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* Division of Structural Biology, Biozentrum, University of Basel, Basel, Switzerland;
Division of Immunopathology, Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Vienna, Austria;
Paul Ehrlich Institut, Department of Allergology, Langen, Germany;
Allergie-Ambulatorium Reumannplatz, Vienna, Austria;
¶ Christian Doppler Laboratory for Allergy Research, Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, 1090 Vienna, Austria
We report the three-dimensional structure of the complex between the major respiratory grass pollen allergen Phl p 2 and its specific human IgE-derived Fab. The Phl p 2-specific human IgE Fab has been isolated from a combinatorial library constructed from lymphocytes of a pollen allergic patient. When the variable domains of the IgE Fab were grafted onto human IgG1, the resulting Ab (huMab2) inhibited strongly the binding of allergic patients’ IgE to Phl p 2 as well as allergen-induced basophil degranulation. Analysis of the binding of the allergen to the Ab by surface plasmon resonance yielded a very low dissociation constant (KD = 1.1 x 10–10 M), which is similar to that between IgE and Fc
;RI. The structure of the Phl p 2/IgE Fab complex was determined by x-ray crystallography to 1.9 Å resolution revealing a conformational epitope (876 Å2) comprised of the planar surface of the four-stranded anti-parallel β-sheet of Phl p 2. The IgE-defined dominant epitope is discontinuous and formed by 21 residues located mostly within the β strands. Of the 21 residues, 9 interact directly with 5 of the 6 CDRs (L1, L3, H1, H2, H3) of the IgE Fab predominantly by hydrogen bonding and van der Waals interactions. Our results indicate that IgE Abs recognize conformational epitopes with high affinity and provide a structural basis for the highly efficient effector cell activation by allergen/IgE immune complexes.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This project was supported by the Swiss National Foundation Grant 31-116804 (to Z.M.-H.), by Grant 813003 of the Austrian Research Promotion Agency and BIOMAY, Vienna, Austria to (S.F.), by Grant F1815 of the Austrian Science Fund (to R.V.), by a research grant from BIOMAY, Vienna, Austria, and the Christian Doppler Research Association, Vienna, Austria.
Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 2vxq and r2vxqsf, respectively.
2 S.P. and S.F. contributed equally to this study.
3 R.V. and Z.M.-H. contributed equally to this study.
4 Address correspondence and reprint requests to Prof. Rudolf Valenta, Christian Doppler Laboratory for Allergy Research, Division of Immunopathology, Department of Pathophysiology, Center for Physiology and Pathophysiology, Vienna General Hospital, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: rudolf.valenta{at}meduniwien.ac.at
5 Abbreviations used in this paper: RU, resonance units; PEG, polyethylene glycol; CEP, conformational epitope prediction; BLG, β-lactoglobulin.
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