Importance of the loop connecting A and B helices of human interferon-gamma in recognition by interferon-gamma receptor.

Abstract

Characterization of murine-human hybrid interferon-gamma (IFN-gamma) molecules suggests that substitution of the peptide connecting the A and B helices in human IFN-gamma with the murine sequence significantly blocks the protein's binding to the human interferon-gamma receptor. Mutagenesis showed that this effect is localized to the central part of this A-B loop peptide, particularly Ser20, Asp21, Val22, and Ala23. One mutant, IFN-gamma/A23E,D24E,N25K, was examined by NMR. This "EEK" mutation does not significantly alter the conformation of interferon-gamma, suggesting that the effects of these mutations are not the result of global conformational changes. The A-B loop is near histidine 111, a residue previously shown to be important in receptor-ligand interaction (Lunn, C. A., Fossetta, J., Dalgarno, D., Murgolo, N., Windsor, W., Zavodny, P. J., Narula, S. K., and Lundell, D. (1992) Protein Eng. 5, 253-257). We show that copper forms a complex between histidine 19 in the A-B loop and histidine 111. This metal complex lacks the ability to interact with the interferon-gamma receptor. These results suggest that the A-B loop contains important structural information needed for receptor-ligand binding and hence biological activity of human interferon-gamma.