Lipid-induced Extension of Apolipoprotein E Helix 4 Correlates with Low Density Lipoprotein Receptor Binding Ability

Apolipoprotein E (apoE) serves as a ligand for the low density lipoprotein receptor (LDLR) only when bound to lipid. The N-terminal domain of lipid-free apoE exists as globular 4-helix bundle that is conferred with LDLR recognition ability after undergoing a lipid binding-induced conformational change. To investigate the structural basis for this phenomenon, site-directed spin label electron paramagnetic resonance spectroscopy experiments were conducted, focusing on the region near the C-terminal end of helix 4 (Ala-164). Using C112S apoE-N-terminal as template, a series of single cysteine substitution variants (at sequence positions 161, 165, 169, 173, 176, and 181) were produced, isolated, and labeled with the nitroxide probe, methane thiosulfonate. Electron paramagnetic resonance analysis revealed that lipid association induced fixed secondary structure in a region of the molecule known to exist as random coil in the lipid-free state. In a complementary approach, site-directed fluorescence analysis using an environmentally sensitive probe indicated that the lipid-induced transition of this region of the protein to α helix was accompanied by relocation to a more hydrophobic environment. In studies with full-length apoE single Cys variants, a similar random coil to stable backbone transition was observed, consistent with the concept that lipid interaction induced an extension of helix 4 beyond the boundary defining its lipid-free conformation. This structural transition likely represents a key conformational change necessary for manifestation of the LDLR recognition properties of apoE. Apolipoprotein E (apoE) serves as a ligand for the low density lipoprotein receptor (LDLR) only when bound to lipid. The N-terminal domain of lipid-free apoE exists as globular 4-helix bundle that is conferred with LDLR recognition ability after undergoing a lipid binding-induced conformational change. To investigate the structural basis for this phenomenon, site-directed spin label electron paramagnetic resonance spectroscopy experiments were conducted, focusing on the region near the C-terminal end of helix 4 (Ala-164). Using C112S apoE-N-terminal as template, a series of single cysteine substitution variants (at sequence positions 161, 165, 169, 173, 176, and 181) were produced, isolated, and labeled with the nitroxide probe, methane thiosulfonate. Electron paramagnetic resonance analysis revealed that lipid association induced fixed secondary structure in a region of the molecule known to exist as random coil in the lipid-free state. In a complementary approach, site-directed fluorescence analysis using an environmentally sensitive probe indicated that the lipid-induced transition of this region of the protein to α helix was accompanied by relocation to a more hydrophobic environment. In studies with full-length apoE single Cys variants, a similar random coil to stable backbone transition was observed, consistent with the concept that lipid interaction induced an extension of helix 4 beyond the boundary defining its lipid-free conformation. This structural transition likely represents a key conformational change necessary for manifestation of the LDLR recognition properties of apoE. Apolipoprotein E (ApoE) 2The abbreviations used are: apoE, apolipoprotein E; LDLR, low density lipoprotein receptor; NT, N-terminal; SDSL, site-directed spin label; EPR, electron paramagnetic resonance; MTS, methane thiosulfonate; DMPC, dimyristoyl-phosphatidylcholine; TBS, Tris-buffered saline; AEDANS, N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine; WT, wild type. 2The abbreviations used are: apoE, apolipoprotein E; LDLR, low density lipoprotein receptor; NT, N-terminal; SDSL, site-directed spin label; EPR, electron paramagnetic resonance; MTS, methane thiosulfonate; DMPC, dimyristoyl-phosphatidylcholine; TBS, Tris-buffered saline; AEDANS, N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine; WT, wild type. is an anti-atherogenic protein that regulates cholesterol levels in plasma by virtue of its ability to serve as a high affinity ligand for members of the low density lipoprotein receptor (LDLR) family (1Brown M. Goldstein J. Science. 1986; 232: 34-47Crossref PubMed Scopus (4332) Google Scholar, 2Herz J. Bock H.H. Annu. Rev. Biochem. 2002; 71: 405-434Crossref PubMed Scopus (351) Google Scholar). Transgenic mice deficient in apoE display severe hypercholesterolemia and develop spontaneous and diet-induced atherosclerosis (3Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Crossref PubMed Scopus (1829) Google Scholar, 4Linton M.F. Atkinson J.B. Fazio S. Science. 