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Conserved lysines and arginines within amino acids 140-150 of apolipoprotein (apo) E are crucial for the interaction between apoE and the low density lipoprotein receptor (LDLR). To explore the roles of amphipathic alpha-helix and basic residue organization in the binding process, we performed site-directed mutagenesis on the 22-kDa fragment of apoE (amino acids 1-191). Exchange of lysine and arginine at positions 143, 146, and 147 demonstrated that a positive charge rather than a specific basic residue is required at these positions. Consistent with this finding, substitution of neutral amino acids for the lysines at positions 143 and 146 reduced the binding affinity to about 30% of the wild-type value. This reduction corresponds to a decrease in free energy of binding of approximately 600 cal/mol, consistent with the elimination of a hydrogen-bonded ion pair (salt bridge) between a lysine on apoE and an acidic residue on the LDLR. Binding activity was similarly reduced when K143 and K146 were both mutated to arginine (K143R + K146R), indicating that more than the side-chain positive charge can be important.Exchanging lysines and leucines indicated that the amphipathic alpha-helical structure of amino acids 140-150 is critical for normal binding to the low density lipoprotein receptor.

The influence of apolipoprotein conformation on the ability of high density lipoprotein (HDL) to remove cellular free cholesterol (FC) has not been studied in detail. To address the effects of amphipathic alpha-helix structure on cellular FC efflux, three class A helical peptides and apolipoprotein (apo) AI were complexed to dimyristoyl phosphatidylcholine (DMPC) to make discoidal complexes that were used as acceptors of cell cholesterol. The peptides consisted of an 18-amino acid, amphipathic, alpha-helical peptide with the sequence DWLKAFYDKVAEKLKEAF (18A), a dimer of 18A covalently linked by a proline residue (37pA), and acetyl-18A-amide (Ac-18A-NH2) that has a higher alpha-helix content than the unblocked 18A molecule. The three peptides strongly mimic the lipid-binding characteristics of the amphipathic segments of apolipoproteins and form discoidal complexes with DMPC that are similar in diameter (11-12 nm) to those formed by human apoAI when reconstituted at a 2.5:1 (w:w) phospholipid to protein ratio. The abilities of these complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts; efflux of FC from both the plasma membrane and the lysosomal pools was examined. For each of the acceptors, the removal of cholesterol from the plasma membrane and lysosomal pools was equally efficient. All four discoidal complexes were equally efficient cell membrane FC acceptors when compared at saturating acceptor concentrations of200 micrograms of DMPC/ml of medium. However, at the same lipid concentration, protein-free DMPC small unilamellar vesicles (SUV) were significantly less efficient. The initial rates of FC removal from cells at saturating concentrations of acceptor particles (Vmax) were 12, 10, 10, and 11% per h, respectively, for the complexes containing either 18A, Ac-18A-NH2, 37pA, or apoAI, but only 1% cellular FC per h for the DMPC SUV. The 10-fold higher Vmax for the apoprotein/peptide-containing acceptors was likely due to a reversible interaction of apoprotein or peptide with the plasma membrane that changed the lipid packing characteristics in such a way as to increase the rate of FC desorption from the cell surface. This interaction required amphipathic alpha-helical segments, but it was not affected by the length, number, or lipid-binding affinity of the helices. Furthermore, the efflux efficiency was not dependent on the amino acid sequence of the helical segments which suggests that this interaction is not mediated by a specific cell surface binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

Spin-spin coupling constants derived from high-resolution 1H NMR spectra of pure 1-myristoyl-sn-glycero-3-phosphocholine (MLPC) micelles and 60:1 mol/mol been analyzed in order to determine the effects of apoprotein on phosphatidylcholine (PC) polar group conformation. The shift ratios of the polar group proton resonances after addition of the paramagnetic shift reagent Fe(CN)6(3-) to the above MLPC systems, egg PC small unilamellar vesicles, and human HDL3 have been used to compare the PC polar group conformations in all systems. The location of the largely alpha-helical apo A-I molecules in the complex with MLPC was deduced from its effects on the chemical shifts and spin-lattice relaxation times (T1) of the well-resolved 1H resonances from the various parts of the lipid molecules. The data are consistent with the apo A-I molecules lying in the surface fo the MLPC micelle with their amphipathic, alpha-helical segments intercalated among the glycerophosphocholine groups of the lipid molecules so that aromatic amino acid side chains are interspersed among the lipid hydrocarbon chains. This leads to a spacing out of the glycerol backbones and immediately adjacent methylene groups of the MLPC molecules, thereby causing an enhancement of the motions affecting T1. The presence of apo A-I at the lipid-water interface apparently does no perturb the PC polar group conformation, indicating that this conformation is determined by intramolecular effects. The preferred conformation of the phosphocholine group (Hauser, H., Pascher, I., Pearson, R. H., & Sundell, S. (1981) Biochim. Biophys, Acta 650, 21-51] is characterized by an almost exclusively gauche conformation of the choline group and predominantly antiperiplanar conformations about the C-C-O-P and P-O-C-C bonds. The PC molecules in MLPC micelles, MLPC-apo A-I complexes, egg PC vesicles, and HDL3 all have this polar group conformation.

