The membrane-integrated synthase FKS is involved in the biosynthesis of β-1,3-glucan, the core component of the fungal cell wall^1,2. FKS is the target of widely prescribed antifungal drugs, including echinocandin and ibrexafungerp^3,4. Unfortunately, the mechanism of action of FKS remains enigmatic and this has hampered development of more effective medicines targeting the enzyme. Here we present the cryo-electron microscopy structures of Saccharomyces cerevisiae FKS1 and the echinocandin-resistant mutant FKS1(S643P). These structures reveal the active site of the enzyme at the membrane–cytoplasm interface and a glucan translocation path spanning the membrane bilayer. Multiple bound lipids and notable membrane distortions are observed in the FKS1 structures, suggesting active FKS1–membrane interactions. Echinocandin-resistant mutations are clustered at a region near TM5–6 and TM8 of FKS1. The structure of FKS1(S643P) reveals altered lipid arrangements in this region, suggesting a drug-resistant mechanism of the mutant enzyme. The structures, the catalytic mechanism and the molecular insights into drug-resistant mutations of FKS1 revealed in this study advance the mechanistic understanding of fungal β-1,3-glucan biosynthesis and establish a foundation for developing new antifungal drugs by targeting FKS.

膜整合合酶 FKS 参与真菌细胞壁核心成分 β-1,3-葡聚糖的生物合成^1,2 。 FKS 是广泛使用的抗真菌药物的靶标，包括棘白菌素和 ibrexafungerp ^3,4 。不幸的是，FKS 的作用机制仍然是个谜，这阻碍了针对该酶的更有效药物的开发。在这里，我们展示了酿酒酵母FKS1 和棘白菌素抗性突变体 FKS1 (S643P) 的冷冻电镜结构。这些结构揭示了膜-细胞质界面上酶的活性位点以及跨越膜双层的葡聚糖易位路径。在 FKS1 结构中观察到多重结合的脂质和显着的膜扭曲，表明 FKS1-膜相互作用活跃。棘白菌素耐药突变聚集在 FKS1 的 TM5-6 和 TM8 附近的区域。 FKS1(S643P) 的结构揭示了该区域脂质排列的改变，表明突变酶的耐药机制。本研究揭示的FKS1的结构、催化机制和耐药突变的分子见解推进了对真菌β-1,3-葡聚糖生物合成机制的理解，并为开发针对FKS的新型抗真菌药物奠定了基础。