Structural-guided identification of two modulators of β−1,3-glucan synthase FKS1

FKS1 is a β−1,3-glucan synthase critical for fungal cell wall formation and a target for antifungal drugs such as echinocandin and ibrexafungerp. However, the mechanisms regulating FKS1 activity remain largely unknown. Here, we reveal that transfer RNA (tRNA) acts as an endogenous inhibitor, whereas GSR1 functions as a stabilizer of FKS1. The cryo-EM structure of FKS1 adopts a tRNA-mediated homodimer configuration, representing a quiescent state of β−1,3-glucan synthase. Unexpectedly, the copurified endogenous tRNA is identified as a potent inhibitor that suppresses FKS1 activity. Moreover, high-resolution cryo-EM density analysis enable the identification of GSR1 as an additional binding partner of FKS1. Mutagenesis experiments confirm the interaction between FKS1 and GSR1. Evolutionarily conserved GSR1 is found to increase the stability of FKS1 in β−1,3-glucan biosynthesis. Collectively, our findings identify both tRNA and GSR1 as intrinsic modulators of β−1,3-glucan biosynthesis, thereby providing opportunities for the further development of FKS1-targeted antifungal drugs. This study discovers that tRNA directly binds and regulates the membrane protein FKS1, a β-1,3-glucan synthase essential for fungal cell wall formation. Cryo-EM and biochemical analyses show how tRNA and GSR1 control FKS1 activity and fungal growth.