No abstract

Type I interferons have pleiotropic effects, including antiviral, antiproliferative and immunomodulatory responses. All type I interferons bind to a shared receptor consisting of the two transmembrane proteins ifnar1 and ifnar2. We used negative stain electron microscopy to calculate a three-dimensional reconstruction of the ternary complex formed by a triple mutant interferon-α2 with the ectodomains of ifnar1 and ifnar2. We present a model of the complex obtained by placing atomic models of the subunits into the density map of the complex. The complex of interferon-α2 with its receptor, a class II cytokine receptor, shows structural similarities to the complexes formed by growth hormone and erythropoietin with their receptors, members of the class I cytokine receptor family. Despite different assembly mechanisms, class I and class II cytokine receptors thus appear to initiate signaling through similar arrangements of the receptors induced by the binding of their respective ligands.

The conformational dynamics of proteins plays a key role in their complex physiological functions. Fluorescence resonance energy transfer (FRET) is a particular powerful tool for studying protein conformational dynamics, but requires efficient site-specific labeling with fluorescent reporter probes. We have employed different tris-NTA/fluorophore conjugates, which bind histidine-tagged proteins with high affinity, for site-specific incorporation of FRET acceptors into proteins, which were covalently labeled with a donor fluorophore. We demonstrate versatile application of this approach for exploring the conformation of the type I interferon receptor ectodomains ifnar1-EC and ifnar2-EC. Substantial ligand-induced conformational changes of ifnar1-EC, but not ifnar2-EC, were observed by monitoring the fluorescence intensity and the fluorescence lifetime of the FRET donor. Time-resolved fluorescence correlation spectroscopy revealed a substantial conformational flexibility of ifnar1-EC and a ligand-induced tightening. Our results demonstrate that protein labeling with tris-NTA/fluorophores enables for efficient quantitative intramolecular FRET analysis.