Extracellular interface between APP and Nicastrin regulates Aβ length and response to γ‐secretase modulators

γ‐Secretase complexes (GSECs) are multimeric membrane proteases involved in a variety of physiological processes and linked to Alzheimer's disease (AD). Presenilin (PSEN, catalytic subunit), Nicastrin (NCT), Presenilin Enhancer 2 (PEN‐2), and Anterior Pharynx Defective 1 (APH1) are the essential subunits of GSECs. Mutations in PSEN and the Amyloid Precursor Protein (APP) cause early‐onset AD. GSECs successively cut APP to generate amyloid‐β (Aβ) peptides of various lengths. AD‐causing mutations destabilize GSEC‐APP/Aβ(n) interactions and thus enhance the production of longer Aβs, which elicit neurotoxic events underlying pathogenesis. Here, we investigated the molecular strategies that anchor GSEC and APP/Aβ(n) during the sequential proteolysis. Our studies reveal that a direct interaction between NCT ectodomain and APP (C99) influences the stability of GSEC‐Aβn assemblies and thereby modulates Aβ length. The data suggest a potential link between single‐nucleotide variants in NCSTN and AD risk. Furthermore, our work indicates that an extracellular interface between the protease (NCT, PSEN) and the substrate (APP) represents the target for compounds (GSMs) modulating Aβ length. Our findings may guide future rationale‐based drug discovery efforts.