Title: Effects of Polymorphism on the Lipid Interaction of Human Apolipoprotein E
Abstract: ApoE exists as three common isoforms, apoE2, apoE3, and apoE4; apoE2 and apoE3 preferentially bind to high density lipoproteins, whereas apoE4 prefers very low density lipoproteins (VLDL). To understand the molecular basis for the different lipoprotein distributions of these isoforms in human plasma, we examined the lipid-binding properties of the apoE isoforms and some mutants using lipid emulsions. With both large (120 nm) and small (35 nm) emulsion particles, the binding affinity of apoE4 was much higher than that of apoE2 and apoE3, whereas the maximal binding capacities were similar among the three isoforms. The 22-kDa N-terminal fragment of apoE4 displayed a much higher binding capacity than did apoE2 and apoE3. The apoE4(E255A) mutant, which has no electrostatic interaction between Arg61 and Glu255, showed binding behavior similar to that of apoE3, indicating that N- and C-terminal domain interaction in apoE4 is responsible for its high affinity for lipid. In addition, the apoE3(P267A) mutant, which is postulated to contain a long α-helix in the C-terminal domain, had significantly decreased binding capacities for both sizes of emulsion particle, suggesting that the apoE4 preference for VLDL is not due to a stabilized long α-helical structure. Isothermal titration calorimetry measurements showed that there is no significant difference in thermodynamic parameters for emulsion binding among the apoE isoforms. However, fluorescence measurements of 8-anilino-1-naphthalenesulfonic acid binding to apoE indicated that apoE4 has more exposed hydrophobic surface compared with apoE3 mainly due to the different tertiary organization of the C-terminal domain. The less organized structure in the C-terminal domain of apoE4 leads to the higher affinity for lipid, contributing to its preferential association with VLDL. In fact, we found that apoE4 binds to VLDL with higher affinity compared with apoE3. ApoE exists as three common isoforms, apoE2, apoE3, and apoE4; apoE2 and apoE3 preferentially bind to high density lipoproteins, whereas apoE4 prefers very low density lipoproteins (VLDL). To understand the molecular basis for the different lipoprotein distributions of these isoforms in human plasma, we examined the lipid-binding properties of the apoE isoforms and some mutants using lipid emulsions. With both large (120 nm) and small (35 nm) emulsion particles, the binding affinity of apoE4 was much higher than that of apoE2 and apoE3, whereas the maximal binding capacities were similar among the three isoforms. The 22-kDa N-terminal fragment of apoE4 displayed a much higher binding capacity than did apoE2 and apoE3. The apoE4(E255A) mutant, which has no electrostatic interaction between Arg61 and Glu255, showed binding behavior similar to that of apoE3, indicating that N- and C-terminal domain interaction in apoE4 is responsible for its high affinity for lipid. In addition, the apoE3(P267A) mutant, which is postulated to contain a long α-helix in the C-terminal domain, had significantly decreased binding capacities for both sizes of emulsion particle, suggesting that the apoE4 preference for VLDL is not due to a stabilized long α-helical structure. Isothermal titration calorimetry measurements showed that there is no significant difference in thermodynamic parameters for emulsion binding among the apoE isoforms. However, fluorescence measurements of 8-anilino-1-naphthalenesulfonic acid binding to apoE indicated that apoE4 has more exposed hydrophobic surface compared with apoE3 mainly due to the different tertiary organization of the C-terminal domain. The less organized structure in the C-terminal domain of apoE4 leads to the higher affinity for lipid, contributing to its preferential association with VLDL. In fact, we found that apoE4 binds to VLDL with higher