Lipid binding-induced conformational changes in the N-terminal domain of human apolipoprotein E

Abstract

The N-terminal domain of human apolipoprotein E3 (apoE3) adopts an elongated, globular four helix bundle conformation in the lipid-free state. Upon lipid binding, the protein is thought to undergo a significant conformational change that is essential for manifestation of its low density lipoprotein receptor recognition properties. We have used fluorescence resonance energy transfer (FRET) to characterize helix repositioning which accompanies lipid interaction of this protein. ApoE3(1–183) possesses a single cysteine at position 112 and four tryptophan residues (positions 20, 26, 34, and 39). Modification of Cys112 with the chromophore, N-iodoacetyl-N′-(5-sulfo-1-naphthyl)etheylenediamine (AEDANS) was specific and did not alter the secondary structure content of the protein. The efficiency of energy transfer from donor Trp residues to the AEDANS moiety was 49% in buffer, consistent with close proximity of the chromophores. Guanidine HCl titration experiments induced characteristic changes in the efficiency of energy transfer, indicating that FRET data faithfully reports on the conformational status of the protein. Interaction of AEDANS-apoE3(1–183) with dimyristoylphosphatidylcholine to form disk particles, or with detergent micelles, resulted in large decreases in the efficiency of energy transfer. Distance calculations based on the FRET measurements revealed that lipid binding increases the average distance between the four Trp donors and the AEDANS acceptor from 23 Å to 44 Å. The results obtained demonstrate the utility of FRET to investigate conformational adaptations of exchangeable apolipoproteins and are consistent with the hypothesis that, upon lipid binding, apoE3(1–183) undergoes conformational opening, repositioning helix 1 and 3 to adopt a receptor-active conformation.—Fisher, C. A., and R. O. Ryan. Lipid binding-induced conformational changes in the N-terminal domain of human apolipoprotein E. J. Lipid Res. 1999. 40: 93–99. The N-terminal domain of human apolipoprotein E3 (apoE3) adopts an elongated, globular four helix bundle conformation in the lipid-free state. Upon lipid binding, the protein is thought to undergo a significant conformational change that is essential for manifestation of its low density lipoprotein receptor recognition properties. We have used fluorescence resonance energy transfer (FRET) to characterize helix repositioning which accompanies lipid interaction of this protein. ApoE3(1–183) possesses a single cysteine at position 112 and four tryptophan residues (positions 20, 26, 34, and 39). Modification of Cys112 with the chromophore, N-iodoacetyl-N′-(5-sulfo-1-naphthyl)etheylenediamine (AEDANS) was specific and did not alter the secondary structure content of the protein. The efficiency of energy transfer from donor Trp residues to the AEDANS moiety was 49% in buffer, consistent with close proximity of the chromophores. Guanidine HCl titration experiments induced characteristic changes in the efficiency of energy transfer, indicating that FRET data faithfully reports on the conformational status of the protein. Interaction of AEDANS-apoE3(1–183) with dimyristoylphosphatidylcholine to form disk particles, or with detergent micelles, resulted in large decreases in the efficiency of energy transfer. Distance calculations based on the FRET measurements revealed that lipid binding increases the average distance between the four Trp donors and the AEDANS acceptor from 23 Å to 44 Å. The results obtained demonstrate the utility of FRET to investigate conformational adaptations of exchangeable apolipoproteins and are consistent with the hypothesis that, upon lipid binding, apoE3(1–183) undergoes conformational opening, repositioning helix 1 and 3 to adopt a receptor-active conformation. —Fisher, C. A., and R. O. Ryan. Lipid binding-induced conformational changes in the N-terminal domain of human apolipoprotein E. J. Lipid Res. 1999. 40: 93–99. Human apolipoprotein E (apoE) is a 34 kDa, 299 amino acid exchangeable apolipoprotein which is a key component of triacylglycerol-rich lipoproteins. ApoE, which exists as one of three predominant isoforms, E2, E3 or E4 (1Weisgraber K.H. Apolipoprotein E: structure–function relationships.Adv. Protein Chem. 1994; 45: 249-302Google Scholar), has been shown to function in lipoprotein metabolism, Alzheimer's disease, and nerve regeneration (2Mahley R.W. