Department of Chemical Sciences, University of Naples Federico II, Naples 80126, Italy.
Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764, Neuherberg, Germany.
Nucleic Acids Res. 2023 Sep 8;51(16):8880-8890. doi: 10.1093/nar/gkad634.
Ligand/protein molecular recognition involves a dynamic process, whereby both partners require a degree of structural plasticity to regulate the binding/unbinding event. Here, we present the characterization of the interaction between a highly dynamic G-rich oligonucleotide, M08s-1, and its target protein, human α-thrombin. M08s-1 is the most active anticoagulant aptamer selected thus far. Circular dichroism and gel electrophoresis analyses indicate that both intramolecular and intermolecular G-quadruplex structures are populated in solution. The presence of thrombin stabilises the antiparallel intramolecular chair-like G-quadruplex conformation, that provides by far the main contribution to the biological activity of the aptamer. The crystal structure of the thrombin-oligonucleotide complex reveals that M08s-1 adopts a kinked structural organization formed by a G-quadruplex domain and a long duplex module, linked by a stretch of five purine bases. The quadruplex motif hooks the exosite I region of thrombin and the duplex region is folded towards the surface of the protein. This structural feature, which has never been observed in other anti-exosite I aptamers with a shorter duplex motif, hinders the approach of a protein substrate to the active site region and may well explain the significant increase in the anticoagulant activity of M08s-1 compared to the other anti-exosite I aptamers.
配体/蛋白质分子识别涉及一个动态过程,其中两个伴侣都需要一定程度的结构可塑性来调节结合/非结合事件。在这里,我们介绍了高度动态的富含 G 的寡核苷酸 M08s-1 与其靶蛋白人α-凝血酶之间相互作用的特征。M08s-1 是迄今为止选择出的最活跃的抗凝适体。圆二色性和凝胶电泳分析表明,在溶液中都存在分子内和分子间的 G-四链体结构。凝血酶的存在稳定了反平行的分子内椅式 G-四链体构象,这为适体的生物学活性提供了迄今为止的主要贡献。凝血酶-寡核苷酸复合物的晶体结构揭示了 M08s-1 采用了一种扭曲的结构组织,由 G-四链体结构域和一个长的双链模块组成,由五个嘌呤碱基的延伸连接。四链体基序钩住凝血酶的外切酶 I 区域,双链区域向蛋白质表面折叠。这种结构特征从未在具有较短双链基序的其他抗外切酶 I 适体中观察到,它阻碍了蛋白质底物进入活性位点区域的接近,并且很可能解释了与其他抗外切酶 I 适体相比,M08s-1 的抗凝活性显著增加的原因。