Characterization of the KRas G12D-inhibitor interactions by HDX-MS and molecular dynamics simulations

Abstract

Hydrogen-deuterium exchange (HDX) combined with mass spectrometry (MS) is a powerful technique for studying changes in protein structure and dynamics upon ligand binding. Protein-ligand complexes can result in increased protection of peptide-bond amides in HDX indicating protein structure stabilization. We have characterized the interaction of lead inhibitor candidates towards the KRas G12D oncoprotein by intact protein and bottom-up HDX-MS in combination with molecular dynamics (MD) simulations. Significant differences in HDX protection were detected upon inhibitor binding in the flexible switch-II pocket of the protein. We identified a correlation between ligand binding affinities and corresponding changes in protection. MD simulations of the free and inhibitor-bound KRas G12D proteins revealed changes in the hydrogen bond network of backbone amides in the switch-II region upon inhibitor binding, explaining the observed HDX protection. This combined HDX-MS and MD analysis approach provides a mechanistic picture of the KRas G12D protein-inhibitor interactions and may be a useful tool for future drug design efforts.