The Lipid-associated Conformation of the Low Density Lipoprotein Receptor Binding Domain of Human Apolipoprotein E

Apolipoprotein E (apoE) is a 34-kDa exchangeable apolipoprotein that regulates metabolism of plasma lipoproteins by functioning as a ligand for members of the LDL receptor family. The receptor-binding region localizes to the vicinity of residues 130–150 within its independently folded 22-kDa N-terminal domain. In the absence of lipid, this domain exists as a receptor-inactive, globular four-helix bundle. Receptor recognition properties of this domain are manifest upon lipid association, which is accompanied by a conformational change in the protein. Fluorescence resonance energy transfer has been used to monitor helix repositioning, which accompanies lipid association of the apoE N-terminal domain. Site-directed mutagenesis was used to replace naturally occurring Trp residues with phenylalanine, creating a Trp-null apoE3 N-terminal domain (residues 1–183). Subsequently, tyrosine residues in helix 2, helix 3, or helix 4 were converted to Trp, generating single Trp mutant proteins. The lone cysteine at position 112 was covalently modified withN-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine, which serves as an energy acceptor from excited tryptophan residues. Fluorescence resonance energy transfer analysis of apoE N-terminal domain variants in phospholipid disc complexes suggests that the helix bundle opens to adopt a partially extended conformation. A model is presented that depicts a tandem arrangement of the receptor-binding region of the protein in the disc complex, corresponding to its low density lipoprotein receptor-active conformation. Apolipoprotein E (apoE) is a 34-kDa exchangeable apolipoprotein that regulates metabolism of plasma lipoproteins by functioning as a ligand for members of the LDL receptor family. The receptor-binding region localizes to the vicinity of residues 130–150 within its independently folded 22-kDa N-terminal domain. In the absence of lipid, this domain exists as a receptor-inactive, globular four-helix bundle. Receptor recognition properties of this domain are manifest upon lipid association, which is accompanied by a conformational change in the protein. Fluorescence resonance energy transfer has been used to monitor helix repositioning, which accompanies lipid association of the apoE N-terminal domain. Site-directed mutagenesis was used to replace naturally occurring Trp residues with phenylalanine, creating a Trp-null apoE3 N-terminal domain (residues 1–183). Subsequently, tyrosine residues in helix 2, helix 3, or helix 4 were converted to Trp, generating single Trp mutant proteins. The lone cysteine at position 112 was covalently modified withN-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine, which serves as an energy acceptor from excited tryptophan residues. Fluorescence resonance energy transfer analysis of apoE N-terminal domain variants in phospholipid disc complexes suggests that the helix bundle opens to adopt a partially extended conformation. A model is presented that depicts a tandem arrangement of the receptor-binding region of the protein in the disc complex, corresponding to its low density lipoprotein receptor-active conformation. apolipoprotein N- iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine dimyristoylphosphatidylcholine fluorescence resonance energy transfer wild type normalized fluorescence intensity efficiency of energy transfer Apolipoprotein E (apoE)1plays a critical role in lipoprotein metabolism via its ability to mediate clearance of remnant particles from circulation (1Mahley R.W. Huang Y. Curr. Opin. Lipidol. 1999; 10: 207-217Crossref PubMed Scopus (325) Google Scholar). In addition, apoE has been implicated in other biological processes including neuronal plasticity (2Poirier J. Minnich A. Davignon J. Ann. Med. 1995; 27: 663-670Crossref PubMed Scopus (84) Google Scholar) and dysfunction (3Weisgraber K.H. Pitas R.E. Mahley R.W. Curr. Opin. Struct. Biol. 1994; 4: 507-515Crossref Scopus (55) Google Scholar), steroidogenesis (4Swarnakar S. Reyland M.E. Deng J. Azhar S. Williams D.L. J. Biol. Chem. 1998; 273: 12140-12147Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), lipase activation (5Thuren T. Wilcox R.W. Sisson P. Waite M. J. Biol. Chem. 1991; 266: 4853-4861Abstract Full Text PDF PubMed Google Scholar), and platelet aggregation (6Riddell D.R. Graham A. Owen J.S. J. Biol. Chem. 1997; 272: 89-95Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). ApoE is a 299-amino acid, 34-kDa glycoprotein existing as one of three major isoforms that differ in the residues at positions 112 and 158. ApoE2 contains a cysteine at both sites; apoE3 (the most common isoform) has an arginine