Transmembrane domain 9 of presenilin determines the dynamic conformation of the catalytic site of γ-secretase

One of the most prominent drug targets for the treatment of Alzheimer disease is gamma-secretase, a multi-protein complex responsible for the generation of the amyloid-beta peptide. The catalytic core of the complex lies on presenilin, a multi-spanning membrane protease, the activity of which depends on two aspartate residues located in transmembrane domains 6 and 7. We have recently shown by cysteine-scanning mutagenesis that these aspartates are facing a water-filled cavity in the lipid bilayer, demonstrating how proteolytic cleavage of the substrates can be taking place within the membrane. Here, we demonstrate that transmembrane domain 9 and hydrophobic domain VII in the large cytoplasmic loop of presenilin are dynamic structural parts of this cavity. Hydrophobic domain VII is associated with transmembrane domain 7 in the membrane, probably facilitating the entrance of water molecules in the catalytic site. Transmembrane domain 9, on the other hand, exhibits a highly flexible structure, potentially involved in the transport of substrates to the catalytic site, as well as in the binding of gamma-secretase inhibitors. The conserved proline-alanine-leucine motif at the cytoplasmic part of this domain is extremely close to the catalytic Asp(257) and is crucial for conformational changes leading to the activation of the catalytic site. We, also, identify a unique mutant in this domain (I437C) that specifically blocks amyloid-beta peptide production without affecting the processing of the physiologically indispensable Notch substrate. Our data are finally combined to propose a model for the architectural organization and activation of the catalytic site of presenilin.