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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 18, 2003 as doi:10.1096/fj.02-1181fje. |
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,2
* Dipartimento di Scienze Ginecologiche, Perinatologia e Puericultura, Università di Roma La Sapienza;
Istituto di Neurobiologia e Medicina Molecolare, CNR;
Dipartimento di Chimica, Università di Roma La Sapienza;
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma Tor Vergata;
|| Istituto Nazionale per la Ricerca sugli Alimenti e sulla Nutrizione, Roma, Italy; and
Dipartimento di Chimica Biologica, Università di Padova, Padova, Italy
2Correspondence: Istituto di Neurobiologia e Medicina Molecolare, CNR, Viale Marx 15-43, 00137 Roma, Italy. E-mail: t.parasassi{at}in.rm.cnr.it
SPECIFIC AIMS
Based on our previous report on structural and conformational modifications of apoB-100 in the presence of 17-ß-estradiol (E2), we characterized the interaction between this hormone and the apoB-100 and explored the induced alterations in terms of the structural arrangement of the whole LDL particle. We report evidence for the existence on the apoB-100 of a single specific and saturable binding site for E2, whose occupancy modifies the overall structure of the protein, inducing an increase in the 
-helix fraction.
PRINCIPAL FINDINGS
1. E2 binds to a single site in apoB-100 that is saturable, specific, and dependent on the maintenance of a native protein structure
Data on the binding between E2 and freshly isolated LDL are satisfactorily fitted according to a single class of equivalent binding site model (Fig. 1
A). A linear Scatchard plot (Fig. 1A
, inset) was obtained by plotting the data as a function of bound E2 (
). 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)x106] two orders of magnitude higher than that between E2 and albumin. The binding specificity for E2 was assessed by a competition experiment using the parent hormone estrone (E1), which 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 (Fig. 1A
). When the electronegative LDL- was used, where the apoB-100 is misfolded, no specific binding of E2 was observed, the plot parallel to the x-axis suggesting instead its hydrophobic partitioning (Fig. 1B
).
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2. E2 binding specifically modifies the structure of apoB-100
In the presence of E2, circular dichroism (CD) spectra of apoB-100 in freshly isolated LDL showed an increase in the -helix fraction that was dose dependent in the range between 1:1 and 10:1 (E2:apoB-100). This agrees with our previous report.
We also assessed E2 specificity 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 is significant when considering that E1 associates to the LDL particle with greater affinity than E2, therefore not to the same E2 site.
The peculiar feature of electronegative LDL- is a dramatically altered protein structure. Indeed, the absolute decrease in 
and the broadening of the dichroic band both indicated an increase in the relative amount of ß-sheet structure. This misfolded apoB-100 was not affected by E2 for molar ratios to apoB-100 up to 100:1. Therefore, the hydrophobic partitioning of E2 could not further modify the protein structure. As expected, E1 could not modify the CD spectra of apoB-100 in LDL-.
3. Modified structure and reduced dimension of LDL upon E2 binding
In Fig. 2
A 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 the E2 to apoB-100 ratio granting the highest structural effect in our CD experiments and corresponding to a protein saturation of 78%. The scattering curve obtained for LDL displays relevant modifications due to the presence of E2. All submaxima appear reduced in intensity.
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The P(r) curves of LDL, with and without E2, are reported in Fig. 2B
. In the absence of E2, the P(r) curve reproduces that previously reported by other groups with 1) low r values, up to 7.5 nm, corresponding to the cholesteryl ester core (region A in Fig. 2B
); 2) a negative region corresponding to the hydrocarbon chain of phospholipids in the outer monolayer (region B in Fig. 2B
); and 3) positive peaks at 21 nm and 23 nm corresponding to the protein and to phospholipid head groups on the surface of LDL (region C in Fig. 2B
). The presence of E2 imposes several modifications to all regions of the P(r) curve, suggesting that upon binding to the protein, the hormone modifies the overall structure of the particle. 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, 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); 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. The calculated value of the radius of gyration, Rg, was unaffected by E2 binding, being 14.1 ± 0.5 nm 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 the thickness.
CONCLUSIONS AND SIGNIFICANCE
The specific binding of a small molecule such as E2 to a single site on a huge multipotential protein represents a surprising finding, especially for its profound consequences in affecting the whole LDL structure.
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.
When E2 is bound to its site in apoB-100, all results converge to a picture of an increased conformational packing of the protein, with a shrinkage that fits the fractional increase in -helix. On the basis of a previous computational modeling study in which particle size can be regulated by a spring-like structure formed by the -helices of the 2 domain, we can hypothesize that E2 binding affects a similar dynamic structure able to influence the overall protein arrangement.
Relevant modifications due to E2 binding can also be observed in the interfacial region B that can be attributed to a modified structure of the protein itself or of the interfacial lipids, possibly through the action of apoB-100 ß-sheets in the lipid core ridges.
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.
The regulation of apoB-100 structure by a physiological ligand opens new perspectives to studying the general pathophysiology of LDL particles. ApoB-100 has been described as a multipotential molecule with a role in signaling processes whose several functional regions 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, we can predict that several of these functions will be affected by E2 binding. The protein’s structural modifications also account for the effect of E2 in delaying the LDL in vitro oxidation. The evidence that E1 does not have any structural effect on the apoB-100 is paralleled by the observation that E1 does not show any antioxidant protection despite its high affinity to LDL and the presence of a phenolic group. The physiological relevance of E2 binding to apoB-100 in the early prevention of atheromatous lesions can be evaluated by considering the local concentration of E2 and LDL in the subendothelial space, i.e., the body district specific for the onset of atherosclerosis. The subendothelial space represents a specific district of E2 synthesis and metabolism, therefore with high E2 concentration.
We can hypothesize that E2 delays the formation of insoluble aggregates composed of oxidatively modified LDL. The increased fraction of -helices may hamper the interaction between ß-sheets that has been proposed to lead to this particle aggregation and to the fusion of lipids into large droplets in the subendothelial space. Given the regulation of LDL uptake by cell receptors through the structure of LDL particle itself, we predict that the shrinkage of the apoB-100 induced by E2 will affect the uptake and metabolic fate of LDL in cells.
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 is significant because no other hormones were reported to modify LDL or, with the exception of thyroxine, specifically bind to apoB-100. Our findings open new perspectives to understanding the complex physiology of the LDL particle that, in 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.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1181fje; doi: 10.1096/fj.02-1181fje 
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). Inset: data were plotted as a function of moles of bound E2 per mole of LDL. B) Data obtained using the in vivo minimally modified electronegative LDL-.