GENERATION OF INTERLEUKIN-6 RECEPTOR ANTAGONISTS BY MOLECULAR-MODELING GUIDED MUTAGENESIS OF RESIDUES IMPORTANT FOR GP130 ACTIVATION

Interleukin-6 (IL-6) drives the sequential assembly of a receptor comp lex formed by the IL-6 receptor (IL-6Ralpha) and the signal transducin g subunit, gp130. A model of human IL-6 (hlL-6) was constructed by hom ology using the structure of bovine granulocyte colony stimulating fac tor. The modeled cytokine was predicted to interact sequentially with the cytokine binding domains of IL-6Ralpha and gp130 bridging them in a way similar to that of the interaction between growth hormone and it s homodimeric receptor. Several residues on helices A and C which were predicted as contact points between IL-6 and gp130 and therefore esse ntial for IL-6 signal transduction, were subjected to site-directed mu tagenesis individually or in combined form. Interestingly, while singl e amino acid changes never produced major alterations in IL-6 bioactiv ity, a subset of double mutants of Y31 and G35 showed a considerable r eduction of biological activity and were selectively impaired from ass ociating with gp130 in binding assays in vitro, while they maintained wild-type affinity towards hIL6-Ralpha. More importantly, we demonstra ted the antagonistic effect of mutant Y31D/G35F versus wild-type IL-6.