SPATIAL ORIENTATION OF THE ALPHA-RECEPTOR AND BETA(C)-RECEPTOR CHAIN BINDING-SITES ON MONOMERIC HUMAN INTERLEUKIN-5 CONSTRUCTS

Interleukin-5 (IL-5), a disulfide-linked homodimer, can be induced to fold as a biological active monomer by extending the loop between its third and fourth helices (Dickason, R, R., and Huston, D, P, (1996) Na ture 379, 652-655), We have designed eight monomeric IL-5 proteins to optimize biological activity and stability of the monomer, This was ac hieved by (i) inserting the joining loop at three different positions, (ii) by introducing an additional intramolecular disulfide bridge ont o these backbones, and (iii) by creating circular permutations to fix the position of the carboxyl-terminal helix relative to the three othe r helices, The proteins dimerize with K-d values ranging from 20 to 20 0 mu M and are therefore monomeric at the picomolar concentrations whe re they are biologically active, Introduction of a second disulfide co nfers increased stability, but this increased rigidity results in lowe r activity of the protein, Contrary to wild type IL-5, mutation of the beta(c) contact residue on the first helix, Glu(12), to Lys, into the circularly permutated constructs, did not abolish TF-1 proliferative and eosinophil activation activities, These results indicate that acti vation of the IL-5 receptor complex is not mediated solely by Glu(12) on the first helix, and alternative mechanisms are discussed.