Replacement of helix 1¢ enhances the lipid binding activity of apoE3 N-terminal domain Katherine A. Redmond 1 , Conrad Murphy 1 , Vasanthy Narayanaswami 1 , Robert S. Kiss 2 , Paul Hauser 1 , Emmanuel Guigard 3 , Cyril M. Kay 3 and Robert O. Ryan 1 1 Lipid Biology in Health and Disease Research Group, Children’s Hospital Oakland Research Institute, CA, USA 2 Lipoprotein and Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa, Ontario, Canada 3 Department of Biochemistry and Protein Engineering Network of Centres of Excellence, University of Alberta, Edmonton, Canada Human apolipoprotein E (apoE) is comprised of two structural domains, a 22 kDa N-terminal (NT) domain and a 10 kDa C-terminal (CT) domain [1,2]. Studies conducted with isolated domains reveal that the NT domain contains amino acids responsible for binding to members of the low-density lipoprotein receptor (LDLR) family [3]. Several lines of evidence have led to a consensus that localizes the receptor binding region to residues 134–150 [4]. In the absence of lipid, however, the isolated NT domain is not recognized by Keywords apolipoprotein E; fluorescence spectroscopy; low-density lipoprotein; low- density lipoprotein receptor; phospholipids Correspondence R. O. Ryan, Children’s Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA Fax: +1 510 450 7910 Tel: +1 510 450 7645 E-mail: rryan@chori.org (Received 3 November 2005, revised 1 December 2005, accepted 5 December 2005) doi:10.1111/j.1742-4658.2005.05089.x The N-terminal domain of human apolipoprotein E (apoE-NT) harbors residues critical for interaction with members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid free apoE-NT adopts a stable four- helix bundle conformation, a lipid binding induced conformational adapta- tion is required for manifestation of LDLR binding ability. To investigate the structural basis for this conformational change, the short helix connect- ing helix 1 and 2 in the four-helix bundle was replaced by the sequence NPNG, introducing a b-turn. Recombinant helix-to-turn (HT) variant apoE3-NT was produced in Escherichia coli, isolated and characterized. Stability studies revealed a denaturation transition midpoint of 1.9 m guanidine hydrochloride for HT apoE3-NT vs. 2.5 m for wild-type apoE3- NT. Wild-type and HT apoE3-NT form dimers in solution via an intermolecular disulfide bond. Native PAGE showed that reconstituted high-density lipoprotein prepared with HT apoE3-NT have a diameter in the range of 9 nm and possess binding activity for the LDLR on cultured human skin fibroblasts. In phospholipid vesicle solubilization assays, HT apoE3-NT was more effective than wild-type apoE3-NT at inducing a time dependent decrease in dimyristoylphosphatidylglycerol vesicle light scatter- ing intensity. In lipoprotein binding assays, HT apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent that wild-type apoE3-NT. The results indicate that a mutation at one end of the apoE3-NT four-helix bundle markedly enhan- ces the lipid binding activity of this protein. In the context of lipoprotein associated full-length apoE, increased lipid binding affinity of the N-ter- minal domain may alter the balance between receptor-active and -inactive conformational states. Abbreviations ANS, 8-anilino-1-naphthalene sulfonate; apo, apolipoprotein; CT, carboxy (C) terminal; DMPC, dimyristoylphosphatidylcholine; DMPG, dimyristoylphosphatidylglycerol; FAFA, fatty acid free albumin; HDL, high-density lipoprotein; HT, helix-to-turn; LDL, low-density lipoprotein; LDLR, low-density lipoprotein receptor; NT, amino (N) terminal; PL-C, phospholipase C; rHDL, reconstituted high density lipoprotein; WT, wild-type. 558 FEBS Journal 273 (2006) 558–567 ª 2006 The Authors Journal compilation ª 2006 FEBS the LDLR. On the other hand, upon association with lipid, apoE-NT binds efficiently to the LDLR [3]. X-ray crystallography of apoE3-NT has yielded a high-resolution structure [5,6]. In the absence of lipid, this domain exists