The effects of cholesterol on the conformation and net charge of apoA-I have been investigated in homogeneous recombinant high density lipoprotein (HDL) particles. ApoA-I charge and structure in discoidal recombinant HDL complexes containing palmitoyloleoylphosphatidylcholine and cholesterol have been quantitated by guanidine HCl denaturation, circular dichroism, electrokinetic analysis, and NMR spectroscopy of [13C]lysine-labeled apoA-I. In a discoidal particle containing 2 molecules of apoA-I and 160 molecules of palmitoyloleoylphosphatidylcholine, apoA-I exhibits an alpha-helix content of 75%, and the particle has a net negative surface charge of -5.2e/mol of apoA-I at pH 8.6. Addition of 2 molecules of cholesterol to this complex has no significant effect upon particle size, but slightly decreases the net charge (-5.0e) and alpha-helix content (68%) of apoA-I and enhances the stability of the helical segments, as reflected by an increase in the free energy of unfolding from 2.9 to 3.5 kcal/mol. In contrast, increasing the cholesterol content to 20 molecules/particle progressively increases particle size and apoA-I net negative charge (-6.1e), and there is a concomitant reduction in the free energy of stabilization of the alpha-helical structure in apoA-I to 2.2 kcal/mol. (13CH3)2-Lys resonances from apoA-I in discoidal recombinant HDL exhibit six chemical shifts at pH 10; these peaks originate from dimethyl-Lys residues that have pKa values ranging from 8.4 to 10.3. The titration behavior of apoA-I Lys residues is generally similar in the presence and absence of cholesterol, except that 4 Lys residues titrate at a significantly higher pH in the presence of cholesterol. These data are consistent with cholesterol having a direct effect on apoA-I conformation and charge in HDL. Structural changes of this magnitude can affect the interactions between HDL and various plasma proteins and cell surfaces. It is therefore likely that the cholesterol content of HDL plays an important role in regulating the metabolism of this lipoprotein.

Several subspecies of human high density lipoprotein (HDL) exist, and particle surface charge is one parameter that can be used to distinguish them. For instance, pre-beta HDL has a lower negative surface charge than the bulk of plasma HDL resulting in slower migration when subjected to electrophoresis in an agarose gel at pH 8.6. The molecular basis for the variations in HDL particle surface charge have been established in this work. Quantitative analysis of electrophoretic mobilities in agarose gels indicates that the surface potentials of reconstituted pre-beta HDL and alpha-HDL3 are -7.6 and -11.4 mV, respectively. The difference of 3.8 mV corresponds to an apparent 1e more net negative charge on alpha-migrating HDL3. The contributions of the lipid and protein components to the charge of HDL3 were defined using reconstituted particles (rHDL) containing various combinations of HDL3 protein and lipid. The surface potentials of spherical and discoidal rHDL are not affected by the particle apoprotein composition. Thin layer chromatography of the HDL3 phospholipid fraction from five normolipidemic subjects revealed that it consists of approximately 84% phosphatidylcholine, 12% sphingomyelin, and 4% phosphatidylinositol (PI) by weight. PI significantly affects the charge of discoidal rHDL and is responsible for approximately 0.7 mV of the surface potential difference between pre-beta- and alpha-HDL. Reconstitution of spherical complexes containing apolipoprotein AI, palmitoyl-oleoyl phosphatidylcholine, and HDL3 neutral lipid esters showed that particles containing a neutral lipid core migrate to an alpha position in an agarose gel. The change in particle shape accounts for approximately 3 mV of the difference in surface potential between pre-beta and alpha-HDL. Overall, the charge of HDL particles is determined by 1) a direct contribution of negative charge from PI molecules in the surface of the lipoprotein and 2) the conformation of the resident apolipoprotein(s) which is influenced by the presence of neutral lipids and the particle shape. This quantitative understanding of surface charge provides a basis for understanding variations in function of HDL subspecies.

High density lipoprotein (HDL) is able to remove unesterified cholesterol from peripheral cells in the process of reverse cholesterol transport by an aