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

• Published in: Acta crystallographica. Section D, Biological crystallography. Biological crystallography., 2000-01, Vol.56 (1), p.14-24
• Main Authors: Randal, Michael, Kossiakoff, Anthony A.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cattle; Crystallization; Crystallography, X-Ray; cytokines; Dimerization; Humans; Interferon-gamma - chemistry; Interferon-gamma - genetics; Interferon-gamma - isolation & purification; interferon-γ (bovine); Mice; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Structure, Quaternary; Rabbits; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Sequence Homology, Amino Acid; Sheep; Species Specificity; Static Electricity; Swine
• ISSN: 1399-0047; 0907-4449; 1399-0047
• DOI: 10.1107/S0907444999014304

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% (Rfree = 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 Rfree 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.