Definition of receptor binding sites on human interleukin-11 by molecular modeling-guided mutagenesis

Interleukin-11 (IL-11) belongs to the interleukin-6 (IL-6)-type subfamily o f long-chain helical cytokines including IL-6, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M, and cardiotrophin-1 , which all share the glycoprotein gp130 as a signal transducing receptor c omponent. IL-11 acts on cells expressing gp130 and the IL-11 receptor (IL-1 1R) alpha-subunit (IL-11R alpha). The structural epitopes of IL-11 required for the recruitment of the individual receptor subunits have not yet been defined. Based on the structure of CNTF, a three-dimensional model of human IL-11 was built, Using this model, 10 surface exposed amino acid residues of IL-11 were selected for mutagenesis using analogies to the well-characte rized receptor recruitment sites of IL-6, CNTF, and LIF. The respective mut ants of human IL-11 were expressed as soluble fusion proteins in bacteria. Their biological activities were determined on HepG2 and Ba/F3-130-11 alpha cells. Several mutants with substantially decreased bioactivity and one hy peragonistic mutant were identified and further analyzed with regard to rec ruitment of IL-11R alpha and gp130. The low-activity mutant I171D still bin ds IL-11R alpha but fails to recruit gp130, whereas the hyperagonistic vari ant R135E more efficiently engages the IL-11R subunits. The low-activity mu tants R190E and L194D failed to bind to IL-11R alpha. These findings reveal a common mechanism of receptor recruitment in the family of IL-6-type cyto kines and offer considerable perspectives for the rational design of IL-11 antagonists and hyperagonists.