Structural and Biochemical Studies on the Activation Mechanism of Lecithin:Cholesterol Acyltransferase

Abstract

Coronary heart disease (CHD) is the leading cause of death in the United States and is strongly associated with low levels of high density lipoprotein‐cholesterol (HDL‐C), thus there is significant clinical interest in methods that raise HDL‐C in order to treat and prevent CHD. Lecithin:cholesterol acyltransferase (LCAT) esterifies cholesterol on high density lipoprotein (HDL), which promotes HDL maturation and reverse cholesterol transport, the process by which cholesterol is transported from arterial plaques to the liver. Mutations in LCAT are known to cause two genetic diseases. Fish eye disease (FED) is characterized by partial loss of LCAT activity, whereas familial LCAT deficiency (FLD) is characterized by complete loss of LCAT. Individuals with either FED or FLD have low HDL‐C levels and corneal opacities, while FLD patients ultimately develop renal failure. Consequently, administration of recombinant LCAT and LCAT‐activating compounds are being investigated as treatments to increase HDL in both CHD and FLD, and have already shown promise in animal models. However, improvement of these therapeutics is limited due to a lack of structural information on LCAT and its interactions with HDL. LCAT is 50% identical to lysosomal phospholipase A2 (LPLA2). We previously solved a 1.84 Å structure of LPLA2, as well as a low‐resolution structure of LCAT (8.7 Å), both of which are in an active “lid open” conformation. In addition, structures of LPLA2 bound to inhibitors that mimic substrate binding led to models for how LPLA2 and LCAT bind to phospholipids and subsequently transfer an acyl group to the appropriate acceptor. Here we present a new “closed” crystal structure of LCAT at 3.3 Å wherein a retractable lid appears to block substrate access to the active site. Interestingly, the lid interacts with residues on the catalytic core that are mutated in LCAT genetic disease and are hypothesized to be important for activation by apolipoprotein A‐I (ApoA‐I), the major protein component of HDL particles. We therefore hypothesized that ApoA‐I binding will compete with the lid and thereby stimulate LCAT activity. We have probed the role of the lid by examining disease‐causing and lid mutations for esterase activity on a soluble substrate, binding to synthetic HDLs, and transacylase activity on HDLs. Overall, our results confirm the importance of the retractable lid and its observed intramolecular contacts in proper HDL binding and cholesterol esterification. Overall, these studies are expected to guide the design of more potent small molecule LCAT activators as well as more catalytically efficient recombinant LCAT that could be used for enzyme replacement therapy.Support or Funding InformationThis work was supported by US National Institutes of Health (NIH) grant F037815, an American Heart Association (AHA) Postdoctoral Fellowship 15POST24870001 (K.A.M.), and a University of Michigan Chemical Biology Postdoctoral Fellowship (A.G.).