Structural modifications of interleukin-2 at positions 47 and 65

Area of Science:

• * Protein Biochemistry
• * Molecular Biology
• * Immunology

Background:

• * Interleukin-2 (IL-2) is a critical cytokine for T-cell growth and proliferation.
• * Proline residues can introduce kinks in alpha-helices, potentially influencing protein structure and function.
• * Previous research mistakenly identified Pro-47 as a key helix-disrupting residue.

Purpose of the Study:

• * To investigate the impact of altering proline residues in IL-2 alpha-helices on protein bioactivity and conformation.
• * To understand the role of Pro-47 and Pro-65 in IL-2 structure-function relationships.

Main Methods:

• * Site-directed mutagenesis was used to create mutants at Pro-47 and Pro-65 positions.
• * Various amino acid substitutions (acidic, neutral, helix-stabilizing) were introduced.
• * Bioactivity and binding affinity (Kd) to high-affinity receptors were measured for the mutants.

Main Results:

• * Mutants Asn-47 and Asp-47 showed significantly reduced bioactivity (50- and 700-fold decreases) and increased Kd to high-affinity receptors (180- and 90-fold increases).
• * These mutations resulted in partial agonists, indicating altered protein conformation and disrupted hydrophobic packing.
• * Gly-47, Gly-65, and Ala-65 mutations had less pronounced effects on bioactivity and binding affinities.

Conclusions:

• * Specific proline substitutions, particularly at position 47, critically impact IL-2 bioactivity and receptor binding by altering protein conformation.
• * Asn-47 and Asp-47 substitutions disrupt the protein's hydrophobic core, leading to partial agonism.
• * Pro-65 appears less critical for IL-2 function, suggesting conservative mutations are tolerated at this site.