Molecular Insights into Fungal Glycosylphosphatidylinositol Transamidase Complex

Abstract The glycosylphosphatidylinositol (GPI) biosynthesis pathway is critical for antifungal drug development. As a key component of this pathway, GPI transamidase (GPIT) catalyzes the attachment of GPI anchors to proteins, a process essential for fungal cell wall integrity and virulence. Despite its biological significance, structural and mechanistic insights into fungal GPIT remain limited. Here, a series of cryo‐electron microscopy structures capturing distinct functional states of Saccharomyces cerevisiae GPIT is reported, including GPIT complexed with a GPI anchor, GPIT bound to a substrate‐mimetic peptide, and an unprecedented dimeric GPIT assembly. These structures reveal the conserved GPI anchor binding site formed by Gab1 and Gpi16, as well as a key protein substrate recognition site, Gpi16 Y550. Comparative structural analyses uncover fungal‐specific adaptations and the dynamic accommodation of catalytic subunit Gpi8. The dimeric GPIT structure exhibits a unique T‐shaped organization unexpectedly mediated by transmembrane helices of Gab1 and Gaa1, a configuration unlikely to form in the human counterpart. This study provides a molecular framework for understanding GPIT function and species‐specific divergences, providing a molecular basis for antifungal drug development.