mutants of surface residues Tyr87, Ser95, Ser133, and Ser147 affect receptor binding and cytotoxic activity but not trimer formation, suggesting that these residues are involved directly in receptor binding. The fact that residues Arg31, Ala35, Tyr87, Ser95, and Ser147, located on the opposite sides of a monomer, are clustered at the intersubunit grooves of TNF trimer supports the current notion that TNF receptor binding sites are trivalent and are located at the three intersubunit grooves. However, our finding that Ser133, which is outside the groove, can also be involved directly in receptor binding suggested that the receptor binding sites of TNF may not be confined to the intersubunit grooves, but extended to include additional surface residues.
Zhang
X M
XM
Department of Molecular Genetics, SmithKline and French Laboratories, King of Prussia, Pennsylvania 19406.
Weber
I
I
Chen
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002470:Cell Survival / Q000187:drug effects; D006801:Humans; D007739:L Cells; D046911:Macromolecular Substances; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology
D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha