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Allosteric modulation plays a critical role in enzyme functionality and requires a deep understanding of the interactions between the active and allosteric sites. γ-Secretase (GS) is a key therapeutic target in the treatment of Alzheimer's disease (AD), through its role in the synthesis of amyloid ß peptides that accumulate in AD patients. This study explores the structure and dynamic effects of GS modulation by E2012 binding, employing well-tempered metadynamics and conventional molecular dynamics simulations across three binding scenarios: (1) GS enzyme with and without L458 inhibitor, (2) the GS-substrate complex together with the modulator E2012 in two different binding modes, and (3) E2012 interacting with a C99 substrate fragment. Our findings reveal that the presence of L458 induces conformational changes that contribute to stabilization of the GS enzyme dynamics, previously reported as a key factor that allowed the resolution of the cryo-EM structure and the enhanced binding of E2012. Furthermore, we identified the most favorable binding site for E2012 within the GS-substrate complex, uncovering significant modulatory effects and a complex network of interactions that influence the position of the substrate for catalysis. In addition, we explore a potential substrate-modulator binding before the formation of the enzyme-substrate complex. The insights gained from our study emphasize the importance of these interactions in the development of potential therapeutic interventions that target the functionality of the GS enzyme in AD.

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IMPORTANCE: Mitochondria constitute major sources of H2O2 and other reactive oxygen species in eukaryotic cells. The division of these organelles is crucial for multiple processes in cell biology and relies on highly regulated mechano-GTPases that are oligomerization dependent and belong to the dynamin-related protein family, like A. nidulans DnmA. Our previous work demonstrated that H2O2 induces mitochondrial constriction, division, and remodeling of the outer membrane. Here, we show that H2O2 also induces a DnmA aggregation consistent with higher-order oligomerization and its recruitment to mitochondria. The study of this response uncovered that H2O2 induces the depolymerization and reorganization of actin as well as the critical role that cysteines 450 and 776 play in DnmA function. Our results provide new insights into the mechanisms of reactive oxygen species cell signaling and how they can regulate the dynamics of the actin cytoskeleton and the division of mitochondria and peroxisomes.

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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-ß (Aß) 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 pKa 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 Aß peptides and the development of Alzheimer's disease.

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Crystallin aggregation in the eye lens is one of the leading causes of cataract formation. The increase in the human γD-crystallin (HγDC) aggregation propensity has been associated with the oligomerization of its partially folded and fully unfolded structure. A recent study demonstrated that the binding of flavonoid morin (MOR) to HγDC inhibits the fibrillation of this protein. In this work, we carry out an exhaustive search for the possible binding site of MOR on HγDC by combining an ensemble docking approach with the Wrap 'N' Shake protocol. In agreement with previous results, we found a potential MOR-binding site in the cleft formed between the N-terminal and C-terminal domains of HγDC. MOR preference for the cleft residues was observed even with the interface-opened intermediate conformers of HγDC. In addition, metadynamics simulations were carried out to corroborate the stabilizing activity of MOR on HγDC structure and to identify the structural regions implicated during the unfolding inhibition. Overall, this study provides relevant insights into the identification of new HγDC aggregation inhibitors.

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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-ß 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.

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DAPT is a potent γ-secretase (GS) inhibitor that blocks the production of short amyloid-ß (Aß) peptides. Aggregation and oligomerization of Aß peptides have been associated with the development and progression of Alzheimer's disease. A recent cryo-electron microscopy density map disclosed DAPT binding at the GS active site. In this study, we employed the density map data to assign a possible binding pose of DAPT to characterize its dynamic behavior through different molecular dynamics simulation approaches. Our simulations showed a high preference of DAPT for the intramembrane region of the protein and that its entry site is located between TM2 and TM3 of PS1. DAPT interaction with the active site led to a decreased flexibility of key PS1 regions related to the recognition and internalization of GS substrates. Moreover, our study showed that the proximity of DAPT to the catalytic aspartic acids should be able to modify its protonation states, preventing the enzyme from reaching its active form. These results provide valuable information toward understanding the molecular mechanism of a GS inhibitor for the development of novel Alzheimer's disease treatments.

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Regulation of microtubule assembly by antimitotic agents is a potential therapeutic strategy for the treatment of cancer, parasite infections, and neurodegenerative diseases. One of these agents is nocodazole (NZ), which inhibits microtubule polymerization by binding to ß-tubulin. NZ was recently co-crystallized in Gallus gallus tubulin, providing new information about the features of interaction for ligand recognition and stability. In this work, we used state-of-the-art computational approaches to evaluate the protonation effects of titratable residues and the presence of water molecules in the binding of NZ. Analysis of protonation states showed that residue E198 has the largest modification in its pKa value. The resulting E198 pKa value, calculated with pH-REMD methodology (pKa =6.21), was higher than the isolated E amino acid (pKa =4.25), thus being more likely to be found in its protonated state at the binding site. Moreover, we identified an interaction between a water molecule and C239 and G235 as essential for NZ binding. Our results suggest that the protonation state of E198 and the structural water molecules play key roles in the binding of NZ to ß-tubulin.

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