Physikalische Biochemie , Universität Potsdam , Karl-Liebknecht-Str. 24-25 , 14476 Potsdam , Germany.
Max Planck Institute of Colloids and Interfaces , Am Mühlenberg 1 , 14476 Potsdam , Germany.
J Am Chem Soc. 2018 Aug 22;140(33):10447-10455. doi: 10.1021/jacs.8b03719. Epub 2018 Aug 9.
The principles of protein-glycan binding are still not well understood on a molecular level. Attempts to link affinity and specificity of glycan recognition to structure suffer from the general lack of model systems for experimental studies and the difficulty to describe the influence of solvent. We have experimentally and computationally addressed energetic contributions of solvent in protein-glycan complex formation in the tailspike protein (TSP) of E. coli bacteriophage HK620. HK620TSP is a 230 kDa native trimer of right-handed, parallel beta-helices that provide extended, rigid binding sites for bacterial cell surface O-antigen polysaccharides. A set of high-affinity mutants bound hexa- or pentasaccharide O-antigen fragments with very similar affinities even though hexasaccharides introduce an additional glucose branch into an occluded protein surface cavity. Remarkably different thermodynamic binding signatures were found for different mutants; however, crystal structure analyses indicated that no major oligosaccharide or protein topology changes had occurred upon complex formation. This pointed to a solvent effect. Molecular dynamics simulations using a mobility-based approach revealed an extended network of solvent positions distributed over the entire oligosaccharide binding site. However, free energy calculations showed that a small water network inside the glucose-binding cavity had the most notable influence on the thermodynamic signature. The energy needed to displace water from the glucose binding pocket depended on the amino acid at the entrance, in agreement with the different amounts of enthalpy-entropy compensation found for introducing glucose into the pocket in the different mutants. Studies with small molecule drugs have shown before that a few active water molecules can control protein complex formation. HK620TSP oligosaccharide binding shows that similar fundamental principles also apply for glycans, where a small number of water molecules can dominate the thermodynamic signature in an extended binding site.
蛋白质-聚糖结合的原理在分子水平上仍未得到很好的理解。试图将聚糖识别的亲和力和特异性与结构联系起来，受到缺乏实验研究模型系统和难以描述溶剂影响的限制。我们通过实验和计算解决了大肠杆菌噬菌体 HK620 的尾刺蛋白 (TSP) 中溶剂在蛋白质-聚糖复合物形成中的能量贡献。HK620TSP 是一种 230 kDa 的天然三聚体，由右手平行β-螺旋组成，为细菌表面 O-抗原多糖提供了扩展的刚性结合位点。一组高亲和力的突变体与六糖或五糖 O-抗原片段具有非常相似的亲和力结合，尽管六糖在被封闭的蛋白质表面腔中引入了额外的葡萄糖支链。然而，对于不同的突变体，发现了非常不同的热力学结合特征；然而，晶体结构分析表明，在复合物形成过程中没有发生主要的寡糖或蛋白质拓扑结构变化。这指向了溶剂的影响。使用基于迁移率的方法进行分子动力学模拟揭示了一个扩展的溶剂位置网络，分布在整个寡糖结合位点上。然而，自由能计算表明，位于葡萄糖结合腔内部的小水分子网络对热力学特征有最显著的影响。从葡萄糖结合口袋中置换水所需的能量取决于入口处的氨基酸，这与在不同突变体中引入葡萄糖到口袋中发现的不同焓熵补偿量一致。以前的小分子药物研究表明，少数活性水分子可以控制蛋白质复合物的形成。HK620TSP 寡糖结合表明，类似的基本原理也适用于聚糖，其中少数水分子可以在扩展的结合位点中主导热力学特征。
