The FASEB Journal express article 10.1096/fj.02-1181fje. Published online Sept. 18, 2003.

One site on the apoB-100 specifically binds 17-β-estradiol and regulates the overall structure of LDL Roberto Brunelli,*,† Giulia Greco,† Mario Barteri,‡ Ewa K. Krasnowska,† Giampiero Mei,§ Fausta Natella,║ Alessandro Pala,* Simona Rotella,‡ Fulvio Ursini,¶ Lucio Zichella,* and Tiziana Parasassi†

*Dipartimento di Scienze Ginecologiche, Perinatologia e Puericultura, Universita’ di Roma La Sapienza, Roma; †Istituto di Neurobiologia e Medicina Molecolare, CNR, Roma; ‡Dipartimento di Chimica, Universita’ di Roma La Sapienza, Roma; §Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Universita’ di Roma Tor Vergata, Roma; ║Istituto Nazionale per la Ricerca sugli Alimenti e sulla Nutrizione, Roma; and ¶Dipartimento di Chimica Biologica, Universita’ di Padova, Padova, Italy

Corresponding author: Tiziana Parasassi, Istituto di Neurobiologia e Medicina Molecolare, CNR, Viale Marx 15-43, 00137 Roma, Italy. E-mail: t.parasassi@in.rm.cnr.it

ABSTRACT

The major protein component (apoB-100) of low-density lipoprotein (LDL) is known as a multipotential molecule the several functional regions of which can all be affected by key structural modifications driven by specific domains. Based on our previous report on structural and conformational modifications of apoB-100 in the presence of 17-β-estradiol (E2), we characterized the interaction between E2 and the apoB-100 and further explored the induced alterations in terms of the structural arrangement of the whole LDL particle. We report evidence for the existence on apoB-100 of a single specific and saturable binding site for E2, the occupancy of which modifies the overall structure of the protein, inducing an increase in the α-helix fraction. As a consequence, the structure of the LDL particle is deeply perturbed, with a change in the arrangement of both the outer shell and lipid core and an overall volume shrinkage. The evidence of a regulation of apoB-100 structure by a physiological ligand opens new perspectives in the study of the biological addressing of the LDL particle and suggests a novel rationale in the search for mechanisms underlying the beneficial role of E2 in decreasing the risk of early lesions in atherosclerosis.

Key words: electronegative LDL • equilibrium dialysis • circular dichroism • SAXS

he protein component in low density lipoprotein (LDL), apoB-100, is a moderately hydrophobic, extraordinarily large protein, consisting of 4536 aminoacid residues, that plays a unique role in lipid transport. In addition, apoB-100 directs the biosynthesis of the

triglyceride rich lipoproteins and regulates the conversion of a portion of the very low-density lipoprotein remnants to LDL (1). During this process, conformational changes in apoB-100 result in the formation of a receptor binding site the recognition of which by the hepatic LDL receptor is critical for the maintenance of serum LDL level.

T

Computational analysis of the apoB-100 revealed a pentapartite structure composed of three α-helical and two β-structured domains (2). The structure of the α1-domain suggests that it may act as an anchor for the protein to the lipid environment at early stages of synthesis (1). The α2-domain has been proposed to be the flexible lipid-associating domain that adjusts to the variation of the particle size (2). The α3-domain seems to regulate the binding to the LDL receptor by masking/unmasking the receptor binding site located in the β2-domain (3). The β-sheet regions of apoB-100 are key structural areas for the integrity of LDL particles and represent lipid-associating domains (4).

Several functional regions not involved in lipid metabolism have been identified in apoB-100 and legitimize the enormity of the molecule, including the attachment sites for apolipoprotein(a) (5), lipoprotein lipase (6), platelet activating factor acetylhydrolase (7), tromboplastin (8), and glycosaminoglycans (9). Evidence was also obtained that apoB-100 has a role in the control of gene expression and as a gene transfer vector (10) and can be a novel member of the SRC protein kinase family (11).

We recently reported evidence for a modification of apoB-100 structure and conformation when incubating LDL with the hormone 17-β-estradiol (E2; ref 12). The present study was designed to characterize the interaction between E2 and the apolipoprotein in LDL and to further explore the consequences on the whole particle arrangement. We used freshly prepared LDL and the in vivo minimally modified electronegative LDL- (13), which shows the loss of most α-helix structures. We found that one E2 molecule binds to each apoB-100 to a specific site and modifies the dimension and the overall structure of the LDL particle, highlighting an endogenous mechanism for the modulation of LDL properties.

