An active covalently linked dimer of human interferon-gamma. Subunit orientation in the native protein.
We have constructed and expressed a covalently linked head to tail dimer of human interferon-gamma (IFN-gamma) in which two monomers are joined head to tail via a rigid peptide hinge using genetic engineering techniques. The hinge was derived from the human immunoglobin IgA1 sequence (Hallewell, R.A., Laria, I., Tabrizi, A., Carlin, G., Getzoff, E.D., Tainer, J.A., Cousens, L.S., and Mullenbach, G.T. (1989) J. Biol. Chem. 264, 5260-5268). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the polypeptide produced by this construction migrates as a 30,000 polypeptide species. The protein elutes as a single species by molecular sieve chromatography under native conditions. The covalently linked dimer exhibits one-half the antiviral activity of native dimeric IFN-gamma; receptor binding assays show the covalently linked dimer binds to the IFN-gamma receptor with one-half the avidity of native IFN-gamma. This difference is not due to conformational differences between the two molecules, as the aromatic region of the NMR spectrum of the purified covalently linked dimer is identical with that of the wild type protein. From these data, we suggest that human IFN-gamma associates in a head to tail dimer in its active configuration. Regions of IFN-gamma are contiguous with the amino and carboxyl termini and are obscured by the hinge peptide in the covalently linked dimer. Our studies demonstrate that these regions may be important for receptor-ligand interaction.
Lunn
C A
CA
Department of Biotechnology/Molecular Biology, Schering-Plough Research Institute, Bloomfield, New Jersey 07003.
Davies
L
L
Dalgarno
D
D
Narula
S K
SK
Zavodny
P J
PJ
Lundell
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D006801:Humans; D007070:Immunoglobulin A / Q000737:chemistry; D007371:Interferon-gamma / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011971:Receptors, Immunologic / Q000378:metabolism*; D017471:Receptors, Interferon; D011994:Recombinant Proteins
D007070:Immunoglobulin A; D046911:Macromolecular Substances; D011971:Receptors, Immunologic; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D007371:Interferon-gamma