Apolipoprotein A-I lysine modification: Effects on helical content, lipid binding and cholesterol acceptor activity

Biochimica et Biophysica Acta 1761 (2006) 64 – 72 http://www.elsevier.com/locate/bba

Apolipoprotein A-I lysine modification: Effects on helical content, lipid binding and cholesterol acceptor activity Gregory Brubaker a , Dao-Quan Peng a , Benjamin Somerlot a , Davood J. Abdollahian a , Jonathan D. Smith a,b,c,⁎ a Department of Cell Biology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA Department of Cardiovascular Medicine, Cleveland Clinic Foundation, Cleveland, OH 44195, USA Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA b

c

Received 17 August 2005; received in revised form 13 January 2006; accepted 17 January 2006 Available online 10 February 2006

Abstract We examined the role of the positively charged lysine residues in apoAI by chemical modification. Lysine modification by reductive methylation did not alter apoAI's net charge, secondary or tertiary structure as observed by circular dichroism and trytophan fluorescence, respectively, or have much impact on lipid binding or ABCA1-dependent cholesterol acceptor activity. Acetylation of lysine residues lowered the isoelectric point of apoAI, altered its secondary and tertiary structure, and led to a 40% decrease in cholesterol acceptor activity, while maintaining 93% of its lipid binding activity. Exhaustive lysine acetoacetylation lowered apoAI's isoelectric point, profoundly disrupted its secondary and tertiary structure, and led to 90% and 82% reductions in cholesterol acceptor and lipid binding activities, respectively. The dose-dependent acetoacetylation of an increasing proportion of apoAI lysine residues demonstrated that cholesterol acceptor activity was more sensitive to this modification than lipid binding activity, suggesting that apoAI lysine positive charges play an important role in ABCA1 mediated lipid efflux beyond the role needed to maintain α-helical content and lipid binding activity. © 2006 Elsevier B.V. All rights reserved. Keywords: Reverse cholesterol transport; Lipid binding; Class A amphipathic helix; Circular dichroism; Alpha helical content

1. Introduction Human plasma apolipoprotein A-I (apoAI) is a 243-residue protein associated primarily with HDL. Analysis of the protein sequence has led to parsing apoAI into an N-terminal globular domain consisting of residues 1–43, with the remainder of the protein consisting of 10 helical regions of 11 or 22 residues each [1]. Alignment of apoAI from residues 44–243 in a helical wheel using 3.67 residues per turn yields a structure with exactly 11 positions and the following properties: (1) the prolines are all aligned in one position; (2) the hydrophobic Abbreviations: apoAI, apolipoprotein A-I; OPA, o-phthaldialdehyde; DGGB, DMEM supplemented with 50 mM glucose, 2 mM glutamine, and 0.2% BSA; PLC, phospholipase C; LDL, low density lipoprotein; WMF, wavelength of maximum fluorescence ⁎ Corresponding author. Department of Cell Biology NC10, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Tel.: +1 216 444 2248; fax: +1 216 444 9404. E-mail address: [email protected] (J.D. Smith). 1388-1981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2006.01.007

residues are overwhelmingly aligned in three consecutive positions to create a hydrophobic face; (3) the positively charged lys and arg residues are heavily concentrated in two consecutive positions on each side of the hydrophobic face; and (4) the negatively charged asp and glu residues are found opposite the hydrophobic face in three positions [2]. This arrangement of hydrophobic, positively, and negatively charged residues has been defined by Segrest and colleagues as a class A amphipathic helix [2]. The 3D structure of lipid-free full-length apoAI has not been determined, yet enough is known about its behavior and structure from biophysical studies as well as crosslinking and mass spectroscopy to propose that it adopts a fourhelix bundle structure with the N- and C-termini of the protein in close proximity, and with its hydrophobic surfaces pointing inward to stabilize its structure [3–7]. ApoAI can bind to dimyristyl phosphatidylcholine dispersions and form stacked phospholipid discs that are 11 nm in diameter [8]. The 3D structure of apoAI in phospholipid discs or in HDL has also not been determined, but enough is known to

G. Brubaker et al. / Biochimica et Biophysica Acta 1761 (2006) 64–72

make good models. The starting point for these models is the crystal structure of the N-terminal deleted Δ1–43 human apoAI [9]. The remarkable structure of the Δ1–43 apoAI crystal revealed a completely open extended α helix that curves into a horseshoe with the N and C termini separated by only 2.3 nm. The structure of the apoAI dimer is composed of two molecules pancaked on top of each other in an offset and antiparallel form to generate an elliptical ring shaped dimer with minimum and maximum diameters of 9 and 13.5 nm, dimensions remarkably similar to apoAI phospholipid discs [9]. The crystal structure, supported by modeling studies and salt bridge calculations from the Segrest lab, led to the antiparallel belt model of apoAI on HDL, replacing the previous picket fence model [9–11]. Nitroxide spin labeling and cross-linking supported the belt model for apoAI phospholipid discs [12,13]. The parallel alignment and salt bridges of apoAI in the belt model is also consistent with a helical hairpin model in which two apoAI hairpins each cover half of the disc perimeter and meet in an head to head or head to tail manner. To distinguish these possibilities, Davidson's lab used cross-linking followed by mass spectrometry, however, the results are equally consistent with both the belt model and the head to head hairpin model, and perhaps both structures can exist [7,14]. Cells expressing ABCA1 can also convert lipid-free apoAI into a nascent lipoprotein-containing cell-derived phospholipids and cholesterol [15]. ApoAI binds directly and specifically to cellular ABCA1 as demonstrated by chemical cross-linking studies [16–19]. Using peptides derived from apoAI helixes, Bielicki's lab has proposed that the alignment of negatively charged residues in the center of the polar face of the amphipathic α-helix is important for ABCA1-mediated cholesterol acceptor activity [20]. There are 21 lys residues and 16 arg residues in apoAI, most of which are highly conserved among different species [1]. Modification of these positively charged residues has been previously associated with structural alterations and loss of activation of lecithin cholesterol acetyltransferase [21]. We had noticed anecdotally that extensive labeling of apoAI on lysine residues often led to its loss of cholesterol acceptor activity. We now extend this observation systematically and present data that specific lysine charge modifications alter apoAI's secondary and tertiary structure, and affect its ability to bind lipids and act as an ABCA1-dependent acceptor of cellular cholesterol. We found that apoAI's ABCA1dependent cholesterol acceptor activity was more sensitive to lysine charge modification than its lipid binding activity, suggesting that apoAI lysine residues may play a specific role during its ABCA1 mediated lipidation. 2. Materials and methods 2.1. Modifications of ApoAI Lysine Residues Human plasma derived apoAI (BioDesign, Saco, ME) was dialyzed against PBS, diluted to 0.5 mg/ml, and stored in frozen aliquots. We determined that the different lysine modifications variously altered protein determination assays. Acetylated apoAI protein concentration was determined using the BCA Protein Assay (Pierce Chemical; Rockford, IL) using native apoAI for the standard curve. Native, reductive methylated, and acetoacetylated apoAI protein

