A structure-differential binding method for elucidating the interactions between flavonoids and cytochrome-c by ESI-MS and molecular docking

The study of noncovalent interactions between pharmaceutical molecules and proteins is essential for understanding molecular mechanisms of protein function, and provides foundations for de novo therapeutic agent design. Electrospray ionization mass spectrometry (ESI-MS) has nowadays become a popular tool for analyzing the noncovalent protein complexes, however it usually has difficulty in determining the interaction sites and binding mechanisms. In this work, a new structure-differential binding (SDB) method, combined with ESI-MS and molecular docking (MD) techniques (SDB–ESIMS–MD), was developed and applied to a study of the binding interactions in noncovalent protein–small drug molecule complexes for the characterization of binding sites and binding modes. Using this developed method, protein complexes of flavonoid and flavonoid glycoside ligands and cytochrome-c (Cyt-c) were studied in detail. ESI-MS was used to determine the relative binding affinities and dissociation constants of flavonoid–Cyt-c complexes, and to measure the changes in the stability of the protein complexes with the structural modifications of the ligands for identifying effective binding functional groups. Molecular docking simulations complemented ESI-MS experiments by providing the protein–ligand interaction profile of each complex and displaying the binding mode for each interaction. This SDB–ESIMS–MD method can be applied to a broad range of protein–drug interactions and used to guide further research in the study of structure–binding relationship between drug molecules and targeted biomacromolecules.