Role of glycans in cholesteryl ester transfer protein revealed by molecular dynamics simulation

Current cholesteryl ester transfer protein (CETP) inhibitors are designed based on the unglycosylated crystal structure, and most of them have failed to cure cardiovascular disease (CVD). It is particularly important for us to investigate the glycosylation structure of CETP (CETP‐G) and effect of glycans on the structure and function of CETP. Here, we used a total of 3.0‐μs molecular dynamics (MD) trajectories of nascent structure of CETP (CETP‐N) and CETP‐G to study their structural differentiations, to shed new light on the CETP‐mediated lipid exchange. In accordance with our simulations and previous mutation studies, relative to CETP‐N, CETP‐G adopts a more stretched shape with higher hydrophobic and hydrophilic solvent‐accessible surface area (SASA) of N‐terminal oscillating with larger amplitude, in which Glycan88 provides partial assistance for CEs through the N‐terminal. Glycan341 reduces the flexibility of neck flap, with the interference of CEs through the neck region. Besides, Glycan240 reduces the flexibility of Helix‐X to interfere the CEs transfer. Glycan396 decreases the flexibility and increases the hydrophobic SASA of C‐terminal. Overall, these glycans affect the dynamics and structure of CETP through forming H‐bonds with surrounding residues, and the sampled conformations of glycan is also affected by its surrounding residues. Thus, glycans are an integral part of CETP, further studies on the CETP inhibition and treatment of CVD should fully consider the effect of glycans.     

Atomistic MD simulation reveals the mechanism by which CETP penetrates into HDL enabling lipid transfer from HDL to CETP

Inhibition of cholesterol ester transfer protein (CETP), a protein mediating transfer of neutral lipids between lipoproteins, has been proposed as a means to elevate atheroprotective HDL subpopulations and thereby reduce atherosclerosis. However, off-target and adverse effects of the inhibition have raised doubts about the molecular mechanism of CETP-HDL interaction. Recent experimental findings have demonstrated the penetration of CETP into HDL. However, atomic level resolution of CETP penetration into HDL, a prerequisite for a better understanding of CETP functionality and HDL atheroprotection, is missing. We constructed an HDL particle that mimics the actual human HDL mass composition and investigated for the first time, by large-scale atomistic molecular dynamics, the interaction of an upright CETP with a human HDL-mimicking model. The results demonstrated how CETP can penetrate the HDL particle surface, with the formation of an opening in the N barrel domain end of CETP, put in evidence the major anchoring role of a tryptophan-rich region of this domain, and unveiled the presence of a phenylalanine barrier controlling further access of HDL-derived lipids to the tunnel of CETP. The findings reveal novel atomistic details of the CETP-HDL interaction mechanism and can provide new insight into therapeutic strategies.     

Borrelia burgdorferi locus BB0795 encodes a BamA orthologue required for growth and efficient localization of outer membrane proteins

The outer membrane (OM) of the pathogenic diderm spirochete, Borrelia burgdorferi, contains integral β‐barrel outer membrane proteins (OMPs) in addition to its numerous outer surface lipoproteins. Very few OMPs have been identified in B. burgdorferi, and the protein machinery required for OMP assembly and OM localization is currently unknown. Essential OM BamA proteins have recently been characterized in Gram‐negative bacteria that are central components of an OM β‐barrel assembly machine and are required for proper localization and insertion of bacterial OMPs. In the present study, we characterized a putative B. burgdorferi BamA orthologue encoded by open reading frame bb0795. Structural model predictions and cellular localization data indicate that the B. burgdorferi BB0795 protein contains an N‐terminal periplasmic domain and a C‐terminal, surface‐exposed β‐barrel domain. Additionally, assays with an IPTG‐regulatable bb0795 mutant revealed that BB0795 is required for B. burgdorferi growth. Furthermore, depletion of BB0795 results in decreased amounts of detectable OMPs in the B. burgdorferi OM. Interestingly, a decrease in the levels of surface‐exposed lipoproteins was also observed in the mutant OMs. Collectively, our structural, cellular localization and functional data are consistent with the characteristics of other BamA proteins, indicating that BB0795 is a B. burgdorferi BamA orthologue.     

Chloroplast outer envelope protein P39 in Arabidopsis thaliana belongs to the Omp85 protein family

Proteins of the Omp85 family chaperone the membrane insertion of β‐barrel‐shaped outer membrane proteins in bacteria, mitochondria, and probably chloroplasts and facilitate the transfer of nuclear‐encoded cytosolically synthesized preproteins across the outer envelope of chloroplasts. This protein family is characterized by N‐terminal polypeptide transport‐associated (POTRA) domains and a C‐terminal membrane‐embedded β‐barrel. We have investigated a recently identified Omp85 family member of Arabidopsis thaliana annotated as P39. We show by in vitro and in vivo experiments that P39 is localized in chloroplasts. The electrophysiological properties of P39 are consistent with those of other Omp85 family members confirming the sequence based assignment of P39 to this family. Bioinformatic analysis showed that P39 lacks any POTRA domain, while a complete 16 stranded β‐barrel including the highly conserved L6 loop is proposed. The electrophysiological properties are most comparable to Toc75‐V, which is consistent with the phylogenetic clustering of P39 in the Toc75‐V rather than the Toc75‐III branch of the Omp85 family tree. Taken together P39 forms a pore with Omp85 family protein characteristics. The bioinformatic comparison of the pore region of Toc75‐III, Toc75‐V, and P39 shows distinctions of the barrel region most likely related to function. Proteins 2017; 85:1391–1401. © 2014 Wiley Periodicals, Inc.     

Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer protein transgenic mice

The human cholesteryl ester transfer protein (CETP) facilitates the exchange of neutral lipids among lipoproteins. In order to evaluate the effects of increased plasma CETP on lipoprotein levels, a human CETP minigene was placed under the control of the mouse metallothionein-I promoter and used to develop transgenic mice. Integration of the human CETP transgene into the mouse genome resulted in the production of active plasma CETP. Zinc induction of CETP transgene expression caused depression of serum cholesterol due to a significant reduction of high density lipoprotein cholesterol. There was no change in total cholesterol content in very low and low density lipoproteins. However, there was a decrease in the free cholesterol/cholesteryl ester ratio in plasma and in all lipoprotein fractions of transgenic mouse plasma, suggesting stimulation of plasma cholesterol esterification. The results suggest that high levels of plasma CETP activity may be a cause of reduced high density lipoproteins in humans.     

Nonuniform chain collapse during early stages of staphylococcal nuclease folding detected by fluorescence resonance energy transfer and ultrarapid mixing methods

The development of tertiary structure during folding of staphylococcal nuclease (SNase) was studied by time‐resolved fluorescence resonance energy transfer measured using continuous‐ and stopped‐flow techniques. Variants of this two‐domain protein containing intradomain and interdomain fluorescence donor/acceptor pairs (Trp and Cys‐linked fluorophore or quencher) were prepared to probe the intradomain and interdomain structural evolution accompanying SNase folding. The intra‐domain donor/acceptor pairs are within the β‐barrel domain (Trp27/Cys64 and Trp27/Cys97) and the interdomain pair is between the α‐helical domain and the β‐barrel domain (Trp140/Cys64). Time‐resolved energy transfer efficiency accompanying folding and unfolding at different urea concentrations was measured over a time range from 30 μs to ～10 s. Information on average donor/acceptor distances at different stages of the folding process was obtained by using a quantitative kinetic modeling approach. The average distance for the donor/acceptor pairs in the β‐barrel domain decreases to nearly native values whereas that of the interdomain donor/acceptor pairs remains unchanged in the earliest intermediate (<500 μs of refolding). This indicates a rapid nonuniform collapse resulting in an ensemble of heterogeneous conformations in which the central region of the β‐barrel domain is well developed while the C‐terminal α‐helical domain remains disordered. The distance between Trp140 and Cys64 decreases to native values on the 100‐ms time scale, indicating that the α‐helical domain docks onto the preformed β‐barrel at a late stage of the folding. In addition, the unfolded state is found to be more compact under native conditions, suggesting that changes in solvent conditions may induce a nonspecific hydrophobic collapse.     

Reduction in the concentration and activity of plasma cholesteryl ester transfer protein by alcohol.