65

concentrations were determined by absorbance at 280 nm, using the molar extinction coefficient of 3.17 × 104. Modifications were performed essentially as previously described [22]. For each of the modifications, the molar ratio of modification reagent to apoAI is given such that a 1:1 ratio implies 1 molecule of modification reagent for each of the 21 lysine residues and the amino terminal amine. For extensive lysine acetylation, 1.0 ml of 0.5 mg/ml apoAI was added to 1.0 ml of saturated sodium acetate in an ice bath. 24 μl of acetic anhydride was added in four equal aliquots over the period of 1 h, mixed, and incubated on ice, followed by dialysis against PBS (molar ratio of acetic anhydride:apoAI primary amines ∼550:1). The sample was concentrated by exposing the dialysis membrane to Sephadex G-100 powder, and passed through a 0.22-μm filter. For extensive lysine acetoacetylation, 1.0 ml of 0.5 mg/ml apoAI was dialyzed overnight against 0.1M borate buffer pH 8.5 at room temperature. 4 μl of diketene (Sigma-Aldrich) was added and incubated at room temperature for 5 min (molar ratio of diketene:apoAI primary amines ∼130:1). In order to hydrolyze acetoacetylated hydroxyl groups, the mixtures were dialyzed against 0.2 M carbonate/bicarbonate buffer, pH 9.5 for 6 h at room temperature and overnight at 4 °C, and then dialyzed against PBS and passed through a 0.22 μm filter. Overnight dialysis against 0.5M hydroxylamine at 37 °C was used in attempt to reverse the acetoacetylation reaction [22]. For extensive lysine reductive methylation, 1.0 ml of 0.5 mg/ml apoAI was dialyzed overnight against 0.1M borate buffer pH 9.0 at room temperature. 1.0 mg of sodium borohydride and 6.0 μl of 7.4% formaldehyde were added to the sample on ice. Four additional aliquots of 7.4% formaldehyde were added every 6 min over a 30-min period. The sodium borohydride and formaldehyde treatments were repeated once and the sample was dialyzed against PBS and at passed through a 0.22-μm filter (molar ratio of formaldehyde:apoAI primary amines ∼400:1). Analysis of the free (unmodified) lysine amines was performed using the sensitive fluorogenic reagent o-phthaldialdehyde (OPA) [23]. Modified apoAI or unmodified apoAI standard (in duplicate) were incubated with 250 μl of complete OPA reagent solution (Sigma-Aldrich; St. Louis, MO) in a 96-well plate while shaking at room temperature for 5 min, and the fluorescence (360 nm excitation and 460 nm emission) was determined in a plate reader. The % lysine modification was calculated relative to unmodified apoAI. The OPA Assay was also performed in the presence of 3M Urea with no significant changes to the results, indicating that all lysine residues in lipid-free native apoAI were exposed to the aqueous environment and accessible to the OPA reagent.

2.2. ApoAI electrophoresis and chromatography Electrophoresis experiments were performed using gels and reagents from Invitrogen (Carlsbad, CA). For Western blot analysis, 250 ng of apoAI per lane was denatured in an SDS sample buffer, run on a 10% Tris glycine gel in the presence of SDS, and the protein was transferred to a PVDF membrane. The membrane was probed sequentially with goat anti-human apoAI primary antibody (1:1,000 dilution, DiaSorin, Stillwater, MN) and rabbit anti-goat-HRP conjugated antibody (1:10,000 dilution), and apoAI was visualized with an enhanced chemiluminescent substrate. For analysis of net charge, 500 ng of apoAI per lane was run on a pH 3–10 isoelectric focusing gel that was electrophoresed for 1 h at 100 V, 1 h at 200 V and 30 min at 500 V. ApoAI in the IEF gel was transferred to a membrane and detected as described above. For the analysis of apoAI:DMPC disc size, 250 ng of apoAI incubated overnight with DMPC dispersions was electrophoresed on a 4–20% Tris– glycine native gel, and the protein was transferred to a PVDF membrane and detected as described above. The recombinant HDL used as a reference for disc size was prepared by cholate dialysis with a 100:10:1 mole ratio of POPC: cholesterol:apoAI, as previously described [24]. Fast performance liquid chromatography (FPLC) using a Superdex 200 column (Amersham Bioscience, Newark, NJ) was employed to approximate the Stokes diameter of the lipid-free apoAI samples. All protein samples and standards were run in PBS, pH 7.0 at a flow rate of 1.0 ml/min. The elution peaks for proteins of known molecular mass and Stokes diameter were used to create a standard curve for approximating the diameter of the modified apoAI samples.

2.3. ABCA1-dependent cholesterol efflux assay RAW 264.7 murine macrophage cells were plated at 225,000 cells per well in a 24-well dish in DMEM supplemented with 10% fetal bovine serum. On the

66

G. Brubaker et al. / Biochimica et Biophysica Acta 1761 (2006) 64–72 Luminescence Spectrometer with an excitation fixed at 295 nm and emission from 300 to 450 nm. The wavelength of maximum fluorescence (WMF) was determined, which is dependent upon the apoAI tryptophan environment, with a red shift observed as tryptophan goes from a hydrophobic to an aqueous environment [6].

following day, the cells were cholesterol labeled by incubation overnight in DMEM containing 1% FBS, which was preincubated with [3H] cholesterol (Amersham Biosciences; Newark, NJ) to yield a final concentration of 0.5 μCi/ ml. On day three, the labeling media was removed and the cells were washed and treated for 16 h in DGGB media (DMEM supplemented with 50 mM glucose, 2 mM glutamine, 0.2% BSA) in the presence or absence of 0.3 mM 8Br-cAMP (Sigma-Aldrich; St. Louis, MO) to induce ABCA1 activity [25,26]. On day four, the cells were washed and chased for 4 h in serum-free DMEM in the presence or absence of various apoAI preparations, in the presence or absence of 0.3 mM 8Br-cAMP. The radioactivity in the chase media was determined after brief centrifugation to pellet debris. Radioactivity in the cells was determined by extraction in hexane:isopropanol (3:2) with the solvent evaporated in a scintillation vial prior to counting. The percent cholesterol efflux was calculated as 100 × (medium dpm) / (medium dpm + cell dpm). ABCA1-dependent cholesterol acceptor activity for each apoAI preparation was calculated as the % cholesterol efflux from the 8Br-cAMP-treated cells minus the % cholesterol efflux from the control treated cells.