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology.Science. 1988; 240: 622-630Google Scholar, 3Weisgraber K.H. Mahley R.W. Human apolipoprotein E: the Alzheimer's disease connection.FASEB J. 1996; 10: 1485-1494Google Scholar). There is extensive evidence that apoE plays a key role in facilitating clearance of lipoprotein particles from plasma via its ability to bind cell surface receptors including the low density lipoprotein (LDL) receptor (2Mahley R.W. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology.Science. 1988; 240: 622-630Google Scholar) and the LDL receptor-related protein (4Kowal R.C. Herz J. Goldstein J.L. Esser V. Brown M.S. Low density lipoprotein receptor-related protein mediates uptake of cholesteryl esters derived from apoprotein E–enriched lipoproteins.Proc. Natl. Acad. Sci. USA. 1989; 86: 5810-5814Google Scholar). Structural studies of apoE (5Aggerbeck L.P. Wetterau J.R. Weisgraber K.H. Wu C-S.C. Lindgren F.T. Human apolipoprotein E3 in aqueous solution II. Properties of the amino- and caboxyl-terminal domains.J. Biol. Chem. 1988; 263: 6249-6258Google Scholar, 6Wetterau J.R. Aggerbeck L.P. Rall Jr., S.C. Weisgraber K.H. Human apolipoprotein E3 in aqueous solution I. Evidence for two structural domains.J. Biol.Chem. 1988; 263: 6240-6248Google Scholar) revealed it is comprised of two independently folded protein domains that can be isolated after treatment of the protein with thrombin. The 22 kDa N-terminal domain (residues 1–191) adopts a water-soluble, monomeric globular conformation that is resistant to denaturation. By contrast, the 10 kDa C-terminal domain (residues 216–299) is less stable to denaturation, has a high lipid binding affinity, and is responsible for self association of apoE in the absence of lipid. In terms of binding to the LDL receptor, several lines of evidence have led to a consensus which localizes the receptor binding region of apoE to the N-terminal domain, specifically between residues 130 and 150 (1Weisgraber K.H. Apolipoprotein E: structure–function relationships.Adv. Protein Chem. 1994; 45: 249-302Google Scholar). This region of the protein is rich in basic amino acids and their proposed role in receptor interactions is consistent with studies demonstrating loss of receptor binding after chemical modification of lysine and arginine residues (7Mahley R.W. Innerarity T.L. Pitas R.E. Weisgraber K.H. Brown J.H. Gross E. Inhibition of lipoprotein binding to cell surface receptors of fibroblasts following selective modification of arginyl residues in arginine-rich and B apoproteins.J. Biol.Chem. 1977; 252: 7279-7287Google Scholar, 8Weisgraber K.H. Innerarity T.L. Mahley R.W. Role of lysine residues in plasma lipoproteins in high affinity binding to cell surface receptors on human fibroblasts.J. Biol. Chem. 1978; 253: 9053-9062Google Scholar). In the absence of lipid, the isolated N-terminal domain is not recognized by the LDL receptor. However, complexation with phospholipids to form discoidal structures results in a particle that binds efficiently to the LDL receptor (9Innerarity T.L. Friedlander B.J. Rall Jr., S.C. Weisgraber K.H. Mahley R.W. The receptor binding domain of human apolipoprotein E. Binding of apolipoprotein E fragments.J. Biol. Chem. 1983; 258: 12341-12347Google Scholar). This behavior is similar to that observed for full-length apoE, which binds to LDL receptors on fibroblasts only after complexation with lipid (10Innerarity T.L. Pitas R.E. Mahley R.W. Binding of arginine rich (E) apolipoprotein after recombination with phospholipid vesicles to low density lipoprotein receptors of fibroblasts.J. Biol. Chem. 1979; 254: 4186-4190Google Scholar). These data support the view that a lipid binding-induced conformational adaptation of apoE, which can be effectively mimicked by the isolated N-terminal domain, is an essential feature of apoE function as a ligand for receptor-mediated endocytosis of plasma lipoproteins. In 1991, Wilson et al. (11Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Three dimensional structure of the LDL receptor-binding domain of human apolipoprotein E.Science. 