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. 1994 Mar 15;13(6):1357–1367. doi: 10.1002/j.1460-2075.1994.tb06389.x

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

R Savino 1, A Lahm 1, A L Salvati 1, L Ciapponi 1, E Sporeno 1, S Altamura 1, G Paonessa 1, C Toniatti 1, G Ciliberto 1
PMCID: PMC394953  PMID: 7511100

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.

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