(B) In the cryogenic structure, the C2 FG loop (yellow) adopts the unique, IgG4-like conformation and forms a 2882interface with a symmetry-related molecule (gray). FG loop in the other chain adopts the conformation previously observed in the cryogenic IgG4-Fc structures, despite both conformations being accessible. The room temperature human IgG4-Fc structure thus provides a more complete and physiologically relevant description of the conformation of this functionally critical C2 FG loop. == 1 . Introduction == IgG effector functions, such as antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and complement activation, are mediated by the antibody Fc region (C2 and C3 domains). The IgG4 subclass binds certain Fc receptors with lower affinity than IgG1 and IgG3 (Bruhns et al., 2009), and does not activate complement (van der Zee et al., 1986). Antibody determinants that influence the affinity for Fc receptors include sequence variation Apocynin (Acetovanillone) in the C2 domain and hinge region, and the composition of the oligosaccharide moiety attached to the C2 domain (Canfield and Morrison, 1991, Shields et al., 2002). The C2 domain FG loop (residues 325331) plays a crucial role in the interaction with Fc receptors, in which Pro329 from the FG loop forms a hydrophobic proline sandwich interaction with two tryptophan residues from the receptor (Sondermann et al., 2000), and is also involved in the Apocynin (Acetovanillone) interaction between IgG1/3 and C1q (Canfield and Rabbit Polyclonal to OR52E5 Morrison, 1991, Tao et al., 1991, Tao et al., 1993, Idusogie et al., 2000). While the conformation of the C2 domain FG loop is conserved in IgG1, high resolution cryogenic crystal structures of IgG4-Fc revealed a different, unique conformation for the C2 FG loop, which would disrupt the interaction with Fc receptors (Davies et al., 2014b). Subsequent cryogenic crystal structures of IgG4-Fc (Davies et al., 2014a) and intact IgG4 (Scapin et al., 2015) revealed that the IgG4 C2 FG loop could also adopt the conserved IgG1-like conformation. However , the role of the unique loop conformation in modulating the biological properties of IgG4, and whether one, or both, conformations could be adopted at physiological temperature, and in solution, remains unclear. Using a technique to collect data from crystalsin situ(Axford et al., 2015), we solved a 2 . 7 resolution room temperature (RT) structure of recombinant human IgG4-Fc. The RT IgG4-Fc structure reveals conformational diversity in the C2 FG Apocynin (Acetovanillone) loop. In contrast to the cryogenic structure, the FG loop adopts the IgG1-like conformation in one C2 domain, with substantial changes to the crystal packing interactions at the higher temperature which would preclude the unique conformation due to steric clashes. On the other hand, the FG loop from the other C2 domain is able to adopt either conformation in fact it adopts the unique, IgG4-like conformation at room temperature, a conformation that would disrupt the interaction with Fc receptors. == 2 . Materials and methods == == 2 . 1 . Protein production and crystallisation == Recombinant, glycosylated human IgG4-Fc was produced and crystals were grown as described previously (Davies et al., 2014b), with the following modification: a Greiner Bio-One CrystalQuick X plate was set up using a reservoir volume of 20 L, and drops comprising 0. 5 L protein (3 mg/mL) and 0. 5 L reservoir. Crystals typically started to appear after one day. == 2 . 2 . Data collection, structure determination and refinement == Data were collected at room temperature (293 K) at beamline I03 at the Diamond Light Source (Harwell, UK) from crystalsin situ. Small wedges (typically 36) of data were collected Apocynin (Acetovanillone) from different crystals, or spatially distinct regions from a single crystal, using an oscillation per image of 0. 2 . For multiple datasets collected from a single.
(B) In the cryogenic structure, the C2 FG loop (yellow) adopts the unique, IgG4-like conformation and forms a 2882interface with a symmetry-related molecule (gray)
