Crystal structure of cholesteryl ester transfer protein reveals a long ...

© 2007 Nature Publishing Group http://www.nature.com/nsmb

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Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules Xiayang Qiu, Anil Mistry, Mark J Ammirati, Boris A Chrunyk, Ronald W Clark, Yang Cong, Jeffrey S Culp, Dennis E Danley, Thomas B Freeman, Kieran F Geoghegan, Matthew C Griffor, Steven J Hawrylik, Cheryl M Hayward, Preston Hensley, Lise R Hoth, George A Karam, Maruja E Lira, David B Lloyd, Katherine M McGrath, Kim J Stutzman-Engwall, Ann K Subashi, Timothy A Subashi, John F Thompson, Ing-Kae Wang, Honglei Zhao & Andrew P Seddon 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 ˚ resolution, revealing a 60-A ˚ -long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic 2.2-A 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.

Low levels of HDL cholesterol (HDL-C) are an independent and important risk factor for cardiovascular disease, the leading cause of death in many countries1. Epidemiological studies suggest that the risk for coronary heart disease is 2%–3% lower for each 0.1 mg l–1 increase in HDL-C2,3. Current HDL-C–elevating drugs have limited efficacy and undesirable side effects4,5, but CETP inhibitors represent a new class of agents6–8. CETP inhibitors have achieved remarkable elevation of HDL-C levels and marked decreases in LDL-C, and early clinical trials gauging their impact on arterial plaque and, ultimately, morbidity and mortality, are ongoing to validate the therapeutic potential of inhibiting CETP for the treatment of cardiovascular disease9. CETP is a hydrophobic glycoprotein that is often bound to HDL in circulation and engages in the transfer of neutral lipids, including cholesteryl ester and triglyceride, among lipoprotein particles10,11. It can facilitate either homoexchange, the bidirectional transfer of the same neutral lipid, or heteroexchange, the net mass transfer of cholesteryl ester and triglyceride between lipoproteins. Heteroexchange can result in the net movement of cholesteryl ester from HDL particles to triglyceride-rich lipoproteins, such as very-lowdensity lipoprotein (VLDL), and in the equimolar transport of triglyceride from VLDL to HDL. Heteroexchange has been implicated in the physiological process of reverse cholesterol transport, by which excess cholesterol is removed from peripheral tissues (for example, the arterial wall) and returned to the liver for elimination. CETP also mediates the homoexchange of phospholipid among lipoproteins,

although the net phospholipid transfer is carried out by the phospholipid transfer protein, PLTP12. Extensive studies of CETP polymorphisms and genetic deficiency suggest direct links between CETP, HDL-C level and cardiovascular disease13. However, many aspects of the CETP biological functions are not fully understood14. The molecular basis for the binding and transfer of the various lipids is also unclear. CETP belongs to a family of proteins that engage in lipid binding (lipopolysaccharide-binding protein (LBP) and bactericidal/ perme ability-increasing protein (BPI)) and lipid transfer (CETP and PLTP)15, but emerging family members may have other functions16. The crystal structure of a member of this family, BPI (22% identity), has revealed two distinct phospholipid-binding cavities and a fold that may be common to the family17. The BPI cavities are similar to those of other lipid-binding and transfer proteins, which have diverse folds but typically contain a small lipid-binding cavity (400–880 A˚3) with a flexible lid that regulates lipid access, as exemplified by fatty acid– binding protein (FABP)18. The cavity alternates between distinct apo (empty) and holo (lipid-bound) states during lipid transfer. Some authors have used the term ‘tunnel’ to describe the cavity—for example, in steroidogenic acute regulatory protein–related lipid transfer (START) domains19, glycolipid transfer protein (GLTP)20 and the oxysterol-binding protein–related protein Osh4 (ref. 21). In GLTP and Osh4, the tunnel is an elongated pocket with a single opening. In the START domains, the tunnel is a valley in the lid-open

Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06430, USA. Correspondence should be addressed to X.Q. ([email protected]). Received 4 November 2006; accepted 29 December 2006; published online 21 January 2007; doi:10.1038/nsmb1197

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a

5

341

A′ B

396 B′

A X 90°


b

Ω2

Ω1

240

A′

Figure 1 Overall structure of CETP. (a) Ribbon diagram of N-terminal (green) and C-terminal (yellow) domains, with linker in red. Whenever possible, N-terminal side is shown on left throughout the figures. CE1 (magenta) and CE2 (cyan) are shown as space fills and phospholipid as black bonds. N-glycosylation sites are shown as blue bonds, with 341 and 396 labeled. ‘5’ marks the observed N terminus. Helices A, B, A¢, B¢ and X are labeled. Helix X belongs to the C-terminal domain but interacts with residues of the N-terminal domain. (b) The view after a 901 rotation. The four structural units shown are barrel N (green), central b-sheet (orange), barrel C (yellow) and helix X (cyan). The O1 flap is in gray. N-glycosylation sites 88 and 240 are labeled. (c,d) Ca traces of CETP (yellow) and BPI (purple) with the barrel Ns (c) or barrel Cs (d) overlaid. CETP-bound phospholipid molecules are in red.

B Ω2 B′ A 88

X

Ω1

c

d

state. A single point of entry and egress is used for lipid exchange in the proposed models17–22; hence, ‘tunnel’ may not be the most precise description. To understand the molecular basis of CETP functions, we solved the crystal structure of human CETP in complex with four bound lipid molecules at 2.2-A˚ resolution. The structure reveals an unprecedented 60-A˚-long lipid-filled tunnel that traverses the core of the molecule and has two distinct openings. These results prompt new ideas as to how CETP accomplishes its principal biological function, and they may also aid the design of new therapeutic agents. RESULTS Overall structure Plasma CETP has 476 amino acid residues and a 74-kDa molecular mass, of which 28% is attributed to N-glycosylation at residues 88, 240, 341 and 396 (ref. 10). The high content of glycosylation, hydrophobic residues (44%) and free cysteines (five) presented significant challenges for structural studies. We solved the first structure of CETP at 3.5 A˚ by multiple isomorphous replacement methods, using crystals of a CETP mutant (C1A S90A S343A) in complex with an antibody Fab fragment (Supplementary Data online). Another fully active mutant (C1A C131A N88D N240D N341D) later yielded superior crystals without Fab. The Asn-Asp mutations in the construct were designed to remove glycosylation


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while preserving protein solubility and improving crystal quality23. The higher-resolution structure was solved by molecular replacement using the 3.5-A˚ CETP structure as the search model. The final model, used in the analysis described herein, has been refined to 2.2-A˚ resolution. CETP has an elongated ‘boomerang’ shape with dimensions of 135 A˚ 30 A˚ 35 A˚ and a fold homologous to that of BPI16. The fold consists of two similar domains connected by a linker, residues 240–259 in CETP (Fig. 1a). The structure of CETP can be divided into four structural units: one barrel at each end of the protein (barrels N and C), a central b-sheet between the two barrels and a C-terminal extension that is not present in BPI (Fig. 1b). Each barrel contains a highly twisted b-sheet and two helices (A and B in barrel N, A¢ and B¢ in barrel C), with helices B and B¢ being longer than A and A¢. The central b-sheet includes six antiparallel strands consisting of residues before and after the barrels (Supplementary Fig. 1 online). These three structural units in CETP overlay well with the homologous units in BPI, giving r.m.s. deviations of 1.6 A˚ for barrel N (137 matching Ca atoms), 1.5 A˚ for the central b-sheets (85 matching Cas) and 2.3 A˚ for barrel C (105 matching Cas). The fourth unit, Glu465–Ser476 at the C terminus of CETP, forms a distorted amphipathic helix, helix X, that unwinds slightly at the end. The structure of CETP confirms the earlier predicted folds for the individual structural units10,15. However, the relative orientations of these units are very different, resulting in a distinct overall structure for CETP. When barrel N of CETP is superimposed on that of BPI, the two barrel Cs are separated by 25 A˚ at the tips (Fig. 1c). When the two barrel Cs are overlaid, the two barrel Ns differ by 25 A˚ at the tips (Fig. 1d). Compared to that of CETP, the concave surface in BPI is almost flat, consistent with its biological function of recognizing lipopolysaccharide on the surfaces of bacteria or in solution16. Four bound lipids in a continuous tunnel The structure of CETP reveals four bound lipid molecules, which must have been incorporated during protein production, as no lipid was introduced after expression. Electron density (Fig. 2a), mass spectrometry (Supplementary Data) and the natural abundance of lipid species in mammalian cells support our model of the lipids as two cholesteryl oleate molecules and two dioleoylphosphatidylcholine molecules (Fig. 2b). Studies using biochemical techniques and proteins from diverse sources have suggested that 1 mol CETP binds 0.9–1.4 mol cholesteryl ester, 0.1–0.5 mol triglyceride and 1.3–1.7 mol phospholipid10,24,25. The variability in these values and the BPI-based model have led to the hypothesis that CETP has one neutral lipid– binding site and one phospholipid-binding site15. Our structure demonstrates for the first time that CETP can accommodate two neutral lipids and two phospholipids, and reveals the binding sites of these lipids.

