title: Molecular basis of the interaction between complement receptor type 2 (CR2/CD21) and Epstein-Barr virus glycoprotein gp350.
abstract: 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.
authors.affiliation: 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
authors.forename: Kendra A
authors.initials: KA
authors.lastname: Young
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Herbert
authors.forename: Paul N
authors.initials: PN
authors.lastname: Barlow
authors.forename: V Michael
authors.initials: VM
authors.lastname: Holers
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Hannan
mesh_terms: 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
chemical_list: C037662:EBV-associated membrane antigen, Epstein-Barr virus; D050505:Mutant Proteins; D017464:Receptors, Complement 3d; D014763:Viral Matrix Proteins
references.citation: Science. 2001 Jun 1;292(5522):1725-8
references.pmid: 11387479
references.citation: J Exp Med. 1991 Dec 1;174(6):1299-311
references.pmid: 1660522
references.citation: J Immunol. 2006 Jul 1;177(1):383-94
references.pmid: 16785534
references.citation: Proc Natl Acad Sci U S A. 1984 Feb;81(3):881-5
references.pmid: 6230668
references.citation: J Virol. 1985 Jun;54(3):665-74
references.pmid: 2987520
references.citation: J Virol. 2000 Jul;74(14):6324-32
references.pmid: 10864642
references.citation: Proteins. 2005 Aug 1;60(2):232-8
references.pmid: 15981252
references.citation: Proc Natl Acad Sci U S A. 1984 Jul;81(14):4510-4
references.pmid: 6087328
references.citation: J Mol Biol. 2007 Jun 1;369(2):567-83
references.pmid: 17434528
references.citation: EMBO J. 1991 Apr;10(4):919-26
references.pmid: 1849076
references.citation: J Biol Chem. 2007 Dec 14;282(50):36614-25
references.pmid: 17925391
references.citation: J Virol. 1987 May;61(5):1416-20
references.pmid: 3033269
references.citation: J Immunol. 2001 Aug 1;167(3):1490-9
references.pmid: 11466369
references.citation: Proc Natl Acad Sci U S A. 1985 Mar;82(5):1490-3
references.pmid: 2983347
references.citation: Vaccine. 1999 Feb 26;17(7-8):660-8
references.pmid: 10067671
references.citation: J Med Virol. 1996 Nov;50(3):263-71
references.pmid: 8923292
references.citation: Proc Natl Acad Sci U S A. 1980 Sep;77(9):5307-11
references.pmid: 6254073
references.citation: Biochemistry. 2001 May 22;40(20):5931-41
references.pmid: 11352728
references.citation: J Virol. 1999 Sep;73(9):7722-33
references.pmid: 10438862
references.citation: Virology. 2001 Nov 10;290(1):106-14
references.pmid: 11882994
references.citation: J Virol. 1988 Dec;62(12):4452-64
references.pmid: 2460635
references.citation: J Immunol. 2001 Nov 15;167(10):5758-66
references.pmid: 11698449
references.citation: J Immunol. 2004 Jun 15;172(12):7537-47
references.pmid: 15187133
references.citation: J Infect Dis. 2007 Dec 15;196(12):1749-53
references.pmid: 18190254
references.citation: J Immunol. 1991 Jul 15;147(2):590-4
references.pmid: 1830068
references.citation: J Mol Biol. 2005 Feb 25;346(3):845-58
references.pmid: 15713467
references.citation: Nat Struct Mol Biol. 2006 Nov;13(11):996-1001
references.pmid: 17072314
references.citation: Proc Natl Acad Sci U S A. 2004 Nov 16;101(46):16286-91
references.pmid: 15534216
references.citation: J Mol Biol. 2005 Feb 25;346(3):859-73
references.pmid: 15713468
references.citation: Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10641-6
references.pmid: 12122212
references.citation: J Virol. 1990 Mar;64(3):1348-52
references.pmid: 2154612
references.citation: Immunol Rev. 2001 Apr;180:146-61
references.pmid: 11414356
references.citation: J Virol. 1989 Dec;63(12):4998-5005
references.pmid: 2555536
references.citation: J Virol. 2007 Aug;81(15):7825-32
references.pmid: 17459936
references.citation: J Immunol. 1994 Jun 15;152(12):5806-13
references.pmid: 7515913
references.citation: Nature. 1992 Aug 6;358(6386):505-7
references.pmid: 1386409
references.citation: J Virol. 1985 Aug;55(2):347-51
references.pmid: 2410629
references.citation: Immunity. 2001 Nov;15(5):775-85
references.pmid: 11728339
references.citation: J Virol. 2000 Nov;74(21):10142-52
references.pmid: 11024143
references.citation: Cell. 1989 Feb 10;56(3):369-77
references.pmid: 2464439
references.citation: J Am Chem Soc. 2003 Feb 19;125(7):1731-7
references.pmid: 12580598
references.citation: J Biol Chem. 2005 Oct 21;280(42):35598-605
references.pmid: 16087675