1991; 252: 1817-1822Google Scholar) reported the X-ray crystal structure of the 22 kDa N-terminal domain of human apoE3 in the lipid-free state. This structure, which is comprised of a bundle of four elongated amphipathic α-helices, bears a remarkable similarity in molecular architecture to full length apolipophorin III (apoLp-III) from Locusta migratoria (12Breiter D.R. Kanost M.R. Benning M.M. Wesenberg G. Law J.H. Wells M.A. Rayment I. Holden H.M. Molecular structure of an apolipoprotein determined at 2.5 Å resolution.Biochemistry. 1991; 30: 603-608Google Scholar) and Manduca sexta (13Wang J. Gagne S. Sykes B.D. Ryan R.O. Insight into lipid surface recognition and reversible conformational adaptations of an exchangeable apolipoprotein by multidimensional heteronuclear NMR techniques.J. Biol. Chem. 1997; 272: 17912-17920Google Scholar). Structural information on these proteins in the absence of lipid has led to the concept that reversible lipid binding is accompanied by a significant conformational change. A model depicting helix bundle apolipoprotein conformational opening is presented in Fig. 1. In this model, proposed by Weisgraber (1Weisgraber K.H. Apolipoprotein E: structure–function relationships.Adv. Protein Chem. 1994; 45: 249-302Google Scholar), the N-terminal domain exposes its hydrophobic interior by ‘opening’ via a putative hinge region located in the loop between helix 2 and helix 3. Inherent in this model is the prediction that helix 1 and 2 move away from helix 3 and 4 as the bundle ‘opens’. Evidence in support of a major conformational change upon lipid binding has been obtained from surface area measurements at the air–water interface which indicate the protein occupies more area per amino acid than can be accounted for if the protein retained its globular structure (14Weisgraber K.H. Lund-Katz S. Phillips M.C. Apolipoprotein E: stucture–function correlations.in: Miller N.E. Tall A.R. High Density Lipoproteins and Atherosclerosis. III. Elsevier, Amsterdam1992: 175-181Google Scholar). In the present study we have used fluorescence resonance energy transfer (FRET; see ref. 15Selvin P.R. Fluorescence resonance energy transfer.Methods Enzymol. 1995; 246: 300-334Google Scholar for a review) to characterize helical repositioning that accompanies lipid binding of this protein domain and compared the data obtained with that predicted by the open conformation model. Recombinant apoE3(1–183) was expressed and purified as described by Fisher et al. (16Fisher C.A. Wang J. Sykes B.D. Kay C.M. Francis G.A. Ryan R.O. Bacterial expression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.Biochem. Cell Biol. 1997; 75: 45-53Google Scholar). Isolated protein was stored lyophilized at −20°C. Five hundred nmoles protein was incubated in 50 mm Tris-HCl, pH 8.0, with 0.1 mm dithiothreitol and 500 nmol N-iodoacetyl-N′-(5-sulfo-1-naphthyl)etheylenediamine (AEDANS; Molecular Probes, Eugene, OR) at 37°C, 2.5 h in the dark. Unreacted AEDANS was separated from labeled protein by passage through a Sepharose CL-6B (Pharmacia) column followed by lyophilization. Incorporation of extrinsic fluorophore was confirmed by electro-spray mass spectrometry and/or absorption spectroscopy. That is: A336ε×MWprotein[protein]=moles fluorophoremoles proteinwhere A is the absorbance and ϵ is the molar extinction coefficient of the fluorophore. The labeled apoE sample displayed characteristic fluorescence properties of AEDANS, including an excitation of and an of FRET protein at and from to on a for excitation and and AEDANS-apoE3(1–183) in 50 mm pH at to to to for 10 from protein and from for was incubated with the of or detergent for at to as described (16Fisher C.A. Wang J. Sykes B.D. Kay C.M. Francis G.A. Ryan R.O. Bacterial expression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.Biochem. Cell Biol. 1997; 75: 45-53Google Scholar). The efficiency (E) of energy transfer was as and is the of the donor in the absence and of The distance between energy donor and acceptor is is the distance at which the transfer efficiency is is the of the donor and In the present the of was used on the of the of of the E. D.R. Fluorescence energy transfer analysis of Scholar) and is the of the between donor and and was to be J.R. of Fluorescence Scholar). is the is the fluorescence of the is the molar extinction coefficient of the and is the in as described R.O. Kay C.M. and properties of Manduca sexta apolipophorin