Nicastrin functions as a molecular gatekeeper to a high-affinity γ-secretase-substrate interaction driven by substrate transmembrane domain

γ-secretase is an intramembrane-cleaving protease (I-CLiP) 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 α- or β-secretases. The mechanism by which γ-secretase selectively recognizes and recruits ectodomain-shed substrates for catalysis remains unclear. Understanding this mechanism is important to Alzheimer's research, as γ-secretase produces amyloid β-peptide critical to pathogenesis. Variations of recombinant substrates were used in purified γ-secretase assays. Most substrates were based on the Notch1 receptor, which was expressed and purified with different permutations from E. coli. Assays were run under detergent-solubilized conditions or with enzyme reconstituted into proteoliposomes. Proteolytic products were detected and quantified by western blot analysis and fluorescence scanning. Co-immunoprecipitations were employed to measure substrate binding to the protease complex. Nicastrin ectodomain was expressed in HEK293 cells as an Fc-fusion protein. The γ-secretase complex and the nicastrin ectodomain were separately subjected to disulfide reduction prior to analysis of substrate processing, as well as for nicastrin stability by limited proteolytic digestion and circular dichroism. The Notch1 neo-ectodomain was completely dispensable for substrate processing by γ-secretase. A substrate composed mostly of the transmembrane domain was effectively cleaved by the protease complex and displayed a low Km (<100 nM). Importantly, this high-affinity interaction between γ-secretase and substrate prevailed in proteoliposomes. Co-immunoprecipitation revealed that poorly cleaved substrates with extended ectodomains bound very weakly to the γ-secretase complex. However, reduction of nicastrin disulfide bonds allowed for better processing by the protease complex. Such reduction disrupted the normal fold of nicastrin, both alone as a recombinant protein or assembled into the γ-secretase complex. Substrate binding to γ-secretase is driven by a high-affinity interaction derived from substrate transmembrane domain, a mechanism in stark contrast to rhomboid—another family of I-CLiPs. Disruption of the nicastrin fold allows for more efficient cleavage of substrates retaining longer ectodomains, suggesting that nicastrin actively excludes larger substrates through steric hindrance, thus serving as a gatekeeper for substrate binding and catalysis.