title: Mutational and structural analysis of the binding interface between type I interferons and their receptor Ifnar2.
abstract: Type I interferons (IFN) exert pleiotropic activities through binding to two cell surface receptors, ifnar1 and ifnar2. We are investigating the biophysical basis of IFN signaling by characterizing the complex of the extra-cellular domain of ifnar2 (ifnar2-EC) with IFNs on the level of purified recombinant proteins in vitro. Here, we present a detailed mutational study on the functional epitopes on both IFN and ifnar2. Kinetic and thermodynamic parameters were determined by label-free heterogeneous phase detection. On IFNalpha2, a relatively small functional epitope comprising ten amino acid residues was localized, which is nearly entirely formed by residues on the AB loop. Two hot-spot residues, L30 and R33, account for two-thirds of the total interaction energy. Comparing the anti-viral potency of the various mutants to the binding affinity towards ifnar2 revealed a proportional correlation between the two, suggesting a rate-limiting role of ifnar2 binding in IFN signaling. On ifnar2, residues T46, I47 and M48 were identified as hot-spots in the interaction with IFNalpha2. For another ten residues on ifnar2, significant contribution of interaction energy was determined. Based on these data, the functional epitope on ifnar2 was defined according to a homology model based on other members of the class II hCR family in good agreement with the complementary functional epitope on IFNalpha2. Although IFNalpha2 and IFNbeta bind competitively to the same functional epitope, mutational analysis revealed distinct centers of binding for these IFNs on ifnar2. This small shift of the binding site may result in different angular orientation, which can be critically coupled to cytoplasmic signaling.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, 76100, Israel.
authors.forename: J
authors.initials: J
authors.lastname: Piehler
authors.forename: G
authors.initials: G
authors.lastname: Schreiber
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D007370:Interferon Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016898:Interferon-alpha / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016899:Interferon-beta / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D010446:Peptide Fragments; D011485:Protein Binding; D053633:Receptor, Interferon alpha-beta; D017471:Receptors, Interferon / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013499:Surface Properties; D013816:Thermodynamics
chemical_list: D000998:Antiviral Agents; D007370:Interferon Type I; D016898:Interferon-alpha; D008565:Membrane Proteins; D010446:Peptide Fragments; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D053633:Receptor, Interferon alpha-beta; D016899:Interferon-beta