The C-terminal PAL motif and transmembrane domain 9 of presenilin 1 are involved in the formation of the catalytic pore of the γ-secretase

gamma-Secretase is an unusual membrane-embedded protease, which cleaves the transmembrane domains (TMDs) of type I membrane proteins, including amyloid-beta precursor protein and Notch receptor. We have previously shown the existence of a hydrophilic pore formed by TMD6 and TMD7 of presenilin 1 (PS1), the catalytic subunit of gamma-secretase, within the membrane by the substituted cysteine accessibility method. Here we analyzed the structure of TMD8, TMD9, and the C terminus of PS1, which encompass the conserved PAL motif and the hydrophobic C-terminal tip, both being critical for the catalytic activity and the formation of the gamma-secretase complex. We found that the amino acid residues around the PAL motif and the extracellular/luminal portion of TMD9 are highly water accessible and located in proximity to the catalytic pore. Furthermore, the region starting from the luminal end of TMD9 toward the C terminus forms an amphipathic alpha-helix-like structure that extends along the interface between the membrane and the extracellular milieu. Competition analysis using gamma-secretase inhibitors revealed that the TMD9 is involved in the initial binding of substrates, as well as in the subsequent catalytic process as a subsite. Our results provide mechanistic insights into the role of TMD9 in the formation of the catalytic pore and the substrate entry, crucial to the unusual mode of intramembrane proteolysis by gamma-secretase.