Biochemical analysis and crystallisation of FcγRIIa, the low affinity receptor for IgG

FcγRIIa is one of a family of specific cell surface receptors for immunoglobulin. FcγRIIa, 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 FcγRIIa have not been reported. This study describes the affinity of the FcγRIIa:IgG interaction as well as biochemical characterisation of recombinant FcγRIIa that has been used to generate high quality crystals. Equilibrium binding analysis of the FcγRII:IgG interaction found, IgG3 binds with an affinity of K D=0.6 μM, as expected. Unlike other FcγR, IgG4 also bound to FcγRIIa, K D=3 μM, clearly establishing FcγRIIa as an IgG4 receptor. Biochemical analysis of mammalian and insect cell derived FcγRIIa 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 FcγRIIa are core mannose oligosaccharide side chains. Finally, we describe the first crystallisation of diffraction quality crystals of soluble FcγRIIa. Orthorhombic crystals diffract X-rays beyond 2.1 Å resolution in the space group P2 12 12 with cell dimensions a=78.8 Å, b=100.5 Å, c=27.8 Å. This marks a significant advance towards understanding the three-dimensional structure of FcγRIIa and related FcR proteins that share high amino acid identity with FcγRIIa.