1995; 267: 1034-1037Crossref PubMed Scopus (409) Google Scholar). Conversely, mice overexpressing apoE display a marked regression of atherosclerotic lesions and resistance to diet-induced atherosclerosis (5Bellosta S. Mahley R.W. Sanan D.A. Murata J. Newland D.L. Taylor J.M. Pitas R.E. J. Clin. Investig. 1995; 96: 2170-2179Crossref PubMed Scopus (250) Google Scholar, 6Boisvert W.A. Spangenberg J. Curtiss L.K. J. Clin. Investig. 1995; 96: 1118-1124Crossref PubMed Scopus (184) Google Scholar). ApoE occurs as three different isoforms, apoE2, apoE3, and apoE4, which differ in residues at positions 112 and 158. ApoE3, the most common isoform, bears a Cys and Arg at these positions, respectively. ApoE is composed of two independently folded structural domains that are linked by a protease sensitive loop (7Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar, 8Morrow J.A. Arnold K.S. Dong J. Balestra M.E. Innerarity T.L. Weisgraber K.H. J. Biol. Chem. 2000; 275: 2576-2580Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The 22-kDa N-terminal (NT) domain bears amino acids critical for LDLR binding, whereas the 10-kDa C-terminal domain manifests high lipid binding affinity and is responsible for apoE self-association in the absence of lipid. X-ray crystallography of apoE3-NT reveals a 4-helix bundle molecular architecture (9Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (596) Google Scholar). The classical LDLR recognition sequence encompasses a cluster of positively charged amino acids in the region of residues 134-150 on helix 4 of the N-terminal domain (7Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). Another positively charged residue, Arg-72, has also been implicated in LDLR binding (8Morrow J.A. Arnold K.S. Dong J. Balestra M.E. Innerarity T.L. Weisgraber K.H. J. Biol. Chem. 2000; 275: 2576-2580Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In addition, lipid association appears to be an essential prerequisite for apoE binding to LDLR. Interestingly, full-length apoE and the NT domain are equivalent in terms of LDLR binding ability (7Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). Reconstituted apoE (full-length or NT domain) phospholipid disks possess full receptor binding activity and provide a convenient model for studies of the lipid-associated conformation of apoE. Infrared spectroscopy studies show that helical segments in apoE3-NT domain align perpendicular to phospholipid fatty acyl chains around the perimeter of bilayer disk complexes (10Raussens 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 (85) Google Scholar). In fluorescence resonance energy transfer studies it has been reported that apoE3-NT adopts an extended conformation wherein the molecule aligns in tandem with a second apoE3-NT around the perimeter of the disk (11Fisher C.A. Ryan R.O. J. Lipid Res. 1999; 40: 93-99Abstract Full Text Full Text PDF PubMed Google Scholar, 12Fisher C.A. Narayanaswami V. Ryan R.O. J. Biol. Chem. 2000; 275: 33601-33606Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Spin labeled fatty acids and single tryptophan variants were used to show that apoE3-NT and full-length apoE adopt similar extended conformations (13Narayanaswami V. Maiorano J.N. Dhanasekaran P. Ryan R.O. Phillips M.C. Lund-Katz S. Davidson W.S. J. Biol. Chem. 2004; 279: 14273-14279Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In another approach, a peptide corresponding to apoE residues 126-183 was generated that possessed lipid and LDLR binding activity (14Raussens V. Mah M. Kay C.M. Sykes B.D. Ryan R.O. J. Biol. Chem. 2000; 275: 38329-38336Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Multidimensional NMR studies of apoE (126-183) associated with dodecylphosphocholine micelles revealed that residues known to exist as random coil in the lipid-free state adopt helical structure in the presence of lipid (15Raussens V. Slupsky C.M. Sykes B.D. Ryan R.O. J. Biol. Chem. 2003; 278: 25998-26006Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Questions emerging from these studies relate to whether the lipid-induced random coil to α-helix transition of this 58-residue peptide fragment also applies to apoE-NT or full-length apoE. To address this question we employed site-directed spin label-electron paramagnetic resonance (SDSL-EPR) spectroscopy to investigate the effect of lipid interaction on spin label probes positioned at regular intervals between amino acids 161 and 181 in apoE-NT (see Fig. 1) and full-length apoE. The data reveal that lipid association induces residues beyond Ala-164 (the termination site of helix 4 in lipid-free apoE) to transition from random coil to a more stable backbone. We conclude that the molecular basis for the divergent behavior of lipid-free and lipid-bound apoE in terms of LDLR binding is related to lipid binding-induced extension of helical structure beyond its termination site in the helix bundle state. Production and Bacterial Expression of apoE Variants—Site-directed mutagenesis was performed using the QuikChange XL kit from Stratagene according to the manufacturer's instructions, and all mutations were verified by dideoxy automated DNA sequencing. ApoE3-NT and full-length variants were expressed in Escherichia coli BL21 cells transformed with an apoE-pET-20b plasmid with His tag and purified on a Hi-Trap chelating nickel column from GE Healthcare as described (16Ryan R.O. Forte T.M. Oda M.N. Protein Expression Purif. 