replacing the cysteine at 158; and apoE4 contains arginine at both 112 and 158. The intact protein is composed of two domains, a 10-kDa C-terminal domain with high lipid binding affinity and a 22-kDa N-terminal domain that is responsible for high affinity low density lipoprotein receptor binding (7Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). The x-ray crystal structure of the N-terminal domain (amino acids 1–191) in the absence of lipid reveals a globular bundle of four antiparallel, amphipathic α-helices (8Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (612) Google Scholar). This structure presents a hydrophilic exterior comprised of polar and charged amino acids that sequesters a hydrophobic core of apolar residues. Studies of the isolated N-terminal domain revealed that the helix bundle conformation is receptor-inactive, while lipid association confers low density lipoprotein receptor binding activity (9Innerarity T.L. Friedlander E.J. Rall Jr., S.C. Weisgraber K.H. Mahley R.W. J. Biol. Chem. 1983; 258: 12341-12347Abstract Full Text PDF PubMed Google Scholar). ApoE-N-terminal domain forms discoidal complexes with phospholipid, and these receptor-active particles provide a model system for studying the lipid-associated state (9Innerarity T.L. Friedlander E.J. Rall Jr., S.C. Weisgraber K.H. Mahley R.W. J. Biol. Chem. 1983; 258: 12341-12347Abstract Full Text PDF PubMed Google Scholar). Phospholipid binding is accompanied by little change in the global α-helix content of the protein (10Aggerbeck L.P. Wetterau J.R. Weisgraber K.H. Wu C.S. Lindgren F.T. J. Biol. Chem. 1988; 263: 6249-6258Abstract Full Text PDF PubMed Google Scholar, 11De Pauw M. Vanloo B. Weisgraber K. Rosseneu M. Biochemistry. 1995; 34: 10953-10960Crossref PubMed Scopus (54) Google Scholar, 12Raussens 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 (87) Google Scholar), suggesting that a tertiary structural alteration occurs in a manner such that helical boundaries that define the bundle are maintained. Studies of the surface properties of apoE N-terminal domain at the air/water interface indicate that the helix bundle can undergo a pronounced conformational change resulting in exposure of its hydrophobic interior (13Weisgraber K.H. Lund-Katz S. Phillips M.C. Miller N.E. Tall A.R. High Density Lipoproteins and Atherosclerosis III. Elsevier, Amsterdam1992: 175-181Google Scholar). Fourier transformed infrared spectroscopy of recombinant apoE3 (N-terminal residues 1–183) showed that the protein orients around the periphery of phospholipid bilayer discs with its α-helical axes perpendicular to the fatty acyl chains (12Raussens 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 (87) Google Scholar). Recent FRET studies of apoE3-(1–183) employing an extrinsic energy acceptor covalently bound to Cys112 revealed that, upon phospholipid binding, helix 1 repositions away from helix 3 (14Fisher C.A. Ryan R.O. J. Lipid Res. 1999; 40: 93-99Abstract Full Text Full Text PDF PubMed Google Scholar). This finding is consistent with a model in which the four-helix bundle opens about a “hinge” located between helices 2 and 3 such that helices 1 and 2 move away from helices 3 and 4 (7Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). The net effect of this putative conformational alteration is exposure of a continuous hydrophobic surface that is available for lipid interaction. In the present study, FRET analysis of apoE3-(1–183) variants containing single Trp residues introduced into different helical segments has permitted more refined mapping of the relative position of helices in apoE3-(1–183)·dimyristoylphosphatidylcholine (DMPC) disc complexes. Site-directed mutagenesis was performed using the Altered Sites II in vitro mutagenesis kit (Promega, Madison, WI). Wild type apoE3-(1–183) contains four Trp residues, each located within or adjacent to helix 1. To generate single Trp proteins, residues at positions 20, 26, and 34 were mutated to Phe, generating apoE3-(1–183)W@39. Subsequently, Trp39 was replaced with a Phe to create Trp-null apoE3-(1–183). Other single Trp mutants were created by replacing naturally occurring Tyr residues with Trp (at positions 74, 118, and 162). These mutations introduce Trp residues into helices 2, 3, and 4, respectively. DNA sequencing was performed to confirm introduction of the intended mutations and the lack of undesired mutations. Recombinant apoE3-(1–183) variants were overexpressed in E. coliBL21(DE3), purified as described previously (15Fisher 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 (43) Google Scholar), lyophilized, and stored at −20 °C. For labeling, 500 nmol of protein was solubilized in 50 mm Tris-HCl, pH 8.0, 0.1 mmdithiothreitol and incubated