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Structural requirements for agonist activity of a murine interferon-gamma peptide
B E Szente1, I J Weiner, M J Jablonsky
1Department of Microbiology and Cell Science, University of Florida, Gainesville 32611, USA.
Insights
Murine interferon-gamma (MuIFN-gamma) C-terminus peptide requires helical structure and a polycationic tail for binding to its receptor. This binding is essential for MuIFN-gamma
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Murine interferon-gamma (MuIFN-gamma) interacts with its receptor via N-terminal and C-terminal domains.
- The C-terminus is hypothesized to bind the receptor's cytoplasmic domain post-endocytosis.
- MuIFN-gamma (95-133) peptide exhibits full agonist activity and is internalized via pinocytosis.
Purpose of the Study:
- To investigate the structural elements of MuIFN-gamma (95-133) essential for its agonist activity.
- To determine the role of helical structure and polycationic sequences in MuIFN-gamma receptor binding and biological function.
Main Methods:
- Peptide truncation and proline substitutions were used to disrupt the alpha-helical structure of MuIFN-gamma (95-133).
- The polycationic sequence (RKRKR) in the peptide tail was removed.
- Binding affinity, antiviral activity, and MHC class II molecule induction were assessed.
Main Results:
- Disruption of the alpha-helical structure led to reduced binding and/or loss of antiviral activity and MHC class II induction.
- Removal of the polycationic RKRKR sequence abolished agonist activity.
- These findings pinpoint the C-terminus as the functional site for MuIFN-gamma's biological activity.
Conclusions:
- The helical structure and polycationic tail of the MuIFN-gamma C-terminus are critical for receptor binding.
- These structural elements are necessary for inducing biological activity, including antiviral effects and MHC class II molecule expression.
- The study successfully isolated the functional site of MuIFN-gamma to its C-terminal region.
Abstract:
We have demonstrated previously that murine interferon-gamma (MuIFN-gamma) binds to the extracellular domain of the receptor alpha chain through its N-terminus and subsequently to the cytoplasmic domain of the receptor via its C-terminus. Binding of the C-terminus to the cytoplasmic domain of the receptor is thought to occur following endocytosis of the IFN-gamma-receptor complex. In fact, the MuIFN-gamma C-terminus peptide, MuIFN-gamma (95-133), has full agonist activity on macrophages where it is internalized through pinocytosis. Here we examine the structural elements required for the agonist activity of MuIFN-gamma (95-133). Disruption of the alpha helical structure of the peptide by proline substitutions or truncation of the helix resulted in significant loss of binding or loss of antiviral activity or both and induction of MHC class II molecules. Further, removal of the polycationic sequence RKRKR in the tail beyond the helical structure also resulted in loss of agonist activity. Thus, we have isolated the functional site on MuIFN-gamma to the C-terminus and have shown that its helical structure and polycationic tail are required for binding to the cytoplasmic domain of the receptor and induction of biologic activity.
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