Structural analysis of lecithin:cholesterol acyltransferase bound to high density lipoprotein particles.
ABSTRACT: Lecithin:cholesterol acyltransferase (LCAT) catalyzes a critical step of reverse cholesterol transport by esterifying cholesterol in high density lipoprotein (HDL) particles. LCAT is activated by apolipoprotein A-I (ApoA-I), which forms a double belt around HDL, however the manner in which LCAT engages its lipidic substrates and ApoA-I in HDL is poorly understood. Here, we used negative stain electron microscopy, crosslinking, and hydrogen-deuterium exchange studies to refine the molecular details of the LCAT-HDL complex. Our data are consistent with LCAT preferentially binding to the edge of discoidal HDL near the boundary between helix 5 and 6 of ApoA-I in a manner that creates a path from the lipid bilayer to the active site of LCAT. Our results provide not only an explanation why LCAT activity diminishes as HDL particles mature, but also direct support for the anti-parallel double belt model of HDL, with LCAT binding preferentially to the helix 4/6 region.
Project description:The product of transesterification of phospholipid acyl chains and unesterified cholesterol (UC) by the enzyme lecithin:cholesterol acyltransferase (LCAT) is cholesteryl ester (CE). Activation of LCAT by apolipoprotein (apo) A-I on nascent (discoidal) high-density lipoproteins (HDL) is essential for formation of mature (spheroidal) HDL during the antiatherogenic process of reverse cholesterol transport. Here we report all-atom and coarse-grained (CG) molecular dynamics (MD) simulations of HDL particles that have major implications for mechanisms of LCAT activation. Both the all-atom and CG simulations provide support for a model in which the helix 5/5 domains of apoA-I create an amphipathic "presentation tunnel" that exposes methyl ends of acyl chains at the bilayer center to solvent. Further, CG simulations show that UC also becomes inserted with high efficiency into the amphipathic presentation tunnel with its hydroxyl moiety (UC-OH) exposed to solvent; these results are consistent with trajectory analyses of the all-atom simulations showing that UC is being concentrated in the vicinity of the presentation tunnel. Finally, consistent with known product inhibition of CE-rich HDL by CE, CG simulations of CE-rich spheroidal HDL indicate partial blockage of the amphipathic presentation tunnel by CE. These results lead us to propose the following working hypothesis. After attachment of LCAT to discoidal HDL, the helix 5/5 domains in apoA-I form amphipathic presentation tunnels for migration of hydrophobic acyl chains and amphipathic UC from the bilayer to the phospholipase A2-like and esterification active sites of LCAT, respectively. This hypothesis is currently being tested by site-directed mutagenesis.
Project description:Lecithin:cholesterol acyltransferase (LCAT) plays a key role in reverse cholesterol transport by transferring an acyl group from phosphatidylcholine to cholesterol, promoting the maturation of high-density lipoproteins (HDL) from discoidal to spherical particles. LCAT is activated through an unknown mechanism by apolipoprotein A-I (apoA-I) and other mimetic peptides that form a belt around HDL. Here, we report the crystal structure of LCAT with an extended lid that blocks access to the active site, consistent with an inactive conformation. Residues Thr-123 and Phe-382 in the catalytic domain form a latch-like interaction with hydrophobic residues in the lid. Because these residues are mutated in genetic disease, lid displacement was hypothesized to be an important feature of apoA-I activation. Functional studies of site-directed mutants revealed that loss of latch interactions or the entire lid enhanced activity against soluble ester substrates, and hydrogen-deuterium exchange (HDX) mass spectrometry revealed that the LCAT lid is extremely dynamic in solution. Upon addition of a covalent inhibitor that mimics one of the reaction intermediates, there is an overall decrease in HDX in the lid and adjacent regions of the protein, consistent with ordering. These data suggest a model wherein the active site of LCAT is shielded from soluble substrates by a dynamic lid until it interacts with HDL to allow transesterification to proceed.
Project description:ApoA-I activates LCAT that converts lipoprotein cholesterol to cholesteryl ester (CE). Molecular dynamic simulations suggested earlier that helices 5 of two antiparallel apoA-I molecules on discoidal HDL form an amphipathic tunnel for migration of acyl chains and unesterified cholesterol to the active sites of LCAT. Our recent crystal structure of ?(185-243)apoA-I showed the tunnel formed by helices 5/5, with two positively charged residues arginine 123 positioned at the edge of the hydrophobic tunnel. We hypothesized that these uniquely positioned residues Arg123 are poised for interaction with fatty acids produced by LCAT hydrolysis of the sn-2 chains of phosphatidylcholine, thus positioning the fatty acids for esterification to cholesterol. To test the importance of Arg123 for LCAT phospholipid hydrolysis and CE formation, we generated apoA-I[R123A] and apoA-I[R123E] mutants and made discoidal HDL with the mutants and WT apoA-I. Neither mutation of Arg123 changed the particle composition or size, or the protein conformation or stability. However, both mutations of Arg123 significantly reduced LCAT catalytic efficiency and the apparent Vmax for CE formation without affecting LCAT phospholipid hydrolysis. A control mutation, apoA-I[R131A], did not affect LCAT phospholipid hydrolysis or CE formation. These data suggest that Arg123 of apoA-I on discoidal HDL participates in LCAT-mediated cholesterol esterification.
