Characterization of a soluble ternary complex formed between human interferon-beta-1a and its receptor chains

The extracellular portions of the chains that comprise the human type I int erferon receptor, IFNAR1 and IFNAR2, have been expressed and purified as re combinant soluble His-tagged proteins, and their interactions with each oth er and with human interferon-beta-1a (IFN-beta-1a) were studied by gel filt ration and by cross-linking. By gel filtration, no stable binary complexes between IFN-beta-1a and IFNAR1, or between IFNAR1 and IFNAR2 were detected. However, a stable binary complex formed between IFN-beta-1a and IFNAR2. An alysis of binary complex formation using various molar excesses of IFN-beta -1a and IFNAR2 indicated that the complex had a 1:1 stoichiometry, and redu cing SDS-PAGE of the binary complex treated with the cross-linking reagent dissucinimidyl glutarate (DSG) indicated that the major cross-linked specie s had an apparent M-r consistent with the sum of its two individual compone nts. Gel filtration of a mixture of IFNAR1 and the LFN-beta-1a/IFNAR2 compl ex indicated that the three proteins formed a stable ternary complex. Analy sis of ternary complex formation using various molar excesses of IFNAR1 and the IFN-beta-1a/IFNAR2 complex indicated that the ternary complex had a 1: 1:1 stoichiometry, and reducing SDS-PAGE of the ternary complex treated wit h DSG indicated that the major cross-linked species had an apparent M-r con sistent with the sum of its three individual components. We conclude that t he ternary complex farms by the sequential association of IFN-beta-1a with IFNAR2, followed by the association of LFNAR1 with the preformed binary com plex. The ability to produce the IFN-beta-1a/IFNAR2 and IFN-beta-1a/IFNAR1/ IFNAR2 complexes make them attractive candidates for X-ray crystallography studies aimed at determining the molecular interactions between IFN-beta-1a and its receptor.