The Structure of Apolipoprotein A-I in High Density Lipoproteins

Not long ago, high density lipoproteins (HDL) 2The abbreviations used are: HDL, high density lipoprotein; LDL, low density lipoprotein; CAD, coronary artery disease; RCT, reverse cholesterol transport; apo, apolipoprotein; ABCA1, ATP-binding cassette A1; FRET, fluorescence resonance energy transfer. were second class citizens with regard to therapeutic strategies for lowering the risk of atherosclerosis and coronary artery disease (CAD). To date, most successful approaches have focused on the better understood pathways of cholesterol synthesis and low density lipoprotein (LDL) production, the “forward” cholesterol transport pathway. For example, the statin class of cholesterol synthesis inhibitors significantly reduces LDL levels resulting in a less atherogenic plasma lipoprotein profile. However, the relatively modest improvements in mortality conferred by these drugs suggest that other factors also play significant roles in defining CAD risk. The recent discoveries of HDL-interacting cell surface proteins such as scavenger receptor BI (SR-BI) and ATP-binding cassette transporters A1 (ABCA1) and G1 (for recent reviews see Refs. 1Lee J.Y. Parks J.S. Curr. Opin. Lipidol. 2005; 16: 19-25Crossref PubMed Scopus (165) Google Scholar and 2Zannis V.I. Chroni A. Krieger M. J. Mol. Med. 2006; 84: 276-294Crossref PubMed Scopus (310) Google Scholar) have helped define the steps of reverse cholesterol transport (RCT), i.e. the movement of cholesterol from the periphery to the liver for catabolism (3Glomset J.A. J. Lipid Res. 1968; 9: 155-167Abstract Full Text PDF PubMed Google Scholar, 4Groen A.K. Oude Elferink R.P. Verkade H.J. Kuipers F. Ann. Med. 2004; 36: 135-145Crossref PubMed Scopus (55) Google Scholar). Additionally, there is growing evidence that HDL anti-inflammatory properties may contribute significant protective effects (5Barter P.J. Nicholls S. Rye K.A. Anantharamaiah G.M. Navab M. Fogelman A.M. Circ. Res. 2004; 95: 764-772Crossref PubMed Scopus (1067) Google Scholar), apparently via specific cell signaling pathways (6Seetharam D. Mineo C. Gormley A.K. Gibson L.L. Vongpatanasin W. Chambliss K.L. Hahner L.D. Cummings M.L. Kitchens R.L. Marcel Y.L. Rader D.J. Shaul P.W. Circ. Res. 2006; 98: 63-72Crossref PubMed Scopus (247) Google Scholar). These discoveries have fueled a new interest in HDL as a target for CAD treatment (7Duffy D. Rader D.J. Circulation. 2006; 113: 1140-1150Crossref PubMed Scopus (98) Google Scholar). Unfortunately, a complete understanding of HDL function has been hampered by a lack of information on its structure and the molecular basis of its interactions with other proteins. This review summarizes the latest efforts in understanding the structure of the defining protein component of HDL, apoA-I, in the various stages of the RCT pathway. ApoA-I comprises roughly 70% of the HDL protein mass and apoA-II another 15–20%. The remainder is made up of amphipathic proteins including the apoCs, apoE, apoD, apoM, apoA-IV, paroxonase and many other proteins as identified in a recent proteomics study (8Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. Chea H. Knopp R.H. Brunzell J. Geary R. Chait A. Zhao X.Q. Elkon K. Marcovina S. Ridker P. Oram J.F. Heinecke J.W. J. Clin. Investig. 2007; 117: 746-756Crossref PubMed Scopus (795) Google Scholar). These lower abundance proteins are not present on all HDL particles and may actually be sequestered on compositionally distinct particles within the density class. These amphipathic proteins form stable micellar complexes with phospholipids, cholesterol, triglycerides, and cholesteryl esters. In humans, HDL exists predominantly as two major density species, HDL2 (d = 1.063–1.125 g/ml) and HDL3 (d = 1.125–1.210 g/ml) with diameters ranging from 70–120 Å. Minor, but clearly important, subspecies include lipid-poor apoA-I and nascent discoidal particles. In addition to density, HDL can be separated by major apolipoprotein species using immunoaffinity chromatography into apoA-I-containing particles that lack apoA-II (LpA-I) and those that contain both apoA-I and apoA-II (LpA-I/A-II) (9Cheung M.C. Albers J.J. J. Biol. Chem. 1984; 259: 12201-12209Abstract Full Text PDF PubMed Google Scholar). Several functional distinctions have been proposed for these species, with apoA-II thought by some to be proatherogenic. However, there are also many examples of antiatherogenic properties of apoA-II (see Ref. 10Tailleux A. Duriez P. Fruchart J.C. Clavey V. Atherosclerosis. 