title: 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 response. Inhibitors of IgE-FcεRI binding have been identified and an anti-IgE therapeutic antibody (omalizumab) is used to treat severe allergic asthma. However, preformed IgE-FcεRI complexes that prime cells before allergen exposure dissociate extremely slowly and cannot be disrupted by strictly competitive inhibitors. IgE-Fc conformational flexibility indicated that inhibition could be mediated by allosteric or other non-classical mechanisms. Here we demonstrate that an engineered protein inhibitor, DARPin E2_79 (refs 9, 10, 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-Fc(3-4) 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. Although the structure is indicative of an allosteric inhibition mechanism, mutational studies and quantitative kinetic modelling 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.
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Beomkyu
authors.initials: B
authors.lastname: Kim
authors.forename: Alexander
authors.initials: A
authors.lastname: Eggel
authors.forename: Svetlana S
authors.initials: SS
authors.lastname: Tarchevskaya
authors.forename: Monique
authors.initials: M
authors.lastname: Vogel
authors.forename: Heino
authors.initials: H
authors.lastname: Prinz
authors.forename: Theodore S
authors.initials: TS
authors.lastname: Jardetzky
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D017089:Ankyrin Repeat; D001665:Binding Sites / Q000187:drug effects; D018360:Crystallography, X-Ray; D005453:Fluorescence; D007073:Immunoglobulin E / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D009154:Mutation; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D017455:Receptors, IgE / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D020349:Surface Plasmon Resonance
chemical_list: C577317:DARPin E2 79; D008024:Ligands; D050505:Mutant Proteins; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E