as an elongated globular four-helix bundle. In this conformation the amphipathic a-helices orient their hydrophobic faces toward the center of the bundle. At the same time, polar faces of individual helical segments interact with the aqueous environ- ment. The interior of the bundle contains several leu- cine residues that align to form a leucine zipper-like motif. In addition to the four helices that comprise the bundle, apoE3-NT possesses a short helix, termed helix 1¢, that connects helix 1 and 2. Despite the fact that the residues comprising this helix are highly conserved across species, the structural or functional role of this region of the protein remains unknown. Weisgraber [4] proposed an open conformation model in which the loop segment connecting helix 2 and 3 in the bundle functions as a hinge about which the protein opens to expose its hydrophobic interior. According to this model, helix 1¢ resides in a location where it could play a role in lipid surface recognition and ⁄ or initiation of lipid binding. Moreover, it is conceivable that helix 1¢ undergoes a lipid dependent conformational change that increases exposure of the hydrophobic interior of the bundle, perhaps functioning in a manner similar to the lid segment of lipases [7]. Studies of the lipid interaction properties of apoE3-NT have been facilitated by its ability to transform dimyristoylphosphatidylcholine (DMPC) bilayer vesicles into discrete disc complexes [3]. Raussens et al. [8] investigated the structural organiza- tion of apoE3-NT in DMPC complexes by infrared spectroscopy. These authors presented a model wherein the NT domain adopts an open conformation, circum- scribing the perimeter of the disc bilayer with its major helical axes aligned perpendicular to the fatty acyl chains of DMPC. Support for this molecular orientation has come from studies employing fluorescence reson- ance energy transfer to evaluate distance relationships between specific sites in the protein as a function of lipid binding [9,10]. Also, Lu et al. [11] reported that apoE- NT dependent transformation of DMPC bilayer vesicles into disc complexes is abolished when helical segments in the bundle are tethered by disulfide bond engineering. An important consideration with regard to the lipid interaction properties of apoE3-NT relates to the high intrinsic stability of this domain. Denaturation studies revealed that the NT domain is exceptionally stable compared to either the C-terminal domain or other apolipoproteins [1]. Likewise, lipoprotein binding stud- ies showed that the isolated NT domain has a low affinity for lipoprotein particles [12]. Several studies have investigated the molecular basis of this property with the general finding that various external factors including solution pH, ionic strength or the presence of chaotropic agents, modulates the lipid binding activ- ity of apoE3-NT [13,14]. In the present study we employed a protein engineering approach, removing helix 1¢ and replacing it with a sequence predicted to adopt a b-turn. The resulting variant protein displays a marked enhancement in lipid binding activity. Results Design of helix-to-turn apoE3-NT On the basis of studies with structurally related helix bundle apolipoproteins [15,16], we hypothesized that residues comprising a short connector helix in apoE3- NT function in recognition or initiation of lipid bind- ing, leading to helix bundle opening and formation of a stable binding interaction. Using X-ray structure data [5] as a guide, residues comprising helix 1¢ [SE- QVQEELLS(44–53)] were deleted and replaced by a sequence predicted to adopt a b-turn, NPNG [17]. We hypothesized that the resulting helix-to-turn (HT) vari- ant apoE3-NT would adopt a stable solution confor- mation that retains the protein’s four-helix bundle molecular architecture and LDLR binding capability, yet would display altered lipid binding activity. A rib- bon diagram of apoE3-NT depicting the introduced change is shown in Fig. 