foods-4288909-zenodo

Molecular Docking and Molecular Dynamics (MD) Simulations The 3D structure of Hi-NRK was modeled using AlphaFold3, selecting the highest-scoring model. Ligand structures were obtained from the Protein Data Bank (PDB). Molecular docking was performed with AutoDock Vina, and interactions were visualized using PyMOL. Multiple sequence alignments were conducted using UniProt and ESPript 3.0. MD simulations were performed using the GROMACS 2025 software package. The docked enzyme-substrate complexes were solvated in a cubic box of TIP3P water molecules, extending at least 1.2 nm from the protein surface, and 0.9% NaCl (w/v) was added to each system for charge neutralization. Energy minimization was conducted using the steepest descent algorithm, followed by a simulated annealing protocol to relieve unfavorable contacts and achieve a stable starting configuration, under the CHARMM36 all-atom force field with CMAP corrections for backbone dihedrals. The ATP cofactor and Mg²⁺ ions were parameterized using the CHARMM36 nucleotide and standard ion parameters, respectively. The system was subsequently equilibrated using NVT (constant particle number, volume, and temperature) and NPT (constant particle number, pressure, and temperature) ensembles. A production MD simulation was then run for 100 ns at 313 K (40 ℃) (to simulate thermal stress) and 1 atm, with a 2 fs time step. Trajectory frames were saved every 100 ps. System stability was evaluated by monitoring the RMSD of Cα atoms, total system energy, and density fluctuations over the simulation time, confirming convergence prior to analysis. The trajectories were processed and analyzed using GROMACS tools to calculate the root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), binding free energy, enzyme-substrate distance variations, and dynamic cross-correlation matrix (DCCM) between protein and ligand .