Abstract
gamma-Secretase is an enzyme known to cleave multiple substrates within their transmembrane domains, with the amyloid precursor protein of Alzheimer’s Disease among the most prominent examples. The activity of gamma-secretase strictly depends on the membrane cholesterol content, yet the mechanistic role of cholesterol in the substrate binding and cleavage remains unclear. In this work, we used all-atom molecular dynamics simulations to examine the role of cholesterol in the initial binding of a direct precursor of beta-amyloid polypeptides by gamma-secretase. We showed that in cholesterol-rich membranes, both the substrate and the enzyme region proximal to the active site induce a local membrane thinning. With the free energy methods we found that in the presence of cholesterol the substrate binds favorably to the identified exosite, while cholesterol depletion completely abolishes the binding. To explain these findings, we directly examined the role of hydrophobic mismatch in the substrate binding to gamma-secretase, showing that increased membrane thickness results in higher propensity of the enzyme to bind substrates. Therefore, we propose that cholesterol promotes substrate binding to gamma-secretase by increasing the membrane thickness, which leads to the negative hydrophobic mismatch between the membrane and binding partners.
Highlights
Łukasz Nierzwicki*, Michał Olewniczak, Paweł Chodnicki & Jacek Czub γ-Secretase is an enzyme known to cleave multiple substrates within their transmembrane domains, with the amyloid precursor protein of Alzheimer’s Disease among the most prominent examples
To investigate how the protein-lipid interplay in cholesterol-rich bilayers affects γ-secretase and its substrate to facilitate the binding, we initially run conventional molecular dynamics (MD) simulations for two systems: a single copy of either (1) γ-secretase complex or (2) its substrate β-CTF, embedded in a lipid bilayer composed of DPPC and cholesterol in 3:2 molar ratio (DPPC/Chl) that resembles the cholesterol-rich microdomains (Fig. 1a,b).Time evolution of root-mean-squared deviation (RMSD) of the protein heavy atoms of γ-secretase showed that the enzyme structure fluctuates around the state identified with cryo-EM (Fig. 1c) and neither transmembrane domains (TMDs) γ-secretase nor the whole enzyme undergo any major conformational change in a timescale of 5μ s, in agreement with previous MD studies[33]
We described the role of cholesterol in substrate recognition by γ-secretase on the example of Amyloid Precursor Protein β-CTF by means of MD simulations
Summary
Łukasz Nierzwicki*, Michał Olewniczak, Paweł Chodnicki & Jacek Czub γ-Secretase is an enzyme known to cleave multiple substrates within their transmembrane domains, with the amyloid precursor protein of Alzheimer’s Disease among the most prominent examples. Maturation of the γ-secretase complex involves autocatalytic cleavage between TM6 and TM7 of presenilin, which results in the production of the N- and C-terminal fragments (NTF and CTF) of the s ubunit[11] Both before and after autoproteolysis, the catalytic residues are buried inside the enzyme and are shielded from the hydrophobic environment of the m embrane[12]. The residues that were found to play an important role in the substrate binding are scattered at TM2, TM6 and TM9 of presenilin and form a putative last external binding site[19,20] Such notion is supported by coarse-grained simulations of γ-secretase with TMD of Notch and APP embedded in POPC membrane, in which the substrates were shown to dock to the surface formed by TM2, TM6 and TM9 of presenilin[21].
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