Assessment of the binding mechanism of ergothioneine to human serum albumin: Multi-spectroscopy, molecular docking and molecular dynamic simulation

Ergothioneine (EGT) has attracted great attention due to its extremely potent antioxidant properties, universally acknowledged as ‘longevity vitamin’. In order to comprehensive understanding of its pharmacodynamics and pharmacokinetics, the binding mechanism of EGT with human serum albumin (HSA) was clarified by cutting-edged multi-spectroscopic approaches and in silico molecular docking coupled with molecular dynamic simulation. Our fluorescence quenching results revealed that the binding of EGT to HSA was in a static quenching mode validated by the descending Stern–Volmer constant (K_sv) values (2.82, 2.36, 1.48 × 10⁴ L mol⁻¹) and biomolecular quenching rate constant (K_q) values (2.82, 2.36, 1.48 × 10¹² L mol⁻¹) at 298 K, 305 K, and 310 K, respectively. van’t Hoff criterion revealed the combination of EGT with HSA was a spontaneous exothermic process (ΔG = −24.16 kJ mol⁻¹) via hydrogen bonding and van der Waals force interactions (ΔH = −60.25 kJ mol⁻¹, ΔS = −129.44 J mol⁻¹ K⁻¹) at 310 K. The analysis of UV–vis absorption spectrum, synchronous fluorescence spectrum, three-dimensional fluorescence spectrum and circular dichroism indicated the addition of EGT affected the microenvironment of Trp214 and rearranged the structure of HSA. The binding replacement assay interpreted their binding site was near the subdomain IIA of HSA (Sudlow’s site I), which was intuitively exhibited by molecular docking. In addition of obvious van der Wall forces, attractive charge and Pi-alkyl interactions, the chiral betaine group (N⁺(CH₃⁺)₃) in the side chain of EGT was inclined to form hydrogen bonds with Lys199, Ser287 and Arg257 in the hydrophobic cavity of albumin. Moreover, the dynamic simulation reinforced the equilibrium and stability of formed docking complex by four indicators (RMSD, RMSF, Rg, SASA) within 100 ns.