1. Bacterial expression and characterization of HT apoE3-NT Wild-type (WT) and HT apoE3-NT were isolated from the supernatant fraction of bacteria cultures as described by Fisher et al. [18]. SDS ⁄ PAGE analysis under reducing conditions revealed that the HT apoE3-NT has a faster mobility than wild-type apoE3-NT, consistent with the changes introduced into the amino acid sequence (Fig. 2). Mass spectro- metry of HT apoE3-NT gave rise to a monomer mass ¼ 20 310 Da (calculated mass ¼ 20 318 Da) versus 21 191 Da for WT apoE3-NT. SDS ⁄ PAGE under nonreducing conditions revealed the presence of disulfide linked homo-dimers. ApoE3-NT contains a single cysteine residue (at position 112) that is known to form disulfide bonds [19]. The data pre- sented indicate that WT and HT apoE3-NT exist in solution as a mixture of monomers and disulfide linked homo-dimers. This finding was corroborated by sedimentation equilibrium experiments conducted in the analytical ultracentrifuge, wherein evidence for K. A. Redmond et al. apoE3-NT domain lipid binding FEBS Journal 273 (2006) 558–567 ª 2006 The Authors Journal compilation ª 2006 FEBS 559 the presence of monomers and dimers was obtained for both WT and HT apoE3-NT. Stability properties of HT apoE3-NT The effect of guanidine hydrochloride concentration on the tryptophan fluorescence emission properties of WT and HT apoE3-NT was monitored by fluorescence spectroscopy. In the absence of guanidine hydrochlo- ride, WT apoE3-NT gave rise to a tryptophan fluo- rescence emission wavelength maximum of 346 nm (excitation 280 nm) while the corresponding value for HT apoE3-NT was 348 nm. Upon exposure to high concentrations of guanidine hydrochloride, both pro- teins displayed an  8 nm red shift in wavelength of tryptophan fluorescence emission maximum. Plots of guanidine hydrochloride concentration vs. the percent maximal change in Trp fluorescence emission wave- length maximum (Fig. 3) revealed a transition mid- point at 2.5 m for WT apoE3-NT and a corresponding transition at 1.9 m guanidine hydrochloride for HT apoE3-NT. Fluorescent dye binding To evaluate the extent to which HT apoE3-NT mani- fests altered exposure of hydrophobic sites in the pro- tein, the effect of HT apoE3-NT and WT apoE3-NT on the fluorescence emission intensity of 8-anilino-1- naphthalene sulfonate (ANS) was examined (Fig. 4). In the absence of protein, ANS has a low quantum yield with an emission wavelength maximum of 515 nm (excitation 395 nm). Introduction of WT apoE3-NT induced a 35 nm blue shift in ANS fluores- cence emission wavelength maximum together with an 1234 Fig. 2. SDS ⁄ PAGE analysis of apolipoproteins. Proteins were elec- trophoresed on a 4–20% (w ⁄ v) acrylamide gradient SDS slab gel under reducing (lanes 1 and 2) or nonreducing (lanes 3 and 4) con- ditions and was stained with Coomassie Blue. Lanes 1 and 3, WT apoE3-NT; lanes 2 and 4, HT apoE3-NT. Fig. 3. Effect of guanidine hydrochloride on apoE3-NT tryptophan fluorescence emission. Indicated amounts of guanidine hydrochlo- ride were added to WT apoE3-NT and HT apoE3-NT in buffer (20 m M sodium phosphate, pH 7.4) and, at each concentration, the wavelength of maximum fluorescence emission (excitation 280 nm) was determined. d, WT apoE3-NT; s, HT apoE3-NT. Fig. 1. Ribbon diagram depicting WT apoE3-NT and HT apoE3-NT. Arrows indicate the position of cysteine 112. The figure was pre- pared using PDB coordinates (code 1lpe) and the PYMOL program (http://www.pymol.org). apoE3-NT domain lipid binding K. A. Redmond et al. 560 FEBS Journal 273 (2006) 558–567 ª 2006 The Authors Journal compilation ª 2006 FEBS enhancement in quantum yield. HT apoE3-NT induced a similar blue shift in ANS fluorescence emission wave- length maximum as well as a greater enhancement in quantum yield. Given that these incubations contained equivalent amounts of apolipoprotein, the data indi- cate that HT apoE3-NT possesses more ANS access- ible hydrophobic binding sites than WT apoE3-NT. When examined