Abstract
Human apolipoprotein E (apoE) mediates high affinity binding to the low density lipoprotein receptor when present on a lipidated complex. In the absence of lipid, however, apoE does not bind the receptor. Whereas the x-ray structure of lipid-free apoE3 N-terminal (NT) domain is known, the structural organization of its lipid-associated, receptor-active conformation is poorly understood. To study the organization of apoE amphipathic alpha-helices in a lipid-associated state, single tryptophan-containing apoE3 variants were employed in fluorescence quenching studies. The relative positions of the Trp residues with respect to the phospholipid component of apoE/lipid particles were established from the degree of quenching by phospholipids bearing nitroxide groups at various positions along their fatty acyl chains. Four apoE3-NT variants bearing Trp reporter groups at positions 141, 148, 155, or 162 within helix 4 and two apoE3 variants containing single Trp at positions 257 or 264 in the C-terminal (CT) domain, were reconstituted into phospholipid-containing discoidal complexes. Parallax analysis revealed that each engineered Trp residue in helix 4 of apoE3-NT, as well as those in the CT domain of apoE, localized approximately 5 A from the center of the bilayer. Circular dichroism studies revealed that lipid association induces additional helix formation in apoE. Protease protection assays suggest the flexible loop segment between the NT and CT domains may transition from unstructured to helix upon lipid association. Taken together, these data support a model wherein the alpha-helices in the receptor-binding region and the CT domain of apoE align perpendicular to the fatty acyl chains of the phospholipid bilayer. In this alignment, the residues of helix 4 are arrayed in a positively charged, curved helical segment for optimal receptor interaction.
Highlights
Human apolipoprotein E mediates high affinity binding to the low density lipoprotein receptor when present on a lipidated complex
To address questions relating to the alignment and orientation of the receptor-binding region of the NT domain and lipidbinding sites in the CT domain of lipid-associated apolipoprotein E (apoE), we examined the relative ability of spatially defined nitroxidelabeled PL to quench the fluorescence of a panel of unique single Trp apoE3 variants in reconstituted high density lipoproteins
Experimental Strategy—Receptor binding analyses of genetically engineered and naturally occurring variants of apoE have demonstrated that residues in the region of residues 134 –150 of apoE3 are indispensable for receptor binding activity [1]
Summary
Amino acid side chains in the receptor-binding region upon interaction of the NT domain with lipid [17]. With regard to the CT domain, the precise molecular features responsible for high affinity lipid binding are not known, it is believed that CT domain interaction with lipids is mediated by putative amphipathic ␣-helices [1]. To address questions relating to the alignment and orientation of the receptor-binding region of the NT domain and lipidbinding sites in the CT domain of lipid-associated apoE, we examined the relative ability of spatially defined nitroxidelabeled PL to quench the fluorescence of a panel of unique single Trp apoE3 variants in reconstituted high density lipoproteins (rHDL). It was determined that the engineered Trp residues in helix 4 align uniformly, close to the center of the PL bilayer, thereby presenting the receptor-binding site as a positively charged, curved helical segment analogous to that displayed on spherical lipoprotein particles
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