Probing conformational hotspots for the recognition and intervention of protein complexes by lysine reactivity profiling.

Probing the conformational and functional hotspot sites within aqueous native protein complexes is still a challenging task. Herein, a mass spectrometry (MS)-based two-step isotope labeling-lysine reactivity profiling (TILLRP) strategy is developed to quantify the reactivities of lysine residues and probe the molecular details of protein-protein interactions as well as evaluate the conformational interventions by small-molecule active compounds. The hotspot lysine sites that are crucial to the SARS-CoV-2 S1-ACE2 combination could be successfully probed, such as S1 Lys417 and Lys444. Significant alteration of the reactivities of lysine residues at the interaction interface of S1-RBD Lys386-Lys462 was observed during the formation of complexes, which might be utilized as indicators for investigating the S1-ACE2 dynamic recognition and intervention at the molecular level in high throughput.