Effects of presenilin-1 familial Alzheimer’s disease mutations on γ-secretase activation for cleavage of amyloid precursor protein

Abstract

Presenilin-1 (PS1) is the catalytic subunit of γ-secretase which cleaves within the transmembrane domain of over 150 peptide substrates. Dominant missense mutations in PS1 cause early-onset familial Alzheimer’s disease (FAD); however, the exact pathogenic mechanism remains unknown. Here we combined Gaussian accelerated molecular dynamics (GaMD) simulations and biochemical experiments to determine the effects of six representative PS1 FAD mutations (P117L, I143T, L166P, G384A, L435F, and L286V) on the enzyme-substrate interactions between γ-secretase and amyloid precursor protein (APP). Biochemical experiments showed that all six PS1 FAD mutations rendered γ-secretase less active for the endoproteolytic (ε) cleavage of APP. Distinct low-energy conformational states were identified from the free energy profiles of wildtype and PS1 FAD-mutant γ-secretase. The P117L and L286V FAD mutants could still sample the “Active” state for substrate cleavage, but with noticeably reduced conformational space compared with the wildtype. The other mutants hardly visited the “Active” state. The PS1 FAD mutants were found to reduce γ-secretase proteolytic activity by hindering APP residue L49 from proper orientation in the active site and/or disrupting the distance between the catalytic aspartates. Therefore, our findings provide mechanistic insights into how PS1 FAD mutations affect structural dynamics and enzyme-substrate interactions of γ-secretase and APP.

Molecular Dynamics Activation of γ-Secretase for Cleavage of Notch1 Substrate

“…The shift of cleavage site in the L36F-like mutation of Notch by γ-secretase has been observed in our previous study of the analogous M51F mutant APP-bound γ-secretase 8 . Similar to the mechanism of γ-secretase activation for cleavage of APP and Aβ peptides uncovered from our previous studies 8,9,20 , two conditions must be met for proper cleavage of Notch by γ-secretase (Figure 2). First, the two catalytic aspartates D257 and D385 in PS1 must be sufficiently close, at ~6-7 Å distance, to recruit a nucleophilic water molecule through waterbridged hydrogen bonds.…”

“…GaMD simulations recorded similar averages and standard deviations of the boost potentials across the simulation systems, i.e., 14.7 ± 4.5 kcal/mol for the cryoEM WT, 14.0 ± 4.4 kcal/mol for the cryoEM L36F, 14.0 ± 4.4 kcal/mol for the model WT, and 14.9 ± 4.5 kcal/mol for the model L36F ( Table S1 ). Based on our previous studies 8, 9, 20 , we chose to protonate PS1 residue D385. Furthermore, the distance between the Cγ atoms of catalytic aspartates D257 and D385 in PS1 and the distance between PS1 residue D385 (protonated oxygen) and either Notch residue V35, G34, or C33 (carbonyl oxygen) were used as reaction coordinates to calculate two-dimensional (2D) potential mean force (PMF) free energy profiles to characterize the cleavage of Notch by γ-secretase in different simulation systems ( Figure 1 ).…”

“…In most previous studies by other groups, only one of the listed conditions here was shown in the simulation analysis results, which resulted from using apo γ-secretase structure without any substrate 44 , focusing on only the catalytic aspartate-substrate residue interaction 41 , etc. Furthermore, by analyzing the low-energy conformational states and Notch secondary structures during the GaMD simulations, we proposed that the β3 β-sheet might not be the critical factor for γ-secretase to determine the proper cleavage site on Notch or other substrates 8,9,20 . This was consistent with our previous study where we observed the cleavage of the Aβ49 peptide by γ-secretase even when the C-terminus of the substrate remained unstructured 20 .…”

“…The L36F Notch mutations for both cryoEM and model Notch-bound γ-secretase were computationally generated using the Mutation function of CHARMM-GUI [26][27][28][29][30][31][32] . We chose to protonate residue D385 in PS1 to simulate the activation of γ-secretase for cleavage of Notch based on our previous study 9 . All heteroatom molecules were removed, and all chain termini were capped with neutral patched (acetyl and methylamide).…”

“…These simulation findings were also highly consistent with biochemical experimental data 20, 21 . Very recently, we performed complimentary GaMD simulations alongside biochemical experiments to determine the effects of PS1 FAD mutations on γ-secretase activation for ε cleavage of APP 9 . In that study, we captured a similar mechanism of WT γ-secretase activation for ε cleavage of APP as Bhattarai et al 8 , even though a different catalytic aspartate was protonated (D385 compared to D257) 9 .…”