Selective Protonation of Catalytic Dyad for γ-Secretase-Mediated Hydrolysis Revealed by Multiscale Simulations

γ-Secretase plays a crucial role in producing disease-related amyloid-β proteins by cleaving the amyloid precursor protein (APP). The enzyme employs its catalytic dyad containing two aspartates (Asp257 and Asp385) to hydrolyze the substrate by a general acid-base catalytic mechanism, necessitating monoprotonation of the two aspartates for efficient hydrolysis. However, the precise protonation states of the aspartates remain uncertain. In this study, we employed a multiscale computational approach to investigate the dependence of the catalytic efficiency of γ-secretase on the protonation states of its catalytic dyad. Over 200 ms unbiased atomistic simulations of the substrate-enzyme complex reveal diverse orientations of the scissile bond of the bound substrate and accessible structural ensembles of the catalytic dyad with Asp257-Asp385 distances fluctuating between 4 and 10 Å. With a quantum mechanics/molecular mechanics (QM/MM) approach accelerated by enhanced sampling techniques, we find that the first step of the hydrolysis reaction, i.e., the formation of a gem-diol intermediate, experiences a higher reaction barrier by ∼2 kcal/mol when Asp385 is protonated. Furthermore, we find that Arg269 of the enzyme is most likely responsible for this preference of the protonation state: its basic side chain is spatially close to that of Asp257 and specifically stabilizes the transition state electrostatically when Asp257 is protonated. Collectively, our study suggests that Asp257 is likely the favored protonation site for APP cleavage by γ-secretase.