Biophysical analysis of the interaction of human ifnar2 expressed in E. coli with IFNalpha2.
Type I interferons are cytokines which activate an anti-viral response by binding to two specific cell surface receptors, ifnar1 and ifnar2. Here, we report purification and refolding of the extracellular part of human ifnar2 (ifnar2-EC) expressed in Escherichia coli and its characterization with respect to its interaction with interferon alpha2 (IFNalpha2). The 25 kDa, non-glycosylated ifnar2-EC is a stable, fully active protein, which inhibits antiviral activity of IFNalpha2. The stoichiometry of binding IFNalpha2 is 1:1, as determined by gel filtration, chemical cross-linking and solid-phase detection. The affinity of this interaction is 10 nM, which is similar to the affinity measured for the cell surface-bound ifnar2 receptor. No difference in affinity was found throughout various assays using optical detection as BIAcore or reflectometric interference spectorscopy. However, the binding kinetics as measured in homogeneous phase by fluorescence de-quenching was about three times faster than that measured on a sensor surface. The rate of complex formation is relatively high compared to other cytokine-receptor interactions. The salt dependence of the association kinetics suggest a limited but significant contribution of electrostatic forces towards the rate of complex formation. The dissociation constant increases with decreasing pH according to the protonation of a base with a pKa of 6.7. The surface properties of the IFNalpha2 binding surface on ifnar2 were interpreted according to the pH and salt dependence of the interaction.
Piehler
J
J
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100, Israel.
Schreiber
G
G
D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D006863:Hydrogen-Ion Concentration; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins; D053633:Receptor, Interferon alpha-beta; D017471:Receptors, Interferon / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014721:Vesicular stomatitis Indiana virus / Q000187:drug effects
D000998:Antiviral Agents; C507964:IFNAR1 protein, human; C508033:IFNAR2 protein, human; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D008565:Membrane Proteins; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D053633:Receptor, Interferon alpha-beta