Side-by-side comparison of Notch- and C83 binding to γ-secretase in a complete membrane model at physiological temperature

Abstract

γ-Secretase cleaves the C99 fragment of the amyloid precursor protein, leading to formation of aggregated β-amyloid peptide central to Alzheimer's disease, and Notch, essential for cell regulation. Recent cryogenic electron microscopy (cryo-EM) structures indicate major changes upon substrate binding, a β-sheet recognition motif, and a possible helix unwinding to expose peptide bonds towards nucleophilic attack. Here we report side-by-side comparison of the 303 K dynamics of the two proteins in realistic membranes using molecular dynamics simulations. Our ensembles agree with the cryo-EM data (full-protein Cα-RMSD = 1.62-2.19 Å) but reveal distinct presenilin helix conformation states and thermal β-strand to coil transitions of C83 and Notch100. We identify distinct 303 K hydrogen bond dynamics and water accessibility of the catalytic sites. The RKRR motif (1758-1761) contributes significantly to Notch binding and serves as a "membrane anchor" that prevents Notch displacement. Water that transiently hydrogen bonds to G1753 and V1754 probably represents the catalytic nucleophile. At 303 K, Notch and C83 binding induce different conformation states, with Notch mostly present in a closed state with shorter Asp-Asp distance. This may explain the different outcome of Notch and C99 cleavage, as the latter is more imprecise with many products. Our identified conformation states may aid efforts to develop conformation-selective drugs that target C99 and Notch cleavage differently, e.g. Notch-sparing γ-secretase modulators.

Fig. 1. Simulated γ-secretase models bound to C83 and Notch100 within a lipid bilayer. (A) Wild-type γ-secretase–C83 complex. (B) Wild-type γ-secretase–Notch100. The subunits nicastrin (green), PS1 (blue), APH1-A (magenta), PEN2 (yellow), and the substrates C83/Notch100 (orange) are coloured differently. The small spheres represent the head groups of the POPC molecules. The trans-membrane sequences of the two substrates are shown below the structures (acc: accessibility and hyd: hydrophobicity). The arrows show the position of the cleavage sites in C83 and Notch100. The images were generated using ENDScript2.0.

Fig. 2. Dynamical stability and comparison of experimental and simulated structures. (A) Backbone Cα-RMSD of triplicate γ-secretase–C83 and Notch100 simulations relative to the initial structure. Blue, purple and cyan lines represent simulation 1, 2, and 3 for γ-secretase–C83. Black, orange, and green lines represent simulation 1, 2, and 3 for γ-secretase–Notch100. (B) RMSD of γ-secretase–C83 and γ-secretase–Notch100. (C) Experimental γ-secretase–C83 (6IYC, green) compared to structures for simulation 1 (blue), 2 (purple), and 3 (cyan). (D) Experimental γ-secretase–Notch100 (6IDF, green) compared to simulation 1 (blue), 2 (purple), and 3 (cyan).

Fig. 3. Main atomic dynamics of γ-secretase–C83 and Notch100 complexes. (A–C) The movement of NCT (green), PS1 (cyan), APH1-A (purple), PEN2 (yellow), and C83 (tint grey) of γ-secretase–C83 for simulation 1 (A), 2, (B), and 3 (C). (D–F) The movement of the NCT, PS1, APH1-A, PEN2, and Notch100 (tint grey) of γ-secretase–Notch100 for simulation 1 (D), 2, (E), and 3 (F). The red arrows represent the direction and amplitude of motion. The TM domains in PS1 are marked 1, 2, 3, 4, 5, 6, 6a, 7, 8 and 9.

Fig. 4. Comparison of 303 K-simulated PS1 and experimental cryo-EM structures. (A) Superimposed view of simulated and experimental (6IYC) structures of γ-secretase–C83. (B–D) Ensemble-representative simulated PS1–C83 structures. (E) Zoomed-in view of the catalytic residues in the three simulated structures of 6IYC (catalytic and PAL residues are shown as sticks). (F) Superimposed view of simulated and experimental (6IDF) γ-secretase–Notch100. (G–I) Ensemble-representative simulated PS1–Notch100 structures. (J) Zoomed-in view of the catalytic residues in the three simulated structures of 6IDF. Blue, purple and cyan represent simulation 1, 2, and 3 (deep salmon color represents the substrates); the experimental structures are shown in green.

