Apolipoprotein E

Apolipoprotein EApoE is a 34 kDa protein that contains 299 amino acids. Structural studies reveal that it is comprised of two independently folded domains. The N-terminal domain contains an amino acid sequence that is recognized by the low density lipoprotein (LDL) receptor family while the C-terminal domain displays a high lipid binding affinity. The two domains may be studied in isolation or in the context of the full length protein (Figure 1). Using the X-ray crystal structure of the N-terminal domain obtained in the absence of lipid as a guide, we are investigating the nature of specific conformational changes that accompany lipid binding. In buffer, the N-terminal domain exists as an elongated bundle of four amphipathic _-helices. Hydrophobic amino acids are sequestered in the bundle interior, providing an explanation for the known water solubility of this domain. It is hypothesized that lipid association requires a major conformational change wherein the hydrophobic interior of the helix bundle becomes available for direct interaction with lipid surface binding sites.

Our laboratory has established an efficient bacterial expression system to produce recombinant apolipoprotein and introduce specific mutations. Fluorescence spectroscopy techniques have been used to characterize lipid association induced conformational changes in apoE. Specifically, fluorescence resonance energy transfer between selected tryptophan residues in apoE and a covalently bound extrinsic acceptor molecule provides accurate, intra-molecular distance information. By creating mutant apoE molecules that contain a single tryptophan at specified locations in the protein, we are testing models of helix repositioning of the N-terminal domain upon lipid association. Information gained from these studies is critical in terms of understanding the molecular basis of apoE function as a ligand for the LDL receptor.

FIGURE 1. Model of full length apoE. The N-terminal domain four helix bundle is adapted from the X-ray crystal structure reported by Wilson et al. (1991). The structures of the C-terminal domain and the protease sensitive linker segment are not known and have been modeled here for illustration. The recognition sequence for members of the LDL receptor family (residues 134 ö 150) is shown in gold and the short helix connecting helix 1 and helix 2 of the bundle is shown in magenta.

In other studies we have investigated N and C terminal domain interactions in apoE. Our strategy is to characterize the nature of domain interactions in the presence and absence of lipid. A mutant apoE, engineered to possess a single tryptophan residue in the C-terminal domain has been used to study energy transfer between this site and an acceptor chromophore attached to cysteine 112 in the N-terminal domain. We have obtained direct evidence that the two domains reside in close proximity in the absence of lipid while evidence indicates that this juxtaposition is lost upon interaction with lipid.

Building on structural data obtained from studies of isolated protein domains, we are currently examining the hypothesis that metabolic regulation of apoE interaction with lipoprotein receptors is modulated by the conformation of the N-terminal domain. We postulate that apoE forms a stable interaction with the surface of circulating lipoproteins via its C-terminal lipid binding domain. This interaction effectively anchors the protein to the lipoprotein particle surface and localizes the N-terminal domain at the lipid interface. Depending on various metabolic factors, the N-terminal domain may retain its receptor-inactive helix bundle conformation or alter its structure to interact directly with the lipoprotein surface, with manifestation of receptor binding ability.

Apolipoprotein A-V

Recent comparative studies of human and mouse genome sequences have led to the discovery of a new member of the exchangeable apolipoprotein family. This protein, termed apolipoprotein A-V (apoA-V), was independently discovered in studies of the effect of partial hepatectomy on the up-regulation of liver genes. Transgenic and gene disruption experiments in mice revealed a correlation between apoA-V and plasma triacylglycerol (TG) levels. Given the strong relationship between elevated plasma TG and risk for cardiovascular disease, apoA-V is a potential target for therapeutic intervention. One obstacle preventing a fuller understanding of apoA-V function in lipoprotein metabolism or otherwise, however, is a lack of basic information about this protein. Up till now, apoA-V has not been isolated and, thus, little is known of its structure, stability, lipid binding properties or potential regulatory effects. Similar studies of other exchangeable apolipoproteins have complemented genetic studies, and provided fundamental insight into the biological role of these proteins in plasma lipid homeostasis. To investigate the structure and function relationship of human apoA-V, we have developed a bacterial expression system for production of recombinant protein. We postulate that knowledge gained from studies of apoA-V proposed herein will provide insight into the mechanism whereby increased apoA-V levels are correlated with decreased plasma TG. Toward this end we are working to 1) Characterize the stability and domain structure of lipid-free apoA-V. Recombinant apoA-V has been subjected to hydrodynamic studies and chemical cross-linking to evaluate its self-association properties. Far U.V. circular dichroism spectroscopy has been performed to characterize the secondary structure content and stability of apoA-V. Limited proteolysis and CNBr digestion studies are ongoing to determine the domain structure of apoA-V; 2) Study the lipid and lipoprotein binding properties of apoA-V. The kinetics of apoA-V interaction with phospholipid bilayer vesicles and the properties of apoA-V reconstituted lipoproteins has been determined by electron microscopy, gradient gel electrophoresis and compositional analysis. The relative lipid binding affinity of apoA-V is under investigation in apolipoprotein displacement experiments; 3) Evaluate apoA-V functional properties. We hypothesize that apoA-V manifests its biological effects on plasma TG levels by modulating lipoprotein lipase activity. Lipase activity assays performed with apoA-V are compared to control assays with the known physiological avtivator, apoC-II. The ability of apoA-V to counteract the inhibitory effects of apoC-III is another area of active pursuit.