Nicastrin Functions To Sterically Hinder Gamma-Secretase-Substrate Interactions Driven By Substrate Transmembrane Domain

gamma-Secretase is an intramembrane-cleaving protease that processes many type-I integral membrane proteins within the lipid bilayer, an event preceded by shedding of most of the substrate's ectodomain by alpha- or beta-secretases. The mechanism by which gamma-secretase selectively recognizes and recruits ectodomain-shed substrates for catalysis remains unclear. In contrast to previous reports that substrate is actively recruited for catalysis when its remaining short ectodomain interacts with the nicastrin component of gamma-secretase, we find that substrate ectodomain is entirely dispensable for cleavage. Instead, gamma-secretase-substrate binding is driven by an apparent tight-binding interaction derived from substrate transmembrane domain, a mechanism in stark contrast to rhomboid-another family of intramembrane-cleaving proteases. Disruption of the nicastrin fold allows for more efficient cleavage of substrates retaining longer ectodomains, indicating that nicastrin actively excludes larger substrates through steric hindrance, thus serving as a molecular gatekeeper for substrate binding and catalysis.