with 1 mm N-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS; Sigma) at 37 °C for 1 h in the dark. Unreacted AEDANS was removed by Sepharose CL-6B gel filtration chromatography. Covalent labeling of Cys112 in the apoE3-(1–183) variants was stoichiometric, as determined by electrospray mass spectrometry (15Fisher 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 (43) Google Scholar) and absorption spectroscopy using a Hewlett Packard 8453 spectrophotometer. Protein concentrations were determined using the bicinchoninic acid assay (Pierce). Protein dissolved in phosphate-buffered saline (150 mm NaCl, 3.4 mmEDTA, 100 mm sodium phosphate, pH 7.0) was added to a thin film of DMPC at a mass ratio of 1:3. Following incubation at 42 °C, the mixture was bath-sonicated until clear and further incubated for 16 h at 24 °C. Discoidal complexes were separated from unbound protein by Sepharose CL-6B size exclusion chromatography, and disc-containing fractions were pooled and concentrated to ∼ 1 ml. The lipid/protein molar ratio of the product particles was ∼150:1. Native PAGE analysis of DMPC discoidal complexes composed of wild type or Trp mutant apoE3-(1–183) revealed the complexes were of similar size, consistent with earlier flotation equilibrium and electron microscopy data (15Fisher 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 (43) Google Scholar). Prior to spectroscopic analysis, sample concentrations were adjusted to ∼0.05 OD at 295 nm. Fluorescence spectra were recorded on a Perkin-Elmer LS50 Luminescence Spectrometer using 3–5 nm for both excitation and emission slit widths at room temperature. For emission spectra, samples were excited at 295 nm. Excitation spectra were recorded from 265 to 400 nm using an emission wavelength of 490 nm. The efficiency of energy transfer (E) was calculated from changes in donor fluorescence as follows, E=1−QDAQDEquation 1 where Q D and Q DAare the quantum yield of the donor in the absence and presence of acceptor, respectively. The distance between energy donor and acceptor (R) is given by the following,R=R01E−116Equation 2 where R 0 is the distance at which the transfer efficiency is 50% such thatRo=9.765×103k2JQDn−416,Equation 3 where k 2 is the orientation factor of the donor and acceptor. The value of 23 fork 2 was used based on the assumption of random rotation of the fluorophores (16Selvin P.R. Methods Enzymol. 1995; 246: 300-334Crossref PubMed Scopus (513) Google Scholar); n is the refractive index of the medium between donor and acceptor (taken to be 1.4; Ref. 17Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1983: 303-339Crossref Google Scholar); and J is the spectral overlap integral, given by the following,J=∑IDɛAλ4Δλ∑IDΔλEquation 4 where I D is the fluorescence intensity of the donor, εA is the molar extinction coefficient of the acceptor, and λ is the wavelength in cm. For distance determinations of lipid-bound protein, R 0 was derived from discs that did not contain Trp-null protein, since the number of molecules present on the discs that were contributing to energy transfer could not be precisely determined. The N-terminal domain of wild type apoE3, with four Trp residues (energy donors) located in the region of helix 1, is amenable to FRET analysis. The single Cys at position 112 (helix 3) provides a convenient site for covalent attachment of an extrinsic energy acceptor. Problems associated with the use of wild type apoE3-(1–183), however, include the presence of multiple energy donors and the unknown contribution of intermolecular energy transfer in disc complexes that possess multiple copies of apoE3-(1–183). To circumvent these complications and to gain insight into the movement of other helices relative to the modified Cys in helix 3, naturally occurring Trp residues in apoE3-(1–183) were changed to Phe by site-directed mutagenesis. Subsequently, we created a Trp-null apoE3-(1–183) and single Tyr → Trp apoE3-(1–183) mutants for FRET studies (TableI). Far UV spectroscopy of revealed an α-helix content of N-terminal apoE3, that the mutations introduced did not the α-helix structure content of the properties of apoE3-(1–183) to a single Trp mutant apoE3-(1–183), and the number the amino acid position of the lone wavelength was 295 yield is based on a value of for mm tryptophan in not to a single Trp mutant apoE3-(1–183), and the number the amino acid position of the lone Excitation wavelength was 295 yield is based on a value of for mm tryptophan in not determined. in a The fluorescence emission 295 of revealed a single with a at nm spectra were for each of the single Trp mutants Trp-null apoE3-(1–183) did not to fluorescence emission upon excitation at 295 nm. Wild type apoE3-(1–183) a fluorescence emission λ of in with previously determined (10Aggerbeck L.P. Wetterau J.R. Weisgraber K.H. Wu C.S. Lindgren F.T. J. Biol. Chem. 1988; 263: 6249-6258Abstract Full Text PDF PubMed Google Scholar, 11De Pauw M. Vanloo B. Weisgraber K. Rosseneu M. Biochemistry. 