Hydrogen‐Deuterium Exchange Mass Spectrometry for Molecular Glue Characterization

Abstract Molecular glues are powerful bioactive molecules that stabilize protein–protein interactions. Yet, the precise mechanisms by which many molecular glues exert their adhesive effects are still not well understood. Native mass spectrometry is an established technique used to monitor the stoichiometry and binding equilibria of molecular glue‐induced protein–protein interactions. However, knowledge is lacking on how protein interaction dynamics change upon molecular glue‐induced stabilization, and what conformational changes occur that enhance the stability of the resulting protein‐protein‐glue ternary complex. Here, hydrogen‐deuterium exchange mass spectrometry (HDX‐MS) is showcased as an analytical tool for molecular glue characterization. Using a broadly applicable molecular glue system involving the eukaryotic regulatory protein 14‐3‐3, its binding partners, and the molecular glue fusicoccin A, the power of HDX‐MS is shown in revealing not only molecular glue binding sites, but also conformational changes upon glue binding that result in differentially stabilized protein‐protein complexes. Overall, the HDX‐MS approach will become highly envisaged and valuable for the characterization of molecular glues, guiding their optimization toward successful design.