Biol. Chem. Scholar), was used to protein in and HCl titration mass spectrometry was as described by Fisher et al. (16Fisher C.A. Wang J. Sykes B.D. Kay C.M. Francis G.A. Ryan R.O. Bacterial expression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.Biochem. Cell Biol. 1997; 75: 45-53Google Scholar). Recombinant apoE3(1–183) was labeled with AEDANS on Cys112 as described in and of chromophore, the labeled protein was to electro-spray mass The revealed a single mass to apoE3(1–183) 1 AEDANS with evidence for of more than one AEDANS per absorption the of was determined to be between and revealed that the labeled protein its secondary structure Fluorescence of apoE3(1–183) and in are shown in Fig. The protein sample to a single as reported by Aggerbeck et al. (5Aggerbeck L.P. Wetterau J.R. Weisgraber K.H. Wu C-S.C. Lindgren F.T. Human apolipoprotein E3 in aqueous solution II. Properties of the amino- and caboxyl-terminal domains.J. Biol. Chem. 1988; 263: 6249-6258Google Scholar). In the of the Trp fluorescence was in and a of was observed This with the properties of AEDANS, absorbance with the of The of an at with the large in Trp at that the AEDANS moiety has been by energy transfer from Trp residues in helix 1. The of the changes observed upon AEDANS energy transfer from the donor Trp residues to AEDANS in the helix bundle conformation. the of the AEDANS was by it can be that energy transfer not to the results based on the observed energy transfer between Trp and AEDANS in the protein are reported in 1 and distance between the donor Trp residues and the acceptor AEDANS moiety of 23 2 for resonance energy transfer in lipid-free and AEDANS labeled transfer E as described in and at are the average of three is based on a of for tryptophan in as described in and in a are the average of three is based on a of for tryptophan in AEDANS protein the of HCl on energy transfer from Trp to AEDANS was HCl to 2 was a in the of the and a in the These data that, in 2 apoE3(1–183) its globular protein consistent with the of J.R. Aggerbeck L.P. Rall Jr., S.C. Weisgraber K.H. Human apolipoprotein E3 in aqueous solution I. Evidence for two structural domains.J. Biol.Chem. 1988; 263: 6240-6248Google Scholar). 2.5 HCl and a significant in the of the AEDANS was is a less in the of the Trp In terms of the Trp fluorescence at HCl is a in the indicating that the Trp residues are more to as a function of HCl the that Trp fluorescence is at 2.5 of Trp fluorescence at HCl A similar behavior was with protein upon titration with HCl not The large in energy transfer observed between 2 and 2.5 HCl with data on the N-terminal domain of apoE3 reported by J.R. Aggerbeck L.P. Rall Jr., S.C. Weisgraber K.H. Human apolipoprotein E3 in aqueous solution I. Evidence for two structural domains.J. Biol.Chem. 1988; 263: 6240-6248Google Scholar) and support for the that AEDANS modification did not alter the properties of the protein. These data for the concept that the efficiency of energy transfer in is to the folded of the protein and that changes in structure or conformation are by changes in energy transfer. In studies as the similar results In studies of helix bundle exchangeable apolipoprotein conformation V. Kay C.M. Ryan R.O. Structural and binding of the of apolipophorin III from Manduca 1994; Scholar), we that of as induced a conformational change similar to that upon lipid This was to be to the ability of to a lipid with hydrophobic of the protein by in the bundle conformation. The of on the efficiency of energy transfer in is shown in Fig. an in energy transfer was compared to as by an in AEDANS with a in Trp fluorescence on this information it be that, in of the protein structure effectively the distance between the the efficiency of energy transfer. This structural from the ability of to secondary structure in including apoE3(1–183) (16Fisher C.A. Wang J. Sykes B.D. Kay C.M. Francis G.A. Ryan R.O. Bacterial expression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.Biochem. Cell Biol. 1997; 75: 45-53Google Scholar). However, in the AEDANS moiety was a significant of the AEDANS was observed not A of tryptophan fluorescence was not in the of which a in tryptophan fluorescence as a function of we that the in the AEDANS observed in be to of helix 3 of helix the AEDANS moiety the hydrophobic interior of the protein. This