A mutant form of human interferon-γ (IFN-γSC1) that binds one IFN-γ receptor α chain (IFN-γRα) has been designed and characterized. IFN-γSC1 was derived by linking the two peptide chains of the IFN-γ dimer by a seven-residue linker and changing His111 in the first chain to an aspartic acid residue. Isothermal titration calorimetry shows that IFN-γSC1 forms a 1:1 complex with its high-affinity receptor (IFN-γRα) with an affinity of 27(±9) nM. The crystal structure of IFN-γSC1 has been determined at 2.9 Å resolution from crystals grown in 1.4 M citrate solutions at pH 7.6. Comparison of the wild-type receptor-binding domain and the Asp111-containing domain of IFN-γSC1 show that they are structurally equivalent but have very different electrostatic surface potentials. As a result, surface charge rather than structural changes is likely responsible for the inability of the His111 → Asp domain of to bind IFN-γRα. The AB loops of IFN-γSC1 adopt conformations similar to the ordered loops of IFN-γ observed in the crystal structure of the IFN-γ/IFN-γRα complex. Thus, IFN-γRα binding does not result in a large conformational change in the AB loop as previously suggested. The structure also reveals the final six C-terminal amino acid residues of IFN-γSC1 (residues 253-258) that have not been observed in any other reported IFN-γ structures. Despite binding to only one IFN-γRα, IFN-γSC1 is biologically active in cell proliferation, MHC class I induction, and anti-viral assays. This suggests that one domain of IFN-γ is sufficient to recruit IFN-γRα and IFN-γRα into a complex competent for eliciting biological activity. The current data are consistent with the main role of the IFN-γ dimer being to decrease the dissociation constant of IFN-γ for its cellular receptors. (C) 2000 Academic Press.