Generation of interleukin-6 receptor antagonists by molecular-modeling guided mutagenesis of residues important for gp130 activation.

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Abstract
Interleukin‐6 (IL‐6) drives the sequential assembly of a receptor complex formed by the IL‐6 receptor (IL‐6R alpha) and the signal transducing subunit, gp130. A model of human IL‐6 (hIL‐6) was constructed by homology using the structure of bovine granulocyte colony stimulating factor. The modeled cytokine was predicted to interact sequentially with the cytokine binding domains of IL‐6R alpha and gp130 bridging them in a way similar to that of the interaction between growth hormone and its homodimeric receptor. Several residues on helices A and C which were predicted as contact points between IL‐6 and gp130 and therefore essential for IL‐6 signal transduction, were subjected to site‐directed mutagenesis individually or in combined form. Interestingly, while single amino acid changes never produced major alterations in IL‐6 bioactivity, a subset of double mutants of Y31 and G35 showed a considerable reduction of biological activity and were selectively impaired from associating with gp130 in binding assays in vitro, while they maintained wild‐type affinity towards hIL‐6‐R alpha. More importantly, we demonstrated the antagonistic effect of mutant Y31D/G35F versus wild‐type IL‐6.