Design of Transmembrane Mimetic Structural Probes to Trap Different Stages of γ-Secretase-Substrate Interaction

The transmembrane domain (TMD) of the amyloid precursor protein of Alzheimers disease is processively cut by {gamma}-secretase through endoproteolysis and tricarboxypeptidase "trimming". We recently developed a prototype substrate TMD mimetic for structural analysis--composed of a helical peptide inhibitor linked to a transition-state analog--that simultaneously engages a substrate exosite and the active site and is pre-organized to trap the carboxypeptidase transition state. Here we developed variants of this prototype designed to allow visualization of transition states for endoproteolysis, TMD helix unwinding, and lateral gating of substrate, identifying potent inhibitors for each class. These TMD mimetics exhibited non-competitive inhibition and occupy both exosite and active site as demonstrated by inhibitor cross competition experiments and photoaffinity probe binding assays. The new probes should be important structural tools for trapping different stages of substrate recognition and processing via ongoing cryo-electron microscopy with {gamma}-secretase, ultimately aiding rational drug design.