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a

b

O O O P OO O O O

O O O OO

O O

N

O Cholesteryl ester

Phospholipid

Triglyceride


c

90°

180°

180°

Lipid-binding sites and specificity CE1 is located between barrel N and the central b-sheet (Fig. 1b). The binding site is lined with mostly hydrophobic residues and only a few polar residues (Fig. 2c). Ser230 is 3.6 A˚ from the CE1 ester oxygen, too far away to permit a hydrogen bond (Fig. 3). His232 forms a p-stacking interaction with CE1 ester, and the oleoyl tails of CE1 and phosphatidylcholine 1 (PC1) stack with Phe265 and Phe263, respectively. The PC1 phosphate moiety forms a salt bridge with Arg201 and the amino group interacts with the C-cap dipole of helix B¢. The accessible surface area for the combined CE1 and PC1 sites is 1,060 A˚2. The site for CE2 and PC2 resides between the central b-sheet and barrel C and is topologically equivalent to that for CE1 and PC1. CE2 binds in a more extended conformation and penetrates deeper into the barrel (Fig. 1b). The accessible surface area of the combined CE2 and PC2 sites is 1,100 A˚2. This site contains even fewer polar groups than that for CE1 and PC1 (Fig. 2c) and has no hydrogen-bonding or p-stacking interactions. The noteworthy size and hydrophobicity of the tunnel suggests a limited capacity to discriminate between different neutral lipids. The structure shows that cholesteryl ester can bind either side of the tunnel. Triglyceride can be readily docked in silico into either the CE1/PC1 or CE2/PC2

C opening

N opening

Neck

The four lipids occupy a 60-A˚-long, continuous tunnel that traverses the core of the protein and has two distinct openings (Fig. 2c). The two cholesteryl esters are buried in the middle of the tunnel and two phospholipids plug the tunnel, one at each end. The presence of two lipids in a single cavity has been seen before18, but to our knowledge there is no precedent for four lipids distributed in a tunnel. The accessible surface area and volume of the CETP tunnel are 2,100 A˚2 and 2,560 A˚3, respectively. These dimensions are comparable to the largest pocket volumes reported for lipid-binding proteins22,26. The 60-A˚ length of the tunnel is the longest dimension reported for known pockets in lipid-binding and lipid-transfer proteins. The CETP tunnel is formed by a wall of b-sheets underneath the bound lipids and a layer of helices above the lipids (Fig. 1b). Cholesteryl esters 1 and 2 (CE1 and CE2) snugly fill the length of the tunnel and bind in the same orientation, with the head of CE2 packed against the tail of CE1 (Fig. 2c). The center of the tunnel, which we call the ‘neck’, narrows to 10 A˚ wide and 5 A˚ high. As the rigid steroid ring of cholesteryl ester is about 6 A˚ wide and 4 A˚ thick, the neck is large enough to allow the passage of a neutral lipid. The tunnel is highly hydrophobic and contains only two bound water molecules. The tunnel openings in the N- and C-terminal domains (N and C openings) are each capped by one phospholipid, and these phospholipids bury their acyl chains inside the tunnel and expose the zwitterionic head groups to solvent. This phospholipid binding mode shelters the hydrophobic moieties and allows the protein to be more compatible with an aqueous environment, reminiscent of the phospholipid binding mode in BPI17, the fatty acid binding mode in serum albumin18 and the glycolipid binding mode in GLTP20. The centers of the two tunnel openings are 25 A˚ apart. The N opening is 10 A˚ wide and 5 A˚ high, and the C opening is 13 A˚ 5 A˚ (Fig. 2c). Both openings are large enough to permit lipid access. The architecture of the tunnel is unique among lipid-binding proteins and provides a new basis for understanding lipid binding and transfer in CETP.

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Figure 2 Bound lipids and the tunnel. (a) Electron density for CE1, with Fo – Fc omit map contoured at 2.5 s. (b) Chemical structures of the CETP-binding lipids. Cholesteryl ester is drawn as cholesteryl oleate, phospholipid as dioleoylphosphatidylcholine. The bound cholesteryl ester and phospholipid contain small amounts of other fatty acyl groups. Triglyceride, shown as triolein, is not observed in our structure but can also bind CETP. (c) Surface of the tunnel colored by atom type (red, oxygen; blue, nitrogen; yellow, carbon and sulfur; green, phosphorus). CE1, CE2 and phospholipid carbon atoms are colored in magenta, cyan and gray, respectively. Bottom, enlarged views (one surface color is used for clarity) directly into the three lipid passages of the tunnel.

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Figure 3 Stereo view of the lipid-binding sites. CE1, CE2 and PC1/PC2 carbon atoms are shown in magenta, cyan and gray, respectively. CETP secondary structures are depicted in translucent ribbons. Side chains of the residues described and subjected to mutagenesis are shown in yellow and labeled, with the four crucial neck residues in red.

