Design, characterization, and structure of a biologically active single-chain mutant of human IFN-gamma

A mutant form of human interferon-gamma (IFN-gamma SC1) that binds one IFN- gamma receptor alpha chain (IFN-gamma R alpha) has been designed and charac terized. IFN-gamma SC1 was derived by linking the two peptide chains of the IFN-gamma dimer by a seven-residue linker and changing His111 in the first chain to an aspartic acid residue. Isothermal titration calorimetry shows that IFN-gamma SC1 forms a 1:1 complex with its high-affinity receptor (IFN -gamma R alpha) with an affinity of 27(+/-9) nM. The crystal structure of I FN-gamma SC1 has been determined at 2.9 Angstrom resolution from crystals g rown in 1.4 M citrate solutions at pH 7.6. Comparison of the wild-type rece ptor-binding domain and the Asp111-containing domain of IFN-gamma SC1 show that they are structurally equivalent but have very different electrostatic surface potentials. As a result, surface charge rather than structural cha nges is likely responsible for the inability of the His111 --> Asp domain o f to bind IFN-gamma R alpha. The AB loops of IFN-gamma SC1 adopt conformati ons similar to the ordered loops of IFN-gamma observed in the crystal struc ture of the IFN-gamma/IFN-gamma R alpha complex. Thus, IFN-gamma R alpha bi nding does not result in a large conformational change in the AB loop as pr eviously suggested. The structure also reveals the final six C-terminal ami no acid residues of IFN-gamma SC1 (residues 253-258) that have not been obs erved in any other reported IFN-gamma structures. Despite binding to only o ne IFN-gamma R alpha, IFN-gamma SC1 is biologically active in cell prolifer ation, MHC class I induction, and anti-viral assays. This suggests that one domain of IFN-gamma is sufficient to recruit IFN-gamma R alpha and IFN-gam ma R beta into a complex competent for eliciting biological activity. The c urrent data are consistent with the main role of the IFN-gamma dimer being to decrease the dissociation constant of IFN-gamma for its cellular recepto rs. (C) 2000 Academic Press.