The long-term goal of our research is to understand how lipid transport and metabolism are regulated by molecular interactions between lipoproteins and cell surface receptors. Knowledge of the metabolic roles of apolipoprotein E (apoE) and the low density lipoprotein receptor (LDLR) will be combined with available structural information in the design of an experimental strategy to dissect the determinants of a productive receptor-ligand interaction. In addition studies will be pursued to elucidate the mechanism of ligand release from the receptor. It is postulated that apolipoprotein E (apoE) undergoes a lipid binding induced conformational change that results in extension of helix 4 beyond the boundary identified in its lipid-free helix bundle state. We hypothesize that this helix extension is a critical step that permits alignment of positively charged amino acids such that they adopt a conformation that promotes interaction with ligand binding repeats of the LDLR. Electron paramagnetic spin resonance and NMR spectroscopy analysis of specifically labeled apoE will be performed in the absence and presence of lipid, permitting direct assessment of a postulated structural transition from random coil (lipid-free state) to alpha helix (lipid associated state) in this region of the protein. It is further postulated that termination of helix 4 in lipid-free apoE is caused by a sequence specific termination signal that can be overcome by lipid association. Site directed mutagenesis studies will be performed to disrupt a putative helix cap motif and the effect on apoE helix 4 termination investigated. The hypothesis that disruption of the helix cap motif will result in helix extension and adoption of a receptor active conformation in the absence of lipid will be tested in receptor binding assays. In other studies a soluble fragment of the LDLR encompassing its seven complement-like ligand binding, LDL-A repeats and the epidermal growth factor precursor homology domain, including its a-propeller segment, will be employed in ligand binding and release studies with apoE. LDL-A repeat swapping experiments and modification of the spacer sequences between repeats will be used to determine the molecular requirements for a productive interaction with apoE containing reconstituted high density lipoprotein ligands. The hypothesis that a pH-dependent intra-molecular interaction between the a-propeller segment and specific LDL-A repeats is responsible for ligand release will be examined. The results of these experiments will extend our understanding of the LDLR pathway, apolipoprotein-mediated lipoprotein metabolism and plasma cholesterol homeostasis. Based on the fact that aberrations in the LDLR pathway are positively correlated to onset of cardiovascular disease, we anticipate that new knowledge gained from these studies will provide insight into molecular mechanisms that regulate vascular cholesterol flux in health and disease.
Showing the most recent 10 out of 60 publications