2003; 1: 98-103Crossref Scopus (131) Google Scholar). Samples were electrophoresed on precast 4-20% acrylamide gradient SDS gels (Invitrogen) at 30-mA constant current and stained with Coomassie Blue. Nitroxide-labeled apoE—Spin labeling was carried out as described earlier (17Oda M.N. Forte T.M. Ryan R.O. Voss J.C. Nat. Struct. Biol. 2003; 10: 455-460Crossref PubMed Scopus (113) Google Scholar). Briefly, 5 mg of apoE in buffer A (20 mm sodium phosphate, 0.5 m NaCl, pH 7.4) containing 3 m guanidine-HCl, and 0.25 mm dithiothreitol was loaded onto a Hi-Trap column. The column was then washed with 3 column volumes of buffer A containing 3 m guanidine-HCl and 100 μm Tris(2-carboxyethyl)phosphine hydrochloride followed by three column volumes of buffer A containing 3 m guanidine-HCl plus 160 μm methane thiosulfonate (MTS) and incubated for 45 min at 25 °C. The column was then washed with 3 column volumes of buffer A and protein eluted with buffer A containing 0.5 m imidazole. Eluted protein was dialyzed to remove imidazole and stored at 4 °C. ApoE·phospholipid Complexes—Dimyristoyl-phosphatidylcholine (DMPC)-apoE disks were prepared as described earlier (18Fisher C.A. Wang J. Francis G.A. Sykes B.D. Kay C.M. Ryan R.O. Biochem. Cell Biol. 1997; 75: 45-53Crossref PubMed Scopus (40) Google Scholar). Briefly, DMPC was dissolved in chloroform:methanol (3:1 v/v) and dried into a thin film in a glass tube. Following dispersion of the DMPC in 10 mm Tris-HCl, 150 mm NaCl, pH 7.2 (TBS), MTS-labeled apoE variants were added (2.5:1 DMPC/apoE weight ratio). This mixture was subjected to bath sonication until clear. Solubilized apoE·DMPC complexes were dialyzed against TBS and stored at 4 °C until use. Native PAGE of DMPC·apoE complexes was performed to evaluate the size of lipoprotein complexes. For UV circular dichroism spectroscopy, analysis of apoE-NT single cysteine variants was conducted on a Jasco-710 spectropolarimeter maintained at 24 °C. The results indicated that α-helix content was not adversely affected by probe attachment. Furthermore, with each variant examined, association with DMPC induced a 2-12% increase in α-helix content. Electron Paramagnetic Resonance Measurements—EPR spectra were recorded at 22 °C in TBS buffer on a JEOL-X band spectrometer equipped with a loop gap resonator in a single scan over 100 G as described earlier (17Oda M.N. Forte T.M. Ryan R.O. Voss J.C. Nat. Struct. Biol. 2003; 10: 455-460Crossref PubMed Scopus (113) Google Scholar). The microwave power was set at 2 milliwatts, and the modulation amplitude was set at 1 G. C112S apoE-NT served as a control for MTS labeling. EPR spectroscopy of this protein yielded no signal, confirming the specificity of label incorporation. LDLR Binding Assay—The ability of variant apoE-NT to interact with recombinant human LDLR was evaluated as described by Yamamoto et al. (19Yamamoto T. Lamoureux J. Ryan R.O. J. Lipid Res. 2006; 47: 1091-1096Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Briefly, C112S apoE-NT (3 μg protein (lipid free or DMPC bound)) was pre-incubated for 5 min with 4 μg of recombinant LDLR (residues 1-699) followed by the addition of 1 μg of tryptophan-null apoE-NT·DMPC that was labeled on Cys-112 with the fluorescent probe, N-(iodoacetyl)-N′-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS). The mixtures were incubated in 20 mm Tris, pH 7.2, 2 mm CaCl2, 90 mm NaCl at 25 °C for 1 h to fluorescence were performed at 25 °C on a spectrometer as described (19Yamamoto T. Lamoureux J. Ryan R.O. J. Lipid Res. 2006; 47: 1091-1096Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). In single Cys substitution variants of C112S apoE-NT were labeled with AEDANS, and fluorescence spectra of the labeled variants μg of were recorded in lipid-free or lipid-bound state as described earlier V. Ryan R.O. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, 12Fisher C.A. Narayanaswami V. Ryan R.O. J. Biol. Chem. 2000; 275: 33601-33606Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The was set at and the fluorescence spectra between and with a of and investigate the that lipid association induces residues beyond the termination site in helix 4 to adopt a helical conformation that LDLR recognition studies were Cys-112 in apoE3-NT was with This substitution was necessary to labeling of the to cysteine C112S apoE-NT was expressed and in terms of secondary structure and its LDLR binding UV circular dichroism spectroscopy that the secondary structure content of the protein was by the C112S Furthermore, (13Narayanaswami V. Maiorano J.N. Dhanasekaran P. Ryan R.O. Phillips M.C. Lund-Katz S. Davidson W.S. J. Biol. Chem. 2004; 279: 14273-14279Abstract Full Text Full Text PDF PubMed Scopus (72) Google in were for spectra in the presence of the helix v/v) or when as DMPC disks not The results of LDLR binding revealed whereas lipid-free C112S apoE-NT is a association with DMPC, this variant manifests LDLR binding activity equivalent to wild on these C112S apoE-NT was used as a to a series of single cysteine substitution apoE variants that were then labeled with MTS and subjected to EPR spectroscopy in lipid-free and lipid-bound of cysteine apoE-NT variants, and were by The revealed a single band properties were from that of apoE3-NT A corresponding electrophoresed revealed the presence of of in apoE3-NT and the different cysteine substitution C112S apoE-NT not show of The band in each of the cysteine substitution apoE3-NT This be related to the that Cys-112 was in a more region of the 4-helix and were to in the presence of SDS with variants with a cysteine in to the of the residues in different apoE-NT variants were then labeled with MTS, into DMPC and by gradient PAGE The that C112S apoE-NT and MTS-labeled apoE-NT variants associated with DMPC to lipid complexes of similar size of this size were reported to possess between 4 and apoE disk C.A. Narayanaswami V. Ryan R.O. J. Biol. Chem. 2000; 275: 33601-33606Abstract Full Text Full Text PDF PubMed Scopus (69) Google PAGE of apoE-NT·DMPC complexes. DMPC complexes were prepared with MTS-labeled apoE-NT variants and electrophoresed for 24 h at V. molecular weight C112S apoE-NT apoE-NT apoE-NT apoE-NT and apoE-NT EPR of MTS-labeled apoE-NT spectroscopy was performed on variants EPR spectra of lipid-free and lipid-bound apoE-NT variants at positions 161, 165, 169, 173, 176, and 181 are In the lipid-free spin at positions 173, 176, and 181 display similar This is of spin undergoing and is of a and state for the backbone of these In the EPR of lipid-free apoE-NT spin labeled at 161 a of a site a region of fixed secondary a spin label at this served as a control analysis that 161 of helix which encompasses residues (9Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (596) Google Scholar). The for this which reveals an in a helical the of this C. Nat. Struct. Biol. 2000; PubMed Scopus Google Scholar). at a similar a helical state for this The of apoE-NT at reveals a of of a more between that for residues and lipid was a in the EPR of all variants, which was with at positions 173, 176, and The is a of consistent with stable interaction of these with the lipid Interestingly, the in the of apoE-NT labeled at is of a this a of stable or in the lipid-bound state. of apoE-NT that labeled residues to a more hydrophobic when the molecule was with DMPC, fluorescence studies were single cysteine variants were labeled with the environmentally sensitive fluorescence probe, G. PubMed Scopus Google and spectra of lipid-free and lipid-bound the of 161, lipid association induced a in of fluorescence with corresponding spectra in the lipid-free state The lipid is consistent with data on the apoE (126-183) In the lipid-associated this was on the of the α In the of and the was the the spectra of and a of in the association with residues that helical secondary structure lipid association also to a more hydrophobic properties of apoE-NT apoE-NT variants also C112S spectra were recorded in TBS, pH at with a of for and is the of fluorescence apoE-NT variants also C112S spectra were recorded in TBS, pH at with a of for and is the of fluorescence in a EPR of apoE question emerging from studies with the NT domain of apoE is whether results be extended to the full-length To address full-length C112S apoE containing the single Cys were and by EPR For each of the labeled variants examined, the in as as to lipid were similar to for apoE-NT data that secondary structure in this region of the protein is not by the C-terminal consistent with the concept of independently folded and C-terminal domains by Weisgraber and Weisgraber K.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Weisgraber K.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of apoE as a of plasma cholesterol has been to its ability to LDLR family studies the classical LDLR binding site of apoE to the region residues 134-150 (7Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). Using it has been that residues are also critical for manifestation of the LDLR binding activity of apoE Mahley R.