Mutational and structural analysis of the binding interface between type Iinterferons and their receptor ifnar2

Type I interferons (IFN) exert pleiotropic activities through binding to tw o cell surface receptors, ifnar1 and ifnar2. We are investigating the bioph ysical basis of IFN signaling by characterizing the complex of the extra-ce llular domain of ifnar2 (ifnar2-EC) with IFNs on the level of purified reco mbinant proteins in vitro. Here, we present a detailed mutational study on the functional epitopes on both IFN and ifnar2. Kinetic and thermodynamic p arameters were determined by label-free heterogeneous phase detection. On I FN alpha 2, 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 corr elation between the two, suggesting a rate-limiting role of ifnar2 binding in IFN signaling. On ifnar2 residues T46, I47 and M48 were identified as ho t-spots in the interaction with IFN alpha 2. For another ten residues on if nar2, 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 ag reement with the complementary functional epitope on IFN alpha 2. Although IFN alpha 2 and IFN beta 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 angul ar orientation, which can be critically coupled to cytoplasmic signaling. ( C) 1999 Academic Press.