Three-dimensional structure of human γ-secretase

Abstract

The γ-secretase complex, comprising presenilin 1 (PS1), PEN-2, APH-1 and nicastrin, is a membrane-embedded protease that controls a number of important cellular functions through substrate cleavage. Aberrant cleavage of the amyloid precursor protein (APP) results in aggregation of amyloid-β, which accumulates in the brain and consequently causes Alzheimer's disease. Here we report the three-dimensional structure of an intact human γ-secretase complex at 4.5 Å resolution, determined by cryo-electron-microscopy single-particle analysis. The γ-secretase complex comprises a horseshoe-shaped transmembrane domain, which contains 19 transmembrane segments (TMs), and a large extracellular domain (ECD) from nicastrin, which sits immediately above the hollow space formed by the TM horseshoe. Intriguingly, nicastrin ECD is structurally similar to a large family of peptidases exemplified by the glutamate carboxypeptidase PSMA. This structure serves as an important basis for understanding the functional mechanisms of the γ-secretase complex.

Extended Data Figure 1. Construction of the pMLink vector for co-expression of the four components of γ-secretase

a, A schematic diagram of the empty pMLink plasmid. The empty pMLink plasmid was created by insertion of the LINK1 and LINK2 sequences^, into the pCAG vector. The two LINK sequences allow insertion of a PacI digested fragment from one plasmid at the SwaI site of a second plasmid by ligation-independent cloning (LIC) method^,. The four components of γ-secretase were individually cloned into the multiple cloning sites of the empty pMLink vector, generating four plasmids. b, Construction of the pMLink vector that contains all four components of γ-secretase. First, pMLink-Pen-2 and pMLink-Nicastrin were combined by the LIC method to generate pMLink-Pen-2-Nicastrin, whereas pMLink-Aph-1aL and pMLink-PS1 gave rise to pMLink-Aph-1aL-PS1. Then, the two plasmids pMLink-Pen-2-Nicastrin and pMLink-Aph-1aL-PS1 were combined to generate the final plasmid pMLink-Pen-2-Nicastrin-Aph-1aL-PS1. EC: expression cassette (containing promoter, target gene and poly A sequence). c, Expression of PS1 alone resulted in the production of the uncleaved PS1 protein. Transfection of pMLink-PS1 into HEK293F cells led to expression of the uncleaved, full-length PS1. Shown here is result of a Western blot using a monoclonal antibody against the NTF of PS1. d, The purified γ-secretase in amphipol A8-35 was proteolytically active against the APP substrate C100. Cleavage of the substrate APP-C100 was blocked by the specific inhibitor III-31C. The same γ-secretase in amphipol A8-35 under the same buffer was used for cryo-EM analysis.

Extended Data Figure 2. Cryo-EM analysis of the human γ-secretase complex

a, Analysis of γ-secretase in digitonin imaged on back-thinned Falcon II. A representative electron micrograph (scale bar, 20 nm) and reference-free 2D class averages are shown in the upper and lower panels, respectively. b, Analysis of γ-secretase in amphipols imaged on back-thinned Falcon II. c, Analysis of γ-secretase in amphipols imaged on K2 Summit. d, Comparison of densities for the three methods of imaging described above. The fewest number of particles (37K) was used for the generation of higher-resolution images for samples in amphipols on the K2 Summit.

Extended Data Figure 3. Density and resolution of the human γ-secretase complex

a, Resolution limit (color-coded) of the cryo-EM density for γ-secretase in amphipols imaged on K2 Summit. The left panel shows a cut-through view on the interior of the maps. The middle panel shows the entire maps. The low-resolution density (reddish color) likely represents that from amphipols. The right panel shows the best density for TM in the map, with some of the side chain features visible. The 4.5-Å EM map was used here. b, Gold-standard FSC curves for the density maps. The resolution limits reached 4.5 Å and 5.4 Å, respectively, after one (red) or two (green) steps of 3D classification. c, The FSC curve between the Nicastrin ECD atomic model and the corresponding part of the cryo-EM map.

Extended Data Figure 4. Tilt-pair validation of the correct handedness of γ-secretase

Tilt-pair validation plots are shown for γ-secretase and 80S ribosome imaged on the same electron microscope at 0° and 20° tilt angles, under the same magnification, and using the same detector. The position of each dot represents the direction and the amount of tilting for a particle pair in polar coordinates. Blue dots correspond to in-plane tilt transformations; red dots to out-of-plane tilt transformations. For both samples blue dots cluster in the same region of the plot at a tilt angle of approximately 20°, which validates the structure and confirms that our γ-secretase map is in the same handedness as the 80S ribosome map. Note that the increased scatter of the points in the γ-secretase plot results from the significantly smaller molecular weight compared to the 80S ribosome, which results in much less accurate orientational assignments.