affinity compared with apoE3. Human apoE, a 34-kDa protein composed of 299 amino acids, plays an important role in lipoprotein metabolism and neurobiology through its interaction with the low density lipoprotein (LDL) 1The abbreviations used are: LDL, low density lipoprotein; VLDL, very low density lipoprotein(s); HDL, high density lipoproteins; PC, phosphatidylcholine; ANS, 8-anilino-1-naphthalenesulfonic acid; ITC, isothermal titration calorimetry. receptor family and cell-surface heparan sulfate proteoglycans (1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3388) Google Scholar, 2Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar, 3Al-Haideri M. Goldberg I.J. Galeano N.F. Gleeson A. Vogel T. Gorecki M. Sturley S.L. Deckelbaum R.J. Biochemistry. 1997; 36: 12766-12772Crossref PubMed Scopus (72) Google Scholar, 4DeKnijff P. Havekes L.M. Curr. Opin. Lipidol. 1996; 7: 59-63Crossref PubMed Scopus (57) Google Scholar). ApoE exists in three major isoforms, apoE2, apoE3, and apoE4, each differing by cysteine and arginine at positions 112 and 158. ApoE3, the most common form, contains cysteine and arginine at these positions, respectively, whereas apoE2 contains cysteine and apoE4 contains arginine at both sites (5Weisgraber K.H. Rall Jr., S.C. Mahley R.W. J. Biol. Chem. 1981; 256: 9077-9083Abstract Full Text PDF PubMed Google Scholar). These differences have profound effects on the biological functions of apoE. Both apoE3 and apoE4 bind to the LDL receptor with high affinity, whereas apoE2 exhibits defective binding to the LDL receptor and is associated with type III hyperlipoproteinemia (6Mahley R.W. Huang Y. Rall Jr., S.C. J. Lipid Res. 1999; 40: 1933-1949Abstract Full Text Full Text PDF PubMed Google Scholar). ApoE4 is associated with high plasma cholesterol level and an increased risk for both coronary heart disease and Alzheimer's disease (7Davignon J. Gregg R.E. Sing C.F. Arteriosclerosis. 1988; 8: 1-21Crossref PubMed Google Scholar, 8Strittmatter W.J. Roses A.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4725-4727Crossref PubMed Scopus (444) Google Scholar, 9Weisgraber K.H. Mahley R.W. FASEB J. 1996; 10: 1485-1494Crossref PubMed Scopus (277) Google Scholar). The apoE isoforms are further distinguished by their preferential distribution among lipoprotein classes: apoE4 prefers very low density lipoproteins (VLDL), whereas apoE2 and apoE3 prefer high density lipoproteins (HDL) (10Weisgraber K.H. J. Lipid Res. 1990; 31: 1503-1511Abstract Full Text PDF PubMed Google Scholar). ApoE contains two independently folded functional domains: a 22-kDa N-terminal domain (residues 1–191) and a 10-kDa C-terminal domain (residues 222–299) (11Wetterau J.R. Aggerbeck L.P. Rall Jr., S.C. Weisgraber K.H. J. Biol. Chem. 1988; 263: 6240-6248Abstract Full Text PDF PubMed Google Scholar, 12Morrow J.A. Segall M.L. Lund-Katz S. Phillips M.C. Knapp M. Rupp B. Weisgraber K.H. Biochemistry. 2000; 39: 11657-11666Crossref PubMed Scopus (267) Google Scholar). The N-terminal domain exists in the lipid-free state as a four-helix bundle of amphipathic α-helices and contains the LDL receptor-binding region (residues 136–150 in helix 4) (13Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (600) Google Scholar). The C-terminal domain has a high affinity for lipid and is responsible for lipoprotein binding (2Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar, 14Westerlund J.A. Weisgraber K.H. J. Biol. Chem. 1993; 268: 15745-15750Abstract Full Text PDF PubMed Google Scholar). In apoE4, these two domains interact in a unique manner unlike in the other isoforms: Arg112 causes a rearrangement of the Arg61 side chain in the N-terminal domain of apoE4, allowing it to interact with Glu255 in the C-terminal domain (15Dong L.M. Wilson C. Wardell M.R. Simmons T. Mahley R.W. Weisgraber K.H. Agard D.A. J. Biol. Chem. 1994; 269: 22358-22365Abstract Full Text PDF