A specific Role for Cholesterol at the Mu Opioid Homodimer Interface

Abstract

Palmitoylation has recently been shown to stabilize μ-opioid receptor (MOR) homodimerization and receptor-G protein coupling. The MOR palmitoylation site is located at the intracellular end of transmembrane helix 3 (TMH3) (C3.55(170)) rather than in the C-terminus. In work reported here, we tested the hypothesis that a specific cholesterol interaction with palmitoylated C3.55(170) may enhance interactions at the MOR homodimer interface. First, interactive docking studies were used to orient two MOR/cholesterol protomers at the symmetric TMH4-TMH5 interface of mouse dark state rhodopsin. Following energy minimization of the complex, the MOR protomers were found to form an interface that involves N4.41, I4.44, C4.48, I4.51, A4.55 and P4.59, with the cholesterol from protomer A interacting with residues on protomer B and vice versa. Cholesterol was found to contribute 24.7% to the total interaction energy at the dimer interface. In order to explore the structure and dynamics of this unique cholesterol binding site, two sets of NAMD molecular dynamics simulations were then performed employing the CHARMM 27 all atom force field. In the first set of simulations in a fully hydrated POPC bilayer, we examined the structure and stability of a cholesterol docked between palmitoylated C3.55(170) and the MOR monomer. The structure, location and motion of this cholesterol was monitored. In a second set of simulations, the uniqueness of this binding site was tested with the MOR monomer model embedded in a fully hydrated 30% cholesterol/POPC lipid bilayer. Here the cholesterol molecules were randomly placed in the simulation cell. In particular these simulations address the specificity (location/duration) of alternate cholesterol-mu opioid contact sites. Our initial results suggest that the docked binding site at palmitoylated C3.55(170) is a location for specific binding of cholesterol at MOR. [Support: KO5 DA021358 (PHR) and RO1 DA023905 (PYL)]