Structure of γ-Secretase and Its Trimeric Pre-activation Intermediate by Single-particle Electron Microscopy

Fabiana Renzi,Xulun Zhang,William J Rice,Celia Torres-Arancivia,Yacob Gomez-Llorente,Ruben Diaz,Kwangwook Ahn,Chunjiang Yu,Yue-Ming Li,Sangram S Sisodia,Iban Ubarretxena-Belandia

doi:10.1074/jbc.m110.193326

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.

Highlights

Alzheimer disease is a prevalent cause of dementia in adults [1]
TMD4 of APH-1 [28, 29], the C terminus of PEN-2 [30, 31], transmembrane domain (TMD) of NCT [32,33,34], and the C terminus of PS appear to be necessary for ␥-secretase assembly, whereas the binding of PEN-2 to TMD4 of PS appears to be essential for PS endoproteolysis [35, 36]
Transient expression of TAP-PS1, NCT, APH-1, and PEN-2 in HEK293 cells led to TAP-PS1 endoproteolysis, a surrogate marker for ␥-secretase activity [22], and the accumulation of high levels of ϳ55-kDa TAP-PS1 N-terminal fragment and ϳ20-kDa PS1 C-terminal fragment

Summary

The abbreviations used are

A␤, amyloid ␤-peptide; APP, amyloid precursor protein; TMD, transmembrane domain; PS, presenilin; NCT, nicastrin; TAP, tandem affinity purification; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; FSC, Fourier shell correlation. The association of PEN-2 with this trimeric pre-activation intermediate is essential for the endoproteolytic cleavage of PS and for conferring ␥-secretase activity to the complex [16, 22,23,24,25]. TMD4 of APH-1 [28, 29], the C terminus of PEN-2 [30, 31], TMD of NCT [32,33,34], and the C terminus of PS appear to be necessary for ␥-secretase assembly, whereas the binding of PEN-2 to TMD4 of PS appears to be essential for PS endoproteolysis [35, 36]. The structural changes associated with the incorporation of PEN-2 into the trimeric pre-activation intermediate have not been visualized. Comparison with a threedimensional reconstruction of a trimeric pre-activation intermediate that lacks PEN-2 provides structural insight into the activation of ␥-secretase

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION