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Physicochemical characterization of an antagonistic human interleukin-6 dimer
J M Matthews1, A Hammacher, G J Howlett
1Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne), Victoria, Australia.
Insights
A dimeric form of interleukin-6 (IL-6D) acts as an antagonist, blocking IL-6 activity by altering receptor binding. This metastable domain-swapped dimer suggests a new mechanism for IL-6 regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Interleukin-6 (IL-6) is a key cytokine involved in immune responses and inflammation.
- Understanding the structural basis of IL-6 activity and its modulation is crucial for therapeutic development.
Purpose of the Study:
- To investigate the structure and function of a noncovalently bound dimeric form of recombinant human IL-6 (IL-6D).
- To elucidate the mechanism by which IL-6D antagonizes IL-6 activity.
Main Methods:
- STAT3 tyrosine phosphorylation assay using HepG2 cells.
- Spectroscopic analysis including fluorescence spectroscopy and circular dichroism (CD).
- Sedimentation velocity analysis and limited proteolytic susceptibility assays.
- Urea-denaturation studies monitored by far-UV CD.
Main Results:
- IL-6D functions as an antagonist for IL-6 activity without dissociating into monomers under assay conditions.
- Trp157 fluorescence is altered in IL-6D, indicating its proximity to the dimer interface.
- Both IL-6D and monomeric IL-6 (IL-6M) exhibit similar structural compactness and stability.
- IL-6D dissociates at lower urea concentrations than complete unfolding, suggesting partial unfolding precedes dimerization.
- A domain-swapped dimer model is proposed, where swapped helices explain the antagonistic activity by blocking gp130 binding.
Conclusions:
- IL-6D is a metastable domain-swapped dimer that antagonizes IL-6 by preventing binding to gp130.
- The dimeric structure, rather than monomeric IL-6, is responsible for the observed antagonistic effect.
- This finding provides insights into IL-6 receptor interactions and potential therapeutic strategies.
Abstract:
A noncovalently bound dimeric form of recombinant human IL-6 interleukin-6 (IL-6D) was shown to be an antagonist for IL-6 activity, in a STAT3 tyrosine phosphorylation assay using HepG2 cells, under conditions where it does not dissociate into monomeric IL-6 (IL-6M). The fluorescence from Trp157, the single tryptophan residue in the primary sequence of IL-6, is altered in IL-6D, where the wavelength maximum is blue-shifted by 3 nm and the emission intensity is reduced by 30%. These data suggest that Trp157 is close to, but not buried by, the dimer interface. Both IL-6D and IL-6M are compact molecules, as determined by sedimentation velocity analysis, and contain essentially identical levels of secondary and tertiary structure, as determined by far- and near-UV CD, respectively. IL-6D and IL-6M show the same susceptibility to limited proteolytic attack, and exhibit identical far-UV CD-monitored urea-denaturation profiles with the midpoint of denaturation occurring at 6.0 +/- 0.1 M urea. However, IL-6D was found to dissociate prior to the complete unfolding of the protein, with a midpoint of dissociation of 3 M urea, suggesting that dissociation and dimerization occur when the protein is in a partially unfolded state. Based on these results, we suggest that IL-6D is a metastable domain-swapped dimer, comprising two monomeric units where identical helices from each protein chain are swapped through the loop regions at the "top" of the protein (i.e., the region of the protein most distal from the N- and C-termini). Such an arrangement would account for the antagonistic activity of IL-6D. In this model, receptor binding site I, which comprises residues in the A/B loop and the C-terminus of the protein, is free to bind the IL-6 receptor. However, site III, which includes Trp157 and residues in the C/D loop and N-terminal end of helix D, and perhaps site II, which comprises residues in the A and C helices, are no longer able to bind the signal transducing component of the IL-6 receptor complex, gp130.
