CHANGES IN CONFORMATION AND STABILITY UPON FORMATION OF COMPLEXES OF ERYTHROPOIETIN (EPO) AND SOLUBLE EPO RECEPTOR

Erythropoietin (EPO) is a glycoprotein hormone which belongs to the fo ur-helical-bundle cytokine family and regulates the level of circulati ng red blood cells. The EPO receptor (EPOR) belongs to the cytokine-re ceptor family of proteins. While many of the downstream events followi ng receptor/ligand interaction have been defined, both ligand-induced receptor dimerization and conformational changes induced by binding ha ve been implicated as the initial step in signal transduction. In a re cent paper [Philo et al. (1996), Biochemistry 38, 1681-1691] we descri bed the formation of both 1:1 and 2:1 EPOR/EPO complexes. In this pape r, we examine changes in protein conformation and stability resulting from the formation of both 1:1 and 2:1 complexes of the soluble extrac ellular domain of EPOR and the recombinant EPO derived from either Chi nese hamster ovary cells or from Escherichia coli cells. Occupation of the first binding site results in a slight conformational change that is apparent in both the far- and near-UV circular dichroism spectra. Formation of the 2:1 complex results in an even greater change in conf ormation which involves the local environment of one or more aromatic amino acids, accompanied perhaps by a small increase in helical conten t of the complex. This change in local conformation could occur in the EPO molecule, in the EPOR, in both EPOR molecules due to dimerization , or in all molecules in the trimer. The 1:1 complex exhibits increase d stability to thermal-induced denaturation relative to the individual protein component; indeed, the E. coli-derived (nonglycosylated) EPO stays folded in the complex at temperatures where the EPO alone would have unfolded and precipitated, Glycosylation of the receptor increase s the reversibility of thermal denaturation, but does not affect the t emperature at which this unfolding reaction occurs.