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Abstract

The Metabotropic glutamate receptor 2 (mGluR2) belongs to the family of G-protein coupled receptors, a specific class of transmembrane proteins involved in cellular signaling. The functionality of such transmembrane proteins has been identified to largely depend on their microenvironment, namely the lipid bilayer surrounding them. However, the regulation of the receptors by their lipid microenvironment remains poorly understood. In particular, it remains unclear how specific protein-lipid interactions may modulate the function of mGluR2. In the last years, general motifs for non-covalent cholesterol and sphingolipid interaction within helical domains of transmembrane proteins have been described. In these motifs, both tryptophan and tyrosine residues have been found to play a decisive role. For that reason, an alanine mutagenesis screening, targeting tryptophan and tyrosine residues at the transmembrane surface, was carried out in the search of specific sphingolipid or cholesterol interaction sites for mGluR2. For the different Y→A and W→A variants, surface biotinylation and co-immunoprecipitation showed that neither trafficking nor dimerization were disturbed by substitution of these aromatic residues. In contrast, cellular photo-crosslinking assays demonstrated that cholesterol binding was compromised if one tyrosine residue located at the helix five or another at the helix six was replaced. Thus, these experiments suggested these two helices to contain specific cholesterol binding sites. To get a better molecular insight into these specific protein-lipid interactions, lipid binding to the transmembrane domain of mGluR2 was investigated in molecular dynamics (MD) simulation. The molecular dynamics simulations in GROMACS were performed in collaboration with the Max Planck tandem group of Dr. Camilo Aponte-Santamaría. All-atom and coarse-grained MD simulations of the mGluR2 transmembrane domain confirmed the experimental observation, by revealing a highly-localized density of cholesterol near these residues in helices five and six, which smeared out when they were changed to alanine in silico. The simulations also revealed flexibility of the protein structure at the exoplasmic end of helix six which changed upon introduction of point mutations. Overall, the work combining functional assays and MD simulations demonstrated the existence of specific cholesterol binding sites in mGluR2. It will be highly interesting to investigate the functional implications of this newly-found specific protein–cholesterol interaction on the activity and conformation of the receptor.