Mechanism of Tripeptide Trimming by γ-Secretase

The membrane-embedded {gamma}-secretase complex processively cleaves within the transmembrane domain of amyloid precursor protein (APP) to produce 37-to-43-residue amyloid {beta}-peptides (A{beta}) of Alzheimers disease (AD). Despite its importance in pathogenesis, the mechanism of processive proteolysis by {gamma}-secretase remains poorly understood. Here, mass spectrometry and western blotting were used to quantify the efficiency of the first tripeptide trimming step (A{beta}49[->]A{beta}46) of wildtype (WT) and familial AD (FAD) mutant APP substrate. In comparison to WT APP, the efficiency of this first trimming step was similar for the I45F, A42T and V46F APP FAD mutants, but substantially diminished for the I45T and T48P mutants. In parallel with biochemical experiments, all-atom simulations using a novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) method were applied to investigate tripeptide trimming of A{beta}49 by {gamma}-secretase. The starting structure was active {gamma}-secretase bound to A{beta}49 and APP intracellular domain (AICD), as generated from our previous study that captured activation of {gamma}-secretase for the initial endoproteolytic cleavage of APP (Bhattarai et al., ACS Cent Sci, 2020, 6:969-983). Pep-GaMD simulations captured remarkable structural rearrangements of both the enzyme and substrate, in which hydrogen-bonded catalytic aspartates and water became poised for tripeptide trimming of A{beta}49 to A{beta}46. These structural changes required a positively charged N-terminus of endoproteolytic coproduct AICD, which could dissociate during conformational rearrangements of the protease and A{beta}49. The simulation findings were highly consistent with biochemical experimental data. Taken together, our complementary biochemical experiments and Pep-GaMD simulations have enabled elucidation of the mechanism of tripeptide trimming by {gamma}-secretase. Significance statementProduction of amyloid {beta}-peptide (A{beta}) of Alzheimers disease (AD) from its precursor protein requires a series of proteolytic events carried out by the membrane-embedded {gamma}-secretase complex. Mutations in the substrate and enzyme that produce A{beta} cause hereditary AD and these mutations affect tripeptide trimming of initially formed long A{beta} peptides by {gamma}-secretase. Little is known about the structural mechanism of this trimming process. Here, we have developed a molecular dynamic model for the first step of trimming (A{beta}49 to A{beta}46) by {gamma}-secretase. Conformational changes in the enzyme-substrate complex to set up this trimming step require the presence of N-terminally charged endoproteolytic cleavage co-product. Computational effects of AD-causing mutations in A{beta}49 on the trimming process were validated through biochemical experiments.