大豆疫霉苏氨酰tRNA合成酶结构与Borrelidin结合位点解析

The aminoacyl-tRNA synthetase enzymes represent ideal targets for drug discovery. Our previous research demonstrated that Borrelidin is the specific antifungal drug against Phytophthora sojae by targeting threonyl-tRNA synthetase(ThrRS). To the best of our knowledge,Borrelidin is the highest antifungal agent and the inhibition effect of Borrelidin is 62.5-fold higher than that of the widely used and commercial fungicide metalaxyl. In order to further investigate the molecular mechanism of Borrelidin against P. sojae, the crystals of ThrRS, ThrRS-ATP and ThrRS-ATP-Borrelidin were prepared by vapor diffusion approach. After the collection of the various X-ray spectra, the structural models were then constructed by using molecular replacement method. On the basis of the crystal structures, the binding site of Borrelidin and the key amino acids that play important roles in the binding of Borrelidin were identified. Furthermore, the interaction between Borrelidin and ThrRS was elucidated by molecular docking, molecular dynamics simulation, and site-directed mutation. These researches not only pave the way for the drug design using ThrRS as target and the structural modification of Borrelisin, but also provide the clue for investigating the differential inhibition of Borrelidin against eukaryotes P. sojae and prokaryotes from the structural point of view.

氨酰tRNA合成酶是理想的药物靶标。本实验室前期研究发现微生物天然产物Borrelidin是以苏氨酰tRNA合成酶（ThrRS）为靶标的大豆疫霉特效药，抑制作用是防治大豆疫霉商品化药物甲霜灵的62.5倍，是现有杀菌活性最高的化合物。为了深入研究Borrelidin抑制大豆疫霉的分子机理，本项目拟通过气相悬滴扩散法制备ThrRS、ThrRS-ATP和ThrRS-ATP-Borrelidin晶体，收集晶体X射线衍射图谱，采用分子置换法构建晶体结构模型，明确ThrRS上Borrelidin的结合位点和关键氨基酸残基组成，结合分子对接、分子动力学模拟和定点突变分析阐明Borrelidin与ThrRS的相互作用。该研究不仅为基于ThrRS为靶标的药物设计和Borrelidin结构改造奠定基础，而且为从蛋白三维结构差异的角度研究Borrelidin对大豆疫霉抑制作用强于原核生物的分子机制提供新的线索。

氨酰tRNA合成酶是理想的药物靶标。本实验室前期研究发现微生物天然产物Borrelidin是以苏氨酰tRNA合成酶（ThrRS）为靶标的大豆疫霉特效药，抑制作用是防治大豆疫霉商品化药物甲霜灵的62.5倍，是现有杀菌活性最高的化合物。为了深入研究Borrelidin抑制大豆疫霉的分子机理，本项目通过气相悬滴扩散法制备了截短ThrRS-Borrelidin复合物晶体，收集晶体X射线衍射图谱，采用分子置换法构建了分辨率达2.5Å的晶体结构模型。截短的大豆疫霉ThrRS由催化结构域和tRNA反密码子结合域所组成，其ThrRS的催化活性位点具有II型氨酰tRNA合成酶的特征，即催化中心由反向平行的β-sheet以及周围的α-helix和loop组成。Borrelidin位于ThrRS的催化活性中心，其18元环结构与催化活性中心的7个β-折叠片垂直。有14个氨基酸残基与Borrelidin相互作用，其中Arg466、Gln484和Tyr566与Borrelidin之间形成氢键；此外，通过分子动力学模拟鉴定出其它6个与Borrelidin结合有关的氨基酸。经定点突变和酶学性质分析对重要氨基酸Arg-466、Arg-478和Tyr-566进行功能验证，这些氨基酸的突变显著提高了ThrRS对Borrelidin的抗性。将大豆疫霉ThrRS-Borrelidin复合物晶体结构与已报道的ThrRS结构比对，显示Borrelidin与ThrRS的3个底物（苏氨酸、ATP和tRNA）之间存在空间位阻，表明Borrelidin可与底物竞争结合ThrRS。虽然Borrelidin与ThrRS的底物之间无结构相似性，但却同时与3个底物之间存在竞争结合关系，这种抑制剂的作用模式在已知的抑制剂-酶-底物作用模式中很少见，也解释了Borrelidin高效抑制大豆疫霉的作用机理。以大豆疫霉ThrRS为靶标进行高通量筛选，获得了4株Borrelidin产生菌株和2株非Borrelidin产生菌株（NEAU-HV41和NEAU-S7GS2）。以上研究不仅深入揭示了Borrelidin高效抑制大豆疫霉的作用机理，还为基于ThrRS的抑制剂设计和Borrelidin的结构改造提供了结构基础。