NEUTRALIZING EPITOPES ON THE EXTRACELLULAR INTERFERON-GAMMA RECEPTOR (IFN-GAMMA-R) ALPHA-CHAIN CHARACTERIZED BY HOMOLOG SCANNING MUTAGENESIS AND X-RAY CRYSTAL-STRUCTURE OF THE A6 FAB-IFN-GAMMA-R1-108 COMPLEX

The extracellular interferon gamma receptor alpha-chain comprises two immunoglobulin-like domains, each with fibronectin type-III topology, which are responsible for binding interferon gamma at the cell surface . The epitopes on the human receptor recognized by three neutralizing antibodies, A6, gamma R38 and gamma R99, have been mapped by homolog s canning mutagenesis. In this way, a loop connecting beta-strands C and C' in the N-terminal domain was identified as a key component of the epitopes bound by A6 and gamma R38, whereas gamma R99 binds to the C-t erminal domain in a region including strands A and B and part of the l arge C'E loop. The epitope for A6 was confirmed in a crystal structure of a complex between a recombinant N-terminal receptor domain and the Fab fragment from A6, determined by X-ray diffraction to 2.8 Angstrom resolution. The antibody-antigen interface buries 1662 Angstrom(2) of protein surface, including 22 antibody residues from five complementa rity determining regions, primarily through interactions with the CC' surface loop of the receptor. The floor of the antigen binding cavity is formed mainly by residues from CDR L3 and CDR H3 while a surroundin g ridge is formed by residues from all other CDRs except L2. Many pote ntial polar Interactions, as well as 13 aromatic side-chains, four in V-L, six in V-H and three in the receptor, are situated at the interfa ce. The surface of the receptor contacted by A6 overlaps to a large ex tent with that contacted by interferon-gamma,in the ligand-receptor co mplex. However, the conformation of this epitope is very different in the two complexes, demonstrating that conformational mobility in a sur face loop on, this cytokine receptor permits steric and electrostatic complementarity to two quite differently shaped binding sites. (C) 199 7 Academic Press Limited.