Mutation of the IFNAR-1 Receptor Binding Site of Human IFN-R2 Generates Type I IFN Competitive Antagonists † Manjing Pan, ‡ Eyal Kalie, § Brian J. Scaglione, | Elizabeth S. Raveche, | Gideon Schreiber, § and Jerome A. Langer* ,‡ Department of Molecular Genetics, Microbiology, and Immunology, UMDNJsRobert Wood Johnson Medical School, Piscataway, New Jersey 08854, Department of Pathology and Laboratory Medicine, UMDNJsNew Jersey Medical School, Newark, New Jersey 07103, and Department of Biological Chemistry, Weizmann Institute of Science, RehoVot IL-76100, Israel ReceiVed August 22, 2008; ReVised Manuscript ReceiVed September 17, 2008 ABSTRACT: Type I interferons (IFNs) are multifunctional cytokines that activate cellular responses by binding a common receptor consisting of two subunits, IFNAR-1 and IFNAR-2. Although the binding of IFNs to IFNAR-2 is well characterized, the binding to the lower affinity IFNAR-1 remains less well understood. Previous reports identified a region of human IFN-R2 on the B and C helices (“site 1A”: N65, L80, Y85, Y89) that plays a key role in binding IFNAR-1 and contributes strongly to differential activation by various type I IFNs. The current studies demonstrate that residues on the D helix are also involved in IFNAR-1 binding. In particular, residue 120 (Arg in IFN-R2; Lys in IFN-R2/R1) appears to be a “hot-spot” residue: substitution by alanine significantly decreased biological activity, and the charge- reversal mutation of residue 120 to Glu caused drastic loss of antiviral and antiproliferative activity for both IFN-R2 and IFN-R2/R1. Mutations in residues of helix D maintained their affinity for IFNAR-2 but had decreased affinity for IFNAR-1. Single-site or multiple-site mutants in the IFNAR-1 binding site that had little or no detectable in Vitro biological activity were capable of blocking in Vitro antiviral and antiproliferative activity of native IFN-R2; i.e., they are type I IFN antagonists. These prototype IFN antagonists can be developed further for possible therapeutic use in systemic lupus erythematosus, and analogous molecules can be designed for use in animal models. Type I interferons are a family of cytokines characterized by their antiviral, antiproliferative, and immunomodulatory activities (4). For mammals, the type I interferons include the IFN-R and IFN- subtypes and may include other subtypes, such as IFN-ω, IFN-κ, IFN-ǫ, IFN-δ, and IFN-τ. Humans express 13 IFN-Rs and 1 each of IFN-, IFN-ω, IFN-ǫ, and IFN-κ. The structures of the type I IFNs 1 are homologous (reviewed in ref 5), consisting of a five-helix bundle (labeled sequentially “A” to “E”) with a functionally important long loop (“AB loop”) connecting helices A and B. In spite of the high homology and sequence conservation of different IFN subtypes, individual subtypes display different profiles of biological activities, including antipro- liferative, antiviral, and immunomodulatory. IFNs are used clinically for the treatment of various pathologies, including virus infections, tumors, and multiple sclerosis. However, there is strong evidence that type I interferons are inappropropriately expressed in individuals with systemic lupus erythematosus (SLE) and are involved in lupus development and/or progression (reviewed, for instance, in refs 6 and 7). IFN antagonists are therefore needed for possible therapeutic application in SLE and for studies in model systems, such as murine strains that develop lupus-like disease. Type I IFNs activate cellular responses by binding a common high-affinity cell surface receptor consisting of two transmembrane protein subunits, IFNAR-1 and IFNAR-2, which make distinct contributions to ligand binding (8, 9; reviewed in ref 10). Binding of interferon to its receptor complex initiates activation of the Jak/STAT pathway and other signal transduction pathways, leading to the regulation of relevant genes. The subunits of IFNAR make distinct contributions to ligand binding. Human and mouse IFNAR-1 have low but varied intrinsic affinity for the various IFNs (K D ∼0.05-5 µM), whereas IFNAR-2 has moderate to high affinity for IFNs (K D 0.1-100 nM) (8, 9, 11, 12). Receptor binding appears to be a sequential process, with IFN first binding to the higher affinity IFNAR-2, followed by recruitment of IFNAR-1, to form the ternary complex, with consequent † Supported by a research award from the Alliance for Lupus Research to J.A.L. and by funding from the Foundation of UMDNJ. This work was also supported by the Israel Science Foundation funded by the Israel Academy of Sciences and Humanities to G.S. (grant 633/ 06). * To whom correspondence should be addressed. E-mail: langer@ umdnj.edu. Phone: 732-235-5224. Fax: 732-235-5223. ‡ UMDNJsRobert Wood Johnson Medical School. § Weizmann Institute of Science. | UMDNJsNew Jersey Medical School. 1 Abbreviations: CRF-2, cytokine receptor family 2; DMEM, Dul- becco’s modified Eagle’s medium; EC 50 , effective concentration for 50% of maximum effect; ECD, extracellular domain; EDC, N-(3- dimethylaminopropyl)-N′-ethylcarbodiimide; EDTA, ethylenediamine- tetraacetic acid; EMCV, encephalomyocarditis virus; FNIII, fibronectin type III domain; GuHCl, guanidine hydrochloride; IB, inclusion body; IFN, interferon; IC 50 , concentration for 50% inhibition; IL-1, interleukin 1; IL-1R, interleukin 1 receptor; IL-1RA, interleukin 1 receptor antagonist; K D , equilibrium dissociation constant; NHS, N-hydroxysuc- cinimide; NMR, nuclear magnetic resonance; PMSF, phenylmethane- sulfonyl fluoride; SPR, surface plasmon resonance; VSV, vesicular stomatitis virus. Biochemistry 2008, 47, 12018–12027 12018 10.1021/bi801588g CCC: $40.75  2008 American Chemical Society Published on Web 10/21/2008