Structural Basis of Multivalent Binding to Wheat Germ Agglutinin
Structural Basis of Multivalent Binding to Wheat Germ Agglutinin
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DOI：
10.1021/ja101646k
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发表时间：
2010-06-30
影响因子：
15
通讯作者：
Wittmann, Valentin
中科院分区：
文献类型：
--
作者：
Schwefel, David;Maierhofer, Caroline;Wittmann, Valentin
通过定制的多价配体抑制碳水化合物蛋白质相互作用是治疗许多人类疾病的有力策略。这种方法的成功的关键是理解如何实现多价配体的结合增强的分子机制。本文合成了一系列N-乙酰氨基葡萄糖（GlcNAc）衍生物，并通过酶联凝集素测定（ELLA）和X射线晶体学方法研究了它们与植物凝集素小麦胚芽凝集素（WGA）的相互作用。通过核磁共振光谱确定了一个配体的溶液构象。采用具有α-构型的GlcNAc氨基甲酸酯基序并通过间隔区长度的系统变化，我们能够鉴定具有前所未有的高WGA结合效力的二价配体。最佳的二价配体具有9.8 μ M（ELLA）的IC（50）值，对应于2350（在价态校正的基础上为1170，即，每摩尔含糖）与游离GlcNAc相比。WGA和第二好的、密切相关的二价配体的复合物的X射线晶体学解释了这种活性。四个二价分子同时与WGA结合，每个配体桥接相邻的结合位点。这首次表明WGA二聚体的所有八个糖结合位点同时起作用。我们还报道了一种四价新糖肽，其IC（50）值为0.9 μ M，比GlcNAc（6400倍）高25500倍，并对其与戊二醛交联的WGA复合物进行了X射线结构分析。新糖肽的晶体结构和溶液核磁共振结构的比较以及ELLA的结果表明，溶液中糖肽的构象已经以支持与蛋白质多价结合的方式进行了预组织。我们的研究结果表明，桥接相邻的蛋白质结合位点的多价配体是一种有效的策略，以找到高亲和力的蛋白质配体，即使是微妙的变化的接头结构可以有显着的影响的结合亲和力。
The inhibition of carbohydrate protein interactions by tailored multivalent ligands is a powerful strategy for the treatment of many human diseases. Crucial for the success of this approach is an understanding of the molecular mechanisms as to how a binding enhancement of a multivalent ligand is achieved. We have synthesized a series of multivalent N-acetylglucosamine (GIcNAc) derivatives and studied their interaction with the plant lectin wheat germ agglutinin (WGA) by an enzyme-linked lectin assay (ELLA) and X-ray crystallography. The solution conformation of one ligand was determined by NMR spectroscopy. Employing a GIcNAc carbamate motif with alpha-configuration and by systematic variation of the spacer length, we were able to identify divalent ligands with unprecedented high WGA binding potency. The best divalent ligand has an IC(50) value of 9.8 mu M (ELLA) corresponding to a relative potency of 2350 (1170 on a valency-corrected basis, i.e., per mol sugar contained) compared to free GIcNAc. X-ray crystallography of the complex of WGA and the second best, closely related divalent ligand explains this activity. Four divalent molecules simultaneously bind to WGA with each ligand bridging adjacent binding sites. This shows for the first time that all eight sugar binding sites of the WGA dimer are simultaneously functional. We also report a tetravalent neoglycopeptide with an IC(50) value of 0.9 mu M being 25 500 times higher than that of GIcNAc (6400 times per contained sugar) and the X-ray structure analysis of its complex with glutaraldehyde-cross-linked WGA. Comparison of the crystal structure and the solution NMR structure of the neoglycopeptide as well as results from the ELLA suggest that the conformation of the glycopeptide in solution is already preorganized in a way supporting multivalent binding to the protein. Our findings show that bridging adjacent protein binding sites by multivalent ligands is a valid strategy to find high-affinity protein ligands and that even subtle changes of the linker structure can have a significant impact on the binding affinity.