Allosteric modulation and structural plasticity of PS2-containing γ-secretase in the production of Aβ and Alzheimer's disease

Alzheimer’s disease (AD) is one of the leading neurodegenerative disorders characterized by amyloid-β (Aβ) plaque accumulation involving the pivotal role of the aspartyl protease γ-secretase. Familial AD (FAD) mutations in the catalytic subunit of γ-secretase, i.e., presenilin 1/2 (PS1/2) alter the substrate processing, thus promoting increased production of toxic β-amyloid peptide (Aβ42) through sequential proteolysis of the amyloid precursor protein (APP). Recent cryo-electron microscopic structures and computational analyses have provided novel insights into the substrate recognition mechanism of γ-secretase containing PS1; however, the key structural and dynamic features of substrate recognition by PS2 containing γ-secretase are lacking. The present study sought to elucidate the molecular determinants of substrate specificity and allosteric regulation of PS2 γ-secretase in realistic membrane mimetic models under relevant physiological conditions by incorporating cryo-EM data from PS1/2, which represent its mature, membrane-embedded, catalytically active state. We specifically developed three different PS2 containing γ-secretase systems bound to the substrate APP-C83 systems considering the protonation states of the catalytic aspartates, i.e., Asp263+, Asp366+ and Asp263+/366+ embedded in lipid bilayers and performed all-atom MD simulations for 1μs. It reveals protonation states of the PS2 catalytic dyad (Asp263/Asp366) critically regulate geometries, water access, and loop dynamics for APP-C83 ε-cleavage. The Asp366+ state optimizes catalytic distances, sustaining persistent hydrolytic waters, and balances HL1 loop flexibility for precise substrate docking via PAL/β-strand contacts. TM6-TM7 gating provides protonation-selective restraint, throttling active-site volume for compact substrate tilt. Taken together, our findings provide mechanistic insights into how mutations and lipids collectively shape γ-secretase function, informing strategies for targeted therapeutic intervention in AD.