Abstract
The Fc region of IgG antibodies (Cγ2 and Cγ3 domains) is responsible for effector functions such as antibody-dependent cell-mediated cytotoxicity and phagocytosis, through engagement with Fcγ receptors, although the ability to elicit these functions differs between the four human IgG subclasses. A key determinant of Fcγ receptor interactions is the FG loop in the Cγ2 domain. High resolution cryogenic IgG4-Fc crystal structures have revealed a unique conformation for this loop, which could contribute to the particular biological properties of this subclass. To further explore the conformation of the IgG4 Cγ2 FG loop at near-physiological temperature, we solved a 2.7Å resolution room temperature structure of recombinant human IgG4-Fc from crystals analysed in situ. The Cγ2 FG loop in one chain differs from the cryogenic structure, and adopts the conserved conformation found in IgG1-Fc; however, this conformation participates in extensive crystal packing interactions. On the other hand, at room temperature, and free from any crystal packing interactions, the Cγ2 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 Cγ2 FG loop.
Highlights
IgG effector functions, such as antibody-dependent cellmediated cytotoxicity, antibody-dependent cellular phagocytosis and complement activation, are mediated by the antibody Fc region (C␥2 and C␥3 domains)
While the conformation of the C␥2 domain FG loop is conserved in IgG1, high resolution cryogenic crystal structures of IgG4-Fc revealed a different, unique conformation for the C␥2 FG loop, which would disrupt the interaction with Fc␥ receptors (Davies et al, 2014b)
The asymmetric unit of the room temperature (RT) recombinant human IgG4-Fc crystal structure solved from crystals in situ contains one Fc molecule, comprising two chains (A and B)
Summary
IgG effector functions, such as antibody-dependent cellmediated cytotoxicity, antibody-dependent cellular phagocytosis and complement activation, are mediated by the antibody Fc region (C␥2 and C␥3 domains). Antibody determinants that influence the affinity for Fc␥ receptors include sequence variation in the C␥2 domain and hinge region, and the composition of the oligosaccharide moiety attached to the C␥2 domain (Canfield and Morrison, 1991; Shields et al, 2002). While the conformation of the C␥2 domain FG loop is conserved in IgG1, high resolution cryogenic crystal structures of IgG4-Fc revealed a different, unique conformation for the C␥2 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 C␥2 FG loop could adopt the conserved IgG1-like conformation. 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
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