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 γ-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 γ-secretase,
ultimately aiding rational drug design.