Project description:LCAT is an enzyme responsible for the formation of cholesteryl esters from unesterified cholesterol (UC) and phospholipid (PL) molecules in HDL particles. However, it is poorly understood how LCAT interacts with lipoproteins and how apoA-I activates it. Here we have studied the interactions between LCAT and lipids through molecular simulations. In addition, we studied the binding of LCAT to apoA-I-derived peptides, and their effect on LCAT lipid association-utilizing experiments. Results show that LCAT anchors itself to lipoprotein surfaces by utilizing nonpolar 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. The transfer free-energy of PL from the lipid bilayer into the active site is consistent with the activation energy of LCAT. Furthermore, the entry of UC molecules into the active site becomes highly favorable by the acylation of SER181.
Project description:Lecithin:cholesterol acyltransferase (LCAT) has been shown to play a role in the depletion of lipid oxidation products, but this has so far not been studied in humans. In this study, we investigated processes and parameters relevant to lipid oxidation in carriers of functional LCAT mutations.In 4 carriers of 2 mutant LCAT alleles, 63 heterozygotes, and 63 family controls, we measured activities of LCAT, paraoxonase 1, and platelet-activating factor-acetylhydrolase; levels of lysophosphatidylcholine molecular species, arachidonic and linoleic acids, and their oxidized derivatives; immunodetectable oxidized phospholipids on apolipoprotein (apo) B-containing and apo(a)-containing lipoproteins; IgM and IgG autoantibodies to malondialdehyde-low-density lipoprotein and IgG and IgM apoB-immune complexes; and the antioxidant capacity of high-density lipoprotein (HDL). In individuals with LCAT mutations, plasma LCAT activity, HDL cholesterol, apoA-I, arachidonic acid, and its oxidized derivatives, oxidized phospholipids on apo(a)-containing lipoproteins, HDL-associated platelet-activating factor-acetylhydrolase activity, and the antioxidative capacity of HDL were gene-dose-dependently decreased. Oxidized phospholipids on apoB-containing lipoproteins was increased in heterozygotes (17%; P<0.001) but not in carriers of 2 defective LCAT alleles.Carriers of LCAT mutations present with significant reductions in LCAT activity, HDL cholesterol, apoA-I, platelet-activating factor-acetylhydrolase activity, and antioxidative potential of HDL, but this is not associated with parameters of increased lipid peroxidation; we did not observe significant changes in the oxidation products of arachidonic acid and linoleic acid, immunoreactive oxidized phospholipids on apo(a)-containing lipoproteins, and IgM and IgG autoantibodies against malondialdehyde-low-density lipoprotein. These data indicate that plasma LCAT activity, HDL-associated platelet-activating factor-acetylhydrolase activity, and HDL cholesterol may not influence the levels of plasma lipid oxidation products.
Project description:The objective of this study was to establish the role of apoA-IV, ABCA1, and LCAT in the biogenesis of apoA-IV-containing HDL (HDL-A-IV) using different mouse models. Adenovirus-mediated gene transfer of apoA-IV in apoA-I(-/-) mice did not change plasma lipid levels. ApoA-IV floated in the HDL2/HDL3 region, promoted the formation of spherical HDL particles as determined by electron microscopy, and generated mostly ?- and a few pre-?-like HDL subpopulations. Gene transfer of apoA-IV in apoA-I(-/-) × apoE(-/-) mice increased plasma cholesterol and triglyceride levels, and 80% of the protein was distributed in the VLDL/IDL/LDL region. This treatment likewise generated ?- and pre-?-like HDL subpopulations. Spherical and ?-migrating HDL particles were not detectable following gene transfer of apoA-IV in ABCA1(-/-) or LCAT(-/-) mice. Coexpression of apoA-IV and LCAT in apoA-I(-/-) mice restored the formation of HDL-A-IV. Lipid-free apoA-IV and reconstituted HDL-A-IV promoted ABCA1 and scavenger receptor BI (SR-BI)-mediated cholesterol efflux, respectively, as efficiently as apoA-I and apoE. Our findings are consistent with a novel function of apoA-IV in the biogenesis of discrete HDL-A-IV particles with the participation of ABCA1 and LCAT, and may explain previously reported anti-inflammatory and atheroprotective properties of apoA-IV.
