Gonadotrophin-releasing hormone (GnRH) regulates the mammalian reproductive system by binding to its receptor (GnRH1R) and is a target for treating reproductive hormone-dependent disorders and cancers. While the inactive structure of GnRH1R is known, the active conformation and GnRH binding mode that lead to receptor activation are not fully understood. The mechanism of GnRH-induced receptor activation remains poorly understood due to the absence of experimental structures of the active GnRH1R-GnRH complex. To address this gap, we employed computational docking simulations using Rosetta, coupled with a custom Python-based elimination protocol, to identify near-native binding poses. This approach yielded two top-ranked candidates, ROS-1 and ROS-2. Molecular dynamics simulations revealed that ROS-1 induced GnRH1R activation within 1.0 \(\mu\) s, characterised by a \(\approx\) 4 Å outward shift of the cytoplasmic end of TM6. Key interactions included \(\pi -\pi\) stacking between GnRH and GnRH1R (notably Y5 with Y283 \(\phantom{0}^{6.51}\) , Y290 \(\phantom{0}^{6.58}\) , and F309 \(\phantom{0}^{7.38}\) ) and hydrogen bonds with L286 \(\phantom{0}^{6.54}\) . Intramolecular \(\pi -\pi\) interactions within GnRH (Y5 and W3) also played a significant role. Two main communication pathways initiated by R8 of GnRH were identified. R8 formed cation- \(\pi\) interactions with W280 \(\phantom{0}^{6.48}\) and communicated with N87 \(\phantom{0}^{2.50}\) and the DPxxY motif via water-mediated hydrogen bonds. Additional interactions involved M125 \(\phantom{0}^{3.36}\) and the PAF and DRS motifs, which are critical for receptor activation. Key differences in \(\pi -\pi\) interactions at they cytosolic end of TM7 between active and inactive states were identified due to the reorganisation of the DPxxY motif. Finally, GnRH1R communication with lipids through hydrogen bonds involving R240 \(\phantom{0}^{5.67}\) , R75 \(\phantom{0}^{2.38}\) , and S140 \(\phantom{0}^{3.51}\) was observed. This study provides insights into the active conformation and binding dynamics of the GnRH-GnRH1R complex, advancing our current understanding by providing a coherent picture that consolidates previous interpretations, thereby paving the way to better therapeutic applications.
