Structure of gamma-secretase and its trimeric pre-activation intermediate by single-particle electron microscopy

Abstract

The γ-secretase membrane protein complex is responsible for proteolytic maturation of signaling precursors and catalyzes the final step in the production of the amyloid β-peptides implicated in the pathogenesis of Alzheimer disease. The incorporation of PEN-2 (presenilin enhancer 2) into a pre-activation intermediate, composed of the catalytic subunit presenilin and the accessory proteins APH-1 (anterior pharynx-defective 1) and nicastrin, triggers the endoproteolysis of presenilin and results in an active tetrameric γ-secretase. We have determined the three-dimensional reconstruction of a mature and catalytically active γ-secretase using single-particle cryo-electron microscopy. γ-Secretase has a cup-like shape with a lateral belt of ∼40-50 Å in height that encloses a water-accessible internal chamber. Active site labeling with a gold-coupled transition state analog inhibitor suggested that the γ-secretase active site faces this chamber. Comparison with the structure of a trimeric pre-activation intermediate suggested that the incorporation of PEN-2 might contribute to the maturation of the active site architecture.

FIGURE 1.

Purification of mature and enzymatically active γ-secretase. A, TAP of γ-secretase from HEK293S cells that stably overexpress TAP-PS1, NCT, APH-1, and PEN-2. The starting membrane solubilisate (Input), flow-through (FT), wash, and eluate from each step were probed by Western blotting against full-length TAP-PS1 and its endoproteolytic derivatives, NCT and PEN-2. Each fraction is indicated at the top. WGA, wheat germ agglutinin; NTF, N-terminal fragment; CTF, C-terminal fragment; CBP, calmodulin-binding protein. B, purified γ-secretase was analyzed by SDS-PAGE and visualized by silver staining. The bands corresponding to the γ-secretase components are indicated. C, the transition state analog inhibitor L-685,458 inhibited production of Aβ40 by purified γ-secretase. DMSO, dimethyl sulfoxide. D, purified γ-secretase catalyzed the in vitro conversion of the substrate C100-FLAG to both Aβ40 and Aβ42 products in a ratio analogous to that found in vivo.

FIGURE 2.

Single-particle electron microscopy of γ-secretase. A, representative CCD image of γ-secretase in vitreous ice. B–F, labeling of γ-secretase complexes by an active site-directed inhibitor (B) and antibodies specific for NCT (C), APH-1 (D), PEN-2 (E), and PS (F). G, determination of the resolution of the final three-dimensional reconstruction. The FSC suggests a resolution of 18 Å using the 0.5 criterion. H, gallery of representative class averages and their corresponding back-projections from the three-dimensional map.

FIGURE 3.

Three-dimensional reconstruction of γ-secretase. Shown are views of the final three-dimensional volume of γ-secretase filtered at 18 Å and set to a threshold equivalent to a molecular mass of ∼200 kDa. The double-headed arrow delimits the proposed location of the transmembrane region of the complex. The asterisks depict the proposed location of the ectodomain of NCT. The arrows mark a cavity (white arrows) in the extracellular region that extends to the transmembrane region ending in a lateral opening (black arrows).

FIGURE 4.

Gold labeling of the γ-secretase active site. A, structure of the active site inhibitor L-685,458 linked to the 39.4-Å biotinylated arm. B, gallery of images of γ-secretase particles labeled with gold-streptavidin complexed to the inhibitor.

FIGURE 5.

Comparison of γ-secretase with a trimeric pre-activation intermediate lacking PEN-2. A, representative CCD image of the trimeric pre-activation intermediate under negative stain. B, for comparison, the three-dimensional reconstruction of the pre-activation intermediate (yellow) was docked manually in the envelope corresponding to γ-secretase (magenta mesh). An asterisk marks differences in density between the two reconstructions. C, cut-through views of γ-secretase (magenta) and the pre-activation intermediate (yellow). The black arrows mark the widening of the mouth and the lateral opening. The double-headed arrow depicts the putative path of substrate/product release/entry. The proposed location of the lipid membrane is depicted as a slab.