Single‐molecule force spectroscopy discovers a dual‐binding mode in the <scp>hCG</scp> ‐mediated activation of <scp>LHCGR</scp>

Human Chorionic Gonadotropin (hCG) mediates the activation of luteinizing hormone/choriogonadotropin receptor (LHCGR) by binding to it, which is critical for human reproduction. However, specific dynamics of such interaction remain ambiguous. Herein, we utilized single-molecule force spectroscopy (SMFS) to carry out quantitative analysis during the binding between hCG and separate LHCGR domains. Two distinct binding mechanisms were detected within the LHCGR extracellular domain (ECD), associated with the hinge loop and leucine-rich repeats (LRRs), respectively. Compared to the hinge loop, the LRRs-hCG interaction exhibited a bimodal energy landscape with an exceptionally stable outer barrier (binding lifetime τ 18.4 vs. 0.67 s). Furthermore, the same dual-binding mode was observed from full-length LHCGR on membranes of living cells, confirming the physiological relevance of these distinct binding mechanisms. To explore beyond the experimental measurements, we performed molecular dynamics simulations. The computational outcomes revealed a sequential, three-step hCG/LHCGR dissociation process during which electrostatic forces could facilitate hCG/hinge loop interactions multiple times. Consequently, the anchorage-dependent activation of LHCGR mediated by hCG was specified. Overall, our SMFS-based strategy deciphers the dynamic recognition code of hCG/LHCGR interaction and provides a new clue for developing therapeutic strategies targeting glycoprotein hormone receptors.