Multivalent interactions between ligands and receptors play pivotal roles in many fundamental biological processes, encompassing viral invasions, pathogenic infections, and targeted therapies. The highly transmissible SARS-CoV-2, responsible for the COVID-19 pandemic, relies on the multivalent interaction between its trimeric spike protein and the angiotensin-converting enzyme 2 (ACE2) as a crucial pathway for cellular invasion. Clarifying the intricate nature of these multivalent interactions is essential for understanding the viral infection and formulating targeted therapeutic strategies. However, real-time detection of multivalent interactions at the single-molecule level is challenging due to their transient feature, spanning microseconds to seconds.
Recently, the research group of Professor Yi-Tao Long & Yi-Lun Ying at Nanjing University introduced a ligand-receptor-anchored nanopore that allowed the protein to maintain structural flexibility and favorable orientations in native states, mapping dynamic multivalent interactions. Using a four-state Markov chain model, two concentration-dependent binding pathways for the Omicron spike protein (Omicron S) and soluble angiotensin-converting enzyme 2 (sACE2): sequential and concurrent, were clarified. Real-time kinetic analysis at the single-monomeric subunit level reveals that three S1 monomers of Omicron S exhibit a consistent and robust binding affinity toward sACE2 (−13.1 ± 0.2 kcal/mol). These results highlight the enhanced infectivity of Omicron S compared to other homologous spike proteins (WT S and Delta S). Notably, the preceding binding of sACE2 to Omicron S facilitates the subsequent binding steps, which was previously obscured in bulk measurements. This single-molecule studies resolve the controversy over the disparity between the measured spike protein binding affinity with sACE2 and the viral infectivity, offering valuable insights for drug design and therapies.
Figure 1. Real-time sensing of multivalent ligand-receptor interactions by a ligand-receptor-anchored nanopore.
Figure 2. Binding pathways for Omicron S and sACE2 at various molar ratios.
Figure 3. Binding kinetics of the homologous spike protein and sACE2.
The related paper entitled “Label-Free Mapping of Multivalent Binding Pathways with Ligand-Receptor-Anchored Nanopores” has been published on Journal of the American Chemical Society on August 24, 2024 (Paper link: https://doi.org/10.1021/jacs.4c04934, DOI: 10.1021/jacs.4c04934). Dr. Hui Ma and Prof. Yi-Lun Ying from our department are the co-corresponding authors. Dr. Hui Ma and Master student Yongyong Wang are the co-first authors. This research was supported by the National Natural Science Foundation of China (22104052 and 32250019).