Combining Small-Molecule Bioconjugation and Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS) to Expose Allostery: the Case of Human Cytochrome P450 3A4

We report a novel approach to study allostery which combines the use of carefully selected bioconjugates and hydrogen–deuterium exchange mass spectrometry (HDX-MS). This strategy avoids issues related weak substrate binding ligand relocalization. The utility our method is demonstrated using human cytochrome P450 3A4 (CYP3A4), most important drug-metabolizing enzyme. Allosteric activation inhibition CYP3A4 by pharmaceuticals an mechanism drug interactions. performed HDX-MS analysis on several CYP3A4-effector bioconjugates, some mimic allosteric effect positive effectors, while others show activity enhancement even though label does not occupy pocket (agonistic) or do still blocking site (antagonistic). allowed us better define position site, protein structural dynamics associated with activation, presence coexisting conformers.