Unveiling the molecular mechanism of Gi protein biased activation at mGlu2 – mGlu4 heterodimers through Gaussian accelerated molecular dynamics simulations

Abstract Metabotropic glutamate (mGlu) receptors play a crucial role in synaptic transmission through homodimeric or heterodimeric assemblies. Despite their dimeric nature, only one subunit within the mGlu dimer engages with G proteins during activation, and the biased activation can be further controlled by allosteric modulators. Considering the related molecular mechanisms remain elusive, we employed Gaussian accelerated molecular dynamics (GaMD) simulations to investigate the regulated mechanisms in mGlu2–mGlu4 heterodimers. Our results demonstrate that the Gi protein exhibits a higher binding affinity for mGlu4 compared to mGlu2 within the mGlu2–mGlu4 heterodimer. Meanwhile, when the positive allosteric modulator (PAM) binds to Gi‐coupled subunits—whether mGlu2 or mGlu4—it can enhance the binding affinity between the Gi protein and the subunits of the mGlu2–mGlu4 heterodimer. However, if the PAM binds to mGlu2 while the Gi protein is coupled to mGlu4, the binding affinity may be reduced. Additionally, our results highlight the crucial role of the ICL2 region and the perturbation of the residue‐residue coupling network involved in the regulatory pathways in mediating the PAM‐induced modulation of Gi protein preference. In conclusion, these findings provide novel insights into the molecular mechanism underpinning the Gi protein's preference for mGlu4 within the mGlu2–mGlu4 heterodimers and the regulatory influence of PAM on Gi protein binding, advancing our understanding of their functional mechanisms.