Spectra for all apoAI samples were collected by CD using a Jasco J810 Spectropolarimeter (Jasco Incorporated, Easton, MD). The samples were read in a quartz cell with a 0.2 cm path length under a constant nitrogen flush at ambient temperature. Five spectra were collected for each sample from 190 to 250 nm in continuous scanning mode at a bandwidth of 1 nm with a 0.2-nm data pitch. The spectra were normalized to mean residue ellipticity (MRE) using 115.5 as the mean residue weight (MRW) for control apoAI. The percent α-helix was predicted using the MRE at 222 nm [28] and by using the K2d neural network algorithm that utilizes the spectral data from 200 to 240 nm [29].

2.4. Lipid binding activity assay

2.7. Statistical analyses

Lipid binding activity of apoAI was assessed via (1) inhibition of phospholipase C (PLC) mediated aggregation of human low density lipoprotein (LDL) [27], and (2) by DMPC dispersion clearance. PLC cleaves the head groups from phosphatidylcholine on the LDL surface making it more hydrophobic and resulting in LDL aggregation that is monitored by increased absorbance. ApoAI binding to the hydrolyzed LDL surface prevents this aggregation. Using NBD-labeled apoAI as a lipid sensitive fluorescent probe, we have verified that apoAI does not bind appreciably to LDL prior to incubation with PLC, and that apoAI inhibits the rate of this aggregation in a dose-dependent fashion (see Supplemental Figure 1). LDL (1.019 N d b 1.063) was isolated from human plasma by ultracentrifugation in a potassium bromide gradient and dialyzed against PBS. 200 μl of LDL (0.5 mg/ml) in reaction buffer (50 mM Tris pH 7.4, 150 mM sodium chloride, 2 mM calcium chloride) was added to wells of a 96-well plate along with 30 μl of a solution containing 3 μg of the various apoAI preparations in PBS. The plate was pre-incubated at 37 °C for 10 min, and 10 μl of PLC (6.6 mU/μl, Sigma-Aldrich catalog #P4039) was added and immediately mixed. Control wells substituted reaction buffer in place of LDL, apoAI, or PLC. The plate was placed in a plate absorbance reader at 37 °C and the optical density at 478 nm was measured at 1-min intervals over 90 min. The initial rate of absorbance increase, after any lag period, was calculated for each well. The final concentration of unmodified apoAI used in this assay (12.5 μg/ml) was sufficient to decrease the initial rate of LDL aggregation by ∼75% in various assays. The efficacy of modified apoAI preparations in inhibiting LDL aggregation was normalized to the effect of unmodified apoAI in each experiment. Each determination is the average of duplicate or triplicate wells. This assay yields results similar to the commonly used DMPC dispersion clearance assay, but it is more sensitive and requires less reagents [27]. For the DMPC clearance assay, DMPC was dissolved in 2:1 (v/v) chloroform:methanol and dried under nitrogen in glass vials. The lipid was resuspended in clearance buffer (D-PBS with 0.1 M guanidine hydrochloride and 0.5 mM EDTA) and vortexed for 1 min. The turbid suspension was diluted to 0.5 mg/ml in clearance buffer resulting in lipid dispersions with an absorbance below 1.0 at 325 nm. Using an absorbance microplate reader, each apoAI sample (50 or 60 μg/ml) was tested in triplicate for their ability to clear the DMPC vesicles by monitoring the absorbance at 325 nm in 15-s intervals over 1 h. Since there was no lag time prior to the initiation of clearance and there was a variable lag time prior to our first reading, we could more accurately measure the plateau absorbance value, which was linearly correlated to apoAI levels in the range from 0 to 100 μg/ml, than the initial rate of clearance (see Supplemental Figure 1). Thus, we normalized the endpoint change in absorbance of the modified apoAI preparations to the change in absorbance obtained with unmodified apoAI in each experiment.

All data are shown as the mean ± standard deviation. Comparison of two samples was performed by a two-tailed t test, and for three or more samples ANOVA was performed with Dunnett's multiple comparison posttest comparing the control with each treatment group. All statistics were performed using Prism 4.01 software (GraphPad, San Diego, CA).

2.6. Far UV spectral analysis by circular dichroism (CD)

3. Results 3.1. Modifications of apoAI lysine residues and their effects on apoAI net charge and structure Acetylation, acetoacetylation, and reductive methylation of apoAI's lysine primary amines were performed using excess modification reagents to yield essentially complete lysine modification. The expected structures of the modified lysines are shown in Fig. 1. The unmodified and modified apoAI samples were assayed by SDS-PAGE and visualized by Coomassie staining and by Western blotting. The modified apoAI samples showed little signs of degradation and had similar Coomassie staining pattern and intensity as unmodified apoAI (Fig. 2A). Some cross-linking was evident in the acetylated apoAI preparation, and densitometry revealed that the cross-linked bands accounted for ∼8% of the total protein staining. However, Western blot analysis of these same samples revealed a significant amount of cross-linking for the acetylated and acetoacetylated samples (Fig. 2B). We speculate that the difference between the Coomassie and Western blot staining patterns is due to the antibody preferentially binding to the

2.5. Fluorescence spectroscopy and guanidine denaturation ApoAI preparations were prepared at 10 μg/ml in increasing amounts of guanidine hydrochloride and incubated overnight at 4 °C. The tryptophan fluorescence spectra of each sample was measured using a Perkin Elmer LS 50 B

Fig. 1. Structure and charge of lysine ε-amino group before and after the specified chemical modification.

