Selective Protonation\nof Catalytic Dyad for γ‑Secretase-Mediated\nHydrolysis Revealed by Multiscale Simulations

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