杀菌剂氰烯菌酯与受体肌球蛋白互作的结构生物学研究

Phenamacril is a novel fungicide developed in China, it shows a highly inhibitory activity towards some Fusarium species and exhibits an excellent efficacy in the control of Fusarium head blight (FHB) in the fields. Previous studies showed that phenamacril targets on Myosin-5 of F. graminearum and residues mutations in Myosin-5 confer resistance to phenamacril. In this study, the wild-type and mutated Myosin-5 will be expressed and purified in vitro. ATPase activity of protein and the affinity of protein binding phenamacril will also be tested. The hydrogen deuterium exchange mass spectrometry (HDX-MS) will be used to study the dynamic-conformation change of Myosin-5 to discover the phenamacril potential binding site. Then we solve three-dimensional complex structure of Myosin-5 and phenamacril by structural biology and elucidate molecular mechanism for the specific interaction between phenamacril and Myosin-5. Meanwhile, we also solve the three-dimensional complex structure of phenamacril and residues mutated Myosin-5 related to phenamacril resistance. This could elucidate the variation interaction between phenamacril and the residues mutated Myosin-5. Overall, our results could reveal the molecular mechanism of this fungicide action and pathogen resistance to phenamacril based on the complex structure. These results would help to develop a strategy to design higher activity, broad-spectrum and new anti-resistance myosin-inhibitors.

氰烯菌酯是我国自主创制的镰刀菌专化性杀菌剂，对小麦赤霉病菌具有特效。前期研究表明氰烯菌酯的靶标是肌球蛋白Myosin-5，且Myosin-5氨基酸点突变会引起镰刀菌对该药剂产生抗药性。本项目将通过体外表达和纯化Myosin-5蛋白，检测其ATP酶活性并测定其与氰烯菌酯的亲和性，并利用氢氘交换质谱法研究Myosin-5蛋白的空间构象，寻找Myosin-5蛋白与氰烯菌酯的结合位点；进一步采用结构生物学方法解析Myosin-5蛋白与氰烯菌酯的复合体三维结构，以阐明氰烯菌酯与Myosin-5的特异性互作机制；解析与抗药性相关的Myosin-5点突变蛋白与氰烯菌酯的复合体三维结构，分析点突变后蛋白与药剂的互作变化。研究结果通过蛋白三维构象直观并真实地揭示氰烯菌酯的作用机制及病原菌的抗药性机制，为创制活性更高、抑菌谱更广及反抗性新型肌球蛋白抑制剂提供理论基础。

氰烯菌酯是我国自主创制的镰刀菌专化性杀菌剂，对小麦赤霉病菌具有特效。前期研究表明氰烯菌酯的靶标是肌球蛋白Myosin-5，且Myosin-5氨基酸点突变会引起病原菌对该药剂产生抗药性。本项目建立了体外表达和纯化Myosin-5蛋白的昆虫细胞表达体系，得到足够量的蛋白，用于检测其ATP酶活性并测定其与氰烯菌酯的亲和性，氰烯菌酯对Myosin-5的ATP酶的抑制中浓度为0.36µM，证实异源表达得到的Myosin-5具有生物活性。利用结构生物学方法解析Myosin-5蛋白与氰烯菌酯的复合体三维结构（PDB ID：6UI4），分辨率为2.65Å，体外突变和体内突变氰烯菌酯结合腔中的关键氨基酸，病原菌均对氰烯菌酯产生了不同程度的抗性，阐明了氰烯菌酯与Myosin-5的特异性互作机制。尝试解析与抗药性相关的Myosin-5点突变蛋白与氰烯菌酯的复合体三维结构，但未成功。已报道的对小麦赤霉病菌表现为高抗或中抗的6个点突变位点K216E/R、S217P/L、E420K/G/D、I424R和A577G均位于氰烯菌酯的口袋腔中，这些突变会直接破坏结合腔与氰烯菌酯之间的作用力，导致病原菌对氰烯菌酯产生高抗性。已报道的S418位点在结构中与氰烯菌酯不直接互作，但能够稳定氰烯菌酯与其它氨基酸残基的相互作用，突变后会破坏原有的互作，导致抗性的产生。其它已报道的对氰烯菌酯产生轻度抗性的点突变也直接或间接削弱了结合腔与药剂的相互作用，使病原菌对氰烯菌酯产生弱的抗性。发现了重要的与氰烯菌酯结合的氨基酸位点M375。将稻瘟病菌对应的该位点K突变为M，K375M突变蛋白对氰烯菌酯的敏感性由野生型突变蛋白的不敏感变为敏感，表明氨基酸位点375在不同真菌中的遗传分化可能决定了病原菌对氰烯菌酯的敏感性。以上研究结果将为创制活性更高、抑菌谱更广及反抗性新型肌球蛋白抑制剂的分子设计提供理论基础。