Cryoelectron Microscopy Structure of Purified γ-Secretase at 12 Å Resolution

Abstract

γ-Secretase, an integral membrane protein complex, catalyzes the intramembrane cleavage of the β-amyloid precursor protein (APP) during the neuronal production of the amyloid β-peptide (Aβ). As such, the protease has emerged as a key target for developing agents to treat and prevent Alzheimer's disease. Existing biochemical studies conflict on the oligomeric assembly state of the protease complex, and its detailed structure is not known. Here, we report that purified active human γ-secretase in digitonin has a total molecular mass of ~230 kDa when measured by scanning transmission electron microscopy. This result supports a complex that is monomeric for each of the four component proteins. We further report the 3-dimensional structure of the γ-secretase complex at 12 Å resolution, as obtained by cryo-EM and single particle image reconstruction. The structure reveals several domains on the extracellular side, three solvent-accessible low-density cavities and a potential substrate-binding surface groove in the transmembrane region of the complex.

Presenilin Transmembrane Domain 8 Conserved AXXXAXXXG Motifs Are Required for the Activity of the γ-Secretase Complex

“…Nondenaturating electrophoresis analyses were carried out (blue native PAGE), with HEK293 taken as a reference for the formation of the native ␥-secretase complex (70). MEF ϩ/ϩ and HEK293 cells, but not MEFPSdKO, showed two high molecular mass bands, between 242 and 480 kDa, both positive for PS1 and PS2, compatible with the previously described high molecular mass band (ϳ400 kDa) observed for fully assembled ␥-secretase (45, 70) (Fig.…”

“…The fact that different amino acid substitutions on the same structural determinant result in opposite effects on PS activity while generally preserving the maturation of the complex suggests that the PS AXXXAXXXG motif could be important for the conformation of the mature ␥-secretase complex (70). Nondenaturating electrophoresis analyses were carried out (blue native PAGE), with HEK293 taken as a reference for the formation of the native ␥-secretase complex (70).…”

Nicastrin functions to sterically hinder γ-secretase–substrate interactions driven by substrate transmembrane domain

“…This result suggests a γ-secretase component other than the catalytic presenilin is responsible for this selective cleavage function. Several EM structures of γ-secretase dating back as far as a decade have demonstrated that the ectodomain of nicastrin sits on top of the extracellular side of the γ-secretase TMDs (24,(49)(50)(51). It would therefore stand to reason that nicastrin itself might act to sterically block substrates with large ectodomains from entering the γ-secretase complex for catalysis.…”

γ-Secretase: Successive Tripeptide and Tetrapeptide Release from the Transmembrane Domain of β-Carboxyl Terminal Fragment

“…The characteristics revealed here should be considered when studying the structure-function relationship of ␥-secretase complex (Lazarov et al, 2006;Osenkowski et al, 2009). …”