Characterization of a soluble ternary complex formed between human interferon‐β‐1a and its receptor chains

Abstract

The extracellular portions of the chains that comprise the human type I interferon receptor, IFNAR1 and IFNAR2, have been expressed and purified as recombinant soluble His‐tagged proteins, and their interactions with each other and with human interferon‐β‐1a (IFN‐β‐1a) were studied by gel filtration and by cross‐linking. By gel filtration, no stable binary complexes between IFN‐β‐1a and IFNAR1, or between IFNAR1 and IFNAR2 were detected. However, a stable binary complex formed between IFN‐β‐1a and IFNAR2. Analysis of binary complex formation using various molar excesses of IFN‐β‐1a and IFNAR2 indicated that the complex had a 1:1 stoichiometry, and reducing SDS‐PAGE of the binary complex treated with the cross‐linking reagent dissucinimidyl glutarate (DSG) indicated that the major cross‐linked species had an apparent M r consistent with the sum of its two individual components. Gel filtration of a mixture of IFNAR1 and the IFN‐β‐1a/IFNAR2 complex indicated that the three proteins formed a stable ternary complex. Analysis of ternary complex formation using various molar excesses of IFNAR1 and the IFN‐β‐1a/IFNAR2 complex indicated that the ternary complex had a 1:1:1 stoichiometry, and reducing SDS‐PAGE of the ternary complex treated with DSG indicated that the major cross‐linked species had an apparent M r consistent with the sum of its three individual components. We conclude that the ternary complex forms by the sequential association of IFN‐β‐1a with IFNAR2, followed by the association of IFNAR1 with the preformed binary complex. The ability to produce the IFN‐β‐1a/IFNAR2 and IFN‐β‐1a/IFNAR1/IFNAR2 complexes make them attractive candidates for X‐ray crystallography studies aimed at determining the molecular interactions between IFN‐β‐1a and its receptor.