A discontinuous eight amino acid epitope in human interleukin-3 binds the alpha-chain of its receptor

We have previously reported that, within the first helix of human interleukin (IL)-3, residues Asp(21) and Glu(22) are important for interaction with the alpha- and beta-chains of the IL-3 receptor, respectively, In order to define more precisely the sites of interaction with the receptor, we have performed molecular modeling of the helical core of IL-3 and single amino acid substitution mutagenesis of residues predicted to lie on the surfaces of the A, C, and D helices. The resulting analogues were characterized for their abilities to stimulate proliferation of TF-l cells and for binding to the high affinity (alpha- and beta-chain; IL-3R alpha/R beta) or low affinity (alpha-chain alone; IL-3R alpha) IL-3 receptor. We found that in addition to Asp(21), residues Ser(17), Asn(18), and Thr(25) within the A helix and Arg(108), Phe(113), Lys(116), and Glu(119) within the D helix of IL-3 were important for biological activity, Analysis of their binding characteristics revealed that these analogues were deficient in binding to both the IL-3R alpha/R beta and the IL-3R alpha forms of the receptor, consistent with a selective impairment of interaction with IL-3R alpha. Molecular modeling suggests that these eight amino acid residues are adjacent in the tertiary structure, consistent with a discontinuous epitope interacting selectively with IL-3R alpha. On the other hand, Glu(22) of IL-3 was found to interact preferentially with the beta-chain with bulky and positively charged substitutions causing greater than 10,000-fold reduction in biological activity. These results show fundamental differences between IL-3 and granulocyte-macrophage colony-stimulating factor in the structural basis for recognition of their receptors that has implications for the construction of novel analogues and our understanding of receptor activation.