2003; 164: 1-13Abstract Full Text Full Text PDF Scopus (112) Google Scholar for a review). As it comprises the majority of the protein mass, structural studies of human plasma HDL must first focus on apoA-I. ApoA-I is a 243-amino acid, 28-kDa single polypeptide that lacks glycosylation or disulfide linkages. Aside from the N-terminal 44 amino acids, the apoA-I sequence appears to be organized into eight α-helical segments of 22 amino acids and two 11-mer repeats that are frequently separated by proline residues (11Brouillette C.G. Anantharamaiah G.M. Biochim. Biophys. Acta. 1995; 1256: 103-129Crossref PubMed Scopus (167) Google Scholar). These helices are predicted to be amphipathic, with a hydrophobic face that likely mediates lipid interactions and a polar face that interacts with water. The thermodynamic drive to minimize the aqueous exposure of these hydrophobic surfaces is probably the major mediator of protein folding, whether these surfaces are present in a lipid-free state in which the nonpolar helical faces sequester within the protein or in the lipidated state where they likely contact aliphatic regions of lipid assemblies. More information on how amphipathic helices mediate apoA-I lipid binding can be found in recent work from the Phillips laboratory (12Tanaka M. Dhanasekaran P. Nguyen D. Ohta S. Lund-Katz S. Phillips M.C. Saito H. Biochemistry. 2006; 45: 10351-10358Crossref PubMed Scopus (68) Google Scholar) and in the excellent review series by Brouillette et al. (13Brouillette C.G. Anantharamaiah G.M. Engler J.A. Borhani D.W. Biochim. Biophys. Acta. 2001; 1531: 4-46Crossref PubMed Scopus (229) Google Scholar). About 5–10% of human plasma apoA-I exists in a lipoprotein-unassociated state. This fraction likely derives from direct secretion by the liver/intestine or by dissociation from HDL or triglyceride-rich lipoproteins (14Rye K.A. Barter P.J. Arterioscler. Thromb. Vasc. Biol. 2003; 24: 421-428Crossref PubMed Scopus (263) Google Scholar). Lipid-free apoA-I has garnered significant interest of late because the absence of lipid appears to be a requirement for the interaction of apoA-I with the ABCA1 transporter, a key reaction for the maintenance of plasma HDL levels (2Zannis V.I. Chroni A. Krieger M. J. Mol. Med. 2006; 84: 276-294Crossref PubMed Scopus (310) Google Scholar). Currently, there is no strong consensus as to whether this fraction is truly lipid-free or if it contains trace amounts of lipid (lipid-poor). Most structural studies have focused on apoA-I that has been completely delipidated by various means; it can thus be studied in aqueous solution. Under these conditions, apoA-I forms a heterogeneous population of oligomers from monomers to pentamers in a concentrationdependent manner. As a further complication, apoA-I has been proposed to exhibit characteristics of a “molten globule” with defined elements of secondary structure, but it may contain regions lacking defined tertiary structure (15Gursky O. Atkinson D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2991-2995Crossref PubMed Scopus (173) Google Scholar, 16Rogers D.P. Roberts L.M. Lebowitz J. Datta G. Anantharamaiah G.M. Engler J.A. Brouillette C.G. Biochemistry. 1998; 37: 11714-11725Crossref PubMed Scopus (83) Google Scholar). The reader is referred to the recent works of the Atkinson (17Gorshkova I.N. Liu T. Kan H.Y. Chroni A. Zannis V.I. Atkinson D. Biochemistry. 2006; 45: 1242-1254Crossref PubMed Scopus (41) Google Scholar) and Gursky laboratories (18Gursky O. Protein Pept. Lett. 2007; 14: 171-174Crossref PubMed Scopus (2) Google Scholar) for information on apoA-I folding thermodynamics. Despite the challenges posed by apoA-I dynamics, two groups have successfully crystallized apoA-I. In 1997, Borhani et al. (19Borhani D.W. Rogers D.P. Engler J.A. Brouillette C.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12291-12296Crossref PubMed Scopus (414) Google Scholar) crystallized a deletion mutant lacking the N-terminal 43 amino acids. The structure at 4 Å resolution showed a ringshaped assembly of four apoA-I molecules arranged in extended, kinked α-helices. However, because of the missing N terminus and the tetramerization, the structure appeared to be more applicable to lipid-bound rather than lipid-free apoA-I (see below under “Discoidal HDL”). In 2006, full-length lipidfree apoA-I was crystallized by Ajees et al. (20Ajees A.A. Anantharamaiah G.M. Mishra V.K. Hussain M.M. Murthy H.