Fig. 6: Structural and functional characterization of GPAA1 reveals a protease-like domain.

a Side (i) and normal (ii) view of GPAA1. Numbers indicate transmembrane helices (TMHs) and AH1-3 label the three amphipathic helices (AH). The soluble domain is colored pink and the membrane-associated domain is rainbow-colored (blue, N-terminal; red, C-terminal). b The soluble domain of GPAA1 (red/pink, cylinder) is structurally similar to a Zn-protease AM-1 (cyan, cartoon, PDB ID 2EK8 https://doi.org/10.2210/pdb2EK8/pdb) with a Z-score of 20.6 and Cα RMSD of 3.2 Å (from a DALI search)⁴⁹. c The Zn-binding site of AM-1 (expanded view of the boxed region in b) consists of two each of aspartate, glutamate, and histidine residues that are not fully conserved in GPAA1 (in brackets). d Arrangement of GPAA1 aspartate/glutamate/histidine (D/E/H) residues in the region corresponding to the Zn-binding site in AM-1. Despite having the same composition, these residues are unlikely to form a Zn-binding site because of different spatial arrangements, especially for the two histidine residues (gray text). e Mutation of the D/E/H residues in (d) did not reduce CD59 staining in the flow cytometry assay. The function of wild-type GPAA1 and mutants were assessed by the surface expression of the GPI-AP reporter (CD59) in GPAA1 knockout cells transfected with appropriate plasmids. Cells were gated by TGP fluorescence⁴¹ for GPAA1 expression and analyzed for CD59 staining using phycoerythrin (PE)-conjugated antibodies. The dotted line (gray) indicates the CD59-staining background level from cells expressing an unrelated TGP-tagged membrane protein (negative control). Solid lines indicate staining of cells transfected with the wild-type (black) or mutant genes (red). A vertical dash line marks the threshold (CD59-gating) determined from the negative control. Shown is a representative result from three independent experiments. Data for all the three experiments are included in the Source Data file.