under reducing conditions, the same trend in ANS accessibility between WT and HT apoE3-NT was observed. LDLR binding activity of the HT apoE3-NT To evaluate the ability of WT and HT apoE3-NT to serve as ligands for the LDLR on cultured human skin fibroblasts, lipid associated apolipoproteins were employed. HT and WT apoE3-NT were complexed with DMPC and the resulting particles characterized by native gradient PAGE and tryptophan fluorescence quenching. The reconstituted high-density lipoproteins (rHDL) generated with WT or HT apoE3-NT migra- ted as homogeneous populations of particles with diameters in the range of 9 nm. Likewise, potassium iodide quenching studies of the four tryptophan resi- dues in apoE3-NT gave rise to Stern–Volmer quench- ing constant (Ksv) values of 4.62 m )1 ± 0.1 and 4.77 m )1 ± 0.7 for WT and HT apoE3-NT rHDL, respectively, indicating similar solvent exposure of Trp residues in these lipid particles. Human skin fibroblasts were grown to confluence in lipoprotein deficient serum, transferred to 4 °C and incubated with 125 I-labeled LDL in the absence or presence of competitor ligand. 125 I-labeled LDL binding in the absence of competitor (labeled LDL alone) was taken as 100% (Fig. 5). Inclusion of a 50-fold excess of unlabeled LDL (50· unlabeled LDL) resulted in a marked decrease in 125 I-labeled LDL binding. Like- wise, WT apoE3-NT–DMPC was shown to be an effective competitor of 125 I-labeled LDL binding. Given that the level of reduction of 125 I-labeled LDL binding observed with HT apoE3-NT–DMPC com- plexes at 50 lgÆmL )1 was similar to that observed with WT apoE3-NT–DMPC, we conclude that the HT mutation does not compromise the LDLR binding activity of this protein. Dimyristoylphosphatidylglycerol vesicle solubilization studies A hallmark feature of exchangeable apolipoproteins is their ability to solubilize certain phospholipid bilayer Fig. 4. Effect of apolipoproteins on ANS fluorescence emission. ANS (1 m M)in10mM sodium phosphate, pH 7.0, was excited at 395 nm and emission was monitored from 405 to 600 nm. Curve (a) ANS in buffer at pH 7.0; curve (b) ANS plus 5 l M WT apoE3-NT; curve (c) ANS plus 5 l M HT apoE3-NT. 0 20 40 60 80 100 Labeled LDL alone 50X unlabeled LDL W T apoE3-NT DMPC HT apoE3-NT DMPC 125 I-LDL bound (%) Fig. 5. LDLR binding activity of apoE3-NT. Human skin fibroblasts were incubated with DMEM containing 1 mgÆmL )1 FAFA and 2 lgÆmL )1 125 I-labeled LDL in the absence or presence of competi- tors at 4 °Cfor2h. 125 I-labeled LDL binding to fibroblasts treated with serum free medium in the absence of competitor ligand (cor- responding to 23 742 c.p.m. per mg cell protein) was taken as 100% (bar 1). Binding in the presence of competitors is expressed as percentage of control. Incubations of cells with 125 I-labeled LDL were conducted with the following competitors: a 50-fold excess of unlabeled LDL; 50 lg WT apoE3-NT–DMPC complexes and 50 lg HT apoE3-NT–DMPC complexes. Values reported are the average of three determinations ± SD. K. A. Redmond et al. apoE3-NT domain lipid binding FEBS Journal 273 (2006) 558–567 ª 2006 The Authors Journal compilation ª 2006 FEBS 561 vesicles, transforming them into discoidal complexes. To determine the effect of the amino acid sequence alteration introduced into HT apoE3-NT on the kinet- ics of apoE3-NT lipid binding activity, apolipoprotein dependent dimyristoylphosphatidylglycerol (DMPG) vesicle solubilization was monitored as a function of time (Fig. 6). Whereas DMPG vesicle light scattering intensity did not change upon incubation at 23 °C in buffer alone, inclusion of WT apoE3-NT induces a time dependent reduction in light scattering intensity (T 1 ⁄ 2 ¼ 75 s). By comparison, HT apoE3-NT dis- played a marked enhancement in lipid binding activity, inducing clearance of the turbid vesicle substrate with aT 1 ⁄ 2 < 10 s. The same differences in lipid binding activity were observed when disulfide bonds in WT and HT apoE3-NT were reduced with 1 