PubMed Google Scholar, 16Dong L.M. Weisgraber K.H. J. Biol. Chem. 1996; 271: 19053-19057Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). This domain interaction in human apoE4 is responsible for the preferential association with VLDL and has been suggested to contribute to the accelerated catabolism of this isoform and, consequently, the increased cholesterol and LDL levels in plasma (9Weisgraber K.H. Mahley R.W. FASEB J. 1996; 10: 1485-1494Crossref PubMed Scopus (277) Google Scholar, 16Dong L.M. Weisgraber K.H. J. Biol. Chem. 1996; 271: 19053-19057Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). Association of apoE with lipid is required for its high affinity binding to the LDL receptor (17Innerarity T.L. Pitas R.E. Mahley R.W. J. Biol. Chem. 1979; 254: 4186-4190Abstract Full Text PDF PubMed Google Scholar). A number of recent studies carried out to understand the molecular basis for this phenomenon indicated that the four-helix bundle in the N-terminal domain undergoes a conformational opening upon lipid binding, leading to the receptor-active conformation of apoE (18Raussens V. Fisher C.A. Goormaghtigh E. Ryan R.O. Ruysschaert J.M. J. Biol. Chem. 1998; 273: 25825-25830Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 19Fisher C.A. Ryan R.O. J. Lipid Res. 1999; 40: 93-99Abstract Full Text Full Text PDF PubMed Google Scholar, 20Lu B. Morrow J.A. Weisgraber K.H. J. Biol. Chem. 2000; 275: 20775-20781Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 21Fisher C.A. Narayanaswami V. Ryan R.O. J. Biol. Chem. 2000; 275: 33601-33606Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In this conformation, the positive electrostatic potential in the receptor-binding region of apoE is enhanced, probably allowing its high affinity binding to the LDL receptor (22Lund-Katz S. Zaiou M. Wehrli S. Dhanasekaran P. Baldwin F. Weisgraber K.H. Phillips M.C. J. Biol. Chem. 2000; 275: 34459-34464Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 23Lund-Katz S. Wehrli S. Zaiou M. Newhouse Y. Weisgraber K.H. Phillips M.C. J. Lipid Res. 2001; 42: 894-901Abstract Full Text Full Text PDF PubMed Google Scholar). In addition, we have recently shown that the two domains in apoE4 lead to two different lipid-bound conformations (open or closed four-helix bundle) on emulsion particles (24Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), providing a structural rationale for the variable receptor-binding activity displayed by lipoprotein-associated apoE (25Narayanaswami V. Ryan R.O. Biochim. Biophys. Acta. 2000; 1483: 15-36Crossref PubMed Scopus (158) Google Scholar). Because the three isoforms of apoE exhibit different thermal and chemical stabilities (apoE4 < apoE3 < apoE2) (12Morrow J.A. Segall M.L. Lund-Katz S. Phillips M.C. Knapp M. Rupp B. Weisgraber K.H. Biochemistry. 2000; 39: 11657-11666Crossref PubMed Scopus (267) Google Scholar, 26Acharya P. Segall M.L. Zaiou M. Morrow J. Weisgraber K.H. Phillips M.C. Lund-Katz S. Snow J. Biochim. Biophys. Acta. 2002; 1584: 9-19Crossref PubMed Scopus (65) Google Scholar), the lipid-binding activity of these isoforms is expected to be different. Indeed, the reactivity to dimyristoylphosphatidylcholine liposomes of the 22-kDa N-terminal fragments of the three isoforms tends to vary inversely with the stabilities of these fragments (27Segall M.L. Dhanasekaran P. Baldwin F. Anantharamaiah G.M. Weisgraber K.H. Phillips M.C. Lund-Katz S. J. Lipid Res. 2002; 43: 1688-1700Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In this study, we examined further the lipid interaction of the three isoforms of apoE using lipoprotein-like emulsion particles to understand the molecular basis for the different lipoprotein distribution of apoE isoforms. In addition, to test the hypothesis that the domain interaction in apoE4 stabilizes an extended helical structure in the C terminus that targets a less curved VLDL surface (16Dong L.M. Weisgraber K.H. J. Biol. Chem. 1996; 271: 19053-19057Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 28Segrest J.P. Jones M.K. De Loof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. J. Lipid Res. 1992; 33: 141-166Abstract Full Text PDF PubMed Google Scholar), the lipid-binding properties of two apoE mutants, apoE3(P267A) and apoE4(E255A), were determined. Materials—Egg yolk phosphatidylcholine (PC) and triolein were purchased from Sigma, and stock solutions were stored in chloroform/methanol (2:1) under nitrogen at -20 °C. [14C]Formaldehyde (40–60 Ci/mol) in distilled water was purchased from PerkinElmer Life Sciences. NaCNBH3 (Aldrich) was recrystallized from methylene chloride before use. 8-Anilino-1-naphthalenesulfonic acid (ANS) was purchased from Molecular Probes, Inc. (Eugene, OR). Ultrapure guanidine hydrochloride was from ICN Pharmaceuticals (Costa Mesa, CA). Bacteriological media were obtained from Fisher. The prokaryotic expression vector pET32a was from Novagen (Madison, WI), and the competent Escherichia coli strains BL21(DE3) and DH5α were from Invitrogen. PCR supplies and DNA purification kits were from QIAGEN Inc. (Chatsworth, CA). Restriction enzymes were purchased from Promega (Madison, WI). Isopropyl-β-d-thiogalactopyranoside, β-mercaptoethanol, aprotinin, and ampicillin were from Sigma. Oligonucleotides were from IDT (Coralville, IA). All other salts and reagents were analytical grade. Lipoprotein and Apolipoproteins—VLDL was isolated from fasting normolipidemic human plasma by ultracentrifugation at a density cut of 1.006 g/ml. Examination by agarose gel electrophoresis showed that the VLDL had pre-β mobility and that it was not contaminated with either chylomicrons or LDL. SDS-PAGE showed the expected presence of apoB-100, apoC, and apoE. Gel filtration chromatography (29Boyle K.E. Phillips M.C. Lund-Katz S. Biochim. Biophys. Acta. 1999; 1430: 302-312Crossref PubMed Scopus (15) Google Scholar) demonstrated that the VLDL particles had diameters in the range of 25–60 nm. Full-length human apoE2, apoE3, and apoE4 and their 22- and 10-kDa fragments were expressed and purified as described (24Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 27Segall M.L. Dhanasekaran P. Baldwin F. Anantharamaiah G.M. Weisgraber K.H. Phillips M.C. Lund-Katz S. J. Lipid Res. 2002; 43: 1688-1700Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The mutations in apoE3(P267A) and apoE4(E255A) were introduced using PCR to create DNA inserts that were ligated into a thioredoxin fusion expression vector (pET32a) as described (30Morrow J.A. Arnold K.S. Weisgraber K.H. Protein Expression Purif. 1999; 16: 224-230Crossref PubMed Scopus (91) Google Scholar, 31Saito H. Dhanasekaran P. Nguyen D. Baldwin F. Weisgraber K.H. Wehrli S. Phillips M.C. Lund-Katz S. J. Biol. Chem. 2003; 278: 14782-14787Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The mutation, sequence, and cDNA orientation were confirmed by restriction enzyme analysis and double-stranded DNA sequencing. The resulting fusion proteins were expressed in E. coli, cleaved, and purified as described (30Morrow J.A. Arnold K.S. Weisgraber K.H. Protein Expression Purif. 1999; 16: 224-230Crossref PubMed Scopus (91) Google Scholar). The purity of the proteins was monitored by SDS-PAGE (8–25% gradient) with an Amersham Biosciences Phast electrophoresis system. Protein concentrations were determined by the procedure of Lowry et al. (32Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Binding of ApoE to Emulsion or VLDL Particles—Triolein/egg PC emulsion particles were prepared by sonication and purified by ultracentrifugation as described (24Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The average particle diameter determined by quasi-elastic light scattering measurements was 120 ± 10 nm for large emulsions and 35 ± 5 nm for small emulsions. The binding of apoE