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2126-2131Crossref PubMed Scopus (194) Google Scholar) in the presence of chromium tris-acetylacetonate. The structure (Fig. 1A) indicated that the N-terminal two-thirds of the molecule is involved in an intramolecular four-helix bundle organization, reminiscent of the crystal structures of monomeric apoE (21Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (601) Google Scholar) and the insect apolipophorin III (22Wang J. Sykes B.D. Ryan R.O. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1188-1193Crossref PubMed Scopus (103) Google Scholar). However, the apoA-I four-helix bundle is likely of moderate stability, as the number of H-bonds per residue is only 0.71 compared with 1.03 for apoE. Interestingly, the C-terminal 50 amino acids form an independent hairpin domain that interacts with the corresponding regions of two other apoA-I molecules in the crystal. This organization appears to confirm previous proposals that apoA-I adopts a structural and functional domain organization in which the C terminus mediates lipid interactions with subsequent unfolding of an N-terminal helical bundle (23Davidson W.S. Hazlett T. Mantulin W.W. Jonas A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13605-13610Crossref PubMed Scopus (134) Google Scholar, 24Saito H. Dhanasekaran P. Nguyen D. Holvoet P. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2003; 278: 23227-23232Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Furthermore, the structure is generally consistent with one proposed a year earlier using homology modeling and chemical cross-linking (25Silva R.A. Hilliard G.M. Fang J. Macha S. Davidson W.S. Biochemistry. 2005; 44: 2759-2769Crossref PubMed Scopus (96) Google Scholar). The homology model proposed a four-helix bundle in the N terminus with turns between helices 1, 2, and 3 that are quite similar to the crystal structure (Fig. 1B). Both models also clearly show that the helices of the bundle are not always punctuated by the proline residues originally thought to demarcate the 22-amino acid amphipathic helices. Helix 4 in the homology model is terminated near residue 165 and doubles back on the helical bundle, whereas the crystal structure shows one contiguous helix. Beyond that, both models show the C-terminal residues from about 186 to 191 form a separate C-terminal domain, albeit much less organized in the homology model. Although the Ajees crystal structure appears to be consistent with some data generated via lower resolution techniques as described above, it should be pointed out that the model is inconsistent with several well established observations. One of the obvious differences between the models in Fig. 1 is the total helical content. The crystal structure puts apoA-I at about 83% helical, a much higher content than the 50–57% typically found for monomeric apoA-I by circular dichroism (26Leroy A. Jonas A. Biochim. Biophys. Acta. 1994; 1212: 285-294Crossref PubMed Scopus (34) Google Scholar, 27Saito H. Lund-Katz S. Phillips M.C. Prog. Lipid Res. 2004; PubMed Scopus Google Scholar). The homology model puts apoA-I at a more some were as because of a lack of a homology for those The crystal structure shows that the N-terminal 43 residues are helical, whereas most of this is in the homology model. similar can be made for the 50 or residues at the C recent study by et al. J.C. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar) the secondary structure of the first residues of lipid-free apoA-I by that this contain helical but the helical are with in to the crystal Interestingly, these showed evidence for a of between residues and this they an model on the crystal structure with the N terminus in an (Fig. This that the C-terminal residues were also significantly less helical than by the crystal structure, with only about 43 of those residues in helices Ryan R.O. J.C. Biol. 2003; PubMed Scopus Google Scholar). second of in the C-terminal was also not in the crystal Additionally, the crystal structure shows that the four residues present in the N terminus of apoA-I are to and This is in to fluorescence data with monomeric apoA-I under W.S. K. A. J. Hazlett Jonas A. Biochemistry. PubMed Scopus Google Scholar) a hydrophobic for these of the crystal structure shows that the C-terminal of the helical bundle significant hydrophobic consistent with a stable the N-terminal 1, that are at these data suggest that the of the four helical bundle is probably in the homology and the crystal structure models in Fig. However, in the and C are probably not into the helical in the crystal key that is whether the two to the of apoA-I to as by Rogers et al. D.P. Roberts L.M. Lebowitz J. Datta G. Anantharamaiah G.M. Engler J.A. Brouillette C.G. Biochemistry. 