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Intermolecular interactions in a 44 kDa interferon-receptor complex detected by asymmetric reverse-protonation and two-dimensional NOESY.

ABSTRACT: Type I interferons (IFNs) make up a family of homologous helical cytokines initiating strong antiviral and antiproliferative activity. All type I IFNs bind to a common cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. We studied intermolecular interactions between IFNAR2-EC and IFNalpha2 using asymmetric reverse-protonation of the different complex components and two-dimensional homonuclear NOESY. This new approach revealed with an excellent signal-to-noise ratio 24 new intermolecular NOEs between the two molecules despite the low concentration of the complex (0.25 mM) and its high molecular mass (44 kDa). Sequential and side chain assignment of IFNAR2-EC and IFNalpha2 in their binary complex helped assign the intermolecular NOEs to the corresponding protons. A docking model of the IFNAR2-EC-IFNalpha2 complex was calculated on the basis of the intermolecular interactions found in this study as well as four double mutant cycle constraints, previously observed NOEs between a single pair of residues and the NMR mapping of the binding sites on IFNAR2-EC and IFNalpha2. Our docking model doubles the buried surface area of the previous model and significantly increases the number of intermolecular hydrogen bonds, salt bridges, and van der Waals interactions. Furthermore, our model reveals the participation of several new regions in the binding site such as the N-terminus and A helix of IFNalpha2 and the C domain of IFNAR2-EC. As a result of these additions, the orientation of IFNAR2-EC relative to IFNalpha2 has changed by 30 degrees in comparison with a previously calculated model that was based on NMR mapping of the binding sites and double mutant cycle constraints. In addition, the new model strongly supports the recently proposed allosteric changes in IFNalpha2 upon binding of IFNAR1-EC to the binary IFNalpha2-IFNAR2-EC complex.

SUBMITTER: Nudelman I

PROVIDER: S-EPMC2901802 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Intermolecular interactions in a 44 kDa interferon-receptor complex detected by asymmetric reverse-protonation and two-dimensional NOESY.

Nudelman Ilona I Akabayov Sabine R SR Schnur Einat E Biron Zohar Z Levy Rina R Xu Yingqi Y Yang Daiwen D Anglister Jacob J

Biochemistry 20100601 25

Type I interferons (IFNs) make up a family of homologous helical cytokines initiating strong antiviral and antiproliferative activity. All type I IFNs bind to a common cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. We studied intermolecular interactions between IFNAR2-EC and IFNalpha2 using asymmetric reverse-protonation of the different complex components and two-dimensional homonuclear NOESY. This new approach revealed with an excel ...[more]

PMID: 20496919

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Project description:Cellular intrinsic immunity, mediated by the expression of an array of interferon-stimulated antiviral genes, is a vital part of host defense. We have previously used a bioinformatic screen to identify two interferon-stimulated genes (ISG) with poorly characterized function, interferon-induced protein 44 (IFI44) and interferon-induced protein 44-like (IFI44L), as potentially being important in respiratory syncytial virus (RSV) infection. Using overexpression systems, CRISPR-Cas9-mediated knockout, and a knockout mouse model, we investigated the antiviral capability of these genes in the control of RSV replication. Overexpression of IFI44 or IFI44L was sufficient to restrict RSV infection at an early time postinfection. Knocking out these genes in mammalian airway epithelial cells increased levels of infection. Both genes express antiproliferative factors that have no effect on RSV attachment but reduce RSV replication in a minigenome assay. The loss of Ifi44 was associated with a more severe infection phenotype in a mouse model of infection. These studies demonstrate a function for IFI44 and IFI44L in controlling RSV infection.IMPORTANCE RSV infects all children under 2 years of age, but only a subset of children get severe disease. We hypothesize that susceptibility to severe RSV necessitating hospitalization in children without predefined risk factors is, in part, mediated at the antiviral gene level. However, there is a large array of antiviral genes, particularly in the ISG family, the mechanism of which is poorly understood. Having previously identified IFI44 and IFI44L as possible genes of interest in a bioinformatic screen, we dissected the function of these two genes in the control of RSV. Through a range of overexpression and knockout studies, we show that the genes are antiviral and antiproliferative. This study is important because IFI44 and IFI44L are upregulated after a wide range of viral infections, and IFI44L can serve as a diagnostic biomarker of viral infection.

Dataset Information

Intermolecular interactions in a 44 kDa interferon-receptor complex detected by asymmetric reverse-protonation and two-dimensional NOESY.

Publications

Intermolecular interactions in a 44 kDa interferon-receptor complex detected by asymmetric reverse-protonation and two-dimensional NOESY.

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