Molecular Dynamics Simulation and Photo-Crosslinking Reveal a Specific Cholesterol Binding Site for the Metabotropic Glutamate Receptor 2

The Metabotropic glutamate receptor 2 (mGluR2) belongs to the family of G-protein coupled receptors, a large class of transmembrane proteins whose function has been shown to be regulated by their surrounding lipid environment, especially by the cholesterol content in the membrane. Here, we hypothesize that specific protein-lipid interactions contribute to the dynamics and regulation of mGluR2. Both tryptophan and tyrosine have been found in specific protein-lipid binding motifs. In the search of a sphingolipid or cholesterol binding sites for this protein, an alanine mutagenesis screening was carried out, targeting tryptophan and tyrosine residues at the transmembrane surface of mGluR2. Surface biotinylation and co-immunoprecipitation showed that neither trafficking nor dimerization are disturbed by substitution of these aromatic residues. In contrast, cellular photo-crosslinking assays demonstrated that cholesterol binding is compromised if one tyrosine residue located at the helix five or another at the helix six are replaced. Thus, these experiments suggest these two helices to contain specific cholesterol binding sites. All-atom and coarse-grained molecular dynamics (MD) simulations of the mGluR2 transmembrane domain confirmed this observation, by revealing a highly-localized density of cholesterol near these residues, which smeared out when they were changed to alanine in silico. The simulations also revealed an important collective motion of the helices which seems to be correlated with the presence of cholesterol. Notably of this motion was the tilt of helix six in the outer leaflet close to one of the cholesterol binding sites. Overall, our study combining functional assays and multiscale MD simulations demonstrate the existence of a specific cholesterol binding site in mGluR2. It will be highly interesting to investigate the functional implications of this newly-found specific protein-cholesterol interaction.