A Molecular Model of High Density Lipoproteins

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 71, No. 4, pp , April 1974 A Molecular Model of High Density Lipoproteins (assembly of proteins and lipids/amphipathic a-helical regions/hydrophobicity of apoproteins) GERD ASSMANN AND H. BRYAN BREWER, JR. Molecular Disease Branch, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland Communicated by Donald S. Fredrickson, September 28, 1973 ABSTRACT Based on the analysis of recombined lipidapoprotein complexes by C-13 and P-31 nuclear magnetic resonance spectroscopy and circular dichroism [Assmann, G., Sokoloski, E. A. & Brewer, H. B., Jr. (1974) Proc. Nat. Acad. Sci. USA 71, ; Assmann, G., Highet, R. J., Sokoloski, E. A. & Brewer, H. B., Jr. (1974) Proc. Nat. Acad. Sci. USA 71, in press; Assmann, G. & Brewer, H. B., Jr. (1974) Proc. Nat. Acad. Sci. USA 71, and the identification of conformational amphipathic regions in apoproteins, a new model for human high density lipoproteins is proposed. This model is analogous to membrane models proposed by Singer, in that protein "icebergs" are embedded in a "sea" of lipid. Determination of the mode of interaction between proteins and lipids in lipoprotein macromolecules is of fundamental importance for an understanding of structure and function. The particular properties and specific structural requirements enabling lipoprotein apoproteins to bind lipid molecules are not known. The purpose of the studies reported in the accompanying papers (1-3) was to evaluate some aspects of interaction between the major aproproteins and lipids in the high density lipoprotein (HDL) macromolecule. On the basis of the experimental data obtained, we have proposed a new model for HDL. Previous studies on the structure of HDL by x-ray techniques indicated that HDL has an organized symmetrical structure with an electron-rich (polar) outer region and a relatively electron-poor central (nonpolar) region (4). Enzymatic studies of HDL had also suggested that the majority of phospholipids were located on the surface of the HDL particles (5-7). On the other hand, data derived from electron microscopic studies of HDL have led to the proposal of a subunit structure for HDL (8). In this model, HDL has an electron dense central core surrounded by four to five subunits, each with its own complement of protein, phospholipid, and neutral lipids (9). The axis of the protein molecules was presumed to be perpendicular to the long axis of the phospholipid molecules (9). We have investigated the molecular organization of HDLphospholipid by phosphorus-31 nuclear magnetic resonance spectroscopy. These studies, which are detailed in a separate report (1), indicated that the spectrum of native HDL was characterized by two major resonances separated by 0.5 ppm which could be assigned to phosphatidylcholine (PC) (high field signal) and sphingomyelin (SPM) (low field signal). These experiments included the titration of native HDL with Abbreviations: HDL, high density lipoproteins; PC, phosphatidylcholine; SPM, sphingomyelin; LCAT, lecithin-cholesterolacyltransferase. increasing concentrations of europium. As the Eu+++ concentration was increased, the phosphorus resonance was both broadened and shifted. No unshifted components corresponding to the original peaks were left, indicating that essentially all phospholipid phosphorus was located at the outer surface of the HDL particles (1), i.e., accessible for chelation with Eu These findings are, therefore, consistent with the x-ray diffraction and enzymatic studies, which indicated that HDL is a spherical micelle with the phospholipid polar head groups located on the surface of the HDL particle. The nature of the interaction between the lipids and apoproteins in HDL was also evaluated by carbon-13 nuclear magnetic resonance spectroscopy of native and recombined lipoproteins. These studies, which are described in detail in a separate report (2), involved the recombination of HDL apoproteins with [methyl-13c]phosphatidylcholine, [methyl- '3C]sphingomyelin, and 1,2-[l-oleoyl-'3C]-sn-phosphatidylcholine which had been enriched with 90% carbon-13 by chemical synthesis. Chemical shifts and spin-lattice relaxation times were obtained for the '3C nuclei in the polar head groups of PC and SPM alone in organic solvents or D20, and recombined with