Chitin is an essential component of the fungal cell wall. Chitin synthases (Chss) catalyze chitin formation and translocation across the membrane and are targets of antifungal agents, including nikkomycin Z and polyoxin D. Lack of structural insights into the action of these inhibitors on Chs has hampered their further development to the clinic. We present the cryo-EM structures of Chs2 from Candida albicans (CaChs2) in the apo, substrate-bound, nikkomycin Z-bound, and polyoxin D-bound states. CaChs2 adopts a unique domain-swapped dimer configuration where a conserved motif in the domain-swapped region controls enzyme activity. CaChs2 has a dual regulation mechanism where the chitin translocation tunnel is closed by the extracellular gate and plugged by a lipid molecule in the apo state to prevent non-specific leak. Analyses of substrate and inhibitor binding provide insights into the chemical logic of Chs inhibition, which can guide Chs-targeted antifungal development.

几丁质是真菌细胞壁的重要组成部分。几丁质合酶 (Chss) 催化几丁质形成和跨膜转运，并且是抗真菌剂（包括尼可霉素 Z 和多氧菌素 D）的靶标。缺乏对这些抑制剂对 Chs 作用的结构洞察阻碍了它们在临床上的进一步发展。我们展示了来自白色念珠菌( Ca Chs2) 的 Chs2 在载脂蛋白、底物结合、尼可霉素 Z 结合和多氧霉素 D 结合状态下的冷冻电镜结构。Ca Chs2 采用独特的域交换二聚体配置，其中域交换区域中的保守基序控制酶活性。钙Chs2 具有双重调节机制，其中几丁质易位隧道被细胞外门关闭，并被 apo 状态的脂质分子堵塞，以防止非特异性泄漏。对底物和抑制剂结合的分析提供了对 Chs 抑制的化学逻辑的见解，这可以指导 Chs 靶向抗真菌药物的开发。