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 Alzheimer's disease is cut processively 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 analogue-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 the substrate, identifying potent inhibitors for each class. These TMD mimetics exhibited non-competitive inhibition and occupy both the exosite and the 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.

Automated summary

The study describes a prototype substrate TMD mimetic that can trap transition states for endoproteolysis, TMD helix unwinding, and lateral gating of the substrate. The developed variants of this prototype are designed to allow visualization of different transition states, with the TMD mimetics exhibiting non-competitive inhibition and occupying both the exosite and the active site. These new probes serve as important structural tools for trapping various stages of substrate recognition and processing, aiding in rational drug design through cryo-electron microscopy studies with gamma-secretase.