Human LDL core cholesterol ester packing: three-dimensional image reconstruction and SAXS simulation studies

Abstract

Human LDL undergoes a reversible thermal order-disorder phase transition associated with the cholesterol ester packing in the lipid core. Structural changes associated with this phase transition have been shown to affect the resistance of LDL to oxidation in vitro studies. Previous electron cryo-microscopy studies have provided image evidence that the cholesterol ester is packed in three flat layers in the core at temperatures below the phase transition. To study changes in lipid packing, overall structure and particle morphology in three dimensions (3D) subsequent to the phase transition, we cryo-preserved human LDL at a temperature above phase transition (53°C) and examined the sample by electron microscopy and image reconstruction. The LDL frozen from 53°C adopted a different morphology. The central density layer was disrupted and the outer two layers formed a “disrupted shell”-shaped density, located concentrically underneath the surface density of the LDL particle. Simulation of the small angle X-ray scattering curves and comparison with published data suggested that this disrupted shell organization represents an intermediate state in the transition from isotropic to layered packing of the lipid. Thus, the results revealed, with 3D images, the lipid packing in the dynamic process of the LDL lipid-core phase transition. Human LDL undergoes a reversible thermal order-disorder phase transition associated with the cholesterol ester packing in the lipid core. Structural changes associated with this phase transition have been shown to affect the resistance of LDL to oxidation in vitro studies. Previous electron cryo-microscopy studies have provided image evidence that the cholesterol ester is packed in three flat layers in the core at temperatures below the phase transition. To study changes in lipid packing, overall structure and particle morphology in three dimensions (3D) subsequent to the phase transition, we cryo-preserved human LDL at a temperature above phase transition (53°C) and examined the sample by electron microscopy and image reconstruction. The LDL frozen from 53°C adopted a different morphology. The central density layer was disrupted and the outer two layers formed a “disrupted shell”-shaped density, located concentrically underneath the surface density of the LDL particle. Simulation of the small angle X-ray scattering curves and comparison with published data suggested that this disrupted shell organization represents an intermediate state in the transition from isotropic to layered packing of the lipid. Thus, the results revealed, with 3D images, the lipid packing in the dynamic process of the LDL lipid-core phase transition. Human low density lipoprotein: the mystery of core lipid packingJournal of Lipid ResearchVol. 52Issue 2PreviewLDL particles are very fascinating macromolecular assemblies of apolipoprotein B-100 and lipids and their structural features have attracted the attention of scientists for decades. There is a general consensus in the literature that LDL particles are organized into two major compartments, namely an apolar lipid core, comprised primarily of cholesteryl esters, triglycerides, and some free unesterified cholesterol and an outer amphipathic shell that surrounds the apolar core. This outer shell is composed of a phospholipid monolayer containing most of the free unesterified cholesterol and one single copy of apolipoprotein B-100 [for review, see Ref. Full-Text PDF Open Access LDL is a micro emulsion that transports lipid, mainly cholesterol ester, from liver to the peripheral tissues (1Hevonoja T. Pentikainen M.O. Hyvonen M.T. Kovanen P.T. Ala-Korpela M. Structure of low density lipoprotein (LDL) particles: basis for understanding molecular changes in modified LDL.Biochim. Biophys. Acta. 2000; 1488: 189-210Crossref PubMed Scopus (330) Google Scholar, 2Mahley R.W. Innerarity T.L. Rall Jr, S.C. Weisgraber K.H. Plasma lipoproteins: apolipoprotein structure and function.J. Lipid Res. 1984; 25: 1277-1294Abstract Full Text PDF PubMed Google Scholar). LDL particles are taken up by the cells through the LDL receptor and its cholesterol ester content enters the cell’s metabolic pathways (3Brown M.S. Goldstein J.L. Familial hypercholesterolemia: defective binding of lipoproteins to cultured fibroblasts associated with impaired regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.Proc. Natl. Acad. Sci. USA. 1974; 71: 788-792Crossref PubMed Scopus (391) Google Scholar, 4Brown M.S. Goldstein J.L. Receptor-mediated endocytosis: insights from the lipoprotein receptor system.Proc. Natl. Acad. Sci. 