How Mutations Perturb γ-Secretase Active Site Studied by Free Energy Simulations

γ-Secretase is involved in processing of the amyloid precursor protein (APP) and generation of short Aβ peptides that may play a key role in neurodegenerative diseases such as Alzheimer's disease (AD). Several mutations in γ-secretase influence its activity, resulting in early AD onset (Familial AD or FAD mutations). The molecular details of how mutations, not located close to the active site, can affect enzyme activity is not understood. In molecular dynamics simulations of γ-secretase in the absence of substrate (apo), we identified two active site conformational states characterized by a direct contact between catalytic Asp residues (closed state) and an open water-bridged state. In the presence of substrate, only conformations compatible with the open active site geometry are accessible. Systematic free energy simulations on wild type and FAD mutations indicate a free energy difference between closed and open states that is significantly modulated by FAD mutations and correlates with the corresponding experimental activity. For mutations with reduced activity, an increased penalty for open-state transitions was found. Only for two mutations located at the active site a direct perturbation of the open-state geometry was observed that could directly explain the drop of enzyme activity. The simulations suggest that modulation of the closed/open equilibrium and perturbation of the open (active) catalytic geometry are possible mechanisms of how FAD mutations affect γ-secretase activity. The results also offer an explanation for the experimental finding that FAD mutations, although not located at the interface to the substrate, mainly destabilize the enzyme-substrate complex.