Leucine is the native known ligand of Sestrin2 (Sesn2) and its interaction with Sesn2 is particularly noteworthy, as it influences the activity of mTOR in aging and its associated pathologies. It is important to find out how leucine interacts with Sesn2 and how mutations in the binding pocket of leucine affect the binding of leucine. Therefore, this study was committed to investigating the impact of non-synonymous mutations by incorporating a broad spectrum of simulation techniques, from molecular dynamics to free energy calculations. Our study was designed to model the atomic-scale interactions between leucine and mutant forms of Sesn2 already described in the literature.

Due to the crucial role of hydrogen bonding in biological processes, we assessed hydrogen bonds in each trajectory over time. The wild-type displayed a higher average number of hydrogen bonds in comparison to the mutants, particularly evident in T374A, T386A, R390A, and E451Q mutants. Our results demonstrated that the interaction paradigm for the mutants has been altered thus showing a significant decline in the hydrogen bonding network. These mutations compromised dynamic stability by disrupting conformational flexibility, sampling time, and leucine-induced structural constraints, leading to variations in binding affinity and structural stability. The analysis of molecular dynamics-based flexibility highlighted an increased fluctuation in regions 217-339 and 371-380. These regions correspond to a linker and a loop that cover the leucine binding cavity. This cavity is critical for the "latch" mechanism in the N-terminal, which is essential for binding leucine. Specifically, mutations at three threonine residues that show a high degree of conservation, Thr374, Thr377, and Thr386, disrupted interaction with leucine, as these residues are critically located above the binding site. Mutations at these positions (T374A or T386A) lead to the disruption of the interaction with leucine. Further validation of reduced binding and modified internal motions caused by the mutants was obtained through binding free energy calculations, Principal Components Analysis (PCA), and Free Energy Landscape analysis (FEL). The MM/PBSA approach was used to calculate the total binding free energy for wild-type and mutants. The values obtained were as follows: -47.32±0.71 kcal/mol for the wild-type, -31.93±0.73 kcal/mol for T374A, -37.79±0.76 kcal/mol for Y375F, -35.67±0.82 kcal/mol for T386A, -38.13±0.85 kcal/mol for R390A, -35.44±0.84 kcal/mol for W444E, and -32.21±0.77 kcal/mol for E451Q mutants. Similar patterns were noted in the total binding free energy determined by the MM/GBSA approach. The significant drop in total binding free energy in mutants reinforces previous observations suggesting the functional impairment of Sesn2 caused by these mutations.

Through studying the complex molecular interactions between Sesn2 and leucine, as well as their mutations, and identifying specific areas where mutations significantly affect leucine binding. These findings will aid in identifying potential targets for drugs that can control the mTOR pathway. These therapeutic compounds may play a crucial role in treating metabolic diseases, cancer, and neurological disorders, thereby extending the health span, and improving the quality of life for the aging population.