Young Kendra A, Herbert Andrew P, Barlow Paul N, Holers V Michael, Hannan Jonathan P
Institute of Structural Biology and Molecular Biology, School of Biological Sciences, Mayfield Road, University of Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom.
J Virol. 2008 Nov;82(22):11217-27. doi: 10.1128/JVI.01673-08. Epub 2008 Sep 10.
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.
2型补体受体（CR2）与爱泼斯坦-巴尔病毒糖蛋白gp350的结合对于病毒附着于B淋巴细胞至关重要。我们着手通过开发一种酶联免疫吸附测定（ELISA）来检验关于这种相互作用分子性质的假设，该测定法利用重组gp350的野生型和突变形式以及CR2 N端结构域SCR1和SCR2（称为CR2 SCR1-2）来有效分析gp350-CR2相互作用。为了确定gp350上的CR2结合位点，我们针对gp350中一个广泛涉及CR2和主要抑制性抗gp350单克隆抗体（MAb）72A1结合的区域产生了17个gp350单点取代。这些定点突变确定了一个由谷氨酸-21、天冬氨酸-22、谷氨酸-155、天冬氨酸-208、谷氨酸-210和天冬氨酸-296残基描述的新的带负电荷的CR2结合表面。我们还确定了gp350中与CR2进行非电荷依赖性相互作用的氨基酸残基，包括酪氨酸-151、异亮氨酸-160和色氨酸-162。用藻红蛋白偶联的野生型和突变型gp350与表达CR2的K562细胞孵育，并通过流式细胞术评估结合情况的实验支持了这些数据。ELISA进一步用于确定CR2的SCR1-2内几个带正电荷的残基（精氨酸-13、精氨酸-28、精氨酸-36、赖氨酸-41、赖氨酸-57、赖氨酸-67、精氨酸-83和精氨酸-89），它们参与与gp350的结合相互作用。这些实验使得能够比较那些对于结合gp350重要的CR2残基与那些定义有效抑制性抗CR2 MAb 171（天冬酰胺-11、精氨酸-13、丝氨酸-32、苏氨酸-34、精氨酸-36和酪氨酸-64）表位的残基。诱变数据用于使用软对接程序HADDOCK计算CR2-gp350复合物的模型。