1995; 34: 10953-10960Crossref PubMed Scopus (54) Google Scholar, 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 (43) Google Scholar). The wavelength of fluorescence emission for was with the other single Trp that naturally occurring Trp39 is more the single Trp variants created by of Tyr residues in the protein. with based on the x-ray crystal structure of the N-terminal of apoE3, Trp residues introduced at position (helix (helix or (helix was in Trp quantum yield from for to for apoE3-(1–183)W@39. This be to fluorescence as a of the relative exposure of the different Trp residues. Lipid association in an in Q and a nm in the Trp emission wavelength for and Following of the fluorescence properties of the different apoE3-(1–183) the were on cysteine 112 with by fluorescence using and revealed between and similar to that for apoE N-terminal domain J.R. L.P. Rall Jr., S.C. Weisgraber K.H. J. Biol. Chem. 1988; 263: Full Text PDF PubMed Google Scholar). we that of Cys112 with AEDANS the mutations introduced into the protein the four-helix bundle structure or In the of excitation at 295 nm a in Trp fluorescence quantum yield accompanied by the of a fluorescence around nm This to AEDANS emission energy transfer from excited The relative are upon the distance between the donor and acceptor in the folded protein as by the efficiency (E) of energy E derived from donor emission intensity for and phospholipid disc apoE3-(1–183) proteins. For each mutant DMPC in a in the efficiency of energy other in distance of energy transfer for apoE3-(1–183) single Trp are the of three and were determined as described discs were using and a given single Trp apoE3-(1–183) at a ratio in a of resonance energy as described are the of three as described are the of three 3 3 as described are the of three in a are the of three and were determined as described discs were using and a given single Trp apoE3-(1–183) at a ratio was bound to a in the efficiency of energy transfer as by the in Trp emission intensity with a pronounced in AEDANS emission intensity the other fluorescence spectra of discs with Trp-null Trp-null apoE3-(1–183) the lack of Trp in this and that excitation of AEDANS at this wavelength is In the of single Trp is that energy transfer occurs between located on different disc complexes energy To energy transfer to the disc complexes using were with a of discs using apoE3-(1–183). In this energy transfer was that energy transfer in this system is not since multiple copies of apoE3-(1–183) are bound is that intermolecular energy transfer could of FRET data and distance To for this Trp-null apoE3-(1–183) was used to single Trp apoE3-(1–183) variants The of with Trp-null apoE3-(1–183) a in the ratio of Trp to AEDANS fluorescence intensity that the of intermolecular energy transfer between this is In a similar spectra of or revealed in AEDANS fluorescence intensity that were similar to for apoE3-(1–183)W@39. these spectra were by with Trp-null apoE3-(1–183), consistent with earlier lipid of helix 1 and helix 3 (14Fisher C.A. Ryan R.O. J. Lipid Res. 1999; 40: 93-99Abstract Full Text Full Text PDF PubMed Google Scholar). In the of the data the conformation model helix 1 and 2 relative to helix The fluorescence of in the state revealed energy transfer In to with apoE3-(1–183) or mutants with single Trp residues at positions or 74, of with DMPC to discoidal lipid particles did not a change in Trp or AEDANS emission To the contribution of intermolecular energy transfer to the of were performed using Trp-null apoE3-(1–183). In this a in Trp fluorescence intensity was with a corresponding in AEDANS the other single Trp apoE3-(1–183) proteins, that DMPC binding in of to a where is available to an AEDANS located on a apoE3-(1–183) in the disc The energy transfer upon of AEDANS with Trp-null apoE3-(1–183) the distance between Cys112 and in from apoE3-(1–183) single Trp variants are in with from the x-ray crystal structure of this protein. For protein, however, not with from a conformational of the four-helix bundle (7Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). The value for apoE3-(1–183) was 4 that previously (14Fisher C.A. Ryan R.O. J. Lipid Res. 1999; 40: 93-99Abstract Full Text Full Text PDF PubMed Google Scholar). energy transfer with this protein the contribution of the four Trp residues in the vicinity of helix 1, is to is clear that with DMPC in an distance between helix 1 and helix three of the four Trp residues in apoE3-(1–183) were replaced by Phe