Investigating Structure and Sequence Dependence in the Dimerization of G Protein-Coupled Receptors

Abstract

Recent studies have reached mixed conclusions regarding the lifetime and fraction of G Protein-Coupled Receptor (GPCR) oligomers in living cells. Whilst a few, recently published, single-molecule imaging studies suggest transient association between GPCRs, fluorescence recovery after photobleaching (FRAP) has led to variable conclusions. Two closely related GPCRs, the β1-adrenergic receptor (B1AR) and β2- adrenergic receptor (B2AR), were proposed to form transient interactions and stable homomeric complexes, respectively. To obtain a rigorous mechanistic insight into the association of B1AR and B2AR in the cell membrane, at a level of molecular detail beyond that currently attainable by experimental techniques, we have calculated the free energy of association of these receptors using biased molecular dynamics simulations, in particular, a combination of umbrella sampling and metadynamics. Representing explicitly solvated (in a palmitoyl-oleoyl-phosphatidyl-choline (POPC)/10 % cholesterol bilayer) B2AR and B1AR crystal structures using the MARTINI coarse-grained force field, we studied their homo-dimerization at symmetric interfaces formed by transmembrane (TM) helices that have been implicated in GPCR association (i.e., TM1, TM4, and TM4/5). Reconstruction of the free-energy surfaces as a function of the inter-protomeric distance demonstrates different relative stability of the B1AR or B2AR dimers depending on the sequence and/or the different structural features at the interface. Specifically, we observe that: a) For both receptors, dimers interacting at interfaces defined by TM1 are more stable than TM4 or TM4/5 dimers; and b) Between the two receptors, the B2AR dimers appear to be generally - albeit not significantly - more stable than the B1AR dimers. Our calculations can be generally applied across family A GPCRs, and offer a novel insight into the mechanism of GPCR dimerization in the cell membrane.