1998; 37: 11714-11725Crossref PubMed Scopus (83) Google Scholar) and by Fang et al. Gursky O. Atkinson D. Biochemistry. 2003; PubMed Scopus Google Scholar). should be in that the crystal was generated at high in with and a is likely that the crystal structure a that apoA-I is of at under However, it not the of regions under The homology generated from data under conditions, from its on a of and a of structural Despite both models are as a for in molecular studies to more that the of apoA-I the of a single structure The protein likely exists as a of and The of the models in Fig. 1 not in defining some of residue but in which regions are most likely to in to lipid or contact with cell surface proteins such as HDL are generated by exposure of lipid-free apoA-I to the are excellent for and the resulting of cholesterol to cholesteryl the to the forms found in of apoA-I structure in is for understanding the molecular of they are not from amounts can be in such as have been to in with high and Jonas A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the resulting particles have been studied The particles have a of Å two molecules of apoA-I with molecules of Chem. PubMed Scopus Google Scholar) proposed in the late that the of apoA-I the of discoidal of a with the long helical to the i.e. the model. the model that the 22-amino acid helical punctuated by turns on the proline the to the Chem. PubMed Scopus Google Scholar, J. Jonas A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The Borhani crystal structure (19Borhani D.W. Rogers D.P. Engler J.A. Brouillette C.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12291-12296Crossref PubMed Scopus (414) Google Scholar), lacking apoA-I in a with no of hairpin that apoA-I may a in there have been two studies on particles that the of apoA-I helices in to The first was by et al. V. Engler J.A. Brouillette C.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that clearly the model in particles. More this that apoE V. Lund-Katz S. Phillips M.C. Biochemistry. 2005; 44: PubMed Scopus Google Scholar) and apoA-II R.A. Davidson W.S. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar) also a in particles of similar The second study involved fluorescence of on that also the model for apoA-I Davidson W.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) as well as for apoE V. Dhanasekaran P. Ryan R.O. Phillips M.C. Lund-Katz S. Davidson W.S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the of helical focused on the between molecules of apoA-I on a The model with the is the model in which of two apoA-I molecules a of on its in an (13Brouillette C.G. Anantharamaiah G.M. Engler J.A. Borhani D.W. Biochim. Biophys. Acta. 2001; 1531: 4-46Crossref PubMed Scopus (229) Google Scholar, M. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Biochemistry. 2002; PubMed Scopus Google Scholar). predicted a between the monomers with similar by the Borhani crystal In this of apoA-I molecule in direct Fig. model (11Brouillette C.G. Anantharamaiah G.M. Biochim. Biophys. Acta. 1995; 1256: 103-129Crossref PubMed Scopus (167) Google Scholar) proposed two hairpin where molecule interacts with both a This interactions similar to the they are intramolecular in the The in between the hairpin and models to some in defining tertiary using fluorescence resonance energy clearly out the model but were consistent with contact between the molecules in the or hairpin model H. W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). studies using similar of and A.K. S.A. J. Jonas A. Biochemistry. 2001; PubMed Scopus Google Scholar). More a cross-linking to discoidal HDL particles two molecules of apoA-I R.A. Hilliard G.M. Davidson W.S. Biochemistry. 