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.

• PMID: 41085069
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Journal Article

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Hua Z, Ding X, Wu Y, Zhang D, Hu X, Yang P, Li J, Tan Y, Liu J, Zhang M, ... Show all

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