PHYSICOCHEMICAL CHARACTERIZATION OF AN ANTAGONISTIC HUMAN INTERLEUKIN-6 DIMER

A noncovalently bound dimeric form of recombinant human IL-6 interleuk in-6 (IL-6(D)) was shown to be an antagonist for IL-6 activity, in a S TAT3 tyrosine phosphorylation assay using HepG2 cells, under condition s where it does not dissociate into monomeric IL-6 (IL-6(M)). The fluo rescence from Trp157, the single tryptophan residue in the primary seq uence of IL-6, is altered in IL-6(D), where the wavelength maximum is blue-shifted by 3 nm and the emission intensity is reduced by 30%. The se data suggest that Trp157 is close to, but not buried by, the dimer interface. Both IL-6(D) and IL-6(M) are compact molecules, as determin ed by sedimentation velocity analysis, and contain essentially identic al levels of secondary and tertiary structure, as determined by far- a nd near-UV CD, respectively. IL-6(D) and IL-6(M) show the same suscept ibility to limited proteolytic attack, and exhibit identical far-UV CD -monitored urea-denaturation profiles with the midpoint of denaturatio n occurring at 6.0 +/- 0.1 M urea. However, IL-6(D) was found to disso ciate prior to the complete unfolding of the protein, with a midpoint of dissociation of 3 M urea, suggesting that dissociation and dimeriza tion occur when the protein is in a partially unfolded state. Based on these results, we suggest that IL-6(D) is a metastable domain-swapped dimer, comprising two monomeric units where identical helices from ea ch protein chain are swapped through the loop regions at the ''top'' o f the protein (i.e., the region of the protein most distal from the N- and C-termini). Such an arrangement would account for the antagonisti c activity of IL-6(D). In this model, receptor binding site I, which c omprises residues in the A/B loop and the C-terminus of the protein, i s free to bind the IL-6 receptor. However, site III, which includes Tr p157 and residues in the C/D loop and N-terminal end of helix D, and p erhaps site II, which comprises residues in the A and C helices, are n o longer able to bind the signal transducing component of the IL-6 rec eptor complex, gp130.