Plasma cholesteryl esters, synthesized within high density lipoproteins (HDL), may be transferred from HDL particles to other lipoproteins by plasma cholesteryl ester transfer protein (CETP). Alcohol consumption is associated with increased HDL cholesterol concentration and reduced plasma CETP activity. The alcohol-induced decrease in CETP activity may be due to a low concentration of CETP in plasma or the inhibition of CETP by specific inhibitor proteins or alterations in the composition of plasma lipoproteins. The first two possibilities are studied further in this paper using data on 47 alcohol abusers and 31 control subjects. The activity of CETP was measured as the rate of cholesteryl ester transfer between radio-labeled low density lipoproteins and unlabeled HDL using an in vitro method independent of endogenous plasma lipoproteins. Plasma CETP concentration was determined by a Triton-based radioimmunoassay. The alcohol abusers consuming alcohol (on average 154 g/day) had 28% higher HDL cholesterol (P less than 0.01), 27% lower plasma CETP concentration (P less than 0.001), and 22% lower plasma CETP activity (P less than 0.001) than the controls. Plasma CETP concentration showed a negative correlation with HDL cholesterol among all the subjects (r = -0.317, P less than 0.01) but not among the alcohol abusers alone (r = -0.102, N. S.). During 2 weeks of alcohol withdrawal, plasma CETP concentration and activity of 8 subjects increased, whereas HDL cholesterol decreased by 42% (P less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Looks can be deceiving: recent insights into the mechanism of protein secretion by the autotransporter pathway

Autotransporters are a large superfamily of cell surface proteins produced by Gram‐negative bacteria that consist of an N‐terminal extracellular domain (‘passenger domain’) and a C‐terminal β‐barrel domain that resides in the outer membrane (OM). Although it was originally proposed that the passenger domain is translocated across the OM through a channel formed exclusively by the covalently linked β‐barrel domain, this idea has been strongly challenged by a variety of observations. Recent experimental results have suggested a new model in which both the translocation of the passenger domain and the membrane integration of the β‐barrel domain are facilitated by the Bam complex, a highly conserved heteroligomer that plays a general role in OM protein assembly. Other factors, including periplasmic chaperones and inner membrane proteins, have also recently been implicated in the biogenesis of at least some members of the autotransporter superfamily. New results have raised intriguing questions about the energetics of the secretion reaction and the relationship between the assembly of autotransporters and the assembly of other classes of OM proteins. Concomitantly, new mechanistic and structural insights have expanded the utility of the autotransporter pathway for the surface display of heterologous peptides and proteins of interest.     

Crystal structures of OprN and OprJ,outer membrane factors of multidrug tripartite efflux pumps of Pseudomonas aeruginosa

The genome of Pseudomonas aeruginosa encodes tripartite efflux pumps that extrude functionally and structurally dissimilar antibiotics from the bacterial cell. MexAB‐OprM, MexCD‐OprJ, MexEF‐OprN, and MexXY‐OprM are the main tripartite efflux pumps responsible for multidrug resistance in P. aeruginosa. The outer membrane factors OprN, OprJ, and OprM are essential components of functional tripartite efflux pumps. To elucidate the structural basis of multidrug resistance, we determined the crystal structures of OprN and OprJ. These structures revealed several features, including tri‐acylation of the N‐terminal cysteine, a small pore in the β‐barrel domain, and a tightly sealed gate in the α‐barrel domain. Despite the overall similarity of OprN, OprJ, and OprM, a comparison of their structures and electrostatic distributions revealed subtle differences at the periplasmic end of the α‐barrel domain. These results suggested that the overall structures of these outer membrane factors are specifically optimized for particular tripartite efflux pumps. Proteins 2016; 84:759–769. © 2016 Wiley Periodicals, Inc.     

Mammalian adipose tissue and muscle are major sources of lipid transfer protein mRNA

