Physiological function and pathology of the Alzheimer’s disease causing amyloid precursor protein (APP) are correlated with its cytosolic adaptor Fe65 encompassing a WW and two phosphotyrosine-binding domains (PTBs). The C-terminal Fe65-PTB2 binds a large portion of the APP intracellular domain (AICD) including the GYENPTY internalization sequence fingerprint. AICD binding to Fe65-PTB2 opens an intra-molecular interaction causing a structural change and altering Fe65 activity. Here we show that in the absence of the AICD, Fe65-PTB2 forms a homodimer in solution and determine its crystal structure at 2.6 Å resolution. Dimerization involves the unwinding of a C-terminal α-helix that mimics binding of the AICD internalization sequence, thus shielding the hydrophobic binding pocket. Specific dimer formation is validated by nuclear magnetic resonance (NMR) techniques and cell-based analyses reveal that Fe65-PTB2 together with the WW domain are necessary and sufficient for dimerization. Together, our data demonstrate that Fe65 dimerizes via its APP interaction site, suggesting that besides intra- also intermolecular interactions between Fe65 molecules contribute to homeostatic regulation of APP mediated signaling.

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Abstractγ-Secretase is an aspartyl intramembrane protease that cleaves the amyloid precursor protein (APP) involved in Alzheimer’s disease pathology and other transmembrane proteins. Substrate-bound structures reveal a stable hybrid β-sheet immediately following the substrate scissile bond consisting of β1 and β2 from the enzyme and β3 from the substrate. Molecular dynamics simulations and enhanced sampling simulations demonstrate that the hybrid β-sheet stability is strongly correlated with the formation of a stable cleavage-compatible active geometry and it also controls water access to the active site. The hybrid β-sheet is only stable for substrates with 3 or more C-terminal residues beyond the scissile bond. The simulation model allowed us to predict the effect of Pro and Phe mutations that weaken the formation of the hybrid β-sheet which were confirmed by experimental testing. Our study provides a direct explanation why γ-secretase preferentially cleaves APP in steps of 3 residues and how the hybrid β-sheet facilitates γ-secretase proteolysis.

Cleavage of membrane proteins in the lipid bilayer by intramembrane proteases is crucial for health and disease. Although different lipid environments can potently modulate their activity, how this is linked to their structural dynamics is unclear. Here we show that the carboxy-peptidase-like activity of the archaeal intramembrane protease PSH, a homolog of the Alzheimer's disease-associated presenilin/γ-secretase is impaired in micelles and promoted in a lipid bilayer. Comparative molecular dynamics simulations revealed that important elements for substrate binding such as transmembrane domain 6a of PSH are more labile in micelles and stabilized in the lipid bilayer. Moreover, consistent with an enhanced interaction of PSH with a transition-state analog inhibitor, the bilayer promoted the formation of the enzyme´s catalytic active site geometry. Our data indicate that the lipid environment of an intramembrane protease plays a critical role in structural stabilization and active site arrangement of the enzyme-substrate complex thereby promoting intramembrane proteolysis.

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