Accelerated disassembly of IgE–receptor complexes by a disruptive macromolecular inhibitor

Abstract

IgE antibodies bind the high affinity IgE Fc receptor (FcεRI), found primarily on mast cells and basophils, and trigger inflammatory cascades of the allergic response1,2. Inhibitors of IgE:FcεRI binding have been identified and an anti-IgE therapeutic antibody (omalizumab) is used to treat severe allergic asthma3,4. However, preformed IgE:FcεRI complexes that prime cells prior to allergen exposure dissociate extremely slowly5 and cannot be disrupted by strictly competitive inhibitors. IgE-Fc conformational flexibility indicated that inhibition could be mediated by allosteric or other non-classical mechanisms6–8. Here we demonstrate that an engineered protein inhibitor, DARPin E2_799–11, acts through a non-classical inhibition mechanism, not only blocking IgE:FcεRI interactions, but actively stimulating the dissociation of preformed ligand-receptor complexes. The structure of the E2_79:IgE-Fc3-4 complex predicts the presence of two non-equivalent E2_79 sites in the asymmetric IgE:FcεRI complex, with Site 1 distant from the receptor and Site 2 exhibiting partial steric overlap. While the structure is suggestive of an allosteric inhibition mechanism, mutational studies and quantitative kinetic modeling indicate that E2_79 acts through a facilitated dissociation mechanism at Site 2 alone. These results demonstrate that high affinity IgE:FcεRI complexes can be actively dissociated to block the allergic response and suggest that protein:protein complexes may be more generally amenable to active disruption by macromolecular inhibitors.

SplitCore Technology Allows Efficient Production of Virus-Like Particles Presenting a Receptor-Contacting Epitope of Human IgE

“…Two subdomains of the one heavy chain, namely the FG loop apical residues (H 424 LPR 427 ) and the Ce2-Ce3 linker, bind to P426 pocket, whereas four subdomains of the other heavy chain, namely the FG loop, the Ce2-Ce3 linker, BC and DE loops bind to the Y131 receptor pocket. Involvement of the FG loop in the receptor recognition is underscored by the fact that the binding site for the blocking antibody Omalizumab maps to the FG loop [32,33]. Another demonstration of utility of the FG loop for the development of anti-IgE vaccines came from mapping of the IgE epitopes using synthetic peptides [24,34].…”

Facilitated Dissociation of a Nucleoid Protein from the Bacterial Chromosome

“…However, this picture has been challenged by a number of recent in vitro experiments which have shown off-rates of proteins from duplex DNA that depend on bulk protein concentrations (2)(3)(4)(5). Similar effects have been observed for singlestranded DNA (ssDNA)-binding proteins (6,7), antibody-ligand binding (8), and ssDNA-ssDNA interactions (9).…”

Effects of electrostatic interactions on ligand dissociation kinetics

“…The free ligands can be identical to the initially bound ligand [21,22,[27][28][29][30]. However, different ligand molecules [27,30,31] or even nucleic acid fragments [20] can lead to the facilitated dissociation (FD). According to the proposed molecular mechanism for FD, a free ligand binds to an already occupied binding site and destabilizes the complex by forming a ternary complex (i.e., two ligands on the same site) [19,23,27].…”

“…The free ligands can be identical to the initially bound ligand [21,22,[27][28][29][30]. However, different ligand molecules [27,30,31] or even nucleic acid fragments [20] can lead to the facilitated dissociation (FD).…”