METHODS

Preparation of LDL

Venous blood was obtained from normal fasting adult volunteers, collected into vacutainer test tubes containing ethylenediamine tetraacetic acid (EDTA, Sigma Chemical Co., St. Louis, MO), and immediately centrifuged at 1500 g for 10 min at 4°C. LDL was separated by a short run ultracentrifugation following described procedures (12) and dialyzed overnight against Chelex-treated argon-purged phosphate buffer, pH 7.4 (PBS, Sigma). After dialysis, LDL was immediately used. The purity of the isolated LDL was checked by SDS gradient (3-15%) PAGE under nonreducing conditions and stained with Comassie brilliant blue.

Separation of LDL minus (LDL-) from bulk LDL was accomplished using anion exchange high-pressure liquid chromatography (Perkin Ellmer Series 4 HPLC), as described previously (13). Fractions containing LDL- were pooled and concentrated, and salts were removed by overnight dialysis against Chelex-treated argon-purged PBS and immediately used.

Equilibrium dialysis

Purity of 2,4,6,7-3H(N)17-β-estradiol ([3H]E2, specific activity 4.4 TBq/mmol, NEN, Boston, MA) was assessed by liquid chromatography through a 1 × 15 cm column of Sephadex LH-20 (Amersham Biosciences, Germany) equilibrated and eluted in methanol/toluene 15/85 (v/v).

Peak fraction was dried under nitrogen, dissolved in ethanol, and stored at –15°C. The E2 (Sigma) stock solution in ethanol (1 mg/ml) was diluted with argon-purged PBS to final concentrations ranging from 0.02 to 1 µM. Aliquots of [3H]E2 for a total radioactivity of 20,000 dpm were dried into test tubes, then the different concentrations of unlabeled E2 in PBS were added, and the volume was adjusted to 2.5 ml. Freshly prepared LDL (0.1 µM in 2.5 ml of argon-purged PBS) was dialyzed against the various [3H]E2/E2 samples, for 72 h, at 4°C, under mild shaking. Specificity of the binding was assessed by diluting the 0.1 µM E2 solution with estrone (E1, Sigma) solutions ranging from 0.02 to 5 µM. As detected by SDS PAGE, there was no detectable degradation of apoB-100 during equilibrium binding. The [3H]E2 concentration was determined by scintillation counting. The data have been represented as Scatchard plots, fitted according to one class of equivalent binding site model (14)

][1][ Lknk

L +=

ν (1)

where ν represents the moles of bound E2 per mole of protein, [L] is the free E2 concentration, n is the number of binding sites, and k is the binding constant of each class of sites. The fits were performed using the SPW 1.0 version of the Sigma Plot scientific graphing software.

Circular dichroism

Circular dichroism (CD) spectra of LDL and of LDL- (0.1 µM) were recorded on a Jasco J-710 (Jasco International, Tokyo, Japan) spectropolarimeter equipped with a software for data processing in the far UV (200-250 nm), using 50 mdegree cm-1 sensitivity and 0.1 cm path-length quartz cuvette. To increase the signal-to-noise ratio, six spectra were averaged for each measurement and the blank was subtracted. The cell holder compartment was maintained at 37 ± 0.1°C by a circulating water bath.

Synchrotron radiation small angle X-ray scattering

Samples of 2.4 µM LDL, with or without 24 µM E2, were maintained under argon atmosphere until used for synchrotron radiation small angle X-ray scattering (SR-SAXS) experiments. LDL was prepared from pooled blood of three donors. SR-SAXS data were recorded at the beam line D24 of the DCI Synchrotron Facility of L.U.R.E. (Orsay, France), at 25°C. The storage ring was operated with electron beam energy of 1.85 GeV and an injected current of 200 mA. The X-ray wavelength (λ=0.1488 nm, K-edge of nickel; ∆λ/λ =10−3) was selected with a Ge (111) bent crystal. The position-sensitive proportional detector was 1881 mm away from the sample with a corresponding channel width of ∆k = 9.5 10−2 nm-1. Scattering intensities were determined as a function of the modulus of the scattering vector k = 4πsinθ/λ, where θ is the scattering angle and λ is the X-ray wavelength. Due to a possible damaging effect of the X-ray beam, we collected scattering data using a flux cell that reduces the sample exposure to few seconds. Scattering data were acquired in the angular range of 0.09 < k < 3 nm-1, while our calculations were limited to the range 0.3 < k < 2 nm-1 to avoid misleading contributions due to interparticle interactions at low k values.