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Small D.M. Shipley G.G. Structure of human plasma low-density lipoproteins: molecular organization of the central core.Proc. Natl. Acad. Sci. USA. 1977; 74: 1042-1046Crossref PubMed Scopus (101) Google Scholar, 8Deckelbaum R.J. Shipley G.G. Small D.M. Structure and interactions of lipids in human plasma low density lipoproteins.J. Biol. Chem. 1977; 252: 744-754Abstract Full Text PDF PubMed Google Scholar, 9Laggner P. Kostner G.M. Degovics G. Worcester D.L. Structure of the cholesteryl ester core of human plasma low density lipoproteins: selective deuteration and neutron small-angle scattering.Proc. Natl. Acad. Sci. USA. 1984; 81: 4389-4393Crossref PubMed Scopus (28) Google Scholar). It also has been shown that the phase transition and lipid packing may affect the rate of LDL oxidation (10Schuster B. Prassl R. Nigon F. Chapman M.J. Laggner P. Core lipid structure is a major determinant of the oxidative resistance of low density lipoprotein.Proc. Natl. Acad. Sci. USA. 1995; 92: 2509-2513Crossref PubMed Scopus (39) Google Scholar). Thus, the structure of the LDL lipid core has been intensively studied to shed light on its involvement in the processes of the early atherogenic events. In the 1970s, a model for the cholesterol ester packing was proposed primarily based on X-ray and neutron scattering studies. These studies suggested that the cholesterol ester packed in concentric shells in the core at a temperature below the phase transition, and in an isotropic state at a temperature above the phase transition with the underlying assumption that the LDL is a spherical particle for both core phases (7Atkinson D. Deckelbaum R.J. Small D.M. Shipley G.G. Structure of human plasma low-density lipoproteins: molecular organization of the central core.Proc. Natl. Acad. Sci. USA. 1977; 74: 1042-1046Crossref PubMed Scopus (101) Google Scholar, 8Deckelbaum R.J. Shipley G.G. Small D.M. Structure and interactions of lipids in human plasma low density lipoproteins.J. Biol. Chem. 1977; 252: 744-754Abstract Full Text PDF PubMed Google Scholar, 9Laggner P. Kostner G.M. Degovics G. Worcester D.L. Structure of the cholesteryl ester core of human plasma low density lipoproteins: selective deuteration and neutron small-angle scattering.Proc. Natl. Acad. Sci. USA. 1984; 81: 4389-4393Crossref PubMed Scopus (28) Google Scholar, 11Prassl R. Schuster B. Abuja P.M. Zechner M. Kostner G.M. Laggner P. A comparison of structure and thermal behavior in human plasma lipoprotein(a) and low-density lipoprotein. Calorimetry and small-angle X-ray scattering.Biochemistry. 1995; 34: 3795-3801Crossref PubMed Scopus (15) Google Scholar). More recent work from electron cryo-microscopy (cryo-EM) studies have shown in three-dimensional (3D) images that the LDL particle exhibits a disk shape instead of a sphere at temperatures below the phase transition, and the cholesterol ester forms a flat layered density (12Liu Y. Atkinson D. Enhancing the contrast of apoB to locate the surface components in the 3D density map of human LDL.J. Mol. Biol. 2010; 405: 274-283Crossref PubMed Scopus (14) Google Scholar, 13Orlova E.V. Sherman M.B. Chiu W. Mowri H. Smith L.C. Gotto Jr, A.M. Three-dimensional structure of low density lipoproteins by electron cryomicroscopy.Proc. Natl. Acad. Sci. USA. 1999; 96: 8420-8425Crossref PubMed Scopus (115) Google Scholar, 14Ren G. Rudenko G. Ludtke S.J. Deisenhofer J. Chiu W. Pownall H.J. Model of human low-density lipoprotein and bound receptor based on cryoEM.Proc. Natl. Acad. Sci. USA. 2010; 107: 1059-1064Crossref PubMed Scopus (60) Google Scholar) as opposed to concentric shells. However, the LDL lipid core structure above the phase transition, a more physiologically relevant state, remains controversial from image evidence. Two cryo-EM studies that attempted to freeze the LDL sample from a temperature above the phase transition have reached different conclusions about the changes of the cholesterol ester packing upon the phase transition (15Coronado-Gray A. van Antwerpen R. Lipid composition influences the shape of human low density lipoprotein in vitreous ice.Lipids. 2005; 40: 495-500Crossref PubMed Scopus (11) Google Scholar, 16Sherman M.B. Orlova E.V. Decker G.L. Chiu W. Pownall H.J. Structure of triglyceride-rich human low-density lipoproteins according to cryoelectron microscopy.Biochemistry. 2003; 42: 14988-14993Crossref PubMed Scopus (28) Google Scholar). This discrepancy probably reflects the difficulty to capture the fast speed of this phase transition (17Prassl R. Pregetter M. Amenitsch H. Kriechbaum M. Schwarzenbacher R. Chapman J.M. Laggner P. Low density lipoproteins as circulating fast temperature sensors.PLoS ONE. 2008; 3: e4079Crossref PubMed Scopus (18) Google Scholar) and limitations of performing only two-dimensional (2D) projection image analysis because the LDL particle orientation and imaging defocus will affect the appearance of the striped images (12Liu Y. Atkinson D. Enhancing the contrast of apoB to locate the surface components in the 3D density map of human LDL.J. Mol. Biol. 2010; 405: 274-283Crossref PubMed Scopus (14) Google Scholar). In our previous studies (12Liu Y. Atkinson D. Enhancing the contrast of apoB