Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity
Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity
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DOI：
10.1093/glycob/cws126
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发表时间：
2013-01-01
期刊：
影响因子：
4.3
通讯作者：
Barbirz, Stefanie
中科院分区：
文献类型：
--
作者：
Broeker, Nina K.;Gohlke, Ulrich;Barbirz, Stefanie
噬菌体HK620识别和切割O18A1群大肠杆菌的O抗原多糖及其尾钉蛋白(TSP)。HK620TSP以低亲和力结合六糖片段，但单一氨基酸交换产生了一组具有亚微摩尔解离常数的高亲和力突变体。等温滴定量热法表明，碳水化合物和蛋白质之间通过大量的直接和溶剂介导的氢键形成络合物时只释放了少量的热。在室温下，缔合既是由焓驱动的，也是由熵驱动的，强调了主要的溶剂重排对络合物的形成。晶体结构分析表明，无论六糖亲和力如何，TSP复合体中的蛋白质和糖的构象都是相同的。仅在一种情况下，TSP突变体结合了不同的六糖构象。延伸的糖结合部位可以被分成两个区域：第一，在还原端有一个疏水口袋，亲和力贡献很小。通过单一的天冬氨酸到天冬酰胺的交换可以阻断对这个位点的访问，而不会对六糖的亲和力造成重大损失。第二，谷氨酸与谷氨酰胺的特定交换为额外的水分子创造了一个位置。糖结合时的侧链重排导致脱溶和额外的氢键，这将结合部位的这一区域定义为高亲和力支架。
Bacteriophage HK620 recognizes and cleaves the O-antigen polysaccharide of Escherichia coli serogroup O18A1 with its tailspike protein (TSP). HK620TSP binds hexasaccharide fragments with low affinity, but single amino acid exchanges generated a set of high-affinity mutants with submicromolar dissociation constants. Isothermal titration calorimetry showed that only small amounts of heat were released upon complex formation via a large number of direct and solvent-mediated hydrogen bonds between carbohydrate and protein. At room temperature, association was both enthalpy- and entropy-driven emphasizing major solvent rearrangements upon complex formation. Crystal structure analysis showed identical protein and sugar conformers in the TSP complexes regardless of their hexasaccharide affinity. Only in one case, a TSP mutant bound a different hexasaccharide conformer. The extended sugar binding site could be dissected in two regions: first, a hydrophobic pocket at the reducing end with minor affinity contributions. Access to this site could be blocked by a single aspartate to asparagine exchange without major loss in hexasaccharide affinity. Second, a region where the specific exchange of glutamate for glutamine created a site for an additional water molecule. Side-chain rearrangements upon sugar binding led to desolvation and additional hydrogen bonding which define this region of the binding site as the high-affinity scaffold.