Glatter’s procedure was used to compute the distance distribution function P(r) of the samples under investigation. P(r) is the inverse Fourier transform of the scattering intensities and can be calculated by

dkkrkrkIrP )sin()(2

1)(02 ∫∞

=π

(2)

where r is the distance between electron pairs. This function gives further information on the overall structure of the particle, particularly on the distribution of electrons (15).

The radius of gyration (Rg) was calculated by (15)

drrP

drrrPR

D

D

g)(2

)(

max

max

0

202

∫

∫= (3)

RESULTS

E2 binds to a single site in apoB-100 that is saturable, specific, and dependent on the maintenance of a native protein structure

The data on the binding between E2 and freshly isolated LDL are satisfactorily fitted according to a single class of equivalent binding site model (Eq. 1; Fig. 1A). A linear Scatchard plot (inset of Fig. 1A) was indeed obtained by plotting the data as a function of bound E2 (ν). The fit yielded one binding site per LDL particle (n=0.8±0.1) with a relatively high binding constant [k=(1.7 ± 0.2)×106]. The binding specificity for E2 was assessed by a competition experiment using the parent hormone E1 that differs from E2 for the substitution of a ketone residue to the 17-hydroxyl group of the D ring. Increasing concentrations of E1 did not displace E2 up to a molar ratio between E1 and E2 of 50:1 (open square in the plot of Fig. 1A). When the electronegative LDL- was used, where the apoB-100 is misfolded (see below), no specific binding of E2 was observed and the plot was parallel to the x-axis, suggesting instead its hydrophobic partitioning (Fig. 1B; refs 16–17).

E2 binding specifically modifies the structure of apoB-100

When in the presence of E2, CD spectra of apoB-100 in freshly isolated LDL showed an increase in the α-helix fraction, these changes being dose dependent (Fig. 2). This result was in agreement with our previous report (12).

Also in the CD measurements, E2 specificity was assessed by using E1. We could not observe any modification in the CD spectra when using E1:apoB-100 molar ratio up to 100:1. This result is particularly significant when considering that E1 associates to the LDL particle with greater affinity than E2 (18), therefore, again, not to the same E2 site.

The peculiar feature of electronegative LDL- is a dramatically altered protein structure (Fig. 2). Indeed, both the absolute decrease in θ and the broadening of the dichroic band indicated an increase in the relative amount of β-sheet structure. This misfolded apoB-100 was not affected by E2 for molar ratios to apoB-100 up to 100:1, i.e., 10 times higher than the ratio that granted the greatest effect with LDL (Fig. 2). Therefore, the hydrophobic partitioning of E2 into LDL could not further modify the protein structure. As expected, E1 could not modify the CD spectra of apoB-100 in LDL- (not shown).

Modified structure and reduced dimension of LDL upon E2 binding

The present technology in synchrotron X-ray scattering allows the achievement of highly defined data on dilute particles. As for experiments on LDL particles, X-ray scattering can monitor the structure of the lipid core, of the interfacial shell, and of the protein and phospholipid head groups surface shell (19–20), without radiation damage effects (21). In Fig. 3A, we report the experimental small angle X-ray scattering data of a 2.4 µM LDL solution with and without 24.0 µM E2. We used an E2 to apoB-100 ratio that granted the highest structural effect in our CD experiments and corresponded to a protein saturation of 78%, as determined by the calculated binding constant. The scattering curve obtained for LDL displays relevant modifications due to the presence of E2. In particular, all submaxima appear reduced in intensity.

The P(r) curves of LDL, with and without E2, are reported in Fig. 3B. In the absence of E2, the P(r) curve well reproduces that previously reported by other groups (19–21) with 1) low r values, up to 7.5 nm, corresponding to the cholesteryl ester core (region A in Fig. 3B); 2) a negative region, corresponding to the hydrocarbon chain of phospholipids in the outer monolayer (region B in Fig. 3B); and, finally, 3) the positive peaks at 21 and 23 nm, corresponding to the protein and to phospholipid head groups on the surface of LDL (region C in Fig. 3B). The presence of E2 causes several modifications to all regions of the P(r) curve, suggesting that the hormone, upon binding to the protein, modifies the overall structure of the particle. In particular, the region C shows a better peak resolution and noticeable shape modifications, with a general shift toward lower r values. The interfacial region B is modified by the appearance of a relatively intense peak, centered at ~14 nm. Surprisingly, the core region A also shows different relative intensities of the peaks.

These modifications in the P(r) plot well fit the variation of the particle volume. From the scattering data, we derived the plot of I(k)k2 vs. k (Kratky plot; ref 15, not shown), and its integral was used to determine the particle volume. In our conditions, corresponding to 78% of the protein carrying bound E2, we obtained a 12% decrease of the LDL volume. On the opposite, the calculated Rg value was unaffected by E2 binding, being 14.1 ± 0.5 and 13.8 ± 0.5 nm in the absence and presence of the hormone, respectively. Apparently, the structural modifications of this nonspherical particle are not dependent on the Rg but on the cross section and on the thickness.

DISCUSSION

The analysis of our equilibrium dialysis data show that, at the equilibrium, each LDL particle binds one E2 molecule through a protein site that is saturable, specific, and dependent on the maintenance of the structural integrity of the apoB-100. The binding constant is relatively high,

i.e., two orders of magnitude higher than that between E2 and albumin (16). This specific binding of a small molecule to a single site on a huge multipotential protein certainly represents a surprising finding, particularly for its profound consequences in affecting all LDL structures. A previous equilibrium dialysis study on the interaction between E2 and LDL suggested that E2 simply partitioned into LDL due to its hydrophobicity (16). However, these results suffered from methodological problems related to the long isolation procedure, to the incubation performed at 37°C that may have altered the protein structure for the occurrence of oxidative modifications (22), and to the high concentration of [3H]E2 that considerably contributed to the total E2 concentration, therefore decreasing the sensitivity of the system with respect to the tracer displacement. Consistently, in our experiments no binding was observed when using the misfolded LDL-.

The structure of apoB-100 is specifically modified as a consequence of E2 site occupancy with a dose-dependent fractional increase in the α-helix structure. Indeed, 1) no structural effect could be observed on the apoB-100 when using E1, and 2) the site loss in LDL- precluded any structural effect by the hydrophobically partitioned E2.

SR-SAXS offers detailed information on the structure of LDL particles in solution. We obtained well-resolved data, in good agreement with previous reports. Following the most recent consensus model for the LDL structure (4), the protein component of LDL, apoB-100, resides in the outer surface LDL layer together with phospholipid polar heads (Fig. 3B, region C) and seeps through the interfacial layer down to the core (Fig. 3B, region B) where its amphipathic β-sheet domains organize the so called lipid-core ridges. These structures can drive phase changes in the cholesterol ester core (Fig. 3B, region A; ref 4).

E2 binding to apoB-100 induces several modifications in all LDL layers. Although the equilibrium dialysis experiment showed that E2 binds to a single site in the apoB-100, the SAXS profile shows that the overall outer layer is affected. In Fig. 3B, we evidenced in pink all areas with an r reduction in the region C, this modification being in agreement with the reduction in volume. Apparently, when E2 is bound to its site in apoB-100, all results converge to a picture of an increased conformational packing of the particle, with a shrinkage that well fits the fractional increase in α-helix observed by CD. Indeed, published data show that with the reduction in particle size there is an increase in the number of amphipathic helices of the α2- and α3-domains (4). Based on the model proposed by Hevonoja et al. (2), in which the particle size can be regulated by a spring-like structure formed by the α-helices of the α2 domain, we can hypothesize that E2 binding affects a similar dynamic structure.

Relevant modifications due to E2 binding can also be observed in the interfacial region B, which can be either attributed to a modified structure of the protein itself or of the interfacial lipids, possibly through the action of apoB-100 β-sheets in the lipid-core ridges (4).

Finally, modifications of the lipid core after E2 binding to the protein demonstrate that the regulatory role of apoB-100 extends to the most internal LDL layer, as previously suggested (4).

ApoB-100 is a multipotential molecule with a role in signaling processes (10) and has several functional regions (8, 23–26) that can all be affected by key modifications of selected protein domains. We did not explore the functional consequences of the new structural arrangement of

the protein. Nevertheless, for the relevance of the observed changes we can predict that several of these functions will be affected by E2 binding.