2005; 44: PubMed Scopus (98) Google Scholar). The was that the model in particles with two molecules of apoA-I. This study also that a of the apoA-I molecules also (Fig. in with the The functional for such a are and the that may with distinct plasma factors to HDL subsequent study used a similar to S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), two of which were consistent with the model. Interestingly, the a to that the apoA-I N terminus forms a hairpin residue 44 in to with the C-terminal of the second apoA-I molecule that has also back on (Fig. This organization in that it no forms a the lipid the two molecules form an structure reminiscent of a of This on the model may have for the addition of other to HDL particles. that this is also quite consistent with the proposed by et al. R.A. Hilliard G.M. Davidson W.S. Biochemistry. 2005; 44: PubMed Scopus (98) Google Scholar), in Fig. a et al. Ryan R.O. J.C. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) used to study apoA-I with single and to residues in HDL The data clearly showed that apoA-I forms an with a consistent with the model. In the resonance further that may be a that may in an consistent with previous proposals of a domain near this The of the data on these shows that apoA-I can clearly an organization that the model as in Fig. However, it is also that there is for significant within this at the and in the of the apoA-I Although the model appears to have out of the hairpin models of one that there is for only two helices the of a the addition of a molecule of apoA-I to these that it a distinct from the first to this is to the hairpin organization for at one apoA-I molecule in that contain apoA-I M. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). More work be to this HDL particles contain a lipid of cholesteryl and there is no a to the as in the the surface molecules form a The apoA-I helices likely the molecules with hydrophobic faces the to with the V.K. Anantharamaiah G.M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). no the protein be to out the the in may be from those in the Unfortunately, much less is about the of apoA-I in particles they up the majority of HDL found in et al. D.W. Brouillette C.G. Anantharamaiah G.M. Protein Chem. 1994; 45: PubMed Google Scholar) have that if apoA-I exists in a model in discoidal the interactions of apoA-I helices with the should not significantly with the addition of the lipid Borhani et al. (19Borhani D.W. Rogers D.P. Engler J.A. Brouillette C.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12291-12296Crossref PubMed Scopus (414) Google Scholar) have also the in apoA-I structure between the two These are by circular dichroism and fluorescence studies that apoA-I secondary structure content and the exposure of residues not a discoidal to a in the presence of A. K.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). However, other studies have differences in apoA-I on studies using resonance have indicated that the N-terminal of the molecule that Phillips M.C. Lund-Katz S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Furthermore, in are from those in W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus (68) Google Scholar). study by et al. D.J. Rye K.A. Biochemistry. PubMed Scopus Google Scholar) not differences between and but to the of the of the lipid within particles. The showed that the lipid specific within apoA-I. apoA-I interacts to some with the lipid the that its can on the lipid of a Unfortunately, the resolution of these techniques was not high to between relatively for example, a model or a completely in the More information is can the most between the models in the to the within can be that one of the in is how the structure of apoA-I HDL and This molecule is of an of the RCT pathway. is the between this structural and the for functional that understanding apoA-I structure a The crystal structures and models have a for understanding apoA-I As as these have a key component in recent has been the of to these models to As a that the has a understanding of at the of apoA-I organization in its lipid-free form and in discoidal particles. However, it is that more work to be to the of the more within these structural As above, there is a to a similar of understanding of apoA-I in particles. This may be relatively for but a be to the studies to the heterogeneous particles from apoA-I a organization in human HDL forms of HDL have been under study for 3 the has to that to particles that in the the of human plasma HDL, a major to these of be with the presence of proteins including These may not only with apoA-I on the HDL but they may also its and thus the function of a HDL These a significant from techniques that on a population of particles. these challenges be if are to the to HDL