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ARTICLES Figure 4 The mobile helix X and the O1 flap. (a) Stereo view of helix X (cyan) and the N opening. The Ca trace is shown as yellow ribbons; side chain carbon atoms are in green; bound PC1 carbon atoms are in gray. A quintet of aromatics forms the mouth of the N opening (Phe197, Phe441, Phe461, Phe463 and Phe471), potentially aiding lipid access to the tunnel. (b) Stereo view of the O flaps near the C opening, with the barrel Cs of BPI (purple) and CETP (yellow) overlaid. The fatty acyl tail of CE2 is shown as a cyan space-filling model. O1 and O2 indicate the flaps in CETP and BPI (only O2 is labeled in BPI).


a

b

site, consistent with the notion that CETP has two neutral lipid– binding sites for binding either two cholesteryl esters, one cholesteryl ester and one triglyceride, or possibly two triglycerides. In the presence of other lipids (for example, cholesterol or fatty acids), the tunnel might be able to hold additional combinations of lipids, which may explain the previous difficulties in calculating lipid stoichiometry. Mobile structures near tunnel openings The B-factor increases to 56 A˚2 at Gly462 and B80 A˚2 for most of helix X (Fig. 4a). The head and tail of helix X, Glu465 and Ser476, interact with His25 and Arg37, respectively, both of which show alternative side chain conformations. These observations suggest that helix X is flexible, with Gly462-Phe463-Pro464 being a potential hinge point for movements. The helix is located at the N opening of the tunnel, with the hydrophobic face making contacts with PC1 and forming an apolar path for neutral lipids’ access to the tunnel (Fig. 4a). This proposed role of mobile helix X is similar to those of helices found in other lipid-binding proteins that form flexible lids governing lipid access to lipid-binding pockets. The proposal agrees with the report that mutations on the hydrophobic face of the amphipathic helix reduce transfer activities, whereas mutations on the polar side do not10. Indeed, the 470–475 deletion mutant binds lesser amounts of both cholesteryl ester and triglyceride and has reduced transfer activity compared to wild-type protein27, demonstrating the importance of

helix X in transferring neutral lipid from lipoprotein to CETP, a step that may be rate determining in lipid transfer15. Another region that has elevated B-factors includes the flaps O1 (residues 288–320) and O2 (350–360) (Fig. 4b). These two flaps are adjacent and structurally linked—for example, through the Phe292 and Phe350 stacking interaction. Unlike helix X, O1 does not bind PC2 but rather interacts with the CE2 oleoyl tail and shelters the lipid from aqueous solvent exposure. The corresponding BPI flaps adopt distinct and more open conformations, further suggesting flexibility of O1 and O2. As shown by fluorescence techniques28, CETP undergoes changes that expose additional tryptophan residues upon binding to a phospholipid emulsion. There are five tryptophans in CETP: Trp105 and Trp106 are fully exposed already, and Trp162 and Trp264 are buried in rigid structural units. This leaves Trp299, at the edge of the mobile O1 flap and partially buried (Fig. 4b), as the likely source for the observed conformational changes. As Trp299 is not conserved in other mammalian species with active CETPs (Supplementary Fig. 1), the importance of the mobile loop is unclear. We produced mutations at four additional CE2-facing positions. F292D was assayed because it was not secreted, which could imply negative effects on protein folding. L296Q, a naturally occurring variant29, showed about 40% of the wild-type cholesteryl ester transfer activity, whereas F301D retained 80% of the activity. Val416 makes contacts with both Phe301 and Trp299, which anchor the closed O1 flap (Fig. 4b). V416R was designed to open up the entire flap but was about 40% as active as the

a

X 10 nm

b

Asp208 Trp 105

Lys56 Lys98

Ser476

Asp114 Lys94

Figure 5 Concave surface and HDL binding. (a) The concave surface complements a 10-nm-diameter sphere. Helix X (labeled as X) protrudes into the lipoprotein surface in this view. (b) Electrostatic potentials of the CETP concave surface. Red, blue and white show potentials at –11, +32 and 0 kBT e–1, respectively. Bound phospholipid moieties are shown as sticks. (c) Proposed conformational change for VLDL binding. Ribbon diagrams of CETP (yellow) and BPI (cyan) with central b-sheets overlaid. Helices may change at the kink points (straight arrows) and enable twisting (curved arrows) of the barrels.


