Development of a receptor peptide antagonist to human gamma-interferon and characterization of its ligand-bound conformation using transferred nuclear Overhauser effect spectroscopy.
Polyclonal anti-idiotypic antibody raised to a synthetic discontinuous peptide derived from the human gamma-interferon (huIFN-gamma) sequence recognizes soluble human gamma-interferon receptor (Seelig, G. F., Prosise, W. W., and Taremi, S. S. (1994) J. Biol. Chem. 269, 358-363). We sought to use this reagent to identify a ligand-binding domain within IFN-gamma-receptor. To do this, the neutralizing anti-idiotypic antibody was used to probe overlapping linear peptide octamers of the extracellular domain of the huIFN-gamma receptor. A 22-amino-acid residue receptor segment 120-141 identified by the antibody was synthesized. CD and NMR analysis indicates that peptide 120-141 has no apparent secondary structure in water or in water containing 50% trifluoroethanol. The synthetic receptor peptide inhibited huIFN-gamma induced expression of HLA/DR antigen on Colo 205 cells with an approximate IC50 of 35 microM. Immobilized peptide specifically bound recombinant huIFN-gamma but did not bind human granulocyte-macrophage colony-stimulating factor on a microtiter plate in a direct binding enzyme-linked immunosorbent assay. The binding results are supported by two-dimensional transferred nuclear Overhauser effect (TRNOE) NMR data obtained on the peptide in the presence of recombinant huIFN-gamma. Characterization of the conformation of the bound peptide by TRNOE suggests that this peptide assumes a distinct conformation. Intramolecular interactions within the bound peptide were detected at two non-contiguous regions and at a third region comprising a beta-turn formed by the sequence DIRK. We believe that this represents the structure of the receptor within the ligand-binding domain.
Seelig
G F
GF
Department of Structural Chemistry, Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539, USA.
Prosise
W W
WW
Hawkins
J C
JC
Senior
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D007371:Interferon-gamma / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D017471:Receptors, Interferon / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D000096743:Interferon gamma Receptor
D000906:Antibodies; D007371:Interferon-gamma; D008024:Ligands; D010446:Peptide Fragments; D017471:Receptors, Interferon; D000096743:Interferon gamma Receptor