Crystallographic and solution studies have shown that IgE molecules are acutely bent in their Fc region. cross-linking by allergen leads to cell degranulation, release of inflammatory mediators and an immediate allergic response. Disruption of the IgE-FcRI conversation is usually a validated strategy for therapeutic intervention in allergic diseases including asthma: an anti-IgE monoclonal IgG antibody, omalizumab (Xolair?, Novartis Pharmaceuticals Ltd), inhibits IgE binding to FcRI and is effective in the Tarafenacin treatment of severe persistent asthma and other allergic diseases2. IgE consists of a dimer of two identical heavy and two identical light chains, but unlike IgG in which the antigen-binding Fab region is separated from the receptor-binding Fc region by a flexible hinge, IgE contains an additional disulphide-linked pair of domains, (C2)2, forming a (C2-C3-C4)2 dimer1. Fluorescence depolarisation studies to assess segmental flexibility have shown IgE to be less flexible than IgG3-6, and F?rster resonance energy transfer (FRET) studies that determined distances both intra-molecular and to the membrane led to a model of a compact, bent structure both for IgE free in solution and when bound to FcRI6-9. Although an extended model was also proposed10, X-ray and neutron scattering studies in solution confirmed that IgE and IgE-Fc adopt a compact, bent structure11,12. Nevertheless no one anticipated the acutely and asymmetrically bent conformation that was subsequently observed in the crystal structure of IgE-Fc (Fig. 1a)13. In this bent structure, the (C2)2 domain name pair folds back onto the C3-C4 domains, Tarafenacin forming an extensive intra-molecular interface (1,520?2). The subsequent structure of the complex of IgE-Fc bound to the extracellular domains of the FcRI -chain (sFcRI) revealed an even more acute bend upon receptor binding14, consistent with FRET and fluorescence depolarisation studies that indicated reduced segmental flexibility6,15,16. At this point the presence of an extended conformation of IgE-Fc was all but dismissed. Physique 1 Bent and extended structures adopted by IgE-Fc. Although the C2 domains are not directly involved in binding FcRI, they do contribute to the kinetics of the conversation, decreasing both the association and dissociation rate constants14,17. Interest in their structural and functional role intensified following the discovery that this Fab fragment of omalizumab binds to a partially unbent conformation of IgE-Fc, as detected in a FRET experiment16. This first indication that IgE-Fc may not always be bent, raises the question of whether the molecule transiently explores more extended conformations, and perhaps even flips between bent structures with the C2 domains folded back on opposite sides of the C3-C4 domains. Trapping of transiently populated conformational says has previously been achieved by antibody binding18, and so to explore the potential conformational diversity of IgE-Fc we generated an IgG antibody Fab fragment that binds to IgE-Fc (anti–chain Fab; aFab) and discovered that it had captured an extended conformation. RESULTS Structure of IgE-Fc bound by two aFab fragments The aFab-IgE-Fc crystal structure was solved at 2.9? resolution (see Table 1 for data collection and refinement statistics). Remarkably, the IgE-Fc adopts a fully extended conformation, with two aFab molecules bound, one on each side of the almost perfectly symmetrical IgE-Fc (aFab1-IgE-Fc-aFab2, Fig. 1b and c). Compared with the structure of IgE-Fc alone, the molecule has undergone a drastic unbending of 120 (Fig. 1a and c), losing completely the extensive intra-molecular interface between the C2 and C3-C4 domains. This Tarafenacin unbending appears to derive largely Rabbit Polyclonal to MLKL. from movements in the C2-C3 linker region, in particular residues Pro333, Arg334 and Gly335 (Supplementary Video 1). While.