to generate the distance between AEDANS and Trp39 upon with This however, is that on the of conformational of the helix bundle. other fluorescence data indicate that Trp39 is to its orientation relative to the AEDANS acceptor be be to distance derived from other variants with single Trp residues in helix 1. In the of DMPC binding in distance between and consistent with of the helix bundle. The change in distance between and the AEDANS on Cys112 upon with phospholipid with Trp-null apoE3-(1–183) suggests that helix 4 repositions relative to helix 3 upon with from FRET analysis of apoE3-(1–183) FRET determined using the II DMPC discs were as (15Fisher 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 (43) Google Scholar). R 0 used in distance was from discs in the absence of Trp-null FRET determined using the II in a DMPC discs were as (15Fisher 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 (43) Google Scholar). R 0 used in distance was from discs in the absence of Trp-null protein. To this intermolecular energy transfer was using discs containing Trp-null apoE3-(1–183) and a given apoE3-(1–183) single Trp mutant In this AEDANS can be excited by energy transfer from a Trp donor present on a apoE3-(1–183) in the disc AEDANS emission was from spectra of discs containing Trp-null apoE3-(1–183) and Trp-null apoE3-(1–183) AEDANS emission intensity from these discs was similar to that for complexes containing or that intermolecular energy transfer between single Trp residues on helix 2 or helix 3 of a given apoE3-(1–183) and AEDANS bound to Cys112 of Trp-null apoE3-(1–183) is disc complexes containing Trp-null apoE3-(1–183) and were however, energy transfer was that, upon with repositions to a where can as an energy donor to an AEDANS present on a apoE3-(1–183) In a similar energy transfer was between and Trp-null apoE3-(1–183). that, in DMPC apoE3-(1–183) can in such a that helix 3 and helix 4 of one are helix 4 and helix 3 of a apoE3-(1–183), respectively. the this not to be the for helix 1 or helix the data presented in this are consistent with an conformation model helix 3 from one with helix 4 of a each a partially extended conformation. This model is consistent with the that between helix 1 or 2 and helix 3 or of the bundle about the helix 2 and helix 3 not the other between residues in helix 3 and helix 4 in the bundle conformation (7Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). be however, that the present model of such could be replaced by similar or intermolecular helix 4 between apoE this conformational change not to be the most is consistent with data other including an arrangement of extended apoE study, including FRET analysis of other apoE variants be to or the model presented in The present model that each disc an number of apoE3-(1–183) of the number of apoE3-(1–183) disc as described by Pitas R.E. T.L. Mahley R.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar), based on an of nm and a lipid/protein molar ratio of a value of apoE3-(1–183) four apoE3-(1–183) these data to a of and of however, a of (15Fisher 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 (43) Google Scholar), that discs with to apoE3-(1–183). This is not with disc size by electron microscopy (15Fisher 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 (43) Google Scholar). of this model is the that presents a tandem arrangement of the receptor-binding region of the protein (residues 130–150 in helix the by arrangement by this this an for earlier that indicate with to apoE number and receptor binding activity J. Weisgraber K.H. Mahley R.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar). a with the presence of independently in the receptor S. 1997; PubMed Scopus Google Scholar). data on these that in to ligand binding S.C. Struct. Biol. PubMed Scopus Google Scholar, S.C. Biochemistry. 1999; Scopus Google Scholar). exists about the of receptor recognition by is that of charged amino acid chains in the protein its receptor recognition (7Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). Other data indicate that a tandem of receptor binding the affinity of the ligand for receptor For Pitas R.E. T.L. Mahley R.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar) showed that the number of receptor-active apoE DMPC disc is with binding is that the present studies were with the isolated receptor-binding domain of on available is not the 10-kDa lipid C-terminal domain of intact apoE be in of the present The that N- and C-terminal domain are to in this protein Weisgraber K.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) suggests that the presence of one domain can the structure of the and Kay for with UV

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