to locate the surface components in the 3D density map of human LDL.J. Mol. Biol. 2010; 405: 274-283Crossref PubMed Scopus (14) Google Scholar), the core cholesterol ester layered packing was reliably observed in 3D image reconstructions of the structure of LDL prepared form a temperature lower than the phase transition. In the current studies, we fast-freeze LDL samples from a state above the phase transition and use 3D image reconstruction analysis to provide 3D image information for the LDL core structure that represents the state near physiological temperatures. All the chemicals used in the experiments were standard and available from popular vendors. DPPC (1,2-Dipalmitoyl-sn-glycero-3-phosphorylcholine) was purchased from Matreya LLC. EYPC (L-α-Phosphatidylcholine ∼99%) was purchased from Sigma. Electron microscope grids were QUANTIFOIL ®R 2/2 on 400 mesh Cu grids, which were purchased from Quantifoil Micro Tools (Jena Germany). Human LDL was isolated and a of LDL was prepared and the (12Liu Y. Atkinson D. Enhancing the contrast of apoB to locate the surface components in the 3D density map of human LDL.J. Mol. Biol. 2010; 405: 274-283Crossref PubMed Scopus (14) Google Scholar). human plasma was prepared from of and with LDL was from the plasma by with the density of This density LDL was with two of density to the sample with the density The density LDL was and used for the lipid in were for the the was in the to in the temperature for DPPC and temperature for the the were used for experiments was with a as by D. Structural basis for thermal of human low-density 2005; PubMed Scopus Google Scholar) LDL in was used as the sample and the was used as the was from to with a rate of the the sample was to and was to the of the phase transition. The was from the sample in data with the The of the sample was in the The sample from was as (12Liu Y. Atkinson D. Enhancing the contrast of apoB to locate the surface components in the 3D density map of human LDL.J. Mol. Biol. 2010; 405: 274-283Crossref PubMed Scopus (14) Google Scholar). LDL sample was to the mesh The was at in the of the at a of The was and into The from 53°C was in the the temperature was to 53°C and the was to The the sample was of the for about to the a mesh was the and to for A was used to sample to the surface through a on the of the the the was to the The was to on the for and the was and frozen in the below the The that the through this process A was used to the a below the and the that the sample was to the above the the the sample at 53°C than To the changes at 53°C are associated only with the reversible lipid phase transition than particle the sample used for 53°C was in an for and the LDL sample from this was frozen at for were on electron microscope with an low The defocus from to The data DPPC were with and on a The image was at the DPPC images, the data were with and on The were with a and on a the image was image particles images were with the from G. W. Ludtke S.J. an image for electron Biol. PubMed Scopus Google Scholar), contrast was with from the The images were to structure reconstruction with the S.J. Chiu W. for Biol. 1999; PubMed Scopus Google Scholar). The image reconstruction image results were with the and to for of molecular 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) The small angle X-ray scattering was with a modified from the in the D. M. A. a for and of Res. 2010; PubMed Scopus Google Scholar). density were by the of the LDL core in density for LDL from shell density for LDL from To a more core were to the structure of LDL from 53°C to the high density in the core in the density the modified the molecular composition the LDL particle based on the density All the with a density above the were as high electron density in our the and the cholesterol All the the LDL particle with a density below the was a low electron density in our represents the of the The electron for LDL outer shell lipid and cholesterol and the of the lipids are (7Atkinson D. Deckelbaum R.J. Small D.M. Shipley G.G. Structure of human plasma low-density lipoproteins: molecular organization of the central core.Proc. Natl. Acad. Sci. USA. 1977; 74: 1042-1046Crossref PubMed Scopus (101) Google Scholar). has an electron density of Thus, the form for high and low density were of and to the electron density of the outer cholesterol and the from the lipids with the in the the form and the 3D was as a particle and used in the based on the D. M. A. a for and of Res. 2010; PubMed Scopus Google Scholar). the LDL as a of The sample was from to The a that at and at This represents the phase transition of the cholesterol ester in LDL core R.J. Shipley G.G. Small D.M. Structure and interactions of lipids in human plasma low density lipoproteins.J. Biol. Chem. 1977; 252: 744-754Abstract Full Text PDF PubMed Google Scholar). the sample reached was to and to The with the that the sample a reversible phase transition this temperature DPPC were at and 53°C