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Localization of the active site of human tumour necrosis factor (hTNF) by mutational analysis
X Van Ostade1, J Tavernier, T Prangé
1Laboratory of Molecular Biology, State University of Ghent, Belgium.
Abstract:
In order to define the active site(s) of human tumour necrosis factor (hTNF), we mutagenized its gene at random and directly screened the resulting population for loss of cytotoxic activity on L929 cells. Four biologically inactive mutant proteins (Arg32----Trp, Leu36----Phe, Ser86----Phe and Ala84----Val) behaved similar to the wild-type in various physico-chemical assays. The residues were positioned on a 3D structural model and were found to cluster together at the base of the molecule at each side of the groove that separates two monomers in the trimeric structure. A very conservative mutation at one of these sites (Ala84----Val) almost completely abolished cytotoxic activity. Amino acid alterations in three other residues in close proximity to this receptor binding site were introduced: replacements at positions 29 and 146 clearly reduced cytotoxicity only when non-conservative alterations were introduced (Leu29----Ser and Glu146----Lys), suggesting an indirect influence on the active site. However, a conservative mutation at position 91 (Val----Ala) caused a significant drop (500-fold) in bioactivity which suggests that Val91 may also play a direct role in receptor recognition. Our results favor a model in which each TNF molecule has three receptor-interaction sites (between the three subunits), thus allowing signal transmission by receptor clustering.
Insights
Researchers identified key sites on human tumour necrosis factor (hTNF) essential for its cytotoxic activity. Mutations at specific residues, particularly Ala84 and Val91, significantly reduced hTNF
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Human tumour necrosis factor (hTNF) is a critical cytokine involved in inflammation and immunity.
- Understanding the active sites of hTNF is crucial for developing targeted therapies.
- Previous studies have suggested a trimeric structure for hTNF, but the precise receptor interaction sites were not fully defined.
Purpose of the Study:
- To identify and characterize the active site(s) of human tumour necrosis factor (hTNF) responsible for its cytotoxic activity.
- To elucidate the role of specific amino acid residues in hTNF's interaction with its receptor.
- To propose a model for hTNF receptor binding and signal transduction.
Main Methods:
- Random mutagenesis of the hTNF gene.
- Screening of mutant proteins for loss of cytotoxic activity on L929 cells.
- Physico-chemical characterization of mutant proteins.
- Analysis of residue positions on a 3D structural model of hTNF.
- Introduction of site-directed mutations at specific residues.
Main Results:
- Four biologically inactive mutant proteins (Arg32Trp, Leu36Phe, Ser86Phe, Ala84Val) were identified.
- Mutations at Ala84 and Val91 significantly abolished or reduced cytotoxic activity, respectively.
- Residues Ala84 and Val91 cluster at the base of the trimeric structure, near the groove separating monomers.
- Conservative mutations at positions 29 and 146 had minimal effect, suggesting indirect influence, while a conservative mutation at Val91 significantly reduced bioactivity.
Conclusions:
- The study identified specific amino acid residues (Ala84, Val91) critical for hTNF's cytotoxic activity and receptor recognition.
- Results support a model where hTNF has three receptor-interaction sites located between subunits in the trimeric structure.
- This interaction model provides insights into hTNF-mediated signal transmission through receptor clustering.