mm dithio- threitol, indicating that the presence of disulfide bon- ded homodimeric apolipoprotein does not interfere with the lipid interaction properties of these proteins. Interaction with lipoproteins To examine the ability of HT apoE3-NT to bind spherical lipoproteins, human LDL was incubated with phospholipase C (PL-C) in the presence or absence of HT apoE3-NT or WT apoE3-NT. PL-C induces hydrolysis of LDL phosphatidylcholine, generating diacylglycerol moieties that destabilize LDL structural integrity, resulting in particle aggregation and sample turbidity development. In studies of this phenomenon Liu et al. [20] showed that exchangeable apolipopro- teins bind to PL-C modified LDL and prevent lipo- protein aggregation. In control incubations lacking exogenous apolipoprotein, PL-C induces a rapid increase in LDL sample turbidity (Fig. 7). WT apoE3- NT showed a limited ability to protect LDL from PL-C induced turbidity development while HT apoE3- NT conferred nearly full protection. These studies were extended by evaluating the effect of apolipoprotein concentration on their ability to protect LDL from PL-C induced aggregation (Fig. 8A). Whereas WT apoE3-NT was unable to fully protect LDL from lipo- lysis-induced aggregation at any concentration exam- ined, HT apoE3-NT was more effective, consistent with formation of a stable binding interaction. In a competition experiment, wherein equal amounts of WT and HT apoE3-NT were incubated with LDL and PL-C, HT apoE3-NT preferentially associated with LDL (Fig. 8B). In the absence of PL-C, no apoE3-NT was recovered in the LDL density range. These data confirm the higher lipid affinity of HT apoE3-NT ver- sus its WT counterpart. Discussion An important aspect of apoE function relates to the fact that it manifests LDLR binding activity only when lipid associated. Early studies showed that apoE conformational status affects clearance of Fig. 6. Effect of apolipoproteins on DMPG vesicle light scattering intensity. DMPG vesicles (600 nmoles phospholipid) were incuba- ted in buffer at 23 °C at pH 7.0. Sample right angle light scatter intensity was monitored as a function of time. Curve (a) DMPG vesicles in buffer; curve (b) DMPG vesicles plus 5 nmoles WT apoE3-NT; curve (c) DMPG vesicles plus 5 nmoles HT apoE3-NT. Fig. 7. Effect of apolipoproteins on PL-C induced aggregation of human LDL. Human LDL (100 lg protein) was incubated at 37 °C in the absence (s) or presence of PL-C (0.9 units) with no apolipo- protein (d), 100 lg WT apoE3-NT ( n) or HT apoE3-NT (h). Sample absorbance at 340 nm was determined after 90 min. Values repre- sent mean ± SD (n ¼ 3). apoE3-NT domain lipid binding K. A. Redmond et al. 562 FEBS Journal 273 (2006) 558–567 ª 2006 The Authors Journal compilation ª 2006 FEBS triacylglycerol-rich lipoproteins [21]. Although apoE may be present, some particles remain receptor inac- tive. Using monoclonal antibodies Krul et al. [22] showed that expression of specific apoE epitopes on lipoprotein particles correlates with LDLR binding ability. When considered in light of available structural data and localization of the LDLR recognition sequence to helix 4 in the NT domain, these observa- tions are consistent with the concept that the conform- ational status of the NT domain modulates the receptor recognition properties of apoE. More specific- ally, a conformational transition in the NT domain from its receptor inactive globular four-helix bundle to an ‘open’ lipid-bound conformation is considered to be necessary and sufficient to confer receptor-recognition properties to the protein. Whereas the precise structure is not known, in the case of reconstituted HDL, evi- dence suggests apoE adopts an extended conformation around the periphery of these discoidal particles [23]. Structural and biophysical data on full-length apoE have led to the concept that the CT domain mediates initial contact with lipoprotein surfaces, effectively anchoring the NT domain at the particle surface [24,25]. In this manner, depending on physiological conditions, the NT domain may exist in one of two alternate con- formational states. Given that the NT domain is an independently folded structural element within apoE that, when lipid associated, possesses full LDLR bind- ing activity, studies of this domain in isolation may pro- vide insight into the conformational transition that occurs upon lipid interaction as well as factors that modulate lipid surface recognition and⁄ or initiation of lipid binding. Based on studies with an unrelated helix bundle apolipoprotein [15] we hypothesized that helix 1¢ may play a role in lipid-induced NT domain conforma- tional opening. Characterization studies revealed that both proteins exist in solution as a population of mono- mers and disulfide-linked homodimers. When exposed to increasing concentrations of guanidine hydrochlo- ride, WT apoE3-NT and HT apoE3-NT denature, indu- cing in a red shift in tryptophan fluorescence emission maximum. Whereas, the transition midpoint observed for WT apoE3 is similar to that reported earlier [1], the corresponding transition for HT apoE3-NT occurred at a lower guanidine hydrochloride concentration (2.5 m versus 1.9 m), indicating structural alteration of the pro- tein reduces its ability to resist guanidine hydrochloride induced denaturation. Despite this difference, HT apoE3-NT adopts a solution conformation that remains far more stable than several other members of the apo- lipoprotein family [1]. When associated with DMPC, HT apoE3-NT competed with 125 I-labeled LDL for binding to the LDLR on cultured human skin fibro- blasts. Taken together, these data indicate that the HT mutation did not compromise the ability of this domain to adopt a stable solution conformation or interfere with its function as a ligand for the LDLR. The results also showed that helix 1¢ is not essential for recognition or initiation of lipid binding. Indeed, HT apoE3-NT displayed enhanced lipid-binding activity compared to the WT protein. This result may be a reflection of muta- tion-induced structural alteration of the protein wherein potential lipid binding sites may be exposed. Thus, it appears that helix 1¢ plays a structural role, serving to maintain the integrity of the helix bundle in the absence of lipid, perhaps by contributing to sequestration of the hydrophobic interior of the protein. A B Fig. 8. ApoE3-NT interaction with PL-C treated LDL. (A) Human LDL (100 lg) and PL-C (0.9 units) were incubated at 37 °C in the presence of specified amounts of WT apoE3-NT (s) or HT apoE3- NT (d). Sample absorbance at 340 nm was determined after 90 min. Values represent mean ± SD (n ¼ 3). (B) SDS ⁄ PAGE analy- sis of apolipoprotein associated with PL-C treated LDL. Human LDL (200 lg) was incubated with 400 lg each of WT apoE3-NT and HT apoE3-NT in the absence and presence of PL-C (1.8 units) at 37 °C. After 90 min the sample was subjected to density gradient ultra- centrifugation and the LDL fraction recovered. The sample was dia- lyzed against deionized water, lyophilized, resuspended in sample treatment buffer (reducing) and separated by SDS ⁄ PAGE. Lane 1, WT apoE3-NT standard; lane 2, HT apoE3-NT standard; lane 3, mix- ture of WT and HT apoE3-NT; lane 4, LDL density fraction from incubation with PL-C; lane 5, LDL density fraction from incubation without PL-C. K. A. Redmond et al. apoE3-NT domain lipid binding FEBS Journal 273 (2006) 558–567 ª 2006 The Authors Journal compilation ª 2006 FEBS 563 It is conceivable that, in WT apoE3-NT, helix 1¢ repositions during lipid interaction to reveal hydropho- bic sites in the protein, facilitating opening of the helix bundle by helix 1 and 2 moving away from helix 3 and 4, as depicted by Weisgraber [4] and in Fig. 9A. Alter- natively, the flexible segment connecting helix 2 and 3 (residues 79–90, termed the 80 s loop) could play a role in apoE-NT interaction with lipid surfaces [6]. In this scheme the helix bundle opens via helix 1 and 4 