Molecular basis of the interaction between complement receptor type 2 (CR2/CD21) and Epstein-Barr virus glycoprotein gp350.
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.
Young
Kendra A
KA
Institute of Structural Biology and Molecular Biology, School of Biological Sciences, Mayfield Road, University of Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom. Jonathan.Hannan@ed.ac.uk
Herbert
Andrew P
AP
Barlow
Paul N
PN
Holers
V Michael
VM
Hannan
Jonathan P
JP
D019943:Amino Acid Substitution; D001665:Binding Sites; D045744:Cell Line, Tumor; D004854:Herpesvirus 4, Human / Q000502:physiology*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D017354:Point Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D017464:Receptors, Complement 3d / Q000235:genetics / Q000378:metabolism*; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D053585:Virus Attachment
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D050505:Mutant Proteins; D017464:Receptors, Complement 3d; D014763:Viral Matrix Proteins
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