The plasma cholesteryl ester transfer protein (CETP) catalyzes the transfer of cholesteryl esters from high density lipoproteins (HDL) to triglyceride-rich lipoproteins and plays a major role in the catabolism of HDL. Lipoprotein lipase (LPL) is the rate-limiting enzyme for hydrolysis of circulating triglyceride and is involved in HDL formation. We show that tissues containing LPL are major sources of CETP mRNA in several mammalian species, including some with low cholesteryl ester transfer activity in plasma. In hamsters, adipose tissue and heart were found to be the richest sources of both CETP and LPL mRNA; in situ hybridization studies showed that the same cell types (i.e. adipocytes or myocytes) contained CETP and LPL mRNA in these tissues. Isolated adipocytes synthesized active CETP. Dietary studies revealed a complex pattern of response of CETP mRNA levels in different tissues, which showed partial similarity to the changes in LPL mRNA abundance. However, high cholesterol diets resulted in increased CETP mRNA abundance in adipose tissue, heart, and skeletal muscle, without equivalent changes in LPL mRNA. Plasma HDL cholesteryl ester levels showed strong inverse correlations with CETP mRNA abundance in adipose tissue. The results suggest a conserved function of CETP in adipose tissue and heart, such as a co-ordinate action with LPL to enhance HDL turnover. Although there is considerable overlap in the tissue- and cell-specific pattern of CETP and LPL gene expression, dietary studies revealed only limited parallelism in response at the mRNA level. The increase in CETP mRNA in peripheral tissues in response to increased dietary cholesterol suggests that local induction of CETP synthesis may help to recycle cholesterol deposited in these tissues during lipolysis of dietary lipoproteins.     

Structural,energetic, and dynamic responses of the native state ensemble of staphylococcal nuclease to cavity‐creating mutations

The effects of cavity‐creating mutations on the structural flexibility, local and global stability, and dynamics of the folded state of staphylococcal nuclease (SNase) were examined with NMR spectroscopy, MD simulations, H/D exchange, and pressure perturbation. Effects on global thermodynamic stability correlated well with the number of heavy atoms in the vicinity of the mutated residue. Variants with substitutions in the C‐terminal domain and the interface between α and β subdomains showed large amide chemical shift variations relative to the parent protein, moderate, widespread, and compensatory perturbations of the H/D protection factors and increased local dynamics on a nanosecond time scale. The pressure sensitivity of the folded states of these variants was similar to that of the parent protein. Such observations point to the capacity of the folded proteins to adjust to packing defects in these regions. In contrast, cavity creation in the β‐barrel subdomain led to minimal perturbation of the structure of the folded state, However, significant pressure dependence of the native state amide resonances, along with strong effects on native state H/D exchange are consistent with increased probability of population of excited state(s) for these variants. Such contrasted responses to the creation of cavities could not be anticipated from global thermodynamic stability or crystal structures; they depend on the local structural and energetic context of the substitutions. © 2012 Wiley Periodicals, Inc.     

Familial cholesteryl ester transfer protein deficiency is associated with triglyceride-rich low density lipoproteins containing cholesteryl esters of probable intracellular origin

The net transfer of core lipids between lipoproteins is facilitated by cholesteryl ester transfer protein (CETP). We have recently documented CETP deficiency in a family with hyperalphalipoproteinemia, due to a CETP gene splicing defect. The purpose of the present study was to characterize the plasma lipoproteins within the low density lipoprotein (LDL) density range and also the cholesteryl ester fatty acid distribution amongst lipoproteins in CETP-deficient subjects. In CETP deficiency, the conventional LDL density range contained both an apoE-rich enlarged high density lipoprotein (HDL) (resembling HDLc), and also apoB-containing lipoproteins. Native gradient gel electrophoresis revealed clear speciation of LDL subclasses, including a distinct population larger in size than normal LDL. Anti-apoB affinity-purified LDL from the CETP-deficient subjects were shown to contain an elevated triglyceride to cholesteryl ester ratio, and also a high ratio of cholesteryl oleate to cholesteryl linoleate, compared to their own HDL or to LDL from normal subjects. Addition of purified CETP to CETP-deficient plasma results in equilibration of very low density lipoprotein (VLDL) cholesteryl esters with those of HDL. These data suggest that, in CETP-deficient humans, the cholesteryl esters of VLDL and its catabolic product, LDL, originate predominantly from intracellular acyl-CoA:cholesterol acyltransferase (ACAT). The CETP plays a role in the normal formation of LDL, removing triglyceride and transferring LCAT-derived cholesteryl esters into LDL precursors.     