apoa-ii. It was found that the polar head groups of PC and SPM had the same hydrophilic environment in sonicated lipid particles as in reassembled lipoproteins, indicating that ionic interactions between lipids and apolipoproteins were of minor importance in the formation of lipoprotein particles. The fact that HDL is usually isolated in the ultracentrifuge at high salt concentration and that detergents or organic solvents are required to separate the lipids and proteins indicates that hydrophobic interactions contribute significantly to the overall structure of these macromolecules. The degree of hydrophobicity of proteins can be estimated by calculation of H DAVE (average hydrophobicity), and NPS (frequency of nonpolar side chains) (10, 11). The values for apoa-ii (H 1'AVE 1451 cal per residue, NPS 0.35) and apoc- III (H 'IAVE 1211 cal per residue, NPS 0.28) are among the highest reported in the literature, suggesting that both apoproteins are very hydrophobic in nature. It has been pointed out, however, that hydrophobicity of a protein per se is not necessarily sufficient to produce a lipid-binding protein (12). Examination of the primary structures of apoa-ii and of apoc-iii, a minor apoprotein constituent of HDL (Fig. 1), reveals no long sequences of hydrophobic or hydrophilic residues. The carboxyterminal end of apoa-ii (residues 49-77), however, contains a notable frequency of hydrophobic amino-acid residues and should be regarded as a possible exception. The lack of spatial distribution of polar and nonpolar groups is in sharp contrast; for example, to the amino-acid 1534

2 Proc. Nat. Acad. Sci. USA 71 (1974) ApoLp-Ala or C-IIl Model of High Density Lipoproteins 1535 H 2 N lu As a eu r y r Met s Ly r a sa N OH \ OH Apoip-Gin-Il or A-II,~~~~~~~~~- SA 50 a Vlul ~~~45 VaV Sr al ~r las r o r O CI (LYS)~~~ r Cys Va Glu Va In hr ASP r GI L eu FIG o Primary structure of apoa-ii and apoc-iii m sequences of glycophorin (13), a protein isolated from erythrocyte membranes, and cytochrome b5 (14), a mitochondrial membrane protein. Both proteins are clearly demarcated into distinct hydrophilic and hydrophobic areas, thus exhibiting linear amphipathic properties. ApoA-II and apoc-iii do not contain similar linear amphipathic regions. Previous studies, however, have indicated that apolipoproteins apoa-i, apoa- II, and apoc-iii, as well as specific peptide fragments, increase their helical content when recombined with phospholipids (3, 15-19). We have confirmed these observations and extended them to the binding of SPM to apolipoproteins (3). Based on these results, we propose that the formation of helical structure induced by phospholipids per se, or, alternatively, the hydrophobic environment, provides hydrophobic and hydrophilic helical surfaces for lipid-protein and (or) protein-protein interactions. Not only the secondary structure, but undoubtedly the tertiary structure of these apolipoproteins, are likely to be involved in the formation of a specific conformation which permits both lipid-protein and proteinprotein interactions. We, therefore, believe that the generation of conformational amphipathic properties is probably a necessary structural prerequisite for the assembly of proteins and lipids in HDL. Conformational amphipathic properties in apoa-ii and apoc-iii could be predicted in areas where apolar aminoacid residues are repeatedly juxtaposed to polar residues, such as residues and in apoa-ii and residues in apoc-iii. Models of these regions in helical conformation (Fig. 2). suggest that one surface is hydrophobic, the other hydrophilic in nature. Cyanogen bromide cleavage at residue 26 of apoa-ii reduces the total lipid-binding capacity of the remaining piece (3, 16), conceivably because the total amphipathic area to be generated is reduced by the disturbance of conformational amphipathic properties. Succinylation of the lysine e-amino groups also reduces the phospholipid binding capacity of apoa-ij (3). Because hydrophilic forces do not appear to be of major importance in the binding of either PC or SPM to A-II, the effect of succinylation is most likely due to interference with the formation of conformational amphipathic properties. The lysine residues in intact HDL are available for succinylation, a modification which does not alter the integrity of the lipoproteins (20).

