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γ-Secretase is a membrane-embedded aspartyl protease complex that cleaves the transmembrane domains (TMDs) of a wide variety of type I integral membrane proteins. The most studied substrate is the amyloid precursor protein (APP) involved in Alzheimer’s disease, which is processed first through an endoprotease (ε) activity followed by a tricarboxypeptidase activity of the enzyme (1). Detailed structures of γ-secretase have been determined through cryoelectron microscopy, but much about the nature of substrate recognition and processing remain unclear (2). To address these issues, we developed substrate-based TMD mimetics as chemical tools for structural analysis of γ-secretase. Recently we reported a stoichiometric γ-secretase inhibitor containing an L-helical peptide inhibitor (HPI) that engages the substrate docking exosite connected through an alkyl linker to a transition-state analogue inhibitor (TSA) targeted to the active site (3). In this design, the TSA moiety has only three P’ residues (P1’, P2’, P3’) and should thereby trap the protease at the transition state of carboxypeptidase trimming. In this study, we developed variants of this prototype that should allow visualization of transition states for ε proteolysis, TMD helix unwinding, and lateral gating of substrate. Extending the C-terminus into P4’, P5’, P6’ should trap the transition state of ε proteolysis. Replacement of the 10-atom linker with the 9-atom Gly-Gly-Gly may provide insight into the nature of helix unwinding, as this region connects the helical N-terminus to the extended C-terminus. Finally, to trap the gating process, we replaced the L-HPI moiety with a potent D-HPI. The D-peptide region can apparently dock, but entry into the active site likely requires right-handed helicity. These second-generation conjugates should be important new tools for probing different stages of substrate recognition and processing by γ-secretase.

Michael Wolfe

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