Project description:We studied the significance of four hydrophobic residues within the 225-230 region of apoA-I on its structure and functions and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of an apoA-I[F225A/V227A/F229A/L230A] mutant in apoA-I?/? mice decreased plasma cholesterol, HDL cholesterol, and apoA-I levels. When expressed in apoA-I?/? × apoE?/? mice, approximately 40% of the mutant apoA-I as well as mouse apoA-IV and apoB-48 appeared in the VLDL/IDL/LDL. In both mouse models, the apoA-I mutant generated small spherical particles of pre-?- and ?4-HDL mobility. Coexpression of the apoA-I mutant and LCAT increased and shifted the-HDL cholesterol peak toward lower densities, created normal ?HDL subpopulations, and generated spherical-HDL particles. Biophysical analyses suggested that the apoA-I[225-230] mutations led to a more compact folding that may limit the conformational flexibility of the protein. The mutations also reduced the ability of apoA-I to promote ABCA1-mediated cholesterol efflux and to activate LCAT to 31% and 66%, respectively, of the WT control. Overall, the apoA-I[225-230] mutations inhibited the biogenesis of-HDL and led to the accumulation of immature pre-?- and ?4-HDL particles, a phenotype that could be corrected by administration of LCAT.
Project description:To develop a detailed double belt model for discoidal HDL, we previously scored inter-helical salt bridges between all possible registries of two stacked antiparallel amphipathic helical rings of apolipoprotein (apo) A-I. The top score was the antiparallel apposition of helix 5 with 5 followed closely by appositions of helix 5 with 4 and helix 5 with 6. The rationale for the current study is that, for each of the optimal scores, a pair of identical residues can be identified in juxtaposition directly on the contact edge between the two antiparallel helical belts of apoA-I. Further, these residues are always in the '9th position' in one of the eighteen 11-mer repeats that make up the lipid-associating domain of apoA-I. To illustrate our terminology, 129j (LL5/5) refers to the juxtaposition of the C? atoms of G129 (in a '9th position') in the pairwise helix 5 domains. We reasoned that if identical residues in the double belt juxtapositions were mutated to a cysteine and kept under reducing conditions during disc formation, we would have a precise method for determining registration in discoidal HDL by formation of a disulfide-linked apoA-I homodimer. Using this approach, we conclude that 129j (LL5/5) is the major rotamer orientation for double belt HDL and propose that the small ubiquitous gap between the pairwise helix 5 portions of the double belt in larger HDL discoidal particles is significantly dynamic to hinge off the disc edge under certain conditions, e.g., in smaller particles or perhaps following binding of the enzyme LCAT. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).
Project description:In the present study we have used adenovirus-mediated gene transfer of apoA-I (apolipoprotein A-I) mutants in apoA-I-/- mice to investigate how structural mutations in apoA-I affect the biogenesis and the plasma levels of HDL (high-density lipoprotein). The natural mutants apoA-I(R151C)Paris, apoA-I(R160L)Oslo and the bioengineered mutant apoA-I(R149A) were secreted efficiently from cells in culture. Their capacity to activate LCAT (lecithin:cholesterol acyltransferase) in vitro was greatly reduced, and their ability to promote ABCA1 (ATP-binding cassette transporter A1)-mediated cholesterol efflux was similar to that of WT (wild-type) apoA-I. Gene transfer of the three mutants in apoA-I-/- mice generated aberrant HDL phenotypes. The total plasma cholesterol of mice expressing the apoA-I(R160L)Oslo, apoA-I(R149A) and apoA-I(R151C)Paris mutants was reduced by 78, 59 and 61% and the apoA-I levels were reduced by 68, 64 and 55% respectively, as compared with mice expressing the WT apoA-I. The CE (cholesteryl ester)/TC (total cholesterol) ratio of HDL was decreased and the apoA-I was distributed in the HDL3 region. apoA-I(R160L)Oslo and apoA-I(R149A) promoted the formation of prebeta1 and alpha4-HDL subpopulations and gave a mixture of discoidal and spherical particles. apoA-I(R151C)Paris generated subpopulations of different sizes that migrate between prebeta and alpha-HDL and formed mostly spherical and a few discoidal particles. Simultaneous treatment of mice with adenovirus expressing any of the three mutants and human LCAT normalized plasma apoA-I, HDL cholesterol levels and the CE/TC ratio. It also led to the formation of spherical HDL particles consisting mostly of alpha-HDL subpopulations of larger size. The correction of the aberrant HDL phenotypes by treatment with LCAT suggests a potential therapeutic intervention for HDL abnormalities that result from specific mutations in apoA-I.
Project description:Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme that catalyzes the esterification of free cholesterol in plasma lipoproteins and plays a critical role in high-density lipoprotein (HDL) metabolism. Deficiency leads to accumulation of nascent pre?-HDL due to impaired maturation of HDL particles, whereas enhanced expression is associated with the formation of large, apoE-rich HDL(1) particles. In addition to its function in HDL metabolism, LCAT was believed to be an important driving force behind macrophage reverse cholesterol transport (RCT) and, therefore, has been a subject of great interest in cardiovascular research since its discovery in 1962. Although half a century has passed, the importance of LCAT for atheroprotection is still under intense debate. This review provides a comprehensive overview of the insights that have been gained in the past 50 years on the biochemistry of LCAT, the role of LCAT in lipoprotein metabolism and the pathogenesis of atherosclerosis in animal models, and its impact on cardiovascular disease in humans.