Most reasonably, the structural modifications of the protein also account for the effect of E2 in delaying the LDL in vitro oxidation (18). Fluorescence spectroscopy (12) and SAXS measurements (21) demonstrated that the initial stages of Cu2+-mediated oxidation occur in the outer interfacial layer that we find deeply modified by the E2 binding to apoB-100. In light of our results, E2 antioxidant effect is seen as a consequence of a modified arrangement of this interface, able to hamper the initial oxidation stages (12). The evidence that E1 does not have any structural effect on the apoB-100 is paralleled by the result that E1 does not show any antioxidant protection (18) despite its high affinity to LDL and the presence of a phenolic group.

We can hypothesize that E2 delays the formation of insoluble aggregates composed of oxidatively modified LDL (27). Indeed, the increased fraction of α-helices may hinder the interaction between β-sheets that was proposed to lead to this particle aggregation and to the fusion of lipids into large droplets in the subendothelial space (28–30). Finally, given the regulation of LDL uptake by cell receptors through the structure of LDL particle itself (31), we predict that the shrinkage of apoB-100 induced by E2 will affect the uptake and the metabolic fate of LDL in cells.

Although quite a limited number of LDL have been calculated to carry E2 in circulating plasma (32), the physiological relevance of E2 binding to apoB-100 in the early prevention of atheromatous lesions cannot be evaluated other than by considering the local concentration of both E2 and LDL in the subendothelial space, i.e., the body district specific for the onset of atherosclerosis. Although a calculation of the actual ratio between E2 and LDL in the subendothelial space is not possible, we can argue that it is most likely much more in favor of E2 than in plasma. The number of LDL particles in the subendothelial compartment must be definitely lower than in plasma since little plasma volume permeates toward the subendothelial compartment in the range between 5 and 100 nl/cm2/h (33). Smooth muscle cells in the media synthesize estrogens from the circulating supply of androgen precursors and yield concentrations of these hormones higher than those present in plasma via the estrogen-producing enzymes, aromatase, and 17-β-hydroxy-steroid dehydrogenase (34–35). The relevance of focusing on local concentrations rather than in plasma can be also evidenced by the reported value of 8 × 10−9 M for the dissociation constant of the estrogen receptor in human subendothelial smooth muscle cells (36). When considering that the concentration of E2 in the adult human serum usually ranges between 3 × 10−11 and 2 × 10−9 M, this last being the transient value in the preovulatory peak, the average estrogen concentration in the blood is obviously not sufficient to efficiently activate the E2 receptor-related functions in vascular cells (37).

In conclusion, we highlighted an endogenous mechanism for the modulation of the LDL structure and dimension through the interaction of E2 with apoB-100. This result is particularly significant because no other hormones were reported to modify LDL or, with the exception of thyroxine (38–39), to specifically bind to apoB-100. Our findings open new perspectives in the understanding of the complex physiology of the LDL particle and, in the view of a beneficial role of E2 in reducing the early onset of atheromatous lesions, may suggest novel routes in the search of underlying mechanisms, with the rationale of a modified LDL metabolism, of modulation of apoB-100 functions, and of a delay in the protein misfolding cascade (27).

ACKNOWLEDGMENTS

This study was supported by INDENA SpA, Milano, Italy (G. Greco, T. Parasassi) and by the Training and Mobility of Researchers Program of the European Community for the SAXS experiments at L.U.R.E., France (M. Barteri, S. Rotella). We thank Dr. Patrice Vachette for help during the SAXS experiments.

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Received February 21, 2003; accepted July 18, 2003.

Fig. 1

Figure 1. Scatchard plots of E2 binding to apoB-100. A) Binding data, averaged over 3 separate experiments obtained using LDL, fitted following Eq. 1. Also plotted is the value obtained in the presence of 5 µM E1 (�). In the inset, the data are plotted as a function of moles of bound E2 per mole of LDL. B) Data obtained using the electronegative subpopulation LDL-.

Fig. 2

Figure 2. Circular dichroism spectra of LDL and LDL- in the presence of hormones. Spectra of 0.1 µM LDL, at 37°C, in the absence (1, ___) and in the presence of 0.25 µM E2 (2, - - -), of 0.6 µM E2 (3, ....), of 1 µM E2 (4, __ __), and of

10 µM E1 (5, ���). Spectra of 0.1 µM LDL- (6, ...) in the presence of 10 µM E2 (7, ���).

Fig. 3

Figure 3. A) Synchrotron radiation small angle X-ray scattering of LDL (black) and of LDL in the presence of E2 (red). B) Distance distribution function [P(r)] of LDL (black) and of LDL in the presence of E2 (red). In A and B, the differences between the functions in the absence and in the presence of E2 are evidenced in pink. See the text for the description of regions A, B, and C.