G. Brubaker et al. / Biochimica et Biophysica Acta 1761 (2006) 64–72

67

Fig. 2. Gel electrophoresis of control and modified apoAI. (A) 3.0 μg each of Control (C), acetylated (Ac), acetoacetylated (AA), and reductively methylated (RM) apoAI, was run on a 14% polyacrylamide Tris–glycine SDS gel and stained with Coomassie Brilliant Blue R-250. (B) Western blot of control and modified apoAI. 250 ng of each sample was run on a 10% Tris–glycine, transferred to a membrane and probed for human apoAI. (C) 500 ng of each sample was run on a pH 3–10 gradient isoelectric focusing gel, which was subsequently transferred to a membrane and probed for human apoAI.

cross-linked protein, and we have observed that this same antibody preferentially binds to myeloperoxidase modified apoAI compared to native apoAI in Western blots (data not shown). Size-exclusion chromatography under non-denaturing conditions by FPLC using a Superdex 200 column was used to examine if modified apoAI forms larger complexes than unmodified apoAI. Native apoAI, which is known to self associate in aqueous solution [30,31], eluted at a Stokes diameter of 4.4 to 5.3 nm, while acetylation shifted it to a slightly larger complex at 5.0 to 6.3 nm, and acetoacetylation led to elution at 6.6 to 7.4 nm (data not shown), consistent with the observed effects on apoAI cross-linking. The samples were run on isoelectric focusing gels to determine the effect of each modification on overall protein charge (Fig. 2C). Acetylation and acetoacetylation, which eliminate lysine's positive charge, reduced the isoelectric point (pI) of apoAI from the unmodified pI of 5.3 to below 4.5 and below 4.0, respectively. As expected, reductive methylation of apoAI, which does not eliminate lysine's positive charge, did not change its pI. 3.2. ApoAI lysine modification, ABCA1-dependent cholesterol acceptor and lipid binding activities Two to three independent preparations of each modification were assayed for the efficiency of lysine modification by a sensitive fluorescent method using the OPA reagent. All modifications of apoAI led to N99% modification of lysine residues (all treatments P b 0.01 vs. control, Fig. 3A). ABCA1-dependent cholesterol acceptor activity of apoAI (at 5 μg/ml) was measured by a 4-h incubation with RAW264.7 cells in which ABCA1 was induced by pre-treatment with a cAMP analogue. Two to three independent preparations of each modification were assayed and compared to unmodified apoAI (Fig. 3B). Acetoacetylation and acetylation led to 90% and 40% decreases in cholesterol acceptor activity (P b 0.01 vs. control). Reductive methylation, on the other hand, did not significantly

inhibit cholesterol acceptor activity. Thus, only the two modifications that altered apoAI's net charge inhibited its ABCA1-mediated cholesterol acceptor activity. We attempted to reverse the acetoacetylation and restore the lysine residues by dialysis against 0.5 M hydroxylamine [22]. However, this treatment restored only a fraction of the cholesterol acceptor activity of acetoacetylated apoAI (data not shown). Our inability to completely restore cholesterol acceptor activity could be accounted for by incomplete lysine restoration (∼22% of the modified lysines were not recovered) due to either incomplete reactivity of acetoacetylated lysine residues or the formation of other irreversible lysine products resulting from extensive diketene modification. Irreversible structural changes in apoAI after extensive dikene modification were previously observed [21]. Lipid binding of apoAI was first assessed by its ability to bind to PLC treated LDL and inhibit its aggregation. Two to three independent preparations of each modification were assayed and compared to unmodified apoAI (Fig. 3C). Acetoacetylation led to ∼82% decrease in lipid binding activity compared to unmodified apoAI (P b 0.01). Acetylation and reductive methylation did not lead to significant inhibition of lipid binding activity (7% and 5% loss, respectively). Thus, acetylation resulted in an apoAI with its lipid binding activity well preserved while its cholesterol acceptor activity was partially lost, suggesting that lipid binding activity alone is not the only determinant of the ABCA1-dependent cholesterol acceptor activity of apoAI. DMPC clearance assays were also performed for independent preparations of acetylated and acetoacetylated apoAI versus the unmodified apoAI. ApoAI acetylation led to a modest 5% decrease in lipid binding activity in this assay, similar to the 8% inhibition of lipid binding activity measured for the same acetylated apoAI preparation in the LDL aggregation assay. ApoAI acetoacetylation led to a 60% inhibition of DMPC clearance activity, similar to the 70% inhibition of the lipid binding activity measured for the same

68

G. Brubaker et al. / Biochimica et Biophysica Acta 1761 (2006) 64–72

discs formed after incubation with DMPC dispersion were 15.2 nm in size, and a similar disc size was observed using acetylated or reductively methylated apoAI. However, the acetoacetylated apoAI yielded larger 18.4 nm particles. These data confirm that acetylated and reductively methylated apoAI bind DMPC similar to unmodified apoAI, but that acetoacetylated apoAI forms larger particles. 3.3. ApoAI lysine modification and protein structure ApoAI has 4 tryptophan residues, one in the N-terminal domain, and one each in helixes 1, 2, and 4. In the helix bundle structure of lipid-free apoAI, these tryptophan residues are largely protected from the aqueous environment [6]. Aqueous exposure of the tryptophan residues can be assessed by a red shift in the peak fluorescent emission wavelength. The tryptophan fluorescence emission spectra of each apoAI preparation was collected at increasing concentrations of guanidine to induce denaturation (Fig. 5A). As the guanidine concentration was increased from 0 to 2.5 M, unmodified apoAI exhibited a wavelength of maximum fluorescence (WMF) shift of from 341.5 nm to 355 nm, with an EC50 of 1.0 M guanidine and a high degree of cooperativity (Hill coefficient = 2.3). Reductive methylation of apoAI did not appreciably change either the basal WMF or its guanidine denaturation profile (EC50 = 1.0 M guanidine, Hill coefficient = 2.2). Acetylation of apoAI did not alter the basal WMF, although it led to less cooperativity in guanidine induced denaturation (EC50 = 1.1 M guanidine, Hill coefficient = 1.1). Exhaustive acetoacetylation of apoAI dramatically increased the basal WMF measurements, indicating that this modification led to protein denaturation and aqueous exposure of the 4 tryptophan residues prior to the addition of guanidine. The native and modified apoAI preparations were assessed by circular dichroism spectroscopy (Fig. 5B). The α-helical content of each sample was calculated using mean residue Fig. 3. Effect of apoAI modification on lysine ε-amino groups, cholesterol acceptor activity, and lipid binding activity. (A) Free amines were determined by the OPA assay for control, acetoacetylated (AcAc), acetylated (Ac), and reductively methylated (Red Meth) apoAI. The data are normalized to control apoAI and show the mean ± S.D. for at least two independent assays performed on two to three independent preparations of each modified form (*P b 0.01 vs. control). (B) ABCA1-dependent cholesterol acceptor activity was determined by incubation with cholesterol loaded RAW264.7 cells. The data are normalized to control apoAI and show the mean ± S.D. for triplicate assays performed on two to three independent preparations of each modified form (*P b 0.01 vs. control). (C) Lipid binding activity was determined by the inhibition of PLCmediated LDL aggregation. The data are normalized to control apoAI and show the mean ± S.D. for two independent assays performed on two to three independent preparations of each modified form (*P b 0.01 vs. control).

