Mechanism of Tripeptide Trimming of Amyloid beta-Peptide 49 by gamma-Secretase

The membrane-embedded gamma-secretase complex proces-sively cleaves within the transmembrane domain of amyloid precursorprotein (APP) to produce 37-to-43-residue amyloid beta-peptides (A beta)ofAlzheimer's disease (AD). Despite its importance in pathogenesis, themechanism of processive proteolysis by gamma-secretase remains poorlyunderstood. Here, mass spectrometry and Western blotting were used toquantify the efficiency of tripeptide trimming of wild-type (WT) andfamilial AD (FAD) mutant A beta 49. In comparison to WT A beta 49, theefficiency of tripeptide trimming was similar for the I45F, A42T, andV46F A beta 49 FAD mutants but substantially diminished for the I45T andT48P mutants. In parallel with biochemical experiments, all-atomsimulations using a novel peptide Gaussian accelerated moleculardynamics (Pep-GaMD) method were applied to investigate thetripeptide trimming of A beta 49 by gamma-secretase. The starting structure was the active gamma-secretase bound to A beta 49 and APP intracellulardomain (AICD), as generated from our previous study that captured the activation of gamma-secretase for the initial endoproteolyticcleavage of APP (Bhattarai, A., et al.ACS Cent. Sci.2020,6, 969-983). Pep-GaMD simulations captured remarkable structuralrearrangements of both the enzyme and substrate, in which hydrogen-bonded catalytic aspartates and water became poised fortripeptide trimming of A beta 49 to A beta 46. These structural changes required a positively charged N-terminus of endoproteolyticcoproduct AICD, which could dissociate during conformational rearrangements of the protease and A beta 49. The simulationfindingswere 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 of A beta 49 by gamma-secretase.