Eukaryotic cells are coated with an abundance of glycosylphosphatidylinositol anchor proteins (GPI-APs) that play crucial roles in fertilization, neurogenesis, and immunity. The removal of a hydrophobic signal peptide and covalent attachment of GPI at the new carboxyl terminus are catalyzed by an endoplasmic reticulum membrane GPI transamidase complex (GPI-T) conserved among all eukaryotes. Here, we report the cryo-electron microscopy (cryo-EM) structure of the human GPI-T at a global 2.53-Å resolution, revealing an equimolar heteropentameric assembly. Structure-based mutagenesis suggests a legumain-like mechanism for the recognition and cleavage of proprotein substrates, and an endogenous GPI in the structure defines a composite cavity for the lipid substrate. This elongated active site, stemming from the membrane and spanning an additional ~22-Å space toward the catalytic dyad, is structurally suited for both substrates which feature an amphipathic pattern that matches this geometry. Our work presents an important step towards the mechanistic understanding of GPI-AP biosynthesis.

Eukaryotic cells are coated with an abundance of glycosylphosphatidylinositol anchor proteins (GPI-APs) that play crucial roles in fertilization, neurogenesis, and immunity. Covalent addition of structurally diverse GPI anchorages at the carboxyl termini of target proteins is catalyzed by an endoplasmic reticulum integral membrane GPI transamidase complex (GPI-T) conserved among all eukaryotes. Despite its important role in developmental and cancer biology, a detailed picture of this intricate multi-component machinery remains elusive. Here, we report the cryo-electron microscopy (EM) structure of the human GPI-T complex at a global 2.53 Å resolution, revealing an assembly mechanism whereby the catalytic subunit PIGK is optimally positioned to accommodate its characteristic amphipathic substrates. Further, structural and functional characterizations suggest a previously unrecognized composite GPI-binding site formed by PIGU/PIGK/PIGT subunits and rationalize genetic diseases associated with GPI-T point mutations. Our work presents an important step towards the mechanistic understanding of GPI-AP biosynthesis.