moving away from helix 2 and 3, with the segments connecting helix 1 and 2 and helix 3 and 4 serving as ‘hinges’ (Fig. 9B). It has been suggested that negatively charged side chains of glutamate residues may be attracted to the quaternary amino group of phosphat- idylcholine at the lipid surface, while the flexibility of this region facilitates the required conformational change [6]. Whereas long-range mutation induced structural alterations could affect the 80 s loop and be responsible for the results presented here, two observa- tions implicate a mechanism whereby the helix bundle opens via the loop connecting helix 2 and 3. First, the enhanced phospholipid vesicle solubilization activity and increased binding to modified lipoproteins of HT apoE3-NT compared to WT apoE3-NT is likely to have arisen from increased exposure of hydrophobic sites in the protein normally protected by helix 1¢ and second, the strong phospholipid vesicle solubilization activity observed with the anionic phospholipid, DMPG, would not be expected if the 80 s loop, which contains a cluster of negatively charged amino acids, initiated contact with the lipid surface. Further work, including mutations within the 80 s loop will be required to elucidate the precise mechanism whereby the NT domain initiates contact with lipid surface to undergo the conformational transition that culminates in LDLR recognition. Another goal will be to evaluate whether the increased lipid binding activity of HT apoE3-NT is maintained in the context of full-length apoE. It is conceivable that an NT domain with increased lipid binding activity will result in a greater proportion of lipoprotein associated full-length apoE molecules that adopt a receptor-active conformation. Experimental procedures Lipoproteins, apoE and site directed mutagenesis Human LDL was obtained from Intracel (Frederick, MD, USA). A plasmid vector encoding HT apoE3-NT was cre- ated by DNA amplification using mutagenic oligonucleotide primers and WT apoE3-NT pET 22b plasmid vector, as described elsewhere [26]. WT and HT apoE3-NT were pro- duced and isolated from Escherichia coli under identical conditions, as described by Fisher et al. [18]. Analytical procedures Protein concentrations were determined by absorbance spectroscopy (280 nm) or the bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL, USA) with bovine serum albumin as the standard. SDS ⁄ PAGE was performed on 4–20% (w ⁄ v) acrylamide slab gels run at a constant 30 mA for 1.5 h. Gels were stained with Gel Code (Pierce Chemical Co.) stain according to the manufacturer’s instructions. Mass spectrometry was performed on a Bruker Autoflex MALDI-TOF (Bruker Daltonics, Billerica, MA, USA) instrument equipped with a SCOUT MTP ion source. Samples were spotted onto a Scout 384 plate using a matrix of sinapinic acid saturated in 30% acetonitrile ⁄ 70% water ⁄ 0.1% trifluoroacetic acid. Ions were accelerated A B Fig. 9. Scheme of possible lipid binding- induced conformational changes in apoE3- NT. Models were adapted from X-ray crystal structure of apoE3-NT using the program PYMOL. Labels denote specific a-helices (H1– H4) identified in the helix bundle structure. apoE3-NT domain lipid binding K. A. Redmond et al. 564 FEBS Journal 273 (2006) 558–567 ª 2006 The Authors Journal compilation ª 2006 FEBS at +20 kV and masses were detected in linear mode with Protein A used as external calibrant. Fluorescence spectroscopy Fluorescence spectra were obtained using a PerkinElmer LS 50B luminescence spectrometer (Boston, MA, USA). For dye binding experiments, incubations were carried out in 400 lL 20 mm sodium phosphate buffer (pH 7.0) containing 1 mm ANS [27], in the absence and presence of 5 lm WT apoE3-NT or HT apoE3-NT. Samples were excited at 395 nm (slit width 3 nm) and emission monitored between 405 and 600 nm (3 nm slit width). For guanidine hydro- chloride unfolding experiments, samples were incubated overnight at given denaturant concentrations in order to