Evidence That the “Lid” Domain of Nicastrin Is Not Essential for Regulating γ-Secretase Activity

Abstract Understanding of the structure of the γ-secretase complex consisting of presenilin (PS), anterior pharynx-defective 1 (APH-1), nicastrin (NCT), and presenilin enhancer 2 (PEN-2) is of significant therapeutic interest for the design of γ-secretase modulators for Alzheimer disease. The structure of γ-secretase revealed by cryo-EM approaches suggested a substrate binding mechanism for NCT, a bilobar structure that involved rotation of the two lobes around a central pivot and opening of a “lid” region that facilitates substrate recruitment. To validate this proposal, we expressed NCT that lacks the lid entirely, or a variety of NCT variants that harbor mutations at highly conserved residues in the lid region in NCT-deficient cells, and then assessed their impact on γ-secretase assembly, activity, and stability. In addition, we assessed the impact of mutating a critical residue proposed to be a pivot around which the two lobes of NCT rotate. Our results show that neither the mutations on the lid tested here nor the entire lid deletion has any significant impact on γ-secretase assembly, activity, and stability, and that NCT with the mutation of the proposed pivot rescues γ-secretase activity in NCT-deficient cells in a manner indistinguishable from WT NCT. These findings indicate that the NCT lid is not an essential element necessary for γ-secretase assembly, activity, and stability, and that rotation of the two lobes appears not to be a prerequisite for substrate binding and γ-secretase function.