The TPR domain of BepA is required for productive interaction with substrate proteins and the β‐barrel assembly machinery complex

BepA (formerly YfgC) is an Escherichia coli periplasmic protein consisting of an N‐terminal protease domain and a C‐terminal tetratricopeptide repeat (TPR) domain. We have previously shown that BepA is a dual functional protein with chaperone‐like and proteolytic activities involved in membrane assembly and proteolytic quality control of LptD, a major component of the outer membrane lipopolysaccharide translocon. Intriguingly, BepA can associate with the BAM complex: the β‐barrel assembly machinery (BAM) driving integration of β‐barrel proteins into the outer membrane. However, the molecular mechanism of BepA function and its association with the BAM complex remains unclear. Here, we determined the crystal structure of the BepA TPR domain, which revealed the presence of two subdomains formed by four TPR motifs. Systematic site‐directed in vivo photo‐cross‐linking was used to map the protein–protein interactions mediated by the BepA TPR domain, showing that this domain interacts both with a substrate and with the BAM complex. Mutational analysis indicated that these interactions are important for the BepA functions. These results suggest that the TPR domain plays critical roles in BepA functions through interactions both with substrates and with the BAM complex. Our findings provide insights into the mechanism of biogenesis and quality control of the outer membrane.     

Molecular biology and pathophysiological aspects of plasma cholesteryl ester transfer protein

Plasma cholesteryl ester transfer protein (CETP) facilitates the transfer of cholesteryl ester (CE) from high density lipoprotein (HDL) to apolipoprotein B-containing lipoproteins. Since CETP regulates the plasma levels of HDL cholesterol and the size of HDL particles, CETP is considered to be a key protein in reverse cholesterol transport, a protective system against atherosclerosis. CETP, as well as plasma phospholipid transfer protein, belongs to members of the lipid transfer/lipopolysaccharide-binding protein (LBP) gene family, which also includes the lipopolysaccharide-binding protein (LBP) and bactericidal/permeability-increasing protein. Although these four proteins possess different physiological functions, they share marked biochemical and structural similarities. The importance of plasma CETP in lipoprotein metabolism was demonstrated by the discovery of CETP-deficient subjects with a marked hyperalphalipoproteinemia (HALP). Two common mutations in the CETP gene, intron 14 splicing defect and exon 15 missense mutation (D442G), have been identified in Japanese HALP patients with CETP deficiency. The deficiency of CETP causes various abnormalities in the concentration, composition, and functions of both HDL and low density lipoprotein. Although the pathophysiological significance of CETP in terms of atherosclerosis has been controversial, the in vitro experiments showed that large CE-rich HDL particles in CETP deficiency are defective in cholesterol efflux. Epidemiological studies in Japanese-Americans and in the Omagari area where HALP subjects with the intron 14 splicing defect of CETP gene are markedly frequent, have shown an increased incidence of coronary atherosclerosis in CETP-deficient patients. The current review will focus on the recent findings on the molecular biology and pathophysiological aspects of plasma CETP, a key protein in reverse cholesterol transport.     

Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein

Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.     

How Anacetrapib Inhibits the Activity of the Cholesteryl Ester Transfer Protein? Perspective through Atomistic Simulations

Cholesteryl ester transfer protein (CETP) mediates the reciprocal transfer of neutral lipids (cholesteryl esters, triglycerides) and phospholipids between different lipoprotein fractions in human blood plasma. A novel molecular agent known as anacetrapib has been shown to inhibit CETP activity and thereby raise high density lipoprotein (HDL)-cholesterol and decrease low density lipoprotein (LDL)-cholesterol, thus rendering CETP inhibition an attractive target to prevent and treat the development of various cardiovascular diseases. Our objective in this work is to use atomistic molecular dynamics simulations to shed light on the inhibitory mechanism of anacetrapib and unlock the interactions between the drug and CETP. The results show an evident affinity of anacetrapib towards the concave surface of CETP, and especially towards the region of the N-terminal tunnel opening. The primary binding site of anacetrapib turns out to reside in the tunnel inside CETP, near the residues surrounding the N-terminal opening. Free energy calculations show that when anacetrapib resides in this area, it hinders the ability of cholesteryl ester to diffuse out from CETP. The simulations further bring out the ability of anacetrapib to regulate the structure-function relationships of phospholipids and helix X, the latter representing the structural region of CETP important to the process of neutral lipid exchange with lipoproteins. Altogether, the simulations propose CETP inhibition to be realized when anacetrapib is transferred into the lipid binding pocket. The novel insight gained in this study has potential use in the development of new molecular agents capable of preventing the progression of cardiovascular diseases.     