the shown in from the a When the DPPC were from the a shape This shape morphology of the DPPC was to the EYPC frozen from major of EYPC has a phase transition temperature below and the is in a state D.M. The of lipids from to of Lipid shown in the projection images of LDL frozen from a striped These are to be from the layered cholesterol ester packing and are most observed as with of the the LDL sample frozen from the density of the particles was observed to be in a disrupted a When the sample was to and the and the density in the LDL particle was particles were from electron of LDL samples prepared from of the three The images of particles were and with the free from S.J. Chiu W. for Biol. 1999; PubMed Scopus Google Scholar). images with the features the particle from are shown for comparison in The LDL frozen from striped for the LDL prepared from only striped features were observed in two The density of the particle was in The particles with the density also a shape as opposed to the more shape observed at The of particles in the that high density features was as shown in Three-dimensional structure reconstructions were for the three with the S.J. Chiu W. for Biol. 1999; PubMed Scopus Google Scholar). The for reconstruction were with in the as the LDL from LDL from LDL to The lipid core of LDL from are in in which the structure were with the to the lipid layer and in in which the structure were with the to the lipid layer at The of the are in The LDL core from three layers of density The LDL core structure from 53°C a “disrupted density, and the central layer density was disrupted The LDL particle from 53°C a more which can be in The from the structure with the a at The disrupted shell structure in this the density in the core of LDL by the density the at The of this was with the shape that the scattering work has shown that the phase transition of the LDL lipid core a in the of (17Prassl R. Pregetter M. Amenitsch H. Kriechbaum M. Schwarzenbacher R. Chapman J.M. Laggner P. Low density lipoproteins as circulating fast temperature sensors.PLoS ONE. 2008; 3: e4079Crossref PubMed Scopus (18) Google Scholar). Thus, a to freeze the cryo-EM and the LDL sample in a state above phase transition. In the cryo-EM the can freeze the sample in Y. The mechanism of phase a study J. Full Text PDF PubMed Scopus (115) Google Scholar), the will through a that above the a the and the sample may a transition temperature in the for a The that the sample a transition temperature may be to the lipid phase transition of this in we attempted to the that the sample a transition temperature The was at which is above the that the LDL lipid core from a to an state LDL particle is and free from thermal at this temperature for a of D. Structural basis for thermal of human low-density 2005; PubMed Scopus Google The the surface and the 53°C was to a and the only the was in the speed was with the DPPC The of a DPPC have a phase transition at the temperature of D.M. The of lipids from to of Lipid Scholar). When we the lipid from the adopted a morphology that the of the DPPC are in the phase When we the lipid from 53°C with our the DPPC a This is to the of EYPC for which the are in the phase D.M. The of lipids from to of Lipid Scholar). Thus, our at 53°C is fast to capture the state of the of the DPPC This phase transition in the of Y. The mechanism of phase a study J. Full Text PDF PubMed Scopus (115) Google Scholar) which is to the of the LDL lipid core phase transition (17Prassl R. Pregetter M. Amenitsch H. Kriechbaum M. Schwarzenbacher R. Chapman J.M. Laggner P. Low density lipoproteins as circulating fast temperature sensors.PLoS ONE. 2008; 3: e4079Crossref PubMed Scopus (18) Google Scholar). images with high were for images taken at defocus at which the striped is most In LDL frozen from the striped particles were The the particles were and and from one to the In for the LDL frozen from striped particles were observed and the images disrupted electron as the particles These form and in some formed a concentric with the layer of the LDL particle These images may capture an intermediate state the phase transition of the particles state from the in The the particle may the that is in lipids to the molecular interactions (1Hevonoja T. Pentikainen M.O. Hyvonen M.T. Kovanen P.T. Ala-Korpela M. Structure of low density lipoprotein (LDL) particles: basis for understanding molecular changes in modified LDL.Biochim. Biophys. Acta. 2000; 1488: 189-210Crossref PubMed Scopus (330) Google Scholar). When the sample was the from were and to and 3D image reconstruction. In the for the sample frozen from the electron density formed that were to and the flat surface the the images from 53°C some images with shape density in the core that was concentric with the surface of the LDL particle. In the particle shape was and was more in morphology The images with density that was in shape were only observed in the LDL frozen from 53°C and the particles that were