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Role of the C-terminus in the activity, conformation, and stability of interleukin-6
L D Ward1, A Hammacher, J G Zhang
1Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research, Parkville, Victoria, Australia.
Abstract:
Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183-187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was < 0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6. Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis. These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding. A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.
Insights
Murine interleukin-6 (mIL-6) variants showed that C-terminal amino acids are crucial for structure and function. Truncation or mutation of this region in mIL-6 significantly impacts its biological activity and stability.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Interleukin-6 (IL-6) is a pleiotropic cytokine involved in immune responses, inflammation, and hematopoiesis.
- The structure-function relationship of IL-6, particularly the role of its C-terminus, is critical for understanding its biological activity.
- Investigating IL-6 variants can elucidate mechanisms of receptor binding and signal transduction.
Purpose of the Study:
- To investigate the role of the C-terminal residues of murine interleukin-6 (mIL-6) in receptor binding, biological activity, and structural integrity.
- To construct and characterize mIL-6 variants with modifications at the C-terminus.
- To determine if the C-terminal seven amino acids of human IL-6 define species specificity for receptor binding.
Main Methods:
- Polymerase chain reaction (PCR) was used to construct mIL-6 variants.
- Cell-based assays (e.g., growth stimulatory activity on hybridoma cell line 7TD1) were employed.
- Surface plasmon resonance (SPR) and biosensor analysis were used to assess receptor binding affinity.
- Nuclear magnetic resonance (NMR) spectroscopy and equilibrium unfolding experiments were utilized for structural and stability analysis.
Main Results:
- A mIL-6 variant lacking the C-terminal residues (pMC5) exhibited significantly reduced (<0.05%) growth stimulatory activity and negligible receptor binding.
- A variant with C-terminal residues substituted by human IL-6 counterparts (pMC5H) retained equipotent activity and comparable receptor binding to wild-type mIL-6.
- Both variants failed to interact with the soluble human IL-6 receptor, suggesting the C-terminus alone does not confer species specificity.
- Structural analyses indicated global fold similarity but revealed minor alterations in pMC5, suggesting long-range effects of C-terminal modifications.
- pMC5 was less stable than mIL-6, while pMC5H was more stable, highlighting the structural importance of the C-terminus.
Conclusions:
- The C-terminal amino acids of mIL-6 are essential for its biological activity and receptor binding.
- Modifications at the mIL-6 C-terminus can lead to significant alterations in protein stability and potentially influence distant regions of the molecule.
- The C-terminal seven amino acids of human IL-6 do not solely determine species-specific receptor interactions.