Fig. 5. Dynamics of the catalytic Asp257–Asp385, large hydrophilic loop 1 (HL1) and PAL motif. Left panel: γ-secretase–C83 complex and right panel: γ-secretase–Notch100 complex. (A) Orientation and Cα–Cα distance between the catalytic aspartates in γ-secretase–C83 compared to the experimental 6IYC. (B) Orientation and Cα–Cα distance between the aspartates in γ-secretase–Notch100 compared to 6IDF (blue: simulation 1, purple: simulation 2 and cyan: simulation 3). (C) Dynamics of the Cα–Cα distance between Asp257 and Asp385 in γ-Secretase–C83. (D) Dynamics of the Cα–Cα distance between Asp257 and Asp385 in γ-secretase–Notch100. The magenta line displays the experimental distance. (E) Minimum distance between the terminal residues of HL1 in γ-secretase–C83. (F) Minimum distance between the terminal residues of HL1 in γ-secretase–Notch100. (G) Minimum distance between the terminal PAL residues (Pro433 and Leu435) in γ-secretase–C83. (H) Minimum distance between Pro433 and Leu435 in γ-secretase–Notch100.

Fig. 6. Simulated minimum distances between catalytic Asp257/Asp385 and cleavage sites of C83 and Notch100. Left panels: average distances between the catalytic residues of PS1 and cleavage sites in C83 (Leu720–Val721 giving the Aβ₄₉ pathway and Thr719–Leu720 giving the Aβ₄₈ pathway). Right panel: average distances between the catalytic residues and cleavage sites of Notch100 (Gly1753 and Val1754).

Fig. 7. Important hydrogen bond dynamics of NCT ECD and PS1 interacting with C83 and Notch100. A: NCT, B: presenilin 1 and E: C83 or Notch100. The residues of C83 and Notch100 are numbered according to their position in corresponding substrates.

Fig. 8. Interactions between NCT, PS1 and C83 obtained from ensemble-representative cluster structures. (A) Simulation 1. (B) Simulation 2. (C) Simulation 3 (NCT: green; PS: blue; and C83: salmon). The hydrogen-bonds (shown in magenta dotted lines) are displayed for clarity. The NCT residues interacting with substrate are green, while PS1 is blue and C83 residues are orange. The interacting residues are shown in ball and stick format.

Fig. 9. Intermolecular contacts between NCT, PS1 and Notch100 obtained from the top-ranked cluster. (A) Simulation 1. (B) Simulation 2. (C) Simulation 3. The hydrogen bonds (shown in magenta dotted lines) are displayed for clarity. The NCT residues interacting with substrate are labelled green, while PS1 is blue and Notch100 residues are orange. The interacting residues are shown in ball and stick format.

Fig. 10. Distribution of tilt angles of transmembrane domains (TMs) of PS1 and C83 and Notch100. The tilt angles were computed using the Cα coordinates of the TMs. (A) Tilt angles in γ-secretase–C83. (B) Tilt angles in γ-secretase–Notch100.

Fig. 11. Computed stability effects (ΔΔG_MUT, kcal mol⁻¹) for 149 pathogenic PS1 mutations of substrate-bound γ-secretase (6IYC and 6IDF). The median is shown as a white dot inside the violin plot. (A and D) Computed ΔΔG_MUT for 6IYC and 6IDF using (A) I-Mutant, (B) FoldX, (C) POPMUSIC and (D) mCSM. (E–H) Linear correlation between ΔΔG_MUT of the 149 mutations for 6IYC and 6IDF computed using (E) I-Mutant, (F) FoldX, (G) POPMUSIC and (H) mCSM.