3 1536 Biochemistry: Assmann and Brewer, Jr. 19 (Is~j 5.4 A pitch m y 3.6 residues 189- ( g 5~~ ~ ~~~I (n) E FIG. 2. a-helical portion of apoa-ii with amphipathic properties. A right-handed a-helix with 3.6 amino-acid residues per turn and a periodicity of 5.4 A between residues 11 and 30 of apoa-ii reveals hydrophobic amino acids (Val, Phe, Leu, Met) on one helix surface area and charged (Lys, Glu, Asp) on the opposite helix surface area. These observations are in accord with the concept that the hydrophobic helix surface areas are in intimate contact with the hydrophobic fatty acid chains or neighboring hydrophobic protein areas, leaving the hydrophilic areas exposed and accessible to chemical modification. The apparent structural similarities between apoa-ii and apoc-iii are expressed by their evolutionary relationship. Barker and Dayhoff have compared their amino-acid sequences by use of the "mutation probability matrix" (21, 22). Their alignment score of 3.46 and probability factor P < 10-3 (probability that the similarities in the sequence of the two polypeptides would occur by chance) indicate that these apoproteins may have arisen from a common ancestral gene (23). Data from several laboratories suggest that the aminoterminal end of apoa-ii is not involved in lipid binding (3, 16, 17, 24). The isolated carboxy-terminal end of the molecule (residues 27-77), however, interacts with lipid (3, 16). The importance of amphipathic areas (residues 39-47) versus hydrophobic areas (residues 56-77) in this part of the molecule with respect to lipid binding properties needs to be elucidated. Another interesting feature in the primary sequence of apoa-ii is the distribution of proline residues (positions 5, 32, 51, 74). The distance between them is 27, 19, and 23 amino Proc. Nat. Acad. Sci. USA 71 (1974) acids, including at least two amphipathic areas. Moreover, if the amino-acid residues in these distinct areas are arranged in an a-helix, a maximum length of approximately i would result, which is equivalent to the dimension of a Ca-fatty acid chain in its fully extended form. It is possible that the specific localization of the proline residues permits the folding of the apoprotein into a three-dimensional form, also important for lipid binding. It is difficult at the present time to decide unambiguously whether conformational amphipathic regions in apolipoproteins primarily permit protein-protein interactions or, alternatively, are specific requirements for protein-lipid interactions. Hydrophobic interaction between amphipathic areas of individual apoproteins might expose their hydrophilic areas to the aqueous medium, thus contributing to both solubility and structural integrity. Further investigation will be required to explore which fragments of apolipoproteins are exposed to the aqueous medium or partly buried in the hydrophobic interior of the lipid moieties of the respective particle. Based on our results (3), apoa-i is thought to be involved to a minor extent in phospholipid binding. Its incorporation into HDL, based on recombination experiments (3), appears to require the presence of apoa-ii, suggesting that proteinprotein interactions are required for the integration of apoa-i into the native particle. The observation that apoa-i may be released from HDL by sonication, centrifugation, or treatment with ethyl ether (25) could be taken to support this concept. However, it has been as yet not established whether A-I is dissociated from the native HDL particle with or without a complement of lipid. The functional role of apoa-i in HDL must, at present, remain speculative. Recent evidence has suggested that apoa-i is an activator of lecithin-cholesterolacyltransferase (LCAT) (26), an enzyme of major importance for the regulation of cholesteryl esters and PC in HDL particles. The importance of a specific interaction between apoa-i and cholesterol and cholesteryl esters in the structural integrity of HDL needs to be elucidated. The importance of the C apoproteins in the molecular structure of HDL is as yet unknown. These proteins appear to be peripheral proteins which are easily dissociable and available for exchange with very low density lipoproteins. The attachment sites for these proteins on the HDL molecule are not yet defined. It is of interest that apoc-ii seems to strongly interact with phospholipid, whereas apoc-ii is known to activate lipoprotein lipase (27, 28). These two apoproteins provide an analogy to the A apoproteins, one of which interacts with phospholipid while the other appears to interact with an enzyme, and many indicate a common structural principle in lipoproteins. Based on experimental data from various laboratories