to emulsion particles at room temperature was assayed with a centrifugation method as described (24Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) using apoE samples 14C-labeled to a specific activity of ∼1 μCi/mg of protein by reductive methylation of lysines with [14C]formaldehyde (22Lund-Katz S. Zaiou M. Wehrli S. Dhanasekaran P. Baldwin F. Weisgraber K.H. Phillips M.C. J. Biol. Chem. 2000; 275: 34459-34464Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 24Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 33Lund-Katz S. Weisgraber K.H. Mahley R.W. Phillips M.C. J. Biol. Chem. 1993; 268: 23008-23015Abstract Full Text PDF PubMed Google Scholar); this trace labeling has no detectable effect on the physical properties of the protein and its interaction with lipid (24Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In VLDL binding experiments, the incubation mixtures of 14C-labeled apoE and human VLDL (0.3 mg/ml phospholipid) were centrifuged under the same conditions as in the case of small emulsions. Binding data were fitted by nonlinear regression to a one-binding site model with the GraphPAD Prism program. Isothermal Titration Calorimetry (ITC) Measurements—Heats of apoE binding to emulsions were measured with a MicroCal MCS isothermal titration calorimeter at 25 °C as described (24Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The reactant was placed in the sample cell (1.35 ml) and titrated with 8–10-μl aliquots of the injectant with continual stirring at 400 rpm. To measure the enthalpy of binding at a low surface concentration, apoE solutions were injected into emulsions in the cell at a PC/protein molar ratio of >10,000, at which the injected protein binds completely to the emulsion surface (normal injection). For reverse injection, emulsions were injected into excess apoE (PC/protein ratio < 40), where the emulsion surface appears to be saturated with apoE. Heats of dilution were determined in control experiments by injecting either apoE solution or emulsions into buffer, and these heats were subtracted from the heats determined in the corresponding apoE/emulsion binding experiments. The decay rate constants for the heats of binding were obtained by fitting the titration curves to a one- or two-phase exponential decay model. ANS Fluorescence Measurements—Fluorescence measurements were obtained with a Hitachi F-4500 fluorescence spectrophotometer at 25 °C. The apoE sample was freshly dialyzed from 6 m guanidine hydrochloride and 1% β-mercaptoethanol solution into Tris buffer (pH 7.4) before use. ANS fluorescence spectra were collected from 400 to 600 nm at an excitation wavelength of 395 nm in the presence of 50 μg/ml protein and 250 μm ANS. Under these conditions, ANS was in at least 100-fold excess to the protein (mol/mol). Binding of ApoE Isoforms to Emulsion Particles—Previously, we determined the parameters for binding of apoE4 and its 22- and 10-kDa fragments to emulsion particles using an ultracentrifugal separation (24Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In this study, we applied this method to compare the lipid-binding properties of the three isoforms. As shown in Fig. 1, both full-length apoE2 and apoE3 displayed saturable binding to large emulsions, similar to the behavior of full-length apoE4, whereas the 22-kDa fragments of apoE2 and apoE3 hardly bound to the emulsion surface. The dissociation constant (K d) and the maximal binding capacity (B max) for small and large emulsion particles are listed in Table I. In the case of the full-length proteins, apoE2 and apoE3 displayed much lower binding affinity for both emulsions compared with apoE4, whereas the binding capacities of the three isoforms were similar for both emulsion particle sizes. The binding parameters for full-length apoE3 were comparable to the previously reported data for human apoE3 (34Yokoyama S. Kawai Y. Tajima S. Yamamoto A. J. Biol. Chem. 1985; 260: 16375-16382Abstract Full Text PDF PubMed Google Scholar, 35Derksen A. Small D.M. Biochemistry. 