into the density were The 3D image reconstructions the in core structure the samples frozen from and In the sample prepared from the lipid core the layered organization However, for the sample prepared from the central density layer and the and lower layers were and to form a disrupted This disrupted shell structure was concentric with the surface of LDL particle and the of the shell was about the as that for the layers to the surface in LDL frozen from This core structure was with the results in the projection images and the Thus, the of the core represents the structure from the major of the particles in the image data than an result of the two and The of the in images and flat layers in 3D the structure changes observed in the images from to 53°C are reversible and are to the lipid phase transition. To the temperature that different LDL we the from the as shown in The curves in shape with the curves (7Atkinson D. Deckelbaum R.J. Small D.M. Shipley G.G. Structure of human plasma low-density lipoproteins: molecular organization of the central core.Proc. Natl. Acad. Sci. USA. 1977; 74: 1042-1046Crossref PubMed Scopus (101) Google Scholar, 8Deckelbaum R.J. Shipley G.G. Small D.M. Structure and interactions of lipids in human plasma low density lipoproteins.J. Biol. Chem. 1977; 252: 744-754Abstract Full Text PDF PubMed Google Scholar, 11Prassl R. Schuster B. Abuja P.M. Zechner M. Kostner G.M. Laggner P. A comparison of structure and thermal behavior in human plasma lipoprotein(a) and low-density lipoprotein. Calorimetry and small-angle X-ray scattering.Biochemistry. 1995; 34: 3795-3801Crossref PubMed Scopus (15) Google Scholar), which that the can the electron density from the structure The curves for the layered structure a at The curves for the disrupted shell structure a at the the of the was This change may result from the central which the of the high density the cholesterol are disrupted (12Liu Y. Atkinson D. Enhancing the contrast of apoB to locate the surface components in the 3D density map of human LDL.J. Mol. Biol. 2010; 405: 274-283Crossref PubMed Scopus (14) Google Scholar). The at this from the disrupted shell structure the data of LDL at temperatures the and the temperature the order-disorder phase transition process R.J. Shipley G.G. Small D.M. Structure and interactions of lipids in human plasma low density lipoproteins.J. Biol. Chem. 1977; 252: 744-754Abstract Full Text PDF PubMed Google Scholar). This probably reflects the the disrupted shell and the LDL particle the density of the LDL structure from 53°C to the high density features the LDL particle to a more state of the core lipid. the density and the core density the at in the also and the high density in the shape was The of the in the is to data for LDL at the temperatures above the of the phase transition R.J. Shipley G.G. Small D.M. Structure and interactions of lipids in human plasma low density lipoproteins.J. Biol. Chem. 1977; 252: 744-754Abstract Full Text PDF PubMed Google Scholar). Thus, the lipid packing in the 3D images are to the temperature in the phase transition process as shown in the phase transition the cholesterol are as the temperature the cholesterol to in probably through the with the of phospholipid monolayer at the LDL particle The interactions the may form and the cholesterol are in the shell the temperature to the cholesterol ester the disrupted shell may form the central layer through the with the of the cholesterol that exist in the The transition process is in a This phase transition also has an on the overall shape of LDL which is by the change in LDL particle shape we the cryo-EM experiments to for image evidence of the lipid packing at a state above the phase transition we that the images are more with LDL particles in an intermediate state the phase transition These of our to the the of the LDL core phase transition than the speed of the current However, the of an intermediate state the two phase transition model and the dynamic process of lipid from isotropic to layered packing the lipid-core phase transition. This lipid phase transition also a change in the LDL which may be to the at the particle surface as changes in and the LDL particle surface and may for the different resistance to oxidation for LDL at different temperatures (10Schuster B. Prassl R. Nigon F. Chapman M.J. Laggner P. Core lipid structure is a major determinant of the oxidative resistance of low density lipoprotein.Proc. Natl. Acad. Sci. USA. 1995; 92: 2509-2513Crossref PubMed Scopus (39) Google Scholar). In the as LDL particles to the peripheral may temperatures in which the phase transition A. Human the temperature of the tissues of the Google Scholar). Thus, the structure changes that have been shown in our study are relevant in The of this phase transition structural changes in LDL on the with molecular The and for their in the LDL for the the and at of for the in contrast two three electron microscopy small angle X-ray scattering