Hannan Jonathan P, Young Kendra A, Guthridge Joel M, Asokan Rengasamy, Szakonyi Gerda, Chen Xiaojiang S, Holers V Michael
Department of Medicine and Immunology, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262, USA.
J Mol Biol. 2005 Feb 25;346(3):845-58. doi: 10.1016/j.jmb.2004.12.007. Epub 2005 Jan 8.
We have characterized the interaction between the first two short consensus repeats (SCR1-2) of complement receptor type 2 (CR2, CD21) and C3d in solution, by utilising the available crystal structures of free and C3d-bound forms of CR2 to create a series of informative mutations targeting specific areas of the CR2-C3d complex. Wild-type and mutant forms of CR2 were expressed on the surface of K562 erythroleukemia cells and their binding ability assessed using C3dg-biotin tetramers complexed to fluorochrome conjugated streptavidin and measured by flow cytometry. Mutations directed at the SCR2-C3d interface (R83A, R83E, G84Y) were found to strongly disrupt C3dg binding, supporting the conclusion that the SCR2 interface reflected in the crystal structure is correct. Previous epitope and peptide mapping studies have also indicated that the PILN11GR13IS sequence of the first inter-cysteine region of SCR1 is essential for the binding of iC3b. Mutations targeting residues within or in close spatial proximity to this area (N11A, N11E, R13A, R13E, Y16A, S32A, S32E), and a number of other positively charged residues located primarily on a contiguous face of SCR1 (R28A, R28E, R36A, R36E, K41A, K41E, K50A, K50E, K57A, K57E, K67A, K67E), have allowed us to reassess those regions on SCR1 that are essential for CR2-C3d binding. The nature of this interaction and the possibility of a direct SCR1-C3d association are discussed extensively. Finally, a D52N mutant was constructed introducing an N-glycosylation sequence at an area central to the CR2 dimer interface. This mutation was designed to disrupt the CR2-C3d interaction, either directly through steric inhibition, or indirectly through disruption of a physiological dimer. However, no difference in C3dg binding relative to wild-type CR2 could be observed for this mutant, suggesting that the dimer may only be found in the crystal form of CR2.
我们利用补体受体2（CR2，CD21）游离形式和C3d结合形式的现有晶体结构，对溶液中CR2的前两个短共有重复序列（SCR1-2）与C3d之间的相互作用进行了表征，以针对CR2-C3d复合物的特定区域创建一系列信息丰富的突变。CR2的野生型和突变型在K562红白血病细胞表面表达，并使用与荧光染料偶联的链霉亲和素复合的C3dg-生物素四聚体评估其结合能力，并通过流式细胞术进行测量。发现针对SCR2-C3d界面的突变（R83A、R83E、G84Y）强烈破坏C3dg结合，支持晶体结构中反映的SCR2界面是正确的这一结论。先前的表位和肽图谱研究也表明，SCR1第一个半胱氨酸间区域的PILN11GR13IS序列对于iC3b的结合至关重要。针对该区域内或空间上与之接近的残基的突变（N11A、N11E、R13A、R13E、Y16A、S32A、S32E），以及主要位于SCR1相邻面上的许多其他带正电荷的残基（R28A、R28E、R36A、R36E、K41A、K41E、K50A、K50E、K57A、K57E、K67A、K67E），使我们能够重新评估SCR1上对CR2-C3d结合至关重要的那些区域。本文广泛讨论了这种相互作用的性质以及直接的SCR1-C3d缔合的可能性。最后，构建了一个D52N突变体，在CR2二聚体界面中心区域引入了一个N-糖基化序列。该突变旨在直接通过空间位阻抑制或间接通过破坏生理性二聚体来破坏CR2-C3d相互作用。然而，该突变体相对于野生型CR2在C3dg结合方面未观察到差异，这表明二聚体可能仅存在于CR2的晶体形式中。
