Journal of Molecular Biology
Volume 385, Issue 2, 16 January 2009, Pages 642-652
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Cryoelectron Microscopy Structure of Purified γ-Secretase at 12 Å Resolution

https://doi.org/10.1016/j.jmb.2008.10.078Get rights and content

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. 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 three-dimensional structure of the γ-secretase complex at 12 Å resolution as obtained by cryoelectron microscopy 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.

Introduction

γ-Secretase is a membrane protein complex composed of presenilin (PS), nicastrin (NCT), Aph-1, and Pen-2.1, 2 The necessity and the sufficiency of these four integral membrane proteins for forming the active protease complex have been established by functional reconstitution of γ-secretase activity in Saccharomyces cerevisiae, which lacks these proteins,3 and by high-grade purification of the proteolytically active human complex from overexpressing mammalian cells.4

Three properties make γ-secretase a highly interesting target for investigation. First, γ-secretase is an unconventional aspartyl protease that resides and cleaves its substrates within the lipid bilayer. It belongs to a unique group of intramembrane-cleaving proteases that includes site 2 protease (S2P), rhomboids, and signal peptide peptidase.5 The intramembrane-cleaving proteases appear to have their catalytic residues located inside the membrane. For γ-secretase, the two catalytic aspartic acids reside in adjacent transmembrane domains at the interface of the PS heterodimer and inside the membrane.6 Second, Alzheimer's disease is believed to be caused by the progressive cerebral accumulation of amyloid β (Aβ), and γ-secretase effects the final cleavage of APP to release Aβ.7 Therefore, partially inhibiting γ-secretase could slow, halt, or prevent Alzheimer's disease. Third, in addition to this pathogenic function, γ-secretase processes a wide range of other type I membrane proteins, such as the receptors Notch and Erb-B4, the cell adhesion molecules N-cadherin and E-cadherin, and the neurotrophin co-receptor p75.8

The recent crystal structures of the bacterial rhomboid homolog GlpG and the archaeal membrane protease mjS2P have begun to shed light on how peptide bond hydrolysis may occur in a lipid environment. GlpG has a core domain composed of six transmembrane helices with the enzyme's active site located 10 Å under the outer surface of the lipid bilayer.9, 10, 11, 12 The archaeal S2P also has a six-transmembrane helix core domain; the enzyme's active site coordinates a Zn atom and is located near the middle of the bilayer.13 Except for the fact that they both have six transmembrane helices, these two enzymes share no common structural features. Nevertheless, the overall architectures of the two proteases allow the binding and potential entry of single-helical substrates and the access of water to the active sites.14 However, the bacterial and archaeal enzymes are unrelated to γ-secretase in both sequence and structure. The recent success in bacterial expression and purification of signal peptide peptidase,15 an aspartyl protease that is related to PS,16 raises the hope that the crystal structure of this protease might be solved soon. In contrast, only modest amounts of active γ-secretase complex can be purified due to its complex maturation and assembly of multiple components having 19 transmembrane domains,17 its requirement for certain lipids for activity,18 and its sensitivity to detergent type.19 These constraints will likely hinder the achievement of an atomic resolution structure for γ-secretase.

We recently reported a low-resolution, 3D structure of γ-secretase purified from overexpressing Chinese hamster ovary cells (the γ-30 line) that was reconstructed from the single-particle electron microscopy (EM) images of uranyl-acetate-stained complexes.20 The structure revealed an irregular low-density interior chamber and apical and basal porelike openings that could allow the entry of water molecules and exit of products. However, the concentration and amount of sample we were able to prepare from the γ-30 cells were not adequate for performing cryo-EM. For structural analyses, cryo-EM is more desirable than negative-stain EM because in cryo-EM, the image contrast arises from the protein itself rather than from a contrast agent that can distort the image obtained, as occurs with negative-stain EM. We have recently generated a new Chinese hamster ovary cell line (called S-20) that overexpresses about five times more γ-secretase than the γ-30 line.21 The increased amount of material, while still modest compared to what can be produced for many bacterial or archaeal membrane proteins, has nevertheless enabled us to measure the mass of the human γ-secretase complex by scanning transmission electron microscopy (STEM) and to further determine its cryo-EM structure at 12 Å resolution.

Section snippets

The purified, proteolytically active γ-secretase is a monomeric complex

The oligomeric state (i.e., the stoichiometry) of the four-component γ-secretase complex has been unclear. Earlier immunoprecipitation experiments raised the possibility that γ-secretase contained two copies of PS.22 This suggestion was supported by the reconstitution of γ-secretase activity with two inactive PS mutants that each had one of the two catalytic aspartates mutated to alanine.23 However, a recent biochemical study has suggested a 1:1:1:1 stoichiometry of the complex.24 In previous

Cell line production and complex purification

The S-1 cell line stably expressing human PS1, FLAG-Pen-2, Aph1α2-HA, and NCT-GST and the S-20 cell line stably expressing human PS1, FLAG-Pen-2, Aph1α2-HA, and NCT-V5/HIS were cultured as previously described.4, 21 Additionally, the γ-30 cell line that stably overexpresses human PS1, FLAG-Pen-2, and Aph1α2-HA was transiently transfected with NCT-MBP in the pcDNA5.1 vector. γ-Secretase complexes were purified from these various cell lines as previously described.4, 20 The quality of the

Acknowledgements

The mass measurement was carried out at the BNL STEM facility, a user facility supported by the US Department of Energy. H.L. was partially supported by BNL LDRD grant 05-111 and by NIH R01 grant GM74985. M.W. and D.J.S. were supported by NIH P01 grant AG15379. P.O. was supported by training grant TE AG00222-15. PCF was supported by the Swiss National Science Foundation grant 310000-116652/1 and by the NCCR “Neural Plasticity and Repair.”

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  • Cited by (0)

    P.O. and H.L. contributed equally to this work.

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