The 2.0 Å structure of bovine interferon-γ; assessment of the structural differences between species

Abstract

The structure of bovine interferon-γ (IFN-γ) was determined by multiple isomorphous replacement at 2.0 Å resolution. Bovine IFN-γ crystallizes in two related crystal forms. Crystal form 1 diffracts to 2.9 Å resolution and is reproducible and stable to derivatization. Crystal form 2 diffracts to 2.0 Å resolution, but shows significant non-isomorphism from crystal to crystal. The previously determined structures of several different species of INF-γ were either at too low a resolution [human, 1hig; Ealick et al. (1991), Science, 252, 698–702] or were too inaccurate [bovine, 1rfb; Samudzi & Rubin (1993), Acta Cryst. D49(6), 505–512; rabbit, 2rig; Samudzi et al. (1991), J. Biol. Chem. 266(32), 21791–21797] for the structure to be solved by molecular replacement. The structure was solved in crystal form 1 using two derivatives produced by chemically modifying two free cysteine residues that were introduced by site-directed mutagenesis (Ser30Cys, Asn59Cys). After model building and refinement, the final R value was 21.8% (R free = 30.9%) for all data in the resolution range 8.0–2.9 Å. The crystal form 1 structure was then used as a molecular-replacement model for crystal form 2 data collected from a flash-cooled crystal. Subsequent model building and refinement, using all data in the resolution range 15.0–2.0 Å, gave an R value of 19.7% and an R free of 27.5%. Pairwise comparison of Cα positions of bovine IFN-γ (BOV) and the previously determined 1rfb and 2rig structures indicated some significant differences in the models (r.m.s.d. values for BOV to 1rfb, 4.3 Å; BOV to 2rig, 4.0 Å). An assessment of the quality of the structures was made using the 3D–1D algorithm [Eisenberg et al. (1992), Faraday Discuss. 93, 25–34]. The resulting statistical scoring indicated that BOV was consistent with expected criteria for a 2.0 Å structure, whereas both 1rfb and 2rig fell below acceptable criteria.