acetoacetylated apoAI preparation in the LDL aggregation assay. Thus, both lipid binding assays gave approximately similar results, and the mild effect of apoAI acetylation on lipid binding was confirmed. We also estimated the size of the apoAI discs formed after overnight incubation of native and modified apoAI with DMPC dispersions by non-denaturing gradient gel electrophoresis (Fig. 4). For reference, recombinant discoidal HDL migrated at 8.9 nm, with a minor band at 11.0 nm. ApoAI

Fig. 4. Effects of apoAI modification on discs formed after incubation with DMPC dispersions. ApoAI incubation with DMPC was carried out exactly as described for the lipid binding assay, except that the incubation period was overnight. 250 ng of apoAI in each sample was run on a 4–20% Tris–glycine native gel, transferred to a PVDF membrane and probed for human apoAI. Lane 1, recombinant HDL discs prepared as described in Materials and methods; lane 2, lipid-free apoAI; lane 3, unmodified apoAI after incubation with DMPC; lane 4, acetoacetylated apoAI with DMPC; lane 5, acetylated AI with DMPC; and lane 6, reductively methylated apoAI with DMPC. The migration of a high molecular weight standard (Amersham Biosciences; Newark, NJ) containing proteins of known Stokes diameter (nm) is shown at the left.

G. Brubaker et al. / Biochimica et Biophysica Acta 1761 (2006) 64–72

69

β-sheet and random coil. Acetylation and acetoacetylation decreased α-helix content to 31% and 8%, respectively. The largest compensatory change for acetylated apoAI was an increase in its random coil content (going from 34% to 53%), while the largest compensatory change for acetoacetylated apoAI was an increase in its β-sheet content (going from 8% to 40%). Overall, acetoacetylation of apoAI led to the most severe changes in its secondary and tertiary structures as determined by circular dichroism and tryptophan fluorescence spectroscopy, respectively.

Fig. 5. Effects of apoAI modification on its tertiary and secondary structure. (A) Guanidine denaturation of control, acetoacetylated, acetylated, and reductively methylated apoAI. The denaturation was monitored by the red shift in maximal wavelength of tryptophan fluorescence. The plotted lines are the results of sigmoidal variable slope non-linear regression analysis, which yielded r2 values of N0.99 for all except for acetoacetylated apoAI with an r2 value of 0.93. Hill slope coefficients were derived from these regressions and are stated in the text. (B) Circular dichroism spectra of control apoAI (solid thick line), acetylated apoAI (dotted line), acetoacetylated apoAI (solid thin line) and reductively methylated apoAI (dashed line).

ellipticity at 222 nm (Table 1). Using the spectra from 200 to 240 nm in the K2d neural network algorithm, we also calculated α-helix content along with β-sheet and random coil content (Table 1). These two calculation methods yielded different αhelix values, and the K2d data are presented here since this method integrates more spectral information. Reductive methylation had the smallest effect on apoAI structure, decreasing αhelix from 58% to 51%, with small compensatory increases in Table 1 Effect of lysine modification on apoAI structure as monitored by circular dichroism AI modification

% α-helix

K2d % β-sheet

% Random

222 nm % α-helix

Control Acetylated Acetoacetylated Reductive Methylation

58 31 8 51

8 16 40 12

34 53 51 37

48.3 43.4 16.5 43.9

Fig. 6. Dose-dependent effect of acetoacetylation on apoAI structure and function. (A) Acetoacetylation was performed at varying molar ratios of diketene:apoAI primary amines. The extent of lysine modification (open circles), ABCA1-dependent cholesterol acceptor activity (open squares), and lipid binding activity (closed triangles) were determined as described in Materials and methods. (B) CD spectra of apoAI with increasing molar ratio of diketene:apoAI primary amines. (C) ABCA1-dependent cholesterol acceptor activity (open squares) and lipid binding activity (closed triangles) plotted against the α-helix content of apoAI subjected to varying extents of acetoacetylation.

70

G. Brubaker et al. / Biochimica et Biophysica Acta 1761 (2006) 64–72

Table 2 Dose-dependent effect of apoAI acetoacetylation on its structure Diketene: apoAI amines

% Amines modified

K2d % α-helix

% β-sheet

% Random

0 0.4 0.8 1.7 3.3 33 130

0 (control) 23.3 38.7 53.8 78.3 98.9 99.9

57 50 43 35 29 24 8

8 13 17 15 15 20 40

35 37 41 49 55 56 51

222 nm % α-helix

48.2 43.3 41.6 37.1 34.8 26.9 16.5

3.4. Dose-dependent effects of apoAI acetoacetylation We then examined the effects of varying the extent of lysine acetoacetylation by modification with increasing molar ratios of diketene:apoAI primary amines. As this ratio increased the level of unmodified lysine amines decreased, along with lipid binding and cholesterol acceptor activities (Fig. 6A). The exhaustive acetoacetylation with a diketene:apoAI amine mole ratio of 130 had the most drastic effects on apoAI's lipid binding (Fig. 6A) and secondary structure (Fig. 6B and Table 2). More modest acetoacetylation at dikene:apoAI amine mole ratios of 33 and 3.3 had more modest effects on lipid binding (∼20% inhibition of the activity of unmodified apoAI) and secondary structure (24% and 29% α-helix content vs. 8% for the exhaustively modified form), while still inhibiting ∼90% of the ABCA1dependent cholesterol acceptor activity of unmodified apoAI (Fig. 6A and Table 2). By plotting the loss of α-helix content (using the K2d method) vs. the ABCA1-dependent cholesterol acceptor and lipid binding activities, it is apparent that ABCA1dependent acceptor activity is more sensitive to loss of α-helical content than lipid binding, the latter of which only markedly decreased when the α-helical content decreased below 10% in the exhaustively acetoacetylated protein (Fig. 6C). The results of the less than exhaustive diketene modifications confirm the finding that ABCA1-dependent cholesterol acceptor activity of apoAI is more sensitive to lysine charge modification than the lipid binding activity of apoAI. Similar findings of dosedependent effects of acetoacetylation on apoAI structure and function were observed when the extent of acetoacetylation was altered by performing the modification at varying pH conditions (data not shown). 4. Discussion We have shown in the present study that apoAI lysine positive charges play a role in its ABCA1-dependent cholesterol acceptor activity. This cholesterol acceptor activity is markedly impaired in acetylated apoAI and in partially acetoacetylated apoAI, even when its lipid binding activity is mostly retained. Thus, we conclude that the mechanism of ABCA1-mediated lipid efflux relies on apoAI lysine positive charges more stringently than this charged residue is required for general lipid binding. Although apoAI's lipid binding activity is most likely required for its ABCA1-dependent cholesterol acceptor activity,