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar). et al. (8Morrow J.A. Arnold K.S. Dong J. Balestra M.E. Innerarity T.L. Weisgraber K.H. J. Biol. Chem. 2000; 275: 2576-2580Abstract Full Text Full Text PDF PubMed Scopus (45) Google that results in of LDLR binding lipid-free apoE also LDLR binding it is that lipid association induces a change in the or secondary structure of this that it adopts a for receptor The concept that of positively charged amino chains of apoE to ligand binding of the LDLR is by the results C. N. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). it has been that binding of an the receptor associated protein to a two of LDLR two wherein residues interact with positively charged amino acids in the receptor associated In addition, binding was to be by interaction of with more binding a for binding of to LDLR family The results are consistent with the concept that lipid extension of helix 4 receptor binding by interaction of the region of apoE residues with ligand binding are also in with studies of a 58-residue peptide that encompasses apoE amino acids this peptide is in it adopts an extended α-helix conformation in the presence of the lipid V. Slupsky C.M. Ryan R.O. Sykes B.D. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google or when to dodecylphosphocholine micelles (15Raussens V. Slupsky C.M. Sykes B.D. Ryan R.O. J. Biol. Chem. 2003; 278: 25998-26006Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). the an model of lipid-bound apoE has C.A. Weisgraber K.H. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus (72) Google Scholar). the basis of crystallography studies of it was that residues between 160 and a loop that for of with the receptor binding site (residues In this it is that a single apoE molecule interact with LDLR ligand binding EPR spectra of apoE variants spin at positions 161, 165, and are consistent with an extended structure as that reported earlier (13Narayanaswami V. Maiorano J.N. Dhanasekaran P. Ryan R.O. Phillips M.C. Lund-Katz S. Davidson W.S. J. Biol. Chem. 2004; 279: 14273-14279Abstract Full Text Full Text PDF PubMed Scopus (72) Google the model reported in C.A. Weisgraber K.H. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus (72) Google is also with the In it is to in the For the was conducted using bilayer disk complexes of DMPC and apoE, whereas et al. C.A. Weisgraber K.H. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus (72) Google complexes generated using Furthermore, in to the which employed an isoform, et al. V. Slupsky C.M. Ryan R.O. Sykes B.D. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google used the that the structure and of this is by and C-terminal domain J. Narayanaswami V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Voss J.C. Weisgraber K.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (69) Google Scholar, R.W. Weisgraber K.H. S. 2006; PubMed Scopus Google it is that adopts an lipid-bound conformation. studies are to in lipid-associated apoE. is known that residues beyond amino are in the electron density of lipid-free apoE3-NT (9Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (596) Google Scholar). provide that amino residues at positions and helical in buffer 169, apoE exists as random structure in in the α-helix structure at each of the was observed, as by the EPR these and the region of in for associated with apoE the structural of LDLR apoE is not data are consistent with the lipid-free apoE is to the of by the high lipid binding affinity of the C-terminal bound to the lipoprotein the NT helix bundle its receptor helix bundle conformation or a conformational change V. Ryan R.O. 2000; PubMed Scopus Google Scholar, Dhanasekaran P. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. J. Biochem. 2002; PubMed Scopus Google that results in stable with the lipid and manifestation of LDLR is that the NT domain between an conformation and a helix bundle state to the lipoprotein its C-terminal a a molecular for of lipoprotein on the conformational of the NT A key question to in the absence of apoE-NT domain adopts a 4-helix bundle in which helix 4 at is likely that of the domain into a globular 4-helix bundle hydrophobic it has also been reported that the region of apoE around to a classical helix C. Protein Scholar, V. Slupsky C.M. Sykes B.D. Ryan R.O. J. Biol. Chem. 2003; 278: 25998-26006Abstract Full Text Full Text PDF PubMed Scopus (26) Google at the end of α the termination of helix 4 in apoE is the of a helix then it is that lipid interaction induces a of the extension of the helix and of key positively charged residues for interaction with ligand binding of the LDLR. We of for to the circular dichroism M. M. for with and Wang for