Abstract
Dysfunction of the intramembrane protease γ-secretase is thought to cause Alzheimer’s disease, with most mutations derived from Alzheimer’s disease mapping to the catalytic subunit presenilin 1 (PS1). Here we report an atomic structure of human γ-secretase at 3.4 Å resolution, determined by single-particle cryo-electron microscopy. Mutations derived from Alzheimer’s disease affect residues at two hotspots in PS1, each located at the centre of a distinct four transmembrane segment (TM) bundle. TM2 and, to a lesser extent, TM6 exhibit considerable flexibility, yielding a plastic active site and adaptable surrounding elements. The active site of PS1 is accessible from the convex side of the TM horseshoe, suggesting considerable conformational changes in nicastrin extracellular domain after substrate recruitment. Component protein APH-1 serves as a scaffold, anchoring the lone transmembrane helix from nicastrin and supporting the flexible conformation of PS1. Ordered phospholipids stabilize the complex inside the membrane. Our structure serves as a molecular basis for mechanistic understanding of γ-secretase function.
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Acknowledgements
We thank S. Chen and C. Savva for support with electron microscopy, and J. Grimmett and T. Darling for support with high-performance computing. This work was supported by funds from the Ministry of Science and Technology (2014ZX09507003006 to Y.S.), the National Natural Science Foundation of China (31130002 and 31321062 to Y.S.), a European Union Marie Curie Fellowship (to X.C.B.), and the UK Medical Research Council (MC_UP_A025_1013, to S.H.W.S.).
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Y.S. initiated and supervised the project. G.Y., P.L., D.M., L.S., and R.Z. prepared the sample and pre-screened samples in various detergents on F20. X.B. prepared grids and collected cryo-EM data. X.B. and S.S. calculated the cryo-EM map. C.Y. built and refined the atomic model. X.B. independently built and refined the atomic model. Y.S., L.S., G.Y., and R.Z. designed and analysed the mutational and biochemical characterizations. L.S., G.Y., and R.Z. performed the biochemical assays. All authors contributed to analysis of the structure. X.B, C.Y., S.S. and Y.S. contributed to manuscript preparation.
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Extended data figures and tables
Extended Data Figure 1 Cryo-EM, single-particle analysis of human γ-secretase.
a, Representative raw particles from an original micrograph. b, Representative reference-free 2D class averages of the γ-secretase particles. Two classes identified by a red rectangle box (lower right corner) may contain some density for the extended cytosolic loop sequences between TM6 and TM7 of PS1, which are disordered in the final maps. c, Resolution estimation of the EM structure. The overall resolution is calculated to be 3.4 Å on the basis of gold-standard FSC curve39. d, Colour-coded resolution variations in the γ-secretase structure as estimated by ResMap44. e, FSC curves of the final, Refmac-refined model versus the map it was refined against (in black); of a model refined in the first of the two independent maps used for the gold-standard FSC versus that same map (in red); and of a model refined in the first of the two independent maps versus the second independent map (in green). The small difference between the red and green curves indicates that the refinement of the atomic coordinates did not suffer from severe overfitting.
Extended Data Figure 2 An atomic model of human γ-secretase.
a, The γ-secretase structure is viewed parallel to the lipid membrane. Shown here is EM density for the entire γ-secretase complex. EM density is coloured blue for PS1, yellow for PEN-2, magenta for APH-1, and green for nicastrin. b, The density map for TM2 of PS1. Among the 20 TMs, TM2 of PS1 shows the highest degree of flexibility and only becomes visible at as rod-shaped density in a 7 Å low-pass filtered map. At this resolution, another rod-shaped density is visible next to TM2 and remains unaccounted for. c, EM density map and the atomic model are shown for all seven TMs of APH-1. Two to three bulky residues are indicated for each TM. d, EM density map and the atomic model are shown for seven TMs of human PS1. TM6 exhibits relatively poor EM density, probably because of its intrinsic flexibility. e, EM density map and the atomic model are shown for the three TMs of PEN-2. f, EM density map and the atomic model are shown for the only TM and select regions of nicastrin. g, EM density map and the atomic model for three representative glycans. h, EM density map and putative assignment for two lipid molecules.
Extended Data Figure 3 Overall structure of human γ-secretase.
a, Structure of human γ-secretase is shown in cartoon representation (top) and surface view (bottom) in four successively perpendicular views. The γ-secretase structure is viewed parallel to the lipid membrane. The colouring scheme is the same as in Fig. 1. Two lipid molecules are shown. Eleven glycosylated Asn residues and their glycans are displayed in stick. b, The γ-secretase structure is represented by electrostatic surface potential.
Extended Data Figure 4 Electrostatic surface potential of PS1.
PS1 exhibits a loosely folded structure, with several large cavities and empty spaces between adjacent TMs.
Extended Data Figure 5 Structural comparison between human nicastrin and nicastrin from D. purpureum (DpNCT).
Individual structures of human nicastrin and DpNCT20 are shown in the left and middle panels, respectively. The overlay is shown in the right panel, with a root mean squared deviation of 2.2 Å. Two perpendicular views for each structure are displayed here.
Extended Data Figure 7 Results of crosslinking experiments corroborate the atomic model of γ-secretase.
a, Crosslinking results for the interface between APH-1 and nicastrin (NCT). Three mutant γ-secretase complexes were examined: APH-1-V147C/NCT-I40C, APH-1-V146C/NCT-A664C, and APH-1-A4C/NCT-L673C. Shown in the upper panel is an SDS–PAGE gel blotted by a monoclonal antibody against the HA tag on APH-1. Only in the absence of DTT did crosslinking lead to high molecular mass complexes for the mutant γ-secretase, but not for the WT γ-secretase. The two bands probably represent APH-1 crosslinked to mature nicastrin (mNCT) and immature nicastrin (iNCT). The structural basis is shown in the lower panel. The distances between the Cα atoms of the two residues targeted for cysteine mutation range from 4.1 to 6.1 Å, which would facilitate convenient crosslinking reactions. b, Crosslinking results for the interface between PS1 and PEN-2. The mutant γ-secretase contains PEN-2-P97C and PS1-N190C. Shown in the upper panel is an SDS–PAGE gel blotted by a monoclonal antibody against the Flag tag on PEN-2. c, Crosslinking results for the interface between PEN-2 and nicastrin. Two γ-secretase mutants were examined: PEN-2-T100C/NCT-V224C and PEN-2-L98C/NCT-H222C. d, Crosslinking results for the interface between APH-1 and PS1. Two γ-secretase mutants were examined: APH-1-T204C/PS1-F465C and APH-1-A76C/PS1-I467C.
Extended Data Figure 8 Implication on substrate access to γ-secretase.
Structure of γ-secretase is displayed in three relevant views: left, electrostatic surface potential from the convex side of γ-secretase; middle, overall structure, with key features labelled; right, suggested putative path for substrate access to the active site of γ-secretase.
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Bai, Xc., Yan, C., Yang, G. et al. An atomic structure of human γ-secretase. Nature 525, 212–217 (2015). https://doi.org/10.1038/nature14892
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DOI: https://doi.org/10.1038/nature14892
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