ABSTRACT
Fc gamma RIIa is one of a family of specific cell surface receptors for immunoglobulin. Fc gamma RIIa, which binds immune complexes of certain IgG isotypes, plays important roles in immune homeostasis. However, the precise characteristics of IgG binding and three-dimensional structure of Fc gamma RIIa have not been reported. This study describes the affinity of the Fc gamma RIIa:IgG interaction as well as biochemical characterisation of recombinant Fc gamma RIIa that has been used to generate high quality crystals. Equilibrium binding analysis of the Fc gamma RII:IgG interaction found, IgG3 binds with an affinity of K(D) = 0.6 microM, as expected. Unlike other Fc gamma R, IgG4 also bound to Fc gamma RIIa, K(D) = 3 microM, clearly establishing Fc gamma RIIa as an IgG4 receptor. Biochemical analysis of mammalian and insect cell derived Fc gamma RIIa established the genuine N-terminus with Q being the first amino acid in the sequence Q, A, A, A, P... extending the N-terminus further than previously thought. Furthermore, both potential N-linked glycosylation sites are occupied. Electrospray ionisation mass spectrometry (ESMS) indicate that the N-glycans of baculovirus derived Fc gamma RIIa are core mannose oligosaccharide side chains. Finally, we describe the first crystallisation of diffraction quality crystals of soluble Fc gamma RIIa. Orthorhombic crystals diffract X-rays beyond 2.1 A resolution in the space group P2(1)2(1)2 with cell dimensions a = 78.8 A, b = 100.5 A, c = 27.8 A. This marks a significant advance towards understanding the three-dimensional structure of Fc gamma RIIa and related FcR proteins that share high amino acid identity with Fc gamma RIIa.
Subject(s)
Antigens, CD/chemistry , Antigens, CD/isolation & purification , Immunoglobulin G/metabolism , Receptors, IgG/chemistry , Receptors, IgG/isolation & purification , Animals , Antibody Affinity , Antigens, CD/metabolism , Binding Sites, Antibody , CHO Cells , Cricetinae , Crystallization , Crystallography, X-Ray , Humans , Mass Spectrometry , Receptors, IgG/metabolism , Recombinant Proteins/isolation & purification , SolubilityABSTRACT
Fc gamma receptors bind IgG to initiate cellular responses against pathogens and soluble antigens. We have determined the three-dimensional structure of the extracellular portion of human Fc gammaRIIa to 2.0 A resolution providing a structural basis for the unique functions of the leukocyte FcR family. The receptor is composed of two immunoglobulin domains and arranged to expose the ligand-binding site at one end of domain 2. Using alanine mutants we find that the binding sites for IgG1 and 2 are similar but the relative importance of specific regions on the receptor varies. In crystals, Fc gammaRIIa molecules associate to resemble V(L)V(H) dimers, suggesting that two Fc gammaRIIa molecules could cooperate to bind IgG in an asymmetric manner.
Subject(s)
Antigens, CD/chemistry , Leukocytes/chemistry , Receptors, IgG/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Binding Sites , COS Cells , Conserved Sequence , Crystallization , Crystallography, X-Ray , Dimerization , Humans , Immunoglobulin G/metabolism , Immunoglobulins/chemistry , Ligands , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Receptors, IgG/genetics , Receptors, IgG/metabolism , TransfectionABSTRACT
HEK is a member of the EPH-like receptor tyrosine kinase family, which appear to have roles in development and oncogenesis. Recently, we purified a soluble HEK ligand which is also a ligand (AL1) for the HEK-related receptor EHK1. Promiscuity appears to be a characteristic feature of interactions between the EPH-like receptors and their ligands, termed ligands for EPH-related kinases (LERKs). This prompted us to analyze the interactions between the HEK exodomain and fusion proteins comprising candidate LERKs and the Fc portion of human IgG1 (Fc) or a FLAGTM-peptide tag by surface plasmon resonance, size exclusion high performance liquid chromatography, sedimentation equilibrium, and transphosphorylation. Our results indicate that AL1/LERK7 is the preferred high-affinity ligand for HEK, forming a stable 1:1 complex with a dissociation constant of 12 nM. As expected the apparent affinities of bivalent fusion proteins of LERKs and the Fc portion of human IgG1 had significantly reduced dissociation rates compared with their monovalent, FLAGTM-tagged derivatives. High-avidity binding of monovalent ligands can be achieved by antibody-mediated cross-linking of monovalent ligands and with LERK7 results in specific phosphorylation of the receptor. By extrapolation, our findings indicate that some of the reported LERK-receptor interactions are a consequence of the use of bivalent ligand or receptor constructs and may be functionally irrelevant.