Unveiling the molecular mechanism of <i>G_i</i> protein biased activation at <scp>mGlu₂</scp>–<scp>mGlu₄</scp> heterodimers through Gaussian accelerated molecular dynamics simulations

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 G_i 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 G_i-coupled subunits-whether mGlu₂ or mGlu₄-it can enhance the binding affinity between the G_i protein and the subunits of the mGlu₂-mGlu₄ heterodimer. However, if the PAM binds to mGlu₂ while the G_i 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 G_i protein preference. In conclusion, these findings provide novel insights into the molecular mechanism underpinning the G_i protein's preference for mGlu₄ within the mGlu₂-mGlu₄ heterodimers and the regulatory influence of PAM on G_i protein binding, advancing our understanding of their functional mechanisms.