Intermolecular Interactions in a 44 kDa Interferon−Receptor Complex Detected by Asymmetric Reverse-Protonation and Two-Dimensional NOESY

Ilona Nudelman, Sabine R. Akabayov, Einat Schnur, Zohar Biron, Rina Levy, Yingqi Xu, Daiwen Yang, Jacob Anglister
2010 Biochemistry  
Type I Interferons (IFNs) are a family of homologous helical cytokines initiating strong anti-viral and anti-proliferative 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 IFNα2 using asymmetric reverse-protonation of the different complex components and 2D homonuclear NOESY. This new approach revealed with excellent signal-to-noise
more » ... atio 24 new intermolecular NOEs between the two molecules despite the low concentration of the complex (0.25 mM) and its high molecular weight (44 kDA). Sequential and side-chain assignment of IFNAR2-EC and IFNα2 in their binary complex helped assign the inter-molecular NOEs to the corresponding protons. A docking model of the IFNAR2-EC/IFNα2 complex was calculated based on the inter-molecular interactions found in the present 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 IFNα2. Our docking model doubles the buried surface area of the previous model and significantly increases the number of inter-molecular hydrogen bonds, salt bridges and Van der-Waals interactions. Furthermore, the current model reveals participation of several new regions in the binding site such as the N-terminus and A-helix of IFNα2 and the C-domain of IFNAR2-EC. As a result of these additions, the orientation of IFNAR2-EC relative to IFNα2 has changed by 30° 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 IFNα2 upon IFNAR1-EC binding to the binary IFNα2/ IFNAR2-EC complex. Type I IFNs are a major component of the innate immune system protecting against viral infection.
doi:10.1021/bi100041f pmid:20496919 pmcid:PMC2901802 fatcat:p6nrhmuz7jec5alkbiwpnxrmoi