it seems as if the lipid binding activity of apoAI is not sufficient to mediate optimal cholesterol acceptor activity. ABCA1 expression promotes apoAI binding and uptake, and there is ample evidence of specific binding of apoAI to ABCA1 via chemical cross-linking [18,16,19,17], and it is possible that the recognition of apoAI by ABCA1 may be facilitated by lysine positive charges. Alternatively, ABCA1 has been shown to promote increased levels of anionic phospholipids, such as phosphatidylserine, on the cell surface [32–34], and apoAI lysine positive charges may play an important role in the specific recognition of negatively charged phospholipids on the surface of an ABCA1 expressing cell. ApoAI binds more rapidly to vesicles that contain acidic phospholipids, compared to vesicles made exclusively of the zwitterionic phosphatidylcholine, particularly at the physiological temperature of 37 °C [35]. It has also been shown that switching the positions of the asp and lys in a class A amphipathic peptide, or otherwise altering the charge distribution, greatly reduced its ability to bind to liposomes [36–38]. Thus, lysine residues in apoAI and model peptides may play an important role in their lipid acceptor activity by either direct interaction with ABCA1 or via binding to acidic phospholipids. Our data with acetylated apoAI tend to support the notion that the class A structure of apoAI plays a more important role in ABCA1 mediated lipid efflux than simply for lipid association. ApoAI acetylation did lead to a loss of cooperativity during guanidine denaturation suggesting that loss of positive charges might decrease repulsive forces that promote cooperative unfolding. However, how the loss of lysine positive charges by acetylation precisely alters the domain structure of apoAI has not been determined. Bielicki has implied that an undisrupted array of the negatively charged residues opposite the hydrophobic face is crucial for ABCA1-dependent lipid acceptor activity of peptides derived from apoAI [20]. This inference was based upon the behavior of a series of 33-mer peptides designed by joining an 11-mer apoAI helix with a 22-mer apoAI helix. The 9/10 and 1/ 9 helix peptides, as well as the 9/1 helix transposition peptide, all possessed ABCA1-dependent cholesterol acceptor activity, while the 2/9 and 4/9 peptides, and the 10/9 helix transposition peptide, did not have this activity [20]. Helical wheel projections (4.5 residues per turn, 18 positions) show that the active 9/10, 1/9, and 9/1 peptides share an alignment of negatively charged residues not interrupted by a positively charged residue; in contrast, the inactive 10/9 and 4/9 peptides have a positively charged residue disrupting this alignment [20]. The inactive 2/9 peptide appears to have the favored negative charge distribution, but it has an overall high net negative charge and does not bind lipid [20]. However, we made a different observation about the structure of these peptides from their helical wheel projections, the three active peptides, 9/10, 1/ 9, and 9/1, have only positively charged or neutral residues in the 2 positions adjacent to each side of the hydrophobic face, while the inactive 10/9, 2/9, and 4/9 peptides all contain at least one negatively charged residue in these positions. The helical wheel projection of the 18A model peptide [36], which has ABCA1-dependent cholesterol acceptor activity, also conforms to this rule of having no negatively charged residues in the 2

G. Brubaker et al. / Biochimica et Biophysica Acta 1761 (2006) 64–72

positions on each side of its hydrophobic face. Thus, we could speculate that it is the disruption of the positively charged surfaces, rather than the negatively charged surface, in the 10/9, 2/9, and 4/9 peptides that is associated with their loss of activity. Additional studies with single residue substitutions will be needed to resolve this issue. Many studies have been performed using wild type and mutant apoAI in order to understand the salient structural features required for apoAI lipidation. Deletion of the Cterminal helix 10 greatly reduces the ability of apoAI to bind lipids in cell-free systems and to act as ABCA1-dependent acceptors of cellular lipids, without altering its susceptibility to guanidine or urea denaturation [6,39,40,17]. The crucial role of the C-terminus in lipid binding and efflux was bolstered from data showing that a peptide of this region binds lipids more rapidly than the other apoAI-derived peptides [41]. Electron paramagnetic resonance studies were performed to probe the secondary, tertiary, and quaternary structure of the C-terminus (helixes 8–10) in the context of lipid-free and lipid associated full-length apoAI [42]. A model for the tertiary structure of the C-terminus was proposed in which a helix–loop–helix structure opens up into an extended helix in the transition from lipid-free to lipid bound, similar to viral proteins that mediate membrane fusion [42]. This model predicts that the C-terminus acts as a “lipid sensitive trigger” that upon sensing lipids opens up like a bent spring, and which could promote the rest of the molecule to open up. The distribution of positive charges in helix 10 may be important in its lipid sensing role; Davidson has shown that substitution of lys 238 with glu led to a large decrease in its lipid binding and cholesterol acceptor activities, while substitution of glu 235 with lys actually led to a small increase in cholesterol acceptor activity [39]. Thus, modification of the lys residues in the C-terminus of apoAI may be responsible for much of the loss of cholesterol acceptor activity that we observed upon acetoacetylation or acetylation of apoAI lys residues. We found that apoAI acetoacetylation dose dependently led to its denaturation. Acetylation had a less dramatic effect on the stability of apoAI's structure, leading to a decrease in the cooperativity of apoAI denaturation by guanidine. The strongly cooperative denaturation of unmodified apoAI may be due in part to the repulsive electrostatic forces in the lipid-free apoAI helix bundle, in which the positively charged residues of adjacent helices would be juxtaposed upon the binding of their hydrophobic faces. This electrostatic repulsion would create potential energy in apoAI's folded state and promote cooperative unfolding, once unfolding is initiated. We speculate that acetylation reduces this repulsive force and thereby decreases the cooperativity of denaturation. We found that reductive methylation using sodium borohydride, did not have any major effects on apoAI's structure or function. This agrees with two prior studies in which lysine methylation of apoAI in phospholipid complexes, performed with either sodium borohydride or the milder sodium cyanoborohydride reagent, had no effect on the protein α-helix content, or the ability of the modified protein to form lipid discs or activate lecithin cholesterol acyltransferase [21,43]. Thus, the formation of tertiary amines from lysine by reductive methylation did not

