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γ-Secretase (GS) is an intramembrane aspartyl protease that participates in the sequential cleavage of C99 to generate different isoforms of the amyloid-β (Aβ) peptides that are associated with the development of Alzheimer's disease. Due to its importance in the proteolytic processing of C99 by GS, we performed pH replica exchange molecular dynamics (pH-REMD) simulations of GS in its apo and substrate-bound forms to sample the protonation states of the catalytic dyad. We found that the catalytic dyad is deprotonated at physiological pH in our apo form, but the presence of the substrate at the active site displaces its monoprotonated state toward physiological pH. Our results show that Asp257 acts as the general base and Asp385 as the general acid during the cleavage mechanism. We identified different amino acids such as Lys265, Arg269, and the PAL motif interacting with the catalytic dyad and promoting changes in its acid-base behavior. Finally, we also found a significant pK a shift of Glu280 related to the internalization of TM6-CT in the GS-apo form. Our study provides critical mechanistic insight into the GS mechanism and the basis for future research on the genesis of Aβ peptides and the development of Alzheimer's disease.

γ-Secretase (GS) is one of the most attractive molecular targets for the treatment of Alzheimer's disease (AD). Its key role in the final step of amyloid-β peptides generation and its relationship in the cascade of events for disease development have caught the attention of many pharmaceutical groups. Over the past years, different inhibitors and modulators have been evaluated as promising therapeutics against AD. However, despite the great chemical diversity of the reported compounds, a global classification and visual representation of the chemical space for GS inhibitors and modulators remain unavailable. In the present work, we carried out a two-dimensional (2D) chemical space analysis from different classes and subclasses of GS inhibitors and modulators based on their structural similarity. Along with the novel structural information available for GS complexes, our analysis opens the possibility to identify compounds with high molecular similarity, critical to finding new chemical structures through the optimization of existing compounds and relating them with a potential binding site.

Anterior pharynx-defective 1A (APH-1A) is a seven transmembrane component of γ-secretase (GS), an aspartyl protease enzyme involved in the production of toxic amyloid-β peptides in Alzheimer's disease patients. Cryo-electron microscopy structures of the enzyme complex revealed a central cavity in its APH-1A component, similar to water-containing cavities in G-protein coupled receptors (GPCRs). In this work, we performed molecular dynamics and umbrella sampling simulations to understand the role of the APH-1A cavity in the GS complex. Our results suggest that APH-1A is able to store water molecules in its inner cavity and transport some of them between cell spaces. Additionally, APH-1A allows the influx of extracellular cations into a central hydrophilic cavity but cannot transport them into the intracellular space. Overall, this study seeks to describe an alternative APH-1A function in GS besides its complex stabilization role and provide novel approaches to understand the functioning of the GS enzyme.