and the data presented in the accompanying reports (1-3), we propose an up-dated and considerably expanded model for HDL (Fig. 3). In this model, HDL is a micellar particle with essentially all polar groups of the constituent molecules oriented into the surrounding aqueous environment. The interiors of the micelles, unlike the peripheries, are not in intimate contact with the aqueous phase, but rather occupy a region more akin to the interior of an oil droplet. Both cholesteryl ester and triglyeeride might be visualized as a spherical nucleus (core location away from the aqueous environment) of neutral lipids in the liquid phase. Cholesterol may be located in the interfacial region. Its amphiphilicity makes it surface

4 Proc. Nat. Acad. Sci. USA 71 (1974) Model of High Density Lipoproteins 1537 FIG. 3. Schematic model of high density lipoprotein (see Text). active and its partition coefficient favors the phospholipid rather than the neutral lipid phase. Based primarily on indirect evidence obtained from carbon-13 nuclear magnetic spectroscopy data, we suggest that the protein molecules are oriented parallel rather than perpendicular to the long axis of the phospholipid fatty acid chains (Fig. 2). It should be noted that the schematic model proposed (Fig. 3) has certain hypothetical features. (i) The distribution of phosphatidylcholine and sphingomyelin is illustrated as though it were random. It may be that these phospholipids are arranged in a more specific way. The observation that specific phospholipids in membranes are required for restoring the activity of several isolated membrane proteins (29) may be interpreted to suggest a segregation of some of the phospholipids in membranes. Similarly, the reaction of LCAT with HDL may require asymmetric and specific arrangements of lecithin and cholesterol. (ii) The structural relationship of cholesterol and cholesteryl esters to phospholipid and protein are as yet unknown. Cholesteryl ester and triglycerides are commonly thought to be core constituents in lipoproteins. (iii) The carboxy-terminal end of apoa-ii is shown to interact hydrophobically with the fatty acid chains of the phospholipid. Its amino-terminal end, not involved in lipid binding, is shown to project from the surface area into the aqueous environment. ApoA-I is illustrated to associate with apoa-ii mainly by protein-protein interaction. The extent to which apoa-i and apoa-ii are embedded in the lipid phase cannot be predicted at the present time. The helical content of apoa-i and apoa-ii is schematic, and does not indicate the location or amount of helix in the proteins. The proposed model is not compatible with the subunit model previously suggested for HDL (8), and, instead, closely resembles the lipid protein architecture in membranes as proposed by Singer (30). In Singer's model, protein "icebergs" are visualized as embedded in a "sea" of lipid. Within the "icebergs" of protein there appear to be specific proteinprotein and lipid-protein interactions. The major force involved in lipid-protein binding appears to be hydrophobic in nature. The precise nature of the structural foldings that give rise to the asymmetry of structure and function in HDL remains unknown. 1. Assmann, G., Sokoloski, E. A. & Brewer, H. B., Jr. (1974) Proc. Nat. Acad. Sci. USA 71, Assnmann, G., Highet, R. J., Sokoloski, E. A. & Brewer, H. B., Jr. (1974) Proc. Nat. Acad. Sci. USA 71, in press. 3. Assmann, G. & Brewer, H. B., Jr. (1974) Proc. Nat. Acad. Sci. USA 71, Shipley, G. G., Atkinson, D. & Scanu, A. M. (1972) J. Supramolec. Struc. 1, Camejo, G. (1969) Biochim. Biophys. Acta 175, Ashworth, L. A. E. & Green, C. (1963) Biochem. J. 89, Scanu, A. M. (1972) Biochim. Biophys. Acta 265, Forte, G. M., Nichols, A. V. & Glaeser, R. M. (1968) Chem. Phys. Lipids 2, Gotto, A. M., Jr. (1969) Proc. Nat. Acad. Sci. USA 64, Bigelow, C. C. (1967) J. Theor. Biol. 16, Goldsack, D. E. (1970) Biopolymers 9, Davis, M. A. F., Hauser, H., Leslie, R. B. & Phillips, M. C. (1973) Biochim. Biophys. Acta 317, Marchesi, V. T., Jackson, R. L., Segrest, J. P. & Kahane, I. (1973) Fed. Proc. 32, Strittmatter, P., Rogers, M. J. & Spak, L. (1972) J. Biol. Chem. 247, Lux, S. E., Hirz, R., Shrager, R. I. & Gotto, A. M. (1972) J. Biol. Chem. 247, Lux, S. E., John, K. M., Fleischer, S., Jackson, R. L. & Gotto, A. M. (1972) Biochem. Biophys. Res. Commun. 49, Jackson, R. L., Morrisett, J. D., Pownall, H. J. & Gotto, A. M., Jr. (1973) J. Biol. Chem. 248, Sparrow, J., Gotto, A. M. & Morrisett, J. D. (1973) Proc. Nat. Acad. Sci. USA 70, Jackson, R. L., Baker H. N., David, J. S. K. & Gotto, A. M. (1972) Biochem. Biophys. Res. Commun. 49,

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