Neutralizing epitopes on the extracellular interferon gamma receptor (IFNgammaR) alpha-chain characterized by homolog scanning mutagenesis and X-ray crystal structure of the A6 fab-IFNgammaR1-108 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, gammaR38 and gammaR99, have been mapped by homolog scanning 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 gammaR38, whereas gammaR99 binds to the C-terminal domain in a region including strands A and B and part of the large 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 A resolution. The antibody-antigen interface buries 1662 A2 of protein surface, including 22 antibody residues from five complementarity 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 surrounding ridge is formed by residues from all other CDRs except L2. Many potential polar interactions, as well as 13 aromatic side-chains, four in VL, six in VH and three in the receptor, are situated at the interface. The surface of the receptor contacted by A6 overlaps to a large extent with that contacted by interferon-gamma, in the ligand-receptor complex. However, the conformation of this epitope is very different in the two complexes, demonstrating that conformational mobility in a surface loop on this cytokine receptor permits steric and electrostatic complementarity to two quite differently shaped binding sites.
Sogabe
S
S
Department of Chemistry, University of Zurich, Winterthurerstrasse 190, Zurich, 8057, Switzerland.
Stuart
F
F
Henke
C
C
Bridges
A
A
Williams
G
G
Birch
A
A
Winkler
F K
FK
Robinson
J A
JA
D000595:Amino Acid Sequence; D000937:Antigen-Antibody Reactions; D015703:Antigens, CD / Q000276:immunology*; D015374:Biosensing Techniques; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D018604:Epitope Mapping; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D007371:Interferon-gamma / Q000276:immunology*; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017471:Receptors, Interferon / Q000276:immunology*; D000096743:Interferon gamma Receptor
D015703:Antigens, CD; D007140:Immunoglobulin Fab Fragments; D007371:Interferon-gamma; D046911:Macromolecular Substances; D017471:Receptors, Interferon; D000096743:Interferon gamma Receptor