NMR Backbone Assignments and Secondary Structure of Human Interferon-$

'H, 13C, and '5N NMR assignments of the protein backbone of human interferon-y, a homodimer of 3 1.4 kDa, have been made using the recently introduced three-dimensional (3D) triple-resonance NMR techniques, It is shown that, despite the -40-50-HZ 13Ca and *Ha line widths of this high molecular weight dimer and the extensive overlap in the 'Ha and I3Ca spectral regions, unique sequential assignments can be made on the basis of combined use of the 3D HNCO, HNCA, HN(CO)CA, and HCACO constanttime experiments, the l5N-separated 3D NOESY-HMQC, and the 3D HOHAHA-HMQC experiments. Analysis of the l5N-separated 3D NOESY-HMQC and l3C/l5N-separated four-dimensional (4D) NOESY HMQC spectra together with the secondary C, and CB chemical shifts yielded extensive secondary structure information. The NMR-derived secondary structure essentially confirms results of a recently published low-resolution crystal structure [Ealick et al. (1991) Science 252,698-7021, Le., six helices in the monomer which are mostly a-helical in nature, no @-sheets, a long flexible loop between helices A and B, and a very hydrophobic helix C. The functionally important carboxy terminus, which was not observed in the X-ray study, does not adopt a rigid conformation in solution. A high degree of internal mobility, starting at Pro123, gives rise to significantly narrower resonance line widths for these carboxy-terminal residues compared to the rest of the protein. Interferon (IFN)' was first discovered in 1957 for its antiviral activity (Isaacs & Lindenmann, 1957). Three functionally related, but structurally different interferons, IFN-a, IFN-8, and IFN-y, have been found in all mammalian species (Ijzermans & Marquet, 1989). IFN-y was first described by Wheelock (1965) and is distinctly different from IFN-a and IFN-j3, whose genes are located on a different chromosome (Shows et al., 1982) and which have very limited homology to IFN-y (Ijzermans & Marquet, 1989). IFN-y exhibits a large number of different biological activities, comprising its antiviral activity [for recent reviews, see Ijzermans and Marquet (1989), Murray (1990), Landolfo and Garotta (1991), and Mosmann et al. (1991)], antitumoral and antimicrobial actions, the activation of macrophages, the enhancement of natural killer cell activity and of T-cell cytotoxicity, and the modulation of the Band T-cell response where it acts in an antagonistic sense to interleukin-4 (Mosmann et al., 1991) as well as the enhancement of MHC class I and I1 expression. During the immune response, IFN-y is produced by activated T-cells and natural killer cells (Nathan etal., 1981;Trinchieriet al., 1984). Incontrast toIFN-aand IFN-j3, the biological active form of IFN-y is a dimer (Pestka et al., 1983; Le et al., 1984), and the human IFN-y receptor This workwas supported by the Intramural AIDS Anti-viral Program of the Office of the Director of the National Institutes of Health. To whom correspondence should be addressed. t National Institutes of Health. 11 F. Hoffmann La Roche Ltd. I Abbreviations: IFN, interferon; IFN-yAl0, recombinant human interferon-y lacking the last 10 amino acids and comprising one additional N-terminal methionine; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; HMQC, heteronuclear multiple-quantum coherence; 3D, three dimensional; 4D, four dimensional. On leave from F. Hoffmann La Roche, Basel, Switzerland. has been identified [for a recent review, see Langer and Pestka