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Lys347

Arg201

Asp470

Asp291 Lys377

Glu297

A′ B

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wild-type protein. The O1 flap may open and allow a wider opening, facilitating lipid passage, and our data suggest that its role is not as crucial as that of helix X. Concave surface and lipoprotein binding The CETP concave surface is the site of the N and C openings, helix X and the O1 flap, making this surface the most likely to bind lipoproteins (Fig. 1a). The bound phospholipid found in our structure supports this view, as the lipoprotein surfaces are covered by a phospholipid monolayer. Other surfaces of CETP do not have the proper curvatures to bind spherical lipoproteins, lack direct access to the tunnel and are heavily glycosylated (Fig. 1a,b), supporting the concept that the concave surface is the only surface that can bind lipoproteins. Biochemical studies have shown that CETP has the highest binding affinity for 10-nm-diameter nascent discoidal HDL (Kd ¼ 20–120 nM)30. This diameter coincides with the observed curvature of CETP’s concave face (Fig. 5a), suggesting that CETP can bind a single HDL particle on this face with modest movements of helix X and the O1 flap. The surface contains numerous charged and hydrophobic residues that are distributed evenly rather than in distinct patterns (Fig. 5b), suggesting evenly distributed interactions with lipoprotein surfaces. The sheer size of the interface and the apparent lack of a ‘hot spot’ suggest that small alterations of the concave surface should not greatly change lipoprotein affinity and explain the lack of detectable activity changes in concave-surface mutants31. Notably, helix X protrudes from the concave surface and may partially insert into lipoprotein in the current model (Fig. 5a). This insertion, similar to the membrane insertion proposed for helix A10 of Sec14 (ref. 22), is consistent with the aforementioned role of helix X in facilitating access of neutral lipids from lipoproteins to the CETP tunnel. Tunnel mutants and activity assay We produced 21 mutants to test whether the observed ‘tunnel’ actually operates as a tunnel (Table 1). Four mutants were not secreted, suggesting that they did not fold properly. Seventeen mutants were secreted at high enough abundances to allow partial protein purification; many retain viable transfer activities, suggesting that they fold correctly. The concave surface mutants K98S and R37S, as expected, show no notable change in activity relative to the control His-tagged wild-type CETP. Most other mutants were designed to modify the tunnel interior and should have little effect on lipoprotein binding; their activities should be attributable to direct effects on lipid binding and movements in the tunnel. The N opening mutant R201S shows a ten-fold drop in triglyceride transfer and a smaller effect on cholesteryl ester transfer (Table 1), similar to the results from the nearby helix X mutants27. In the CE1 site, mutation of neighboring residues has very different effect, exemplified by the switch to CE-specific transfer of V198W and the switch to triglyceride-specific activity of Q199A. T138Y and S230A mutations affect transfer of both cholesteryl ester and triglyceride, with S230A allowing less triglyceride transfer and hence resulting in a higher cholesteryl ester/triglyceride ratio. These data suggest that both cholesteryl ester and triglyceride traverse this path to be successfully transferred between lipoprotein particles. The neck of the tunnel makes marginal contacts with the flexible parts of bound cholesteryl ester and is expected to have only a minor role in lipid-binding affinity. However, if lipid passage through the neck is required for activity, mutations aimed at reducing the size of the neck are expected to impair activity, especially for the larger triglyceride molecule. Indeed, all six neck mutants assayed show markedly reduced transfer activities (Table 1). Most notably, triglyceride transfer is completely

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Table 1 Transfer activity and ratio of CETP mutants in the dual-labeled assay CETP

n

CE/TG

CE

TG

Wild-type R37S

5 2

1 0.9

1 0.9

1 1.1

Control Surface

K98S R201S

2 3

1.2 3.7

1.2 0.5

1.0 0.1

Surface N opening

V198W T138Y

1 1

10 0.9

1.0 0.2

0.1 0.2

CE1 CE1

Q199A I15W

1 4

0.2 0.5

0.25 0.4

1.1 0.8

CE1 CE1

S230A H232A

1 1

2.5 2.1

0.5 0.9

0.2 0.4

CE1 CE1

I443W V198W M433W

1 2

19 2.6

0.3 0.6

0.01 0.2

Neck/CE1 Neck

Y375S F265R

Not secreted Not secreted

F270R

Location

Neck Neck

Not secreted

Neck

I443W L457W L457W M459W

1 1

21 1.7

0.3 0.2

0.01 0.1

Neck Neck

L457W M459W

2 1

1.5 1.1

0.4 0.4

0.3 0.3

Neck Neck

L382W L425W

2

Not secreted 0.8 0.5

0.6

CE2 CE2

V428R

2

1.2

1.1

C opening

1.3

All assays are run in quadruplicate; n is the number of separate experiments. CE and TG are the relative transfer activities with cholesteryl ester and triglyceride, respectively, compared with those of the wild-type control. CE/TG is the ratio of the two transfer rates, normalized to the ratio for the wild-type control. Error (s.d.) of each CE/TG ratio is ±0.1. Results are sorted by the structural location of the mutations.

