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) sequenc e recognizes soluble human gamma-interferon receptor (Seelig, G. F., P rosise, 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 with in IFN-gamma-receptor. To do this, the neutralizing anti-idiotypic ant ibody was used to probe overlapping linear peptide octamers of the ext racellular do main of the huIFN-gamma receptor, A 22-amino acid residu e receptor segment 120-141 identified by the antibody was synthesized, CD and NMR analysis indicates that peptide 120-141 has no apparent se condary structure in water or in water containing 50% trifluoroethanol , The synthetic receptor peptide inhibited huIFN-gamma induced express ion of HLA/DR antigen on Cole 205 cells with an approximate IC50 of 35 mu M. Immobilized peptide specifically bound recombinant huIFN-gamma but did not bind human granulocyte-macrophage colony-stimulating facto r on a microtiter plate in a direct binding enzyme-linked immunosorben t assay. The binding results are supported by two-dimensional transfer red nuclear Overhauser effect (TRNOE) NMR data obtained on the peptide in the presence of recombinant huIFN-gamma. Characterization of the c onformation of the bound peptide by TRNOE suggests that this peptide a ssumes a distinct conformation. Intramolecular interactions within the bound peptide were detected at two non-contiguous regions and at a th ird region comprising a p-turn formed by the sequence DIRK. We believe that this represents the structure of the receptor within the ligand- binding domain.