HDL and reverse cholesterol transport. Role of cholesterol ester transfer protein]

As most of peripheral cells are not able to catabolize cholesterol, the transport of cholesterol excess from peripheral tissues back to the liver, namely "reverse cholesterol transport", is the only way by which cholesterol homeostasis is maintained in vivo. Reverse cholesterol transport pathway can be divided in three major steps: 1) uptake of cellular cholesterol by the high density lipoproteins (HDL), 2) esterification of HDL cholesterol by the lecithin: cholesterol acyltransferase and 3) captation of HDL cholesteryl esters by the liver where cholesterol can be metabolized and excreted in the bile. In several species, including man, cholesteryl esters in HDL can also follow an alternative pathway which consists in their transfer from HDL to very low density (VLDL) and low density (LDL) lipoproteins. The transfer of cholesteryl esters to LDL, catalyzed by the Cholesteryl Ester Transfer Protein (CETP), might affect either favorably or unfavorably the reverse cholesterol transport pathway, depending on whether LDL are finally taken up by the liver or by peripheral tissues, respectively. In order to understand precisely the implication of CETP in reverse cholesterol transport, it is essential to determine its role in HDL metabolism, to know the potential regulation of its activity and to identify the mechanism by which it interacts with lipoprotein substrates. Results from recent studies have demonstrated that CETP can promote the size redistribution of HDL particles. This may be an important process in the reverse cholesterol transport pathway as HDL particles with various sizes have been shown to differ in their ability to promote cholesterol efflux from peripheral cells and to interact with lecithin: cholesterol acyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of the read-through domain from bacteriophage Qβ A1 protein

Bacteriophage Qβ is a small RNA virus that infects Escherichia coli. The virus particle contains a few copies of the minor coat protein A1, a C‐terminally prolonged version of the coat protein, which is formed when ribosomes occasionally read‐through the leaky stop codon of the coat protein. The crystal structure of the read‐through domain from bacteriophage Qβ A1 protein was determined at a resolution of 1.8 Å. The domain consists of a heavily deformed five‐stranded β‐barrel on one side of the protein and a β‐hairpin and a three‐stranded β‐sheet on the other. Several short helices and well‐ordered loops are also present throughout the protein. The N‐terminal part of the read‐through domain contains a prominent polyproline type II helix. The overall fold of the domain is not similar to any published structure in the Protein Data Bank.     

CETP and lipid transfer inhibitor protein are uniquely affected by the negative charge density of the lipid and protein domains of LDL

Lipoprotein surface charge influences cholesteryl ester transfer protein (CETP) activity and its association with lipoproteins; however, the relationship between these events is not clear. Additionally, although CETP and its regulator, lipid transfer inhibitor protein (LTIP), bind to lipoproteins, it is not known how the charge density of lipoprotein protein and lipid domains influences these factors. Here, the electronegativity of the protein (by acetylation) and surface lipid (oleate addition) domains of LDL were modified. LDL-only lipid transfer assays measured changes in CETP and LTIP activities. CETP activity was stimulated by <10 microM oleate but completely suppressed by >20 microM. The same electronegative potential induced by acetylation mildly stimulated CETP. Modification-induced enhanced binding of CETP did not correlate with CETP activity. LTIP activity was completely blocked by approximately 10 microM oleate but only mildly suppressed by acetylation. LTIP binding to LDL was not decreased by oleate. Thus, the negative charge of LDL surface lipids, but not protein, is an important regulator of CETP and LTIP activity. Altered binding could not explain changes in CETP activity, suggesting that the extent of CETP binding is not normally rate limiting to its activity. Physiologic and pathophysiologic conditions that modify the negative charge of lipoprotein surface lipids will suppress LTIP activity first, followed by CETP.     

Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules

Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.2-A resolution, revealing a 60-A-long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic phosphatidylcholine at each end. The two tunnel openings are large enough to allow lipid access, which is aided by a flexible helix and possibly also by a mobile flap. The curvature of the concave surface of CETP matches the radius of curvature of HDL particles, and potential conformational changes may occur to accommodate larger lipoprotein particles. Point mutations blocking the middle of the tunnel abolish lipid-transfer activities, suggesting that neutral lipids pass through this continuous tunnel.