ABSTRACT
Fc receptors play a major role in immune defenses against pathogens and in inflammatory processes. The crystal structure of a human immunoglobulin receptor, FcgammaRIIIb, has been determined to 1.8 A resolution. The overall fold consists of two immunoglobulin-like domains with an acute interdomain hinge angle of approximately 50 degrees. Trp-113, wedged between the N-terminal D1 and the C-terminal D2 domains, appears to further restrict the hinge angle. The putative Fc binding region of the receptor carries a net positive charge complementary to the negative-charged receptor binding regions on Fc. A 1:1 binding stoichiometry between the receptor and Fc was measured by both the equilibrium and nonequilibrium size-exclusion chromatography. Two separate parallel dimers are observed in the crystal lattice, offering intriguing models for receptor aggregation.
Subject(s)
Extracellular Space/chemistry , Extracellular Space/immunology , Receptors, IgG/chemistry , Amino Acid Sequence , Binding Sites , Chromatography, Gel , Conserved Sequence , Crystallization , Crystallography, X-Ray , Dimerization , Extracellular Space/metabolism , Humans , Immunoglobulin Fragments/chemistry , Ligands , Models, Molecular , Molecular Sequence Data , Oligopeptides/chemistry , Oligopeptides/immunology , Oligopeptides/metabolism , Protein Structure, Tertiary , Receptors, IgG/metabolism , Sequence Homology, Amino AcidABSTRACT
Expression systems have been designed to test the suitability of expressing the high cysteine containing extracellular domain (residues 1-136) of human transforming growth factor beta type II receptor (TbetaRII). Receptor expressed using a baculovirus system was functional following both enzymatic deglycosylation and elimination of the N-terminal 22 amino acids by protease degradation. Bacterial expression of a TbetaRII lacking the 26 N-terminal amino acids retained the ability to bind its ligand, TGF-beta1. Receptor expressed in bacteria was sensitive to proteolytic degradation at residue Lys98 but a K98T mutation eliminated degradation and did not disrupt binding. Although several different forms of TbetaRII were expressed, only a fusion with glutathione S-transferase gave soluble TbetaRII, which was purified at a yield of 0.1 mg/10 L of bacterial growth. N-Terminal truncations of TbetaRII (residues 22-136 or 27-136) could be refolded from inclusion bodies and purified to an active form with an efficiency of 10%.