1989; 28: 900-906Crossref PubMed Scopus (35) Google Scholar), and the higher affinity of apoE4 compared with apoE3 was also observed for VLDL-size emulsion particles (16Dong L.M. Weisgraber K.H. J. Biol. Chem. 1996; 271: 19053-19057Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 36Dong J. Balestra M.E. Newhouse Y.M. Weisgraber K.H. J. Lipid Res. 2000; 41: 1783-1789Abstract Full Text Full Text PDF PubMed Google Scholar). In contrast to the 22-kDa fragment of apoE4, the 22-kDa fragments of apoE2 and apoE3 displayed negligible binding capacities for both emulsions.Table IBinding parameters of apoE isoforms and their fragments for emulsion particles35-nm emulsion120-nm emulsionK dBmaxK dBmaxμg/mlamino acids/mol PCμg/mlamino acids/mol PCApoE221.8 ± 4.50.84 ± 0.0718.8 ± 2.90.74 ± 0.05ApoE317.3 ± 2.50.88 ± 0.0519.3 ± 2.30.79 ± 0.04ApoE4aData are from Saito et al. (24)4.2 ± 0.60.82 ± 0.038.1 ± 1.30.77 ± 0.03ApoE2 22-kDa fragment26.2 ± 16.90.05 ± 0.0221.4 ± 22.60.03 ± 0.01ApoE3 22-kDa fragment27.7 ± 38.50.02 ± 0.0138.6 ± 22.10.06 ± 0.03ApoE4 22-kDa fragmentaData are from Saito et al. (24)39.2 ± 12.90.32 ± 0.0528.2 ± 7.10.27 ± 0.03a Data are from Saito et al. (24Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) Open table in a new tab To account for the higher affinity of apoE4 for VLDL compared with apoE3, it has been proposed that a N- and C-terminal domain interaction in apoE4 stabilizes an extended helical structure in the C terminus, thereby promoting its binding to VLDL (16Dong L.M. Weisgraber K.H. J. Biol. Chem. 1996; 271: 19053-19057Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). To explore whether this hypothesis can be applied to the different lipid-binding behaviors of apoE3 and apoE4, we examined the lipid-binding properties of two apoE mutants, apoE3(P267A) and apoE4(E255A). ApoE3(P267A) is postulated to have a long α-helix in its C terminus because the mutation P267A is likely to remove the interruption or kink between predicted helices 225–266 and 268–289 (28Segrest J.P. Jones M.K. De Loof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. J. Lipid Res. 1992; 33: 141-166Abstract Full Text PDF PubMed Google Scholar). The mutation E255A in apoE4 is known to alter the apoE4 preference from VLDL to HDL by disrupting the domain interaction (16Dong L.M. Weisgraber K.H. J. Biol. Chem. 1996; 271: 19053-19057Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). Fig. 2 shows the binding isotherms of these mutants for small emulsions in comparison with the isotherms of the respective wild-type proteins; the binding parameters for both sizes of emulsion particles are summarized in Fig. 3. The mutation E255A in apoE4 reduced the lipid affinity without changing the binding capacity for both emulsions; and, as a result, the apoE4(E255A) mutant bound in a similar manner compared with wild-type apoE3 rather than apoE4, indicating that the domain interaction in apoE4 is responsible for its high affinity for lipid. In contrast, the apoE3(P267A) mutant displayed a much lower binding capacity compared with wild-type apoE3 regardless of the emulsion particle size, suggesting that helical length in the C terminus is not responsible for the different lipid-binding behavior of apoE3 and apoE4.Fig. 3Comparison of binding parameters among full-length apoE variants. A, dissociation constant; B, maximal binding capacity. These parameters were derived from binding isotherms shown in Figs. 1 and 2. a.a., amino acids.