Structural and mechanistic insights into the fungal glycosylphosphatidylinositol mannosyltransferase I complex.

Abstract

Glycosylphosphatidylinositol (GPI) anchoring shapes eukaryotic cell-surface architecture and represents an attractive pathway for antifungal intervention. The first committed mannosylation step in GPI biosynthesis is catalyzed by GPI mannosyltransferase I (GPI-MT-I), a membrane-embedded enzyme complex essential for fungal cell-wall integrity and virulence. Despite its therapeutic potential, the molecular basis and chemical mechanism of this lipid-dependent glycosyltransferase have remained unclear. Here, we combine cryoelectron microscopy, chemical synthesis, and functional analyses to define the architecture, substrate recognition, and catalytic mechanism of fungal GPI-MT-I, the Gpi14-Pbn1 heterodimer. We captured catalytically distinct states of fungal GPI-MT-I, including a ternary complex simultaneously bound to dolichol-phosphate-mannose and GlcN-(acyl)phosphatidylinositol. These reveal a membrane-embedded reaction chamber containing a continuous substrate-binding tunnel, in which two amphipathic lipid substrates are positioned in a head-to-head configuration for glycosyl transfer. Structural and mutational analyses establish GPI-MT-I as a GT-C-fold inverting glycosyltransferase and support a concerted S_N2-like mechanism centered on the conserved catalytic aspartate Asp38. Comparative analyses reveal pronounced fungal-specific structural features with therapeutic potential, explaining the functional incompatibility across species. These findings provide a molecular and chemical blueprint for lipid-linked glycosyl transfer in membranes and a foundation for structure-guided antifungal drug development.