BINDING INTERACTIONS OF HUMAN INTERLEUKIN-5 WITH ITS RECEPTOR-ALPHA SUBUNIT - LARGE-SCALE PRODUCTION, STRUCTURAL, AND FUNCTIONAL, STUDIES OF DROSOPHILA-EXPRESSED RECOMBINANT PROTEINS

Human interleukin 5 (hIL5) and soluble forms of its receptor alpha sub unit were expressed in Drosophila cells and purified to homogeneity, a llowing a detailed structural and functional analysis, B cell prolifer ation confirmed that the hIL5 was biologically active. Deglycosylated hTL5 remained active, while similarly deglycosylated receptor alpha su bunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-Angstrom resolution and found to be similar to t hat of the protein produced in Escherichia coli. Human IL5 was shown b y analytical ultracentrifugation to form a 1:1 complex with the solubl e domain of the hIL5 receptor alpha subunit (shIL5R alpha). Additional ly, the relative abundance of ligand and receptor in the hIL5 . shIL5R alpha complex was determined to be 1:1 by both titration calorimetry and SDS-polyacrylamide gel electrophoresis analysis of dissolved cocry stals of the complex. Titration microcalorimetry yielded equilibrium d issociation constants of 3.1 and 2.0 nM, respectively, for the binding of hIL5 to shIL5R alpha and to a chimeric form of the receptor contai ning shIL5R alpha fused to the immunoglobulin Fc domain (shIL5R alpha- Fc). Analysis of the binding thermodynamics of IL5 and its soluble rec eptor indicates that conformational changes are coupled to the binding reaction, Kinetic analysis using surface plasmon resonance yielded da ta consistent with the K-d values from calorimetry and also with the p ossibility of conformational isomerization in the interaction of hIL5 with the receptor alpha subunit, Using a radioligand binding assay, th e affinity of hIL5 with full-length hIL5R alpha in Drosophila membrane s was found to be 6 nM, in accord with the affinities measured for the soluble receptor forms, Hence, most of the binding energy of the alph a receptor is supplied by the soluble domain. Taken with other aspects of hIL5 structure and biological activity, the data obtained allow a prediction for how 1:1 stoichiometry and conformational change can lea d to the formation of hIL5-receptor alpha beta complex and signal tran sduction.