is by the in AEDANS fluorescence in a in energy transfer can be by the in AEDANS fluorescence with an in the tryptophan fluorescence that in and a significant in the AEDANS is observed that is not accompanied by a in Trp fluorescence The experiments described evidence that FRET measurements can information the between helix 1 and helix 3 in the of apoE N-terminal domain conformational opening it can be predicted that an opening in a efficiency of energy transfer between Trp residues in helix 1 and the AEDANS moiety on helix 3. of energy transfer efficiency in the lipid-free globular conformation and that the protein is to disk of and a significant change in the of the donor and acceptor chromophores. of disk displayed fluorescence in the region of AEDANS was a significant change in tryptophan fluorescence upon binding to The average distance between tryptophan residues in helix 1 and the AEDANS moiety in that in as as that predicted on the of a conformational opening of the helix are reported in that possesses of a of the AEDANS fluorescence in from or energy transfer. the to which energy transfer to the results for lipid experiments detergent as a model lipid a of 1 apoE3(1–183) is per The results obtained a similar less energy transfer apoE3(1–183) was to or measurements derived from FRET analysis of AEDANS labeled apoE3(1–183) in the and absence of FRET distance between as per of Wilson et al. as per Fisher et al. from the in the in Fig. distance calculations determined the FRET distance between as per of Wilson et al. (11Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Three dimensional structure of the LDL receptor-binding domain of human apolipoprotein E.Science. 1991; 252: 1817-1822Google as per Fisher et al. (16Fisher C.A. Wang J. Sykes B.D. Kay C.M. Francis G.A. Ryan R.O. Bacterial expression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.Biochem. Cell Biol. 1997; 75: 45-53Google from the in the in Fig. distance calculations determined the in a Fig. of detergent interaction on the efficiency of energy transfer in of 2 in 50 mm pH in and in 1 mm plays a role in plasma cholesterol it as a ligand for cell surface lipoprotein The receptor binding of apoE has been to residues in the N-terminal domain of the protein (1Weisgraber K.H. Apolipoprotein E: structure–function relationships.Adv. Protein Chem. 1994; 45: 249-302Google Scholar). the receptor recognition properties of this protein are only in a state. The of structural information in the lipid-free (11Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Three dimensional structure of the LDL receptor-binding domain of human apolipoprotein E.Science. 1991; 252: 1817-1822Google Scholar) has for the of lipid binding-induced conformational In the present study we have used FRET to investigate helix repositioning of the N-terminal domain bundle upon lipid evidence in of a conformational of the ApoE3(1–183) four tryptophan residues (positions 20, and a single cysteine at position the X-ray structure of this protein it is that the four tryptophan residues are helix 1 (residues 23 to of the bundle Cys112 is located in the of helix 3. residues can function as energy donors Cys112 a for specific of an extrinsic energy the of it is recognized that Cys112 is for modification K.H. Apolipoprotein E human plasma role of at position Lipid Res. Scholar). In the present study we the of Cys112 with AEDANS, a which has been used in FRET studies J.H. Fluorescence energy transfer distance measurements single cysteine the of Biol. 1991; Scholar, J.H. Fluorescence energy transfer distance The hydrophobic helical of A in the Biol. Scholar, A.R. The of structural changes fluorescence resonance energy 1994; Scholar, V. C. of fluorescence energy transfer to in the 1995; Scholar). AEDANS as a energy acceptor in this its absorption with the of The present is the of tryptophan and cysteine in the folded protein are from the crystal structure, an for FRET studies of lipid binding-induced conformational from the X-ray the average distance from the donor to the acceptor is in with the of 23 Å derived from FRET the helix bundle conformation is monomeric in which to FRET (5Aggerbeck L.P. Wetterau J.R. Weisgraber K.H. Wu C-S.C. Lindgren F.T. Human apolipoprotein E3 in aqueous solution II. Properties of the amino- and caboxyl-terminal domains.J. Biol. Chem. 