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Journal of Virology, November 2008, p. 11217-11227, Vol. 82, No. 22
0022-538X/08/$08.00+0     doi:10.1128/JVI.01673-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Molecular Basis of the Interaction between Complement Receptor Type 2 (CR2/CD21) and Epstein-Barr Virus Glycoprotein gp350{triangledown}

Kendra A. Young,1 Andrew P. Herbert,2 Paul N. Barlow,2,3 V. Michael Holers,1 and Jonathan P. Hannan1,3*

Department of Medicine and Immunology, University of Colorado Denver, 1775 N. Ursula Street, Aurora, Colorado 80045,1 Edinburgh Biomolecular NMR Unit, School of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, Scotland, United Kingdom,2 Institute of Structural Biology and Molecular Biology, School of Biological Sciences, Mayfield Road, University of Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom3

Received 6 August 2008/ Accepted 2 September 2008

The binding of the Epstein-Barr virus glycoprotein gp350 by complement receptor type 2 (CR2) is critical for viral attachment to B lymphocytes. We set out to test hypotheses regarding the molecular nature of this interaction by developing an enzyme-linked immunosorbent assay (ELISA) for the efficient analysis of the gp350-CR2 interaction by utilizing wild-type and mutant forms of recombinant gp350 and also of the CR2 N-terminal domains SCR1 and SCR2 (designated CR2 SCR1-2). To delineate the CR2-binding site on gp350, we generated 17 gp350 single-site substitutions targeting an area of gp350 that has been broadly implicated in the binding of both CR2 and the major inhibitory anti-gp350 monoclonal antibody (MAb) 72A1. These site-directed mutations identified a novel negatively charged CR2-binding surface described by residues Glu-21, Asp-22, Glu-155, Asp-208, Glu-210, and Asp-296. We also identified gp350 amino acid residues involved in non-charge-dependent interactions with CR2, including Tyr-151, Ile-160, and Trp-162. These data were supported by experiments in which phycoerythrin-conjugated wild-type and mutant forms of gp350 were incubated with CR2-expressing K562 cells and binding was assessed by flow cytometry. The ELISA was further utilized to identify several positively charged residues (Arg-13, Arg-28, Arg-36, Lys-41, Lys-57, Lys-67, Arg-83, and Arg-89) within SCR1-2 of CR2 that are involved in the binding interaction with gp350. These experiments allowed a comparison of those CR2 residues that are important for binding gp350 to those that define the epitope for an effective inhibitory anti-CR2 MAb, 171 (Asn-11, Arg-13, Ser-32, Thr-34, Arg-36, and Tyr-64). The mutagenesis data were used to calculate a model of the CR2-gp350 complex using the soft-docking program HADDOCK.

* Corresponding author. Mailing address: Institute of Structural Biology and Molecular Biology, School of Biological Sciences, Mayfield Road, University of Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom. Phone: 131-650-5366. Fax: 131-650-8650. E-mail: Jonathan.Hannan{at}ed.ac.uk

{triangledown} Published ahead of print on 10 September 2008.

Journal of Virology, November 2008, p. 11217-11227, Vol. 82, No. 22
0022-538X/08/$08.00+0     doi:10.1128/JVI.01673-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kovacs, J. M., Hannan, J. P., Eisenmesser, E. Z., Holers, V. M. (2009). Mapping of the C3d Ligand Binding Site on Complement Receptor 2 (CR2/CD21) Using Nuclear Magnetic Resonance and Chemical Shift Analysis. J. Biol. Chem. 284: 9513-9520 [Abstract] [Full Text]  


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