71

seemingly disrupt salt bridges in lipid-free apoAI, as indicated by similar susceptibility to guanidine denaturation, and similar baseline CD and tryptophan fluorescence profiles. This study points out the potential problem of the most commonly used methods of labeling apoAI, which use amine reactive reagents and thus modify apoAI on its lysine residues. As the efficiency of the labeling increases, less biological activity will remain. To overcome this problem, we now prepare recombinant single cysteine substitution mutants of apoAI, which can be efficiently labeled using sulfhydryls reactive reagents, resulting in no loss of cellular lipid acceptor activity [44]. Acknowledgements This work was supported by National Institutes of Health Grant RO1 HL-66082. We thank Dr. Michael Zagorski of the Department of Chemistry at Case Western Reserve University for use of their CD spectropolarimeter. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version at doi:10.1016/j.bbalip.2006.01.007. References [1] C.G. Brouillette, G.M. Anantharamaiah, J.A. Engler, D.W. Borhani, Structural models of human apolipoprotein A-I: a critical analysis and review, Biochim. Biophys. Acta 1531 (2001) 4–46. [2] M.K. Jones, G.M. Anantharamaiah, J.P. Segrest, Computer programs to identify and classify amphipathic alpha helical domains, J. Lipid Res. 33 (1992) 287–296. [3] D.P. Rogers, L.M. Roberts, J. Lebowitz, G. Datta, G.M. Anantharamaiah, J.A. Engler, C.G. Brouillette, The lipid-free structure of apolipoprotein A-I: effects of amino-terminal deletions, Biochemistry 37 (1998) 11714–11725. [4] L.M. Roberts, M.J. Ray, T.W. Shih, E. Hayden, M.M. Reader, C.G. Brouillette, Structural analysis of apolipoprotein A-I: limited proteolysis of methionine-reduced and -oxidized lipid-free and lipid-bound human apo A-I, Biochemistry 36 (1997) 7615–7624. [5] A.K. Behling Agree, M.A. Tricerri, M.K. Arnvig, S.M. Tian, A. Jonas, Folding and stability of the C-terminal half of apolipoprotein A-I examined with a Cys-specific fluorescence probe, Biochim. Biophys. Acta 1594 (2002) 286–296. [6] H. Saito, P. Dhanasekaran, D. Nguyen, P. Holvoet, S. Lund-Katz, M.C. Phillips, Domain structure and lipid interaction in human apolipoproteins A-I and E, a general model, J. Biol. Chem. 278 (2003) 23227–23232. [7] R.A. Gangani, D. Silva, G.M. Hilliard, J. Fang, S. Macha, W.S. Davidson, A three-dimensional molecular model of lipid-free apolipoprotein A-I determined by cross-linking/mass spectrometry and sequence threading, Biochemistry 44 (2005) 2759–2769. [8] Y. Fang, O. Gursky, D. Atkinson, Lipid-binding studies of human apolipoprotein A-I and its terminally truncated mutants, Biochemistry 42 (2003) 13260–13268. [9] D.W. Borhani, D.P. Rogers, J.A. Engler, C.G. Brouillette, Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 12291–12296. [10] A.E. Klon, J.P. Segrest, S.C. Harvey, Comparative models for human apolipoprotein A-I bound to lipid in discoidal high-density lipoprotein particles, Biochemistry 41 (2002) 10895–10905. [11] J.P. Segrest, M.K. Jones, A.E. Klon, C.J. Sheldahl, M. Hellinger, H. De Loof, S.C. Harvey, A detailed molecular belt model for apolipoprotein

72

[12]

[13]

[14]

[15] [16]

[17]

[18]

[19]

[20]

[21]

[22] [23] [24]

[25]

[26]

[27]

[28]

[29]

G. Brubaker et al. / Biochimica et Biophysica Acta 1761 (2006) 64–72 A-I in discoidal high density lipoprotein, J. Biol. Chem. 274 (1999) 31755–31758. J.N. Maiorano, W.S. Davidson, The orientation of helix 4 in apolipoprotein A-I-containing reconstituted high density lipoproteins, J. Biol. Chem. 275 (2000) 17374–17380. H. Li, D.S. Lyles, M.J. Thomas, W. Pan, M.G. Sorci-Thomas, Structural determination of lipid-bound ApoA-I using fluorescence resonance energy transfer, J. Biol. Chem. 275 (2000) 37048–37054. W.S. Davidson, G.M. Hilliard, The spatial organization of apolipoprotein A-I on the edge of discoidal high density lipoprotein particles: a mass spectrometry study, J. Biol. Chem. 278 (2003) 27199–27207. A.R. Tall, Role of ABCA1 in cellular cholesterol efflux and reverse cholesterol transport, Arterioscler. Thromb. Vasc. Biol. 23 (2003) 710–711. N. Wang, D.L. Silver, P. Costet, A.R. Tall, Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1, J. Biol. Chem. 275 (2000) 33053–33058. A. Chroni, T. Liu, M.L. Fitzgerald, M.W. Freeman, V.I. Zannis, Crosslinking and lipid efflux properties of apoA-I mutants suggest direct association between apoA-I helices and ABCA1, Biochemistry 43 (2004) 2126–2139. J.F. Oram, R.M. Lawn, M.R. Garvin, D.P. Wade, ABCA1 is the cAMPinducible apolipoprotein receptor that mediates cholesterol secretion from macrophages, J. Biol. Chem. 275 (2000) 34508–34511. M.L. Fitzgerald, A.L. Morris, J.S. Rhee, L.P. Andersson, A.J. Mendez, M.W. Freeman, Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I, J. Biol. Chem. 277 (2002) 33178–33187. P. Natarajan, T.M. Forte, B. Chu, M.C. Phillips, J.F. Oram, J.K. Bielicki, Identification of an apolipoprotein A-I structural element that mediates cellular cholesterol efflux and stabilizes ATP binding cassette transporter A1, J. Biol. Chem. 279 (2004) 24044–24052. A. Jonas, K.E. Covinsky, S.A. Sweeny, Effects of amino group modification in discoidal apolipoprotein A-I-egg phosphatidylcholine– cholesterol complexes on their reactions with lecithin:cholesterol acyltransferase, Biochemistry 24 (1985) 3508–3513. T.L. Innerarity, R.E. Pitas, R.W. Mahley, Lipoprotein–receptor interactions, Methods Enzymol. 129 (1986) 542–565. C.C. Goodno, H.E. Swaisgood, G.L. Catignani, A fluorimetric assay for available lysine in proteins, Anal. Biochem. 115 (1981) 203–211. C.E. Matz, A. Jonas, Micellar complexes of human apolipoprotein A-I with phosphatidylcholines and cholesterol prepared from cholate-lipid dispersions, J. Biol. Chem. 257 (1982) 4535–4540. Y. Takahashi, M. Miyata, P. Zheng, T. Imazato, A. Horwitz, J.D. Smith, Identification of cAMP analogue inducible genes in RAW264 macrophages, Biochim. Biophys. Acta 1492 (2000) 385–394. P. Zheng, A. Horwitz, C.A. Waelde, J.D. Smith, Stably transfected ABCA1 antisense cell line has decreased ABCA1 mRNA and cAMP-induced cholesterol efflux to apolipoprotein AI and HDL, Biochim. Biophys. Acta 1534 (2001) 121–128. H. Liu, D.G. Scraba, R.O. Ryan, Prevention of phospholipase-C induced aggregation of low density lipoprotein by amphipathic apolipoproteins, FEBS Lett. 316 (1993) 27–33. J.A. Morrow, M.L. Segall, S. Lund-Katz, M.C. Phillips, M. Knapp, B. Rupp, W.H. Weisgraber, Differences in stability among the human apolipoprotein E isoforms determined by the amino-terminal domain, Biochemistry 39 (2000) 11657–11666. M.A. Andrade, P. Chacon, J.J. Merelo, F. Moran, Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network, Protein Eng. 6 (1993) 383–390.

