A point mutation of human interferon gamma abolishes receptor recognition.
We identified a single amino acid mutation that abolished the bioactivity of human IFN gamma. The mutation was identified by screening a mutagenized IFN gamma expression library for molecules with altered biological activity. The mutant protein was expressed at high levels in Escherichia coli, and remained soluble upon purification. However, the protein was completely inactive in all IFN gamma assays investigated, exhibiting less than 0.0006% of the specific activity of native IFN gamma antiviral activity. Sequencing the plasmid DNA encoding this mutant protein showed that the histidine at position 111 of native human IFN gamma is changed to aspartic acid (IFN gamma/H111D). Other mutations at this site showed that only hydrophobic amino acids at position 111 maintain significant, though low, biological activity. Structural characterization of the IFN gamma/H111D protein by NMR as well as CD spectroscopy demonstrated that the protein has limited conformational differences from native IFN gamma. Models of the X-ray crystal structure of human IFN gamma [Ealick, P.E., W.J. Cook, S. Vijay-Kumar, M. Carson, T.L. Nagabhushan, P.P. Trotta and C.E. Bugg (1991) Science, 252, 698-702] suggest that this histidine residue is located at a severe 55 degrees bend in the C-terminal F helix. We conclude that H111 lies within or affects the receptor binding domain of human IFN gamma.
Lunn
C A
CA
Department of Biotechnology/Molecular Biology, Schering-Plough Research Institute, Bloomfield, NJ 07003.
Fossetta
J
J
Dalgarno
D
D
Murgolo
N
N
Windsor
W
W
Zavodny
P J
PJ
Narula
S K
SK
Lundell
D
D
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003588:Cytopathogenic Effect, Viral; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D017471:Receptors, Interferon / Q000378:metabolism; D011994:Recombinant Proteins
D017471:Receptors, Interferon; D011994:Recombinant Proteins; D007371:Interferon-gamma