Cholesterol driven alteration of the conformation and dynamics of phospholamban in model membranes

The effects of cholesterol on various membrane proteins are of long-standing interest in membrane biophysics. Here we present systematic molecular dynamics simulations (totaling 1.4 mu s) of integral protein phospholamban incorporated in POPC/cholesterol bilayers (containing 0, 11.11, 22.03, 33.33, and 50 mol% of cholesterol). Phospholamban is a key regulator of cardiac contractility and has recently emerged as a potential drug target. In agreement with experiments, our results show that in a cholesterol-free pure POPC bilayer, phospholamban exhibits broad conformational distribution, ranging from the closed T-state to the extended R-state, crucial for its functionality. Increasing cholesterol concentration progressively stabilizes the bent conformers of phospholamban over open structures, and favors extensive interactions of its amphipathic N-terminal helix with the bilayer surface. The interaction energies between the N-terminal helix of PLB and different POPC/cholesterol bilayers quantitatively confirm its stronger interaction with a higher cholesterol-containing membrane. Simulation with 50 mol% of cholesterol further supports the above conclusions, where phospholamban undergoes rapid conformational transition from extended to closed form, which remains stable for the rest of the simulation time and exhibits the strongest interaction with the membrane. Cholesterol participates in hydrogen-bonding and p-stacking interactions with polar and/or aromatic residues and favors membrane association of phospholamban. We observed cholesterol-enrichment in the neighborhood of phospholamban. Moreover, as a modulator of membrane biophysical properties, cholesterol modifies the hydrophobic matching and trans-membrane tilting of phospholamban and also hinders its 2D-lateral mobility. Altogether, our results highlight atomistic details of protein-lipid interplay and provide new insights into the possible effects of cholesterol on conformational dynamics of phospholamban in membrane bilayers.