blocked in the double mutant I443W L457W, suggesting that lipid passage through the neck is required for transfer activity. The different effects of the mutations can be attributed to the differences in size, flexibility and binding mode between cholesteryl ester and triglyceride. For example, as triglyceride is larger than cholesteryl ester, introducing bulky tryptophans at the neck (I443W L457W) could block triglyceride transfer while allowing some residual transfer of the smaller cholesteryl ester. The passage of lipid through the neck implies the involvement of the CE2 site in transfer, which is confirmed by the reduced activity of the mutants whose CE2 binding is affected (L425W, L457W and M459W). In conclusion, the structural and mutagenesis studies suggest that the tunnel allows neutral lipid to pass through its entire length rather than operating as two separate pockets. This ‘through-the-tunnel’ model is consistent with known biological data and provides a framework to explain many mechanistic observations. For example, as cholesteryl ester is smaller than triglyceride, it is expected to pass through the tunnel with less resistance, in agreement with the two- to eight-fold faster rate of cholesteryl ester transfer relative to that of triglyceride24,32. To our knowledge, this through-the-tunnel model is unprecedented among the large number of known lipid-binding and lipid-transfer proteins. DISCUSSION Possible conformational changes for binding VLDL As crystallized in the present study, CETP has a radius of curvature that makes it well suited for interaction with HDL particles. A straighter conformation must be adopted when CETP binds the much larger VLDL particles (30–100 nm diameter), and the structure of BPI (Fig. 1c,d) may be an excellent model for this. As shown in the overlay of the central b-sheets (Fig. 5c), a less sharply curved conformation of

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ARTICLES Figure 6 Proposed mechanism for CETP-mediated heteroexchange. VLDL (large circles) is normally triglyceride (TG) rich and HDL (smaller circles) is cholesteryl ester (CE) rich. Step 1, CETP filled with CE (as in the crystal structure) binds VLDL and releases the bound phospholipid (phosphatidylcholine, PC). One or two TGs enter the tunnel and an equal amount of CE is deposited into VLDL. Step 2, the TG-bound CETP dissociates from VLDL carrying two phospholipids from the surface, leaving the VLDL particle with a higher CE content. Step 3, the TG-bound CETP engages HDL and releases the bound phospholipid. One or two new CEs enter the tunnel and an equal amount of TG is deposited into HDL. Step 4, the CE-filled CETP dissociates from HDL carrying two phospholipids from the surface and completes a full cycle of heteroexchange, which results in a lower CE content in HDL.

VLDL pool 1 4 Lower HDL-C

HDL VLDL


3 2

CETP from the lipoprotein, the tunnel openings will need to be refilled with phospholipid to permit the protein to return to the aqueous phase. The identity of the neutral lipids in the tunnel and the diameter of the lipoprotein may affect the rate at which phospholipid is reloaded, a process that is probably followed by movements of helix X and the O1 flap and dissociation of CETP from the lipoprotein.

HDL pool CE

PC

TG

Tunnel

Higher VLDL-C

CETP can be modeled by twisting its barrels around the central b-sheet. The high conservation of the structural units, which persists despite their different sequences and functions, suggests that these units are somewhat rigid. Notably, helix A lacks a kink in BPI but is kinked at His25 in CETP. Helix B has two kinks in BPI but only one in CETP (Fig. 5c). The B301 kink in helix A could be straightened to permit the twisting of barrel N, and helix B could kink and bend B301 around Ile190 to generate a kink matching that of BPI at Glu185. For barrel C, a 151 rotation of helix A¢ is sufficient for the domain twisting, but the entire helix B¢ would have to move laterally B8 A˚. As these helices are involved in cholesteryl ester and phospholipid binding, the nature of the bound lipids could affect the energetics of the proposed barrel twists. The twists only slightly alter the shape of the lipid tunnel, mainly near the C opening. The similar affinities for all lipoproteins and the efficiency of CETP-mediated transfers10,24,33 suggest that necessary structural changes are not impeded by any high-energy barrier. There is no evidence that CETP affects the VLDL surface curvature, as has been observed with other proteins on different surfaces34. As the concave surface is the only surface expected to bind lipoproteins, the proposed barrel twists are a plausible way to achieve the changes in curvature necessary to switch from HDL binding to VLDL binding. Lipid exchange at the CETP-lipoprotein interface Cholesteryl ester and triglyceride can be solubilized in the phospholipid monolayer at up to B3% (molar) concentration, with the concentration reflecting the proportion of cholesteryl ester and triglyceride in the oily core of the lipoprotein particle35–37. As demonstrated by the correlation between the transfer of cholesteryl ester or triglyceride and the surface concentration of cholesteryl ester or triglyceride in donor and acceptor particles38, CETPs probably access the phospholipid-solubilized neutral lipids only during transfer. Previous studies have suggested that cholesteryl ester orients its carbonyl group close to the aqueous interface and its sterol ring and fatty acyl chain approximately parallel to phospholipid fatty acyl chains36. Upon lipoprotein binding, phospholipid bound to the tunnel openings may merge into the phospholipid monolayer and permit access to neutral lipids entering and exiting the tunnel. Access to the N opening may be further helped by helix X inserting into the phospholipid monolayer and facilitating neutral-lipid exchange using its hydrophobic face (Fig. 4a). Likewise, the mobile O1 flap may aid the exchange of lipids through the C opening. Before dissociation of