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ITC Measurements—To obtain thermodynamic information about the lipid interaction of apoE isoforms, we performed ITC measurements of apoE binding to emulsions (24Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Fig. 4 shows the injections of full-length proteins and the 22-kDa fragments of apoE isoforms into large emulsions. Using the binding constants given in Table I and Fig. 3, the thermodynamic parameters for binding of apoE isoforms and mutants to small and large emulsions were obtained (Table II). As previously reported for apoE4 (24Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), binding of apoE isoforms and mutants to large particles was an exothermic process, but binding to small particles was much less exothermic or rather endothermic. As a result, the binding to large particles is enthalpically driven, whereas that to small particles is entropically driven. There was no significant difference in the thermodynamic binding parameters among the full-length apoE isoforms. In contrast, the enthalpies of binding of the 22-kDa fragments of apoE2 and apoE3 to large emulsions were much less exothermic than that of the apoE4 22-kDa fragment. In addition, the slow decay of ITC curves for full-length proteins and the apoE4 22-kDa fragment (half-life = 1.6–2.0 min) that appears to reflect opening of the N-terminal four-helix bundle (24Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) was not observed with the 22-kDa fragments of apoE2 and apoE3 (Fig. 4B). These results suggest that the 22-kDa fragments of apoE2 and apoE3 cannot bind to the emulsion surface with the four-helix bundle in an open conformation (20Lu B. Morrow J.A. Weisgraber K.H. J. Biol. Chem. 2000; 275: 20775-20781Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 24Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Interestingly, the enthalpies of binding of mutants apoE3(P267A) and apoE4(E255A) to large emulsions were much reduced compared with the wild-type proteins, suggesting that proper arrangements of charged or helical residues in the C-terminal domain are critical to the favorable enthalpy of binding to large particles.Table IIThermodynamic parameters of binding of apoE isoforms and variants to emulsion particles at 25 °C35-nm emulsion120-nm emulsionΔHΔG aFree energy was calculated according to ΔG = —RT ln 55.5(1/Kd) using the binding constants given in Table I and Fig. 3.ΔS bThe entropy of binding was calculated from ΔG = ΔH— TΔS.ΔHΔG aFree energy was calculated according to ΔG = —RT ln 55.5(1/Kd) using the binding constants given in Table I and Fig. 3.ΔS bThe entropy of binding was calculated from ΔG = ΔH— TΔS.kcal/molkcal/molcal/mol Kkcal/molkcal/molcal/mol KApoE25.2 ± 1.9—10.8 ± 0.254 ± 7—64.3 ± 5.5—10.9 ± 0.1—177 ± 19ApoE3—0.9 ± 0.5—11.0 ± 0.134 ± 2—66.6 ± 7.1—10.9 ± 0.1—187 ± 23ApoE4cData are from Saito et al. (24)—1.7 ± 1.2—11.8 ± 0.134 ± 4—68.7 ± 3.0—11.4 ± 0.1—192 ± 10ApoE2 22-kDa fragment0.4 ± 0.8—10.5 ± 0.336 ± 4—13.9 ± 2.5—10.6 ± 0.4—11 ± 10ApoE3 22-kDa fragment3.1 ± 0.5—10.4 ± 0.445 ± 3—3.7 ± 1.2—10.2 ± 0.322 ± 5ApoE4 22-kDa fragmentcData are from Saito et al. (24)—2.4 ± 0.7—10.2 ± 0.226 ± 3—43.0 ± 4.6—10.4 ± 0.2—109 ± 16ApoE3(P267A)—2.4 ± 1.9—10.9 ± 0.229 ± 7—7.6 ± 3.4—10.6 ± 0.210 ± 12ApoE4(E255A)—4.6 ± 2.1—11.1 ± 0.122 ± 7—34.0 ± 3.8—11.0 ± 0.1—77 ± 13a Free energy was calculated according to ΔG = —RT ln 55.5(1/Kd) using the binding constants given in Table I and Fig. 3.b The entropy of binding was calculated from ΔG = ΔH— TΔS.c Data are from Saito et al. (24Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) Open table in a new tab Based on the ITC results under the two limiting conditions, we showed recently that apoE4 adopts either the opened or closed conformation of the four-helix bundle depending upon the surface concentration of lipid-bound apoE (24Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Fig. 5 shows the comparison of the enthalpies of binding of full-length apoE isoforms to large emulsions at a low surface concentration of apoE (normal injection) or under a saturated condition (reverse injection). Although the enthalpy of apoE2 binding under a saturated con