Deciphering the molecular interaction between Vitamin D3 and pepsin by in vitro and in silico perspectives

The current study explored the molecular interaction between Vitamin D₃ (Vit D₃) and pepsin using multi-spectroscopic, molecular dynamic simulation (MDS), and molecular docking. The fluorescence emission spectra discovered Vit D₃ interacted with pepsin in a static quenching manner due to the formation of the steady-state complex. Thermodynamic data revealed the spontaneous binding of Vit D₃ on pepsin. The formation of the Pepsin-Vit D₃ complex was also validated by circular dichroism (CD) spectroscopy. The fluorescence and CD spectroscopy results revealed Vit D₃ altered the tertiary and secondary structure of pepsin, respectively. Meanwhile, FTIR spectroscopy results revealed a hypochromic shift in the amide I and II peaks. Kinetic parameters showed Vit D₃ inhibited the activity of pepsin by the uncompetitive process. Applied spectroscopic methods disclosed that Vit D₃ binding to pepsin caused microenvironmental modifications around the aromatic residues of protein and changed its structure and function. Moreover, MD simulation and molecular docking were done to analyze the formation of Pepsin-Vit D₃ complexes. Molecular docking findings demonstrated the interaction of Vit D₃ with pepsin mainly involved van der Waals forces and hydrogen bonds that were in good agreement with the fluorescence results. Finally, MDS findings including RMSD, RMSF, and RG confirmed all the experimental data.