Related Experiment Videos
Structure function analysis of interleukin 7: requirement for an aromatic ring at position 143 of helix D
Johanna C vanderSpek1, John A Sutherland, Brian M Gill
1Evans Department of Clinical Research and the Department of Medicine, Boston Medical Center, Boston, Massachusetts, 02118, USA. johanna.vanderspek@bmc.org
Abstract:
The residues located at the carboxyl terminus of helix D in interleukin-7 (IL-7) have previously been targeted as important for recruitment and binding to the gamma chain component of the IL-7 receptor (IL-7R). In this study, Trp 143 of helix D was mutated to His, Phe, Tyr and Pro and these mutants, along with a W143A mutant previously described, were studied to determine the effects on activation of DNA synthesis and binding affinity to IL-7R positive 2E8 cells. The W143F and W143Y mutants were similar to wild type IL-7 in their binding properties and retained 85% and 74% of their activating properties, respectively. In contrast, the W143H mutant possessed a lower binding affinity and a corresponding decrease in activation, the W143A mutant possessed an over 100-fold decreased binding affinity and some residual activation activity and the W143P mutant possessed a greatly decreased binding affinity and did not activate. These results strongly suggest an aromatic residue is required at position 143 for IL-7R binding and subsequent signal transduction.
Insights
Aromatic residues at position 143 of interleukin-7 (IL-7) are crucial for binding to the IL-7 receptor (IL-7R) and activating DNA synthesis. Mutations to non-aromatic residues impair IL-7 receptor binding and signaling.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Interleukin-7 (IL-7) is vital for lymphocyte development.
- The carboxyl terminus of IL-7 helix D is implicated in IL-7 receptor (IL-7R) gamma chain binding.
- Specific residues at the C-terminus of helix D are critical for IL-7 function.
Purpose of the Study:
- To investigate the role of Tryptophan 143 (Trp 143) in IL-7 helix D.
- To determine the impact of Trp 143 mutations on IL-7R binding affinity and activation of DNA synthesis.
- To elucidate the structural requirements for IL-7 signaling.
Main Methods:
- Site-directed mutagenesis of IL-7 at position 143 (Trp to His, Phe, Tyr, Pro, Ala).
- Assessment of binding affinity to IL-7R-positive 2E8 cells.
- Measurement of DNA synthesis activation by IL-7 mutants.
Main Results:
- W143F and W143Y mutants showed near wild-type binding and retained significant activation (85% and 74%).
- W143H mutant exhibited reduced binding and activation.
- W143A mutant had >100-fold decreased binding affinity but residual activation.
- W143P mutant displayed greatly reduced binding and no activation.
Conclusions:
- An aromatic residue at position 143 is essential for effective IL-7 receptor binding.
- Aromaticity at position 143 is required for IL-7-mediated signal transduction and DNA synthesis activation.
- Specific mutations impact IL-7 binding and signaling differently, highlighting the importance of residue type.