[30] L.B. Vitello, A.M. Scanu, Studies on human serum high density lipoproteins. Self-association of apolipoprotein A-I in aqueous solutions. J. Biol. Chem. 251 (1976) 1131–1136. [31] Y. Huang, A. Von Eckardstein, S. Wu, C. Langer, G. Assmann, Generation of pre-beta 1-HDL and conversion into alpha-HDL. Evidence for disturbed HDL conversion in Tangier disease, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 1746–1754. [32] O. Chambenoit, Y. Hamon, D. Marguet, H. Rigneault, M. Rosseneu, G. Chimini, Specific docking of apolipoprotein A-I at the cell surface requires a functional ABCA1 transporter, J. Biol. Chem. 276 (2001) 9955–9960. [33] J.D. Smith, C. Waelde, A. Horwitz, P. Zheng, Evaluation of the role of phosphatidylserine translocase activity in ABCA1-mediated lipid efflux, J. Biol. Chem. 277 (2002) 17797–17803. [34] S. Roosbeek, F. Peelman, A. Verhee, C. Labeur, H. Caster, M.F. Lensink, C. Cirulli, J. Grooten, C. Cochet, J. Vandekerckhove, A. Amoresano, G. Chimini, J. Tavernier, M. Rosseneu, Phosphorylation by protein kinase CK2 modulates the activity of the ATP binding cassette A1 transporter, J. Biol. Chem. 279 (2004) 37779–37788. [35] W.K. Surewicz, R.M. Epand, H.J. Pownall, S.W. Hui, Human apolipoprotein A-I forms thermally stable complexes with anionic but not with zwitterionic phospholipids, J. Biol. Chem. 261 (1986) 16191–16197. [36] R.M. Epand, A. Gawish, M. Iqbal, K.B. Gupta, C.H. Chen, J.P. Segrest, G.M. Anantharamaiah, Studies of synthetic peptide analogs of the amphipathic helix. Effect of charge distribution, hydrophobicity, and secondary structure on lipid association and lecithin:cholesterol acyltransferase activation, J. Biol. Chem. 262 (1987) 9389–9396. [37] P. Kanellis, A.Y. Romans, B.J. Johnson, H. Kercret, R. Chiovetti Jr., T.M. Allen, J.P. Segrest, Studies of synthetic peptide analogs of the amphipathic helix. Effect of charged amino acid residue topography on lipid affinity, J. Biol. Chem. 255 (1980) 11464–11472. [38] R.M. Epand, W.K. Surewicz, D.W. Hughes, H. Mantsch, J.P. Segrest, T.M. Allen, G.M. Anantharamaiah, Properties of lipid complexes with amphipathic helix-forming peptides. Role of distribution of peptide charges, J. Biol. Chem. 264 (1989) 4628–4635. [39] S.E. Panagotopulos, S.R. Witting, E.M. Horace, D.Y. Hui, J.N. Maiorano, W.S. Davidson, The role of apolipoprotein A-I helix 10 in apolipoproteinmediated cholesterol efflux via the ATP-binding cassette transporter ABCA1, J. Biol. Chem. 277 (2002) 39477–39484. [40] A. Chroni, T. Liu, I. Gorshkova, H.Y. Kan, Y. Uehara, A. Von Eckardstein, V.I. Zannis, The central helices of ApoA-I can promote ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux. Amino acid residues 220–231 of the wild-type ApoA-I are required for lipid efflux in vitro and high density lipoprotein formation in vivo, J. Biol. Chem. 278 (2003) 6719–6730. [41] M.N. Palgunachari, V.K. Mishra, S. Lund-Katz, M.C. Phillips, S.O. Adeyeye, S. Alluri, G.M. Anantharamaiah, J.P. Segrest, Only the two end helixes of eight tandem amphipathic helical domains of human apo A-I have significant lipid affinity. Implications for HDL assembly, Arterioscler. Thromb. Vasc. Biol. 16 (1996) 328–338. [42] M.N. Oda, T.M. Forte, R.O. Ryan, J.C. Voss, The C-terminal domain of apolipoprotein A-I contains a lipid-sensitive conformational trigger, Nat. Struct. Biol. 10 (2003) 455–460. [43] D.L. Sparks, M.C. Phillips, S. Lund-Katz, The conformation of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles, J. Biol. Chem. 267 (1992) 25830–25838. [44] J.D. Smith, W. Le Goff, M. Settle, G. Brubaker, C. Waelde, A. Horwitz, M.N. Oda, ABCA1 mediates concurrent cholesterol and phospholipid efflux to apolipoprotein A-I, J. Lipid Res. 45 (2004) 635–644.