1988; 263: 6249-6258Google Scholar) 1 a model of a putative of apoE3 N-terminal domain helix 1 and 2 move away from helix 3 and 4 by a hinge region located in the loop between helix 2 and 3. an opening of the in of the hydrophobic of the amphipathic which are in the interior of the bundle conformation. These hydrophobic are thought to with lipid a binding model, presented by et al. Weisgraber K.H. of lipid binding and of the amino- and domain of human apolipoprotein 1995; Scholar), that interaction with to form the helical in apoE to which to the phospholipid the of an to be and in helix and the average distance between Trp residues in helix 1 and Cys112 Å. the FRET data reported not this model, this molecular is not consistent with data on V. Fisher C.A. E. Ryan R.O. The low density lipoprotein receptor conformation of apolipoprotein E. in N-terminal Biol. Chem. Scholar) or the predicted that of helix 4 (residues into two have on the position of The of this to the LDL receptor binding region (residues has been shown to be an of in specific receptor binding properties S. Innerarity T.L. Weisgraber K.H. for receptor binding of apolipoprotein in III Biol. 1996; Scholar). Guanidine HCl titration experiments revealed this changes in the efficiency of energy transfer with the properties of this protein J.R. Aggerbeck L.P. Rall Jr., S.C. Weisgraber K.H. Human apolipoprotein E3 in aqueous solution I. Evidence for two structural domains.J. Biol.Chem. 1988; 263: 6240-6248Google Scholar). the folded structure displayed energy transfer, resulted in a in energy transfer it can be that AEDANS modification of apoE3(1–183) not alter the properties of the protein. from changes in energy transfer observed upon protein it is that changes in the conformation of apoE3(1–183) be in FRET from experiments as a energy transfer observed as a function of are consistent with the hypothesis that, conformational opening of the helix bundle through of with In this hydrophobic between in the bundle conformation be with the protein to its globular in the of or HCl by the that tryptophan fluorescence is the from to induced a large in AEDANS this was not accompanied by a in tryptophan fluorescence to a by of Trp By contrast, the of AEDANS fluorescence in was between and excitation at between and distance measurements these into changes in donor and/or acceptor fluorescence which have a on energy transfer. with the fluorescence of tryptophan in apoE3(1–183) was not However, displayed energy transfer compared to the lipid-free in an in the distance between the energy donor and the AEDANS moiety from 23 Å to 44 Å. the secondary structure content of the protein was not by binding, we that interaction of apoE3(1–183) with lipid a conformational change that increases the distance between helix 1 and helix 3. these data are consistent with the open conformation model presented in Fig. a opening of the helix bundle be to a distance between donor and acceptor in this protein experiments be to the of the conformational change and to of in the distance that to the observed results in the of the donor or acceptor in the the of a energy donor four Trp in the or of the of Trp residues from an aqueous to a to the we have to the of in the present the energy donor is by the of the four tryptophan in measurements in the AEDANS that the energy transfer of the tryptophan The by the of a energy donor in this can be by of tryptophan residues in apoE3(1–183) to a protein with a single energy donor In this can be in distance this we have with a by In buffer, an energy transfer efficiency compared to consistent with the that, in the bundle crystal structure, from the of AEDANS modification 112 in helix These which the of FRET measurements to in the donor in this that of a single tryptophan apoE3(1–183) distance measurements to be the of a apoE3(1–183) can be used to apoE3(1–183) an for energy transfer in the of and lipoprotein that a tryptophan the cysteine on in the bundle of helix repositioning which accompanies lipid interaction of a The of FRET to the of amphipathic exchangeable apolipoproteins a to study these proteins in their state. The C. Kay and for for with Fig. 1 and V. for of the and is the of a and is a of the for This was by a from the and of apolipoprotein dimyristoylphosphatidylcholine fluorescence resonance energy transfer