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J Biol Chem. 1995 Apr 21;270(16):9459-71.

Binding interactions of human interleukin 5 with its receptor alpha subunit. Large scale production, structural, and functional studies of Drosophila-expressed recombinant proteins.

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Department of Protein Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.


Human interleukin 5 (hIL5) and soluble forms of its receptor alpha subunit were expressed in Drosophila cells and purified to homogeneity, allowing a detailed structural and functional analysis. B cell proliferation confirmed that the hIL5 was biologically active. Deglycosylated hIL5 remained active, while similarly deglycosylated receptor alpha subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-A resolution and found to be similar to that of the protein produced in Escherichia coli. Human IL5 was shown by analytical ultracentrifugation to form a 1:1 complex with the soluble domain of the hIL5 receptor alpha subunit (shIL5R alpha). Additionally, 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 cocrystals of the complex. Titration microcalorimetry yielded equilibrium dissociation 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 containing shIL5R alpha fused to the immunoglobulin Fc domain (shIL5R alpha-Fc). Analysis of the binding thermodynamics of IL5 and its soluble receptor indicates that conformational changes are coupled to the binding reaction. Kinetic analysis using surface plasmon resonance yielded data consistent with the Kd values from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor alpha subunit. Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5R alpha in Drosophila membranes 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 alpha 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 lead to the formation of hIL5.receptor alpha beta complex and signal transduction.

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