attain equilibrium. Subsequently, the samples were excited at 280 nm and scanned from 300 to 375 nm (3.0 nm slit width). For quenching studies, samples were excited at 295 nm and emission was monitored from 300 to 350 nm. A stock solution of potassium iodide contained 1 mm thiosulfate to prevent formation of free iodine. Quenching data were analy- zed by the Stern–Volmer equation: F 0 ⁄ F ¼ 1 + Ksv [Q] where F 0 and F represent the emission maximum in the absence and presence of quencher, respectively. The collision- al quenching constant, Ksv, was determined from the slope of plots of F 0 ⁄ F versus [Q] (quencher concentration). Analytical ultracentrifugation Sedimentation equilibrium experiments were conducted at 20 °C in a Beckman XL-I analytical ultracentrifuge (Fuller- ton, CA, USA) using absorbance optics, as described by Laue and Stafford [28]. Aliquots (110 lL) of the sample solution were loaded into six sector charcoal filled epon (CFE) sample cells, allowing three concentrations to be run simultaneously. Runs were performed at a minimum of three different speeds and each speed was maintained until there was no significant difference in r 2 ⁄ 2 versus absorbance scans taken 2 h apart to ensure that equilibrium was achieved. Sedimentation equilibrium data were evaluated using the nonlin program (J.W. Lary, Rockville, CT, USA), which employs a nonlinear least squares curve-fitting algorithm described by Johnson et al. [29]. The data set obtained at a protein concentration of 0.25 mgÆmL )1 at 19 000 r.p.m. (rotor type, Beckman An50Ti) was omitted due to unexplained signal noise. The protein’s partial specific volume (0.73 mgÆg )1 ) and the solvent density (1.0047 gÆmL )1 ) were estimated using the sednterp program (University of New Hampshire, Durham, NH, USA) [30]. LDLR binding assay Human skin fibroblasts were grown to approximately 60% confluence in the presence of DMEM with 10% fetal bovine serum. Fibroblasts were then grown to 100% con- fluence in DMEM with 10% lipoprotein-deficient serum. At confluence, cells were cooled on ice for 30 min, washed twice with NaCl ⁄ P i containing 1 mgÆmL )1 fatty acid-free albumin (FAFA), then incubated with DMEM containing 1mgÆmL )1 FAFA, 2 lgÆmL )1 125 I-labeled LDL and differ- ent amounts of receptor binding competitor for 2 h at 4 °C. The medium was removed, and the cells were washed five times with chilled NaCl ⁄ P i -FAFA and two times with chilled NaCl ⁄ P i . Cells were released from the surface of the dishes by incubation with 0.1 m NaOH for 1 h at 24 °C and cell-associated radioactivity was measured on a Cobra II Auto-Gamma Counter (PerkinElmer, Woodbridge, Ontario, Canada). Competitor ligands were prepared by cosonication of DMPC bilayer vesicles and a specified apoE3-NT, resulting in formation of disk complexes. DMPG vesicle solubilization studies DMPG bilayer vesicles were prepared by extrusion through a 200 nm filter as described by Weers et al. [31]. Stock solu- tions of protein and lipid vesicles were prepared in 20 mm sodium phosphate, pH 7.0, in the presence or absence of 1mm dithiothreitol. Six hundred nanomoles DMPG was incubated at 23 °C in a thermostated cell holder in the absence or presence of 5 nmoles apolipoprotein (sample volume ¼ 400 lL). Sample right angle light scattering intensity was monitored on a PerkinElmer LS 50B lumines- cence spectrometer, with the excitation and emission mono- chromaters set at 600 nm (3 nm slit width). Lipoprotein binding assay Human LDL was incubated for 90 min at 37 °Cinthe presence of Bacillus cereus phospholipase C (0.9 U per 100 lg LDL protein). Where indicated, apolipoprotein (0–400 lg per 100 lg LDL protein) was included in the reaction mixture. Incubations were conducted in 50 mm Tris ⁄ HCl, pH 7.5, 150 mm NaCl and 2 mm CaCl 2 in a total sample volume of 200 lL. Sample absorbance at 340 nm was determined on a Spectramax 340 microtiter plate rea- der (Sunnyvale, CA, USA). 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