Apolipoprotein E (apoE) is crucial for lipid transport and cholesterol homeostasis within the plasma and central nervous system. Binding to lipoprotein particles activates apoE allowing it to interact with cell surface receptors such as the low density lipoprotein receptor (LDLr). This mediates the clearance of apoE containing lipoproteins through an endocytosis pathway and therefore reduces plasma cholesterol level, explaining the strong anti-atherogenic effect of apoE. Nevertheless, the active conformation of apoE, critical for the receptor interaction, remains elusive. Since high resolution structure-determination methods (i.e. NMR and X-ray crystallography) are not yet applicable to large protein/lipid complexes, we are actually in need of biophysical techniques to solve the structure-activity relationship of lipidated apoE. The aim of this work was to elucidate the lipidated structure of apoE. Therefore, we reconstituted apoE/lipid particles mimicking a subspecies of high density lipoproteins (HDL) found in the plasma, starting from recombinant apoE and synthetic lipids (POPC). After optimization, a homogeneous population of reconstituted apoE/POPC lipoproteins was obtained. A thorough characterization by different biochemical methods (native PAGE, electron microscopy, FTIR…) showed that these lipoprotein particles bear 2 apoE molecules at their surface and display a discoidal shape with a diameter of 105Å. Further structural information was gathered by using a combination of lysine-directed cross-linking and mass spectrometry. We then developed a two-step modeling procedure using all the structural knowledge acquired at this point enriched with literature data to build 3D atomic resolution structural models of the apoE4/POPC complexes. First, monomeric apoE models were produced in the absence of lipids using a distance calculation program. These initial models, where apoE is folded into a helical hairpin, should reflect the conformation adopted by the protein on a disc-shaped lipoprotein. Secondly, two modeled monomeric conformations were dimerized, associated with a lipid disc, solvated and finally subjected to a molecular dynamics (MD) simulation. Our MD trajectories show an elongation of a helix in the N-terminal domain that redefines the position of Arg172, a key residue for LDLr binding, in regards to the well-defined receptor binding region (residues 136-150) of apoE. The observed elongation explains why the lipidation of apoE is an essential requirement in the activation mechanism and offers an ideal framework for the recognition of two specific LDLr ligand binding repeats needed for full binding to the LDLr. Based on an important structural change observed in our different models, we can also propose an activation mechanism relying on a regulation of the accessibility of the ligand binding region by the protein itself. Thus, the two new presented models provide important structural information at the atomic level and new insights into the mechanisms involved in apoE receptor recognition. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished
| Identifer | oai:union.ndltd.org:ulb.ac.be/oai:dipot.ulb.ac.be:2013/209192 |
| Date | 21 November 2014 |
| Creators | Henry, Nicolas |
| Contributors | Raussens, Vincent, Vandenbussche, Guy, Prévost, Martine, Deroo, Stéphanie, Gilis, Dimitri, Gosselet, Fabien |
| Publisher | Universite Libre de Bruxelles, Université libre de Bruxelles, Faculté des Sciences – Chimie, Bruxelles |
| Source Sets | Université libre de Bruxelles |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:ulb-repo/semantics/doctoralThesis, info:ulb-repo/semantics/openurl/vlink-dissertation |
| Format | 1 v. (124 p.), No full-text files |
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