AbstractLecithin-cholesterol acyltransferase (LCAT) is an enzyme responsible for the formation of cholesteryl esters from cholesterol (CHOL) and phospholipid (PL) molecules in high-density lipoprotein (HDL) particles that play a crucial role in the reverse cholesterol transport and the development of coronary heart disease (CHD). However, it is poorly understood how LCAT interacts with lipoprotein surfaces and how apolipoprotein A-I (apoA-I) activates it. Thus, here we have studied the interactions between LCAT and lipids through extensive atomistic and coarse-grained molecular dynamics simulations to reveal mechanistic details behind the cholesterol esterification process catalyzed by LCAT. In addition, we studied the binding of LCAT to apoA-I derived peptides, and their effect on LCAT lipid association utilizing experimental surface sensitive biophysical methods. Our simulations show that LCAT anchors itself to lipoprotein surfaces by utilizing non-polar amino acids located in the membrane-binding domain and the active site tunnel opening. Meanwhile, the membrane anchoring hydrophobic amino acids attract cholesterol molecules next to them. The results also highlight the role of the lid-loop in the lipid binding and conformation of LCAT with respect to the lipid surface. The apoA-I derived peptides from the LCAT activating region bind to LCAT and promote its lipid surface interactions, although some of these peptides do not bind lipids individually. By means of free-energy calculations we provided a hypothetical explanation for this mechanism. We also found that the transfer free-energy of PL to the active site is consistent with the activation energy of LCAT. Furthermore, the entry of CHOL molecules into the active site becomes highly favorable by the acylation of SER181. The results provide substantial mechanistic insights concerning the activity of LCAT that may lead to the development of novel pharmacological agents preventing CHD in the future.
Reaction of human lecithin cholesterol acyltransferase with synthetic micellar complexes of apolipoprotein A-I, phosphatidylcholine, and cholesterol.
