Fo32931 and FoCII5 are two subtypes of Fusarium oxysporum (Fo), a pathogenic filamentous fungus. Phenamacril (PHA), a Fusarium-specific fungicide that targets myosin I, exhibits significant hyphal growth inhibition in Fo32931 but shows weak resistance in FocII5, despite only two amino acid differences in the PHA-binding pocket of myosin I. In this study, we aim to elucidate the molecular basis for the differential sensitivity ofF. oxysporum myosin I variants (FoMyoI³²⁹³¹ and FoMyoI^cII5) to phenamacril through computational methods and biochemical validation. The results suggest that phenamacril functions as an allosteric inhibitor for FoMyoI³²⁹³¹, inhibiting the large oscillation of the converter lever domain (CLD) upon ATP binding and promoting the opening of the outer cleft, further impairing protein function. PHA significantly reduced the coupling between the CLD, especially the converter, and the catalytic center, diminishing the response of the CLD to the motor domain in FoMyoI³²⁹³¹. From the residue mutation experiment, we found that the S418T substitution in FoMyoI^cII5 is the key to the reduced phenamacril sensitivity of FocII5. According to the microscale thermophoresis (MST) assay and pocket conformation analysis, the S418T mutation disturbs the orientation of pocket residues, especially Lys537, leading to a looser pocket and reduced interaction between Lys537 and phenamacril, which lowers the binding affinity of FoMyoI^cII5 for phenamacril. These findings provide deeper insights into the reasons for the lower sensitivity of FoCII5 to phenamacril from both molecular and structural perspectives and will also guide the design of novel inhibitors against resistant Fusarium spp., like FoCII5.

中文翻译：

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从计算和实验角度揭示尖孢镰刀菌 （Fo32931 和 FocII5） 对杀菌剂 Phenamacril 的不同敏感性


Fo32931 和 FoCII5 是尖孢镰刀菌 （Fo） 的两种亚型，Fo 是一种致病性丝状真菌。Phenamacril （PHA） 是一种靶向肌球蛋白 I 的镰刀菌特异性杀菌剂，在 Fo32931 中表现出显着的菌丝生长抑制，但在 FocII5 中表现出较弱的抗性，尽管肌球蛋白 I 的 PHA 结合口袋中只有两个氨基酸差异。在这项研究中，我们旨在通过计算方法和生化验证阐明尖孢镰刀菌肌球蛋白 I 变体 （FoMyoI³²⁹³¹ 和 FoMyoI^cII5） 对苯那马利差异敏感性的分子基础。结果表明，苯那马利作为 FoMyoI³²⁹³¹ 的变构抑制剂发挥作用，抑制 ATP 结合时转换器杠杆结构域 （CLD） 的大振荡并促进外裂的打开，进一步损害蛋白质功能。PHA 显着降低了 CLD（尤其是转换器）与催化中心之间的耦合，从而降低了 CLD 对 FoMyoI³²⁹³¹ 中运动域的响应。从残基突变实验中，我们发现 FoMyoI^cII5 中的 S418T 取代是 FocII5 非那马利敏感性降低的关键。根据微量热泳 （MST） 测定和口袋构象分析，S418T 突变扰乱了口袋残基的方向，尤其是 Lys537，导致口袋更松，Lys537 和苯那马利之间的相互作用减少，从而降低了 FoMyoI^cII5 对苯那马利的结合亲和力。 这些发现从分子和结构角度更深入地解释了 FoCII5 对 phenamacril 敏感性较低的原因，也将指导针对耐药镰刀菌属的新型抑制剂的设计，如 FoCII5。