Abstact

Invasive fungal infections pose life-threatening risks to the increasing population of immunocompromised patients(1,2). Treatment remains challenging due to limited antifungal drugs and increasing resistance. beta-1,3-D-glucan synthase (GS), comprising the catalytic Fks1 and the regulatory small GTPase, Rho1 (refs. (3,4)), is the target of clinically important echinocandin antifungals. Despite recent studies(5-7), the mechanisms of GS catalysis, Rho1 regulation and echinocandin inhibition and resistance remain elusive. Here we present cryo-electron microscopy structures of native Saccharomyces cerevisiae Fks1 (ScFks1) solved under catalytically relevant conditions, revealing its interactions with the antifungal caspofungin (CFN), glucan product from the translocation channel and Rho1. CFN forms a ternary complex with nascent glucan and Fks1 at the membrane-protein interface, suggesting an unexpected role of CFN in stalling polymer translocation. Our echinocandin-resistant S643P structure suggests a resistance mechanism: the substitution destabilizes CFN and glucan binding through both allosteric structural perturbation and direct steric clash. Rho1 binding induces active site rearrangements essential for catalysis, including that of the 'latch loop' for donor substrate coordination. Furthermore, we identify YMR295C as an auxiliary subunit. These findings elucidate the mechanisms of GS-mediated glucan synthesis and its inhibition and resistance by echinocandins, laying the groundwork for rational antifungal design.