Extended Data Figure 5. Connecting density of the TMs

Seven pairs of TMs are shown for which there is EM density connecting the two TMs. The orientation of these seven pairs of TMs reflects that in the intact γ-secretase complex, with extracellular space on the upper side and cytoplasm on the lower side. The 5.4-Å density map was used here. This figure was prepared using Chimera.

Extended Data Figure 6. The density map for the ECD of Nicastrin in stereo view

The EM density is colored cyan, whereas the built model is displayed in Cα trace. The 4.5-Å density map was used here. This figure was prepared using PyMol.

Extended Data Figure 7. Sequence alignment between Nicastrin ECD and the glutamate carboxyl peptidase PSMA

Identical amino acids between Nicastrin and PSMA are highlighted in red, and conserved residues are colored blue. PSMA residues that coordinate the first and second zinc atoms are identified by red squares and red circles, respectively, above the sequences. Residues in Nicastrin that are aligned to the proximity of zinc-binding sites in PSMA are indicated by blue triangles.

Extended Data Figure 8. Speculative assignment of TMs and implications for γ-secretase function

a, TMs 1-11 are putatively assigned to PS1 (blue) and Pen-2 (purple). This assignment is based on three assumptions: (1) PS1 is structurally homologous to mmPSH that contains three layers of TMs; (2) all TMs in the intact γ-secretase have been identified in the current EM structure; and (3) PS1 does not undergo drastic structural rearrangement upon binding to the other three components. Two perpendicular views and a cut-through section are shown. b, Structural comparison of PS1 (blue) with its archaeal homologue mmPSH (grey). PS1 and mmPSH share 23 percent sequence identity in the TM region. Structure of mmPSH represents the inactive conformation. In this speculative comparison, the two TMs of Pen-2 are inserted between TM7 and TM8 of PS1, likely triggering marked conformational shifts in nearby TMs. c, A contrasting model of TM assignment in γ-secretase. Although less likely, we cannot rule out the possibility that PS1 is located at the thin end of the TM horseshoe. In this case, Aph-1 would be assigned to the thick end.

Figure 1. Expression and purification of active human γ-secretase

a, A schematic diagram of the protocol for the expression and purification of the intact human γ-secretase complex. pMLink is our custom-designed vector for simultaneous co-expression of multiple proteins in mammalian cells. b, A representative gel filtration chromatography of human γ-secretase. The peak fractions were visualized on SDS-PAGE by Coomassie staining. PS1 had been completely auto-proteolyzed into NTF and CTF, whereas Nicastrin (NCT) existed in two forms: immature (iNCT) and mature (mNCT), reflecting differences in glycosylation. c, The purified γ-secretase was proteolytically active against the APP substrate C100. Cleavage of the substrate APP-C100 was blocked by the specific inhibitor III-31C.

Figure 2. Overall structure of the human γ-secretase complex

a, An overall density map for the entire human γ-secretase complex. α-Carbon traces are shown for some of the TMs and the ECD. The 5.4-Å map was used in both panels a and b. b, Overall structure of the human γ-secretase complex. Structure of the γ-secretase is viewed from within the plane of lipid membrane (upper panel). The 19 TMs from the four components of γ-secretase are colored blue, whereas the ECD of Nicastrin is shown in green. A cut-through section of the 19 TMs in γ-secretase is shown in the lower panel. The TMs form a horseshoe-shaped structure, with more TMs concentrated at the thick end. The TMs are numbered. Figs. 2a, 3, and 4b were prepared using PyMol, and Figs. 2b and 4a were made in Chimera.

Figure 3. Structure of the extracellular domain (ECD) of Nicastrin

a, Representative cryo-EM density for β-strands (left panels) and α-helices (right panels) of the Nicastrin ECD. The 4.5-Å map was used here. b, The overall structure of Nicastrin ECD closely resembles that of the glutamate carboxyl peptidase PSMA. The atomic model of Nicastrin ECD is shown in rainbow cartoon, with the N- and C-termini colored blue and red, respectively. The structure of PSMA is displayed in the right panel for comparison. c, Structural comparison between Nicastrin (green) and PSMA (grey) for the large lobe (upper panel) and the small lobe (lower panel). d, Identification of a putative substrate-binding site in Nicastrin ECD. A surface groove on Nicastrin ECD, located 40 Å above the lipid membrane, faces the hollow center of the TM horseshoe. Glu333, which is thought to play an important role in substrate recruitment^,, resides in the groove.