Conformational Changes and Unfolding of β-Amyloid Substrates in the Active Site of γ-Secretase

Abstract

Alzheimer's disease (AD) is the leading cause of dementia and is characterized by a presence of amyloid plaques, composed mostly of the amyloid-β (Aβ) peptides, in the brains of AD patients. The peptides are generated from the amyloid precursor protein (APP), which undergoes a sequence of cleavages, referred as trimming, performed by γ-secretase. Here, we investigated conformational changes in a series of β-amyloid substrates (from less and more amyloidogenic pathways) in the active site of presenilin-1, the catalytic subunit of γ-secretase. The substrates are trimmed every three residues, finally leading to Aβ₄₀ and Aβ₄₂, which are the major components of amyloid plaques. To study conformational changes, we employed all-atom molecular dynamics simulations, while for unfolding, we used steered molecular dynamics simulations in an implicit membrane-water environment to accelerate changes. We have found substantial differences in the flexibility of extended C-terminal parts between more and less amyloidogenic pathway substrates. We also propose that the positively charged residues of presenilin-1 may facilitate the stretching and unfolding of substrates. The calculated forces and work/energy of pulling were exceptionally high for Aβ₄₀, indicating why trimming of this substrate is so infrequent.

Keywords: Alzheimer’s disease; beta-amyloid; gamma-secretase; membrane proteolysis; substrate trimming.

Figure 1

A comparison of cleavage products of amyloid precursor protein (APP). APP-C83 is generated by α-secretase cleavage, while APP-C99 is generated by β-secretase cleavage. These pathways are called nonamyloidogenic and amyloidogenic, respectively. Below are four intermediate products obtained from APP-C99, which are substrates for subsequent cuts by γ-secretase: Aβ₄₈ and Aβ₄₅ leading to Aβ₄₂ (more amyloidogenic product), and Aβ₄₆ and Aβ₄₃ leading to Aβ₄₀ (less amyloidogenic product). Black rectangles denote sequences used to create substrates of the same length for MD simulations.

Figure 2

The structure of γ-secretase (GS). (a) The cryo-EM structure (PDB id:6IYC) of GS with APP-C83 substrate. Colors of subunits: PS-1 in cyan, APH-1 in purple, NCT in green, PEN-2 in yellow, and the Aβ substrate in salmon. (b) Magnification of the catalytic site showing β-sheet formed between a substrate and PS-1. The catalytic residues of PS-1 are shown in red. (c) The substrate after threading of substrate sequence into 6IYC structure to obtain the substrate extended by three residues behind the catalytic residues for trimming.

Figure 3

Two-dimensional scatter plots showing distances between the carboxylic group of one of the catalytic residues, Asp385, to the peptide bond of residue that is to be cleaved (horizontal axes) and distances between carboxylic groups of the catalytic residues (vertical axes). Each panel is averaged from two MD simulations. All points are colored according to the MD simulation time from purple (0 ns) to yellow (500 ns). (a) Aβ₄₆ substrate with a Thr43 scissile bond; (b) Aβ₄₃ substrate with a Val40 scissile bond; (c) Aβ₄₈ substrate with an Ile45 scissile bond; (d) Aβ₄₅ substrate with a Ala42 scissile bond.

Figure 4

Two-dimensional scatter plots showing distances between C_α (CA) atoms of selected residues from the substrate and PS-1: the distance between C_α atoms of the last residue (n) of the substrate and Lys380 of PS-1 (horizontal axes), and the distance between C_α atoms of last but one residue (n − 1) of the substrate and Leu381 of PS-1 (vertical axes). Each panel is averaged from two MD simulations. Red dashed square indicates the shortest distances between the above residues with a possibility of forming a β-sheet composed of four residues: n and n-1 substrate residues and PS-1 residues 380–381. (a) Aβ₄₆ substrate; (b) Aβ₄₃ substrate; (c) Aβ₄₈ substrate; (d) Aβ₄₅ substrate.

Figure 5

Timeline showing the secondary structure of four substrates during 500 ns of MD simulation. Each simulation is presented in a separate graph over time along the horizontal axis. N-terminal part of substrates is not shown. (a) Aβ₄₆ substrate; (b) Aβ₄₃ substrate; (c) Aβ₄₈ substrate; (d) Aβ₄₅ substrate.

Figure 6

Lys380 residue forms a salt bridge with the substrate C-terminus; (a) with Thr43 of Aβ₄₃; (b) with Ile45 of Aβ₄₅; (c) with Val46 of Aβ₄₆; (d) with Thr48 of Aβ₄₈; (e) 3D structure of Aβ₄₈ substrate extended by three residues so the catalytic residues (in red) are localized in proximity of next cut residue Ile45. Red dashed rectangles in panels (a–c) indicate formation of a salt bridge. Results from two independent simulations in panels (a–d) are shown in different colors. The hydrogen bonds are indicated by dashed yellow cylinders while distances are shown in [Å]. The catalytic residues are shown in red, PS-1 in cyan and Aβ in salmon.

Figure 7

Two arginine residues, Arg269 and Arg377, of PS-1 form a salt bridge with C-terminus of the substrate. (a) The distance between the carboxyl terminal group of C-terminus of Aβ₄₈ (atom O) and Arg269 of PS-1 (atom CZ). (b) The distance between the carboxyl terminal group of C-terminus of Aβ₄₈ (atom O) and Arg377 of PS-1 (atom CZ). Results from two simulations are shown in different colors. Red dashed rectangles indicate formation of a salt bridge and a hydrogen bond. (c) Magnification of the area showing C-terminal residues of Aβ₄₈ substrate (in salmon) and residues of PS-1 (in cyan): two arginine residues, Arg269 and Arg377, in orange and two catalytic residues, Asp257 and Asp385 (in red). The hydrogen bonds are indicated by dashed yellow cylinders while distances are shown in [Å]. PS-1 is shown in cyan and Aβ in salmon.

Figure 8

Final representative conformations of substrates after SMD simulations in GS-SMD web server. Unfolding was performed until the conformation ready for next trimming was reached (by three residues). GS is not shown so as not to obscure the substrate. (a) Pulling and unfolding of Aβ₄₉ to prepare the next cleavage event Aβ₄₉ → Aβ₄₆; (b) unfolding of Aβ₄₆ → Aβ₄₃; (c) unfolding of Aβ₄₃ → Aβ₄₀; (d) unfolding of Aβ₄₀ → Aβ₃₇. Borders of the hydrophobic core of the membrane are represented by two planes. The polar and charged residues remain on the extracellular side and pull the substrate back. Colors of residues: green—apolar, blue—polar, red—charged.

Figure 9

Box-plot representing the work/energy required to unfold the substrate by three residues in the active site of GS during SMD simulations. For each substrate, eight independent simulations were conducted. The work/energy was calculated for that frame with the shortest sum of distances of the n − 3 residue to the catalytic residues.

Figure 10

GS-SMD simulations—possible cleavage event. A frame with the shortest distance between GS catalytic residues (Asp257 and protonated Asp385) and the n − 3 substrate residue (Thr30) in the simulation of substrate unfolding Aβ₄₉ → Aβ₄₆. The sequence number of the C-terminal residue in the GS-SMD server is always 33, regardless of the thread sequence. Color scheme: PS-1 in cyan and Aβ in salmon.