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Mechanism for neutral-lipid and phospholipid transfer The structure of the concave surface indicates that CETP can bind only one lipoprotein at a time. This provides strong support for the proposal that CETP operates by a carrier mechanism, in which CETP accepts neutral lipids from a donor particle, shuttles them through the aqueous phase and delivers them to an acceptor lipoprotein10. In the more physiologically relevant heteroexchange33,39,40, CETP filled with cholesteryl ester (Fig. 6, step 1) binds VLDL and releases the bound phospholipid. As VLDL is triglyceride rich35, one or two triglycerides can enter the tunnel and deposit an equal amount of cholesteryl ester into VLDL. The triglyceride-bound CETP departs from VLDL carrying two phospholipids from the surface and travels through the aqueous plasma (step 2). It then engages HDL and releases the bound phospholipid (step 3). As HDL is cholesteryl ester rich, one or two new cholesteryl esters can enter the tunnel and an equal amount of bound triglyceride is deposited into HDL. The cholesteryl ester–filled CETP departs from HDL carrying two phospholipids from the surface (step 4) and hence completes a full cycle of heteroexchange. The nonspecific nature of the tunnel suggests similar binding affinities for cholesteryl ester and triglyceride, consistent with CETP-mediated transfers being determined by the relative lipid compositions in lipoproteins38. Homoexchange occurs when a lipid of the same kind as that bound is loaded into the tunnel, which results in no net change in lipoprotein lipid content. Both types of neutral-lipid exchange require the discharge and reloading of phospholipid, forming a basis for CETP-mediated phospholipid exchange that can also occur independently of triglyceride or cholesteryl ester exchange. Lipid movements in the tunnel The CETP structure reveals the presence of a true tunnel—a tube with two distinct openings. All individual lipid movements are reversible. However, the reduced activities of the neck-blocked mutants suggest a net movement of lipids through the entire tunnel during transfer. Plasma CETP is naturally filled with neutral lipids and probably does not go through an apo state at any stage of transfer. The through-thetunnel model supports this concept: in this model, CETP admits a neutral lipid from one opening and deposits a bound lipid from the opposite opening. The model is consistent with the identical orientation of the two bound cholesteryl esters in our structure. It also explains the tightly linked equimolar heteroexchange of cholesteryl

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ARTICLES ester and triglyceride, which would not necessarily occur if an apo state were involved. This novel model is consistent with the efficient CETP-mediated heteroexchange (kcat ¼ 2 s–1)24, without requiring an intermediate apo state or cholesteryl ester and triglyceride competing for access to the same opening. A classical pocket-based mechanism could operate with normal CETP at low abundances or when the tunnel is artificially blocked by mutagenesis. BPI uses a pocket mechanism, and its N-terminal half is sufficient for activity16. It will be interesting to see whether PLTP is more similar to CETP or BPI. Additional CETP structures, such as a triglyceride-bound or VLDLbound one, will be valuable but difficult to obtain. Inhibitors of CETP have been reported; one such inhibitor stabilizes CETP binding to HDL, and the structural basis for the proposed mechanism25 is the subject of ongoing studies. The structure reported here provides a novel basis for future studies of this important protein and may accelerate efforts to discover new agents for treating cardiovascular disease. METHODS Construct design and protein production. Our general strategy was to reduce glycosylation and surface cysteines while maintaining protein expression and solubility for crystallization. Bacteria, insect cells, yeast and in vitro systems did not yield sufficient expression of active protein, leaving mammalian systems as the choice for CETP expression. The host used was the dihydrofolate reductase– deficient Chinese hamster ovary cell line DG44 (ref. 41). We discovered that at least one glycosylation was required for adequate protein secretion. Glycosylation at Asn240 and Asn396 could be removed by enzyme treatments, whereas such treatment was not effective for Asn88 and Asn341. This enabled us to produce sugar-free CETP by expressing the mutant c434 construct (C1A S90A S343A; Supplementary Data), then removing sugars with enzymes. The unglycosylated protein, in complex with an antibody Fab fragment, gave crystals that diffracted to 3.5-A˚ resolution. Subsequently, the mutant c444 construct (C1A C131A N88D N240D N341D) was obtained, yielding the structure described in this report.

Table 2 Data collection and refinement statistics Data collection Space group Cell dimensions a, b, c (A˚) Resolution (A˚)

P 212121 66.8, 70.3, 187.6

Rmerge

99.0–2.2 (2.26–2.18)a 0.078 (0.378)

I / sI

17.0 (2.1)

Completeness (%) Redundancy

88 (49) 5.3 (3.3)

Refinement Resolution (A˚)

93.7–2.2

No. reflections Rwork / Rfree

52,263 0.217 / 0.265

No. atoms Protein

4,465 3,748

Ligand/ion Water B-factors Protein CE1 / CE2 / PC1 / PC2 Water R.m.s. deviations Bond lengths (A˚) Bond angles (1) aValues

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Structure determination and analysis. The structure was solved by molecular replacement using the program AMORE42 and the low-resolution CETP model as the search model. After solvent flattening, ARP/wARP42 was used to improve the phases. Manual rebuilding and iterative refinement were then carried out. The R-factor of the final model is 0.217 (Rfree ¼ 0.265), using all data from 93.7 to 2.2 A˚ resolution. The model contains amino acid residues 5–476, three ordered sugar residues attached to Asn396, two HEPES buffer ions, two chloride ions, four polyethyleneglycol molecules and 416 water molecules. The average B-factors are 43 A˚2 for protein atoms, 63 A˚2 for ligand atoms and 47 A˚2 for water molecules. The B-factors of CE1 and PC1 are B10 A˚2 higher than those of CE2 and PC2 (Table 2). The r.m.s. bond lengths and bond angles are 0.012 A˚ and 1.61. In the Ramachandran plot, 90.9% of residues are in the most favored regions, one residue is in a generously allowed region and none is disallowed. Activity assay of CETP mutants. His/V5-tagged wild-type and mutant proteins were generated for transient expression in human embryonic kidney (HEK) 293S cells. The tag at the N terminus is located on the convex side of CETP (Fig. 1a) and should not affect lipid transfer. Secreted mutants were partially purified in limited amounts, as described43. Protein concentrations were estimated on the basis of phosphorimaging analysis of the nondenaturing PAGE western blot using the 125I-labelled monoclonal antibody to V5. In the duallabeled [3H]triolein and [14C]cholesteryl oleate assay, 200 ng of wild-type CETP was used. The amount of [3H,14C]LDL was 6.7 nmol per assay, whereas HDL was 10.5 nmol per assay. The HDL contained a 2:1 mix of HDL2 and HDL3 fractions. The effects of residual CETP in the HDL3 fraction were subtracted from transfer values using control assays (0.7% for both [3H]triolein and [14C]cholesteryl oleate transfer in assays in which the mean wild-type transfer was 13% and 23%, respectively, for n ¼ 5 assays). Owing to limited protein purity, the CETP concentrations given are approximate. The specific activities for cholesteryl ester and triglyceride transfer could not be determined with a high degree of certainty. However, the cholesteryl ester/triglyceride ratio should not be affected by differences in assay CETP concentrations, and this ratio had very low variability (cholesteryl ester/triglyceride ¼ 1.8 ± 0.1 for n ¼ 5 wild-type assays). Accession codes. Protein Data Bank: Coordinates have been deposited with accession code 2OBD. Note: Supplementary information is available on the Nature Structural & Molecular Biology website. ACKNOWLEDGMENTS We thank the staffs in the X-ray group and synchrotrons for assistance in data collection, G. Andrews, M. Bamberger, L. Morehouse, D. Perry, R. Ruggeri, K. Ranney, I. Reininger, M. Tu and P. Zagouras for insightful discussions and S. Liu, J. Boyd, D. Cunningham, T. Dickinson, J. Duerr, B. King, T. Lanzetti, W. Lin, P. Loulakis, M. Mansour, A. McColl, T. McLellan, F. Rajamohan, M. Rosner, M. Tardie and Z. Xie for supporting work.

301 416

AUTHOR CONTRIBUTIONS M.C.G., S.J.H., T.A.S., I.-K.W., H.Z., K.M.M., K.J.S.-E., T.B.F., L.R.H., K.F.G., Y.C., G.A.K., B.A.C., J.S.C. and A.K.S. provided key reagents. A.M., M.J.A. and D.E.D. crystallized the proteins. X.Q. solved the structure. M.E.L., D.B.L., J.F.T. and R.W.C. contributed to mutagenesis and assays. X.Q., P.H., C.M.H. and A.P.S coordinated the research and data review. X.Q., J.F.T., K.F.G. and A.P.S. wrote the paper.

43 52 / 42 / 73 / 66 47 0.012 1.6

in parentheses are for highest-resolution shell. The data were obtained using one crystal.

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Crystallization and diffraction data collection. The c444 crystals were obtained by hanging drop vapor diffusion, using 10 mg ml–1 protein mixed 1:1 with a well solution of 0.1 M HEPES (pH 7.5), 0.2 M MgCl2 and 27%–35% (w/v) PEG 400 at 4 1C. Crystals appeared in 3 d and grew to 0.1 mm 0.05 mm 0.2–0.7 mm in 15 d. Crystals were flash-frozen in liquid nitrogen in the mother liquor. Data were collected at the Industrial Macromolecular Crystallographic Association beamline at Argonne National Laboratory. The crystal belongs to space group P212121, with a ¼ 66.8 A˚, b ¼ 70.3 A˚ and c ¼ 187.6 A˚ (Table 2). The 2.2-A˚ data set is 88% complete ,with mosaicity 0.31 and Rmerge of 0.078.

COMPETING INTERESTS STATEMENT The authors declare competing financial interests (see the Nature Structural & Molecular Biology website for details).

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