Fig. 3: Characterization of a composite GPI-binding site.

a Cryo-EM density that fills a nearly complete GPI was observed in the membrane cavity “underneath” the catalytic dyad. b Expanded view of the composite site encompassed by the indicated TMHs from GPAA1 (G) /PIGU (U) /PIGT (T) /PIGS (S). Evolutionarily conserved residues (Supplementary Fig. 6c, d) in the vicinity are colored marine (PIGT) and blue (PIGK). Subunits are shown as surfaces except that PIGK was additionally shown as ribbon representations with the catalytic dyad and S1/S1′/S2′ residues highlighted in indicated colors. c Interaction between the partial GPI (green) and GPI-T (colored-coded as indicated). Distances (Å) are either indicated by numbers for H-bonding interactions or omitted for hydrophobic interactions (within 5 Å of GPI). A vertical line marks the membrane boundary. Although the major form of the phosphatidyl moiety in mammal cells contains 1-alkyl-2-acyl-glycerol, a diacylglycerol was modeled based on the density. d Apparent activity of GPI-T mutants relative to the wild-type (WT). GPI-T KO cells were gated by TGP fluorescence⁴¹ for subunit expression and analyzed for surface staining of the reporter GPI-AP (CD59) by flow cytometry. Bar graph is color-coded to match the coloring for subunits in (b). Data represent mean ± s.e.m. from three independent experiments. Source data are provided.