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

Abstract

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.

Fig. 1. The workflow of the mass spectrometry-based two-step isotope labeling-lysine reactivity profiling (TILLRP) strategy for probing the conformational hotspots of the dynamic interaction and pharmaceutical intervention of SARS-CoV-2 S1 with ACE2.

Fig. 2. Lysine reactivity profiling of bovine and human serum albumins by using the TILLRP strategy. (A) The N_LE values of 23 BSA lysine residues; (B) the conformational distribution of the quantified lysine sites; (C–F) the proximal microenvironments of Lys⁷⁶, Lys³²², and Lys⁴⁹⁹ in BSA (PDB: 4f5s), and Lys⁵⁰⁰ in HSA (PDB: 1ao6).

Fig. 3. Quantitative lysine reactivity profiling of S1–ACE2 before and after the formation of the complex. (A) The profiles of the lysine N_LE values of S1, ACE2 and the S1–ACE2 complex; (B) the δN_LE values of the lysine residues in S1 and ACE2 between free and complex states.

Fig. 4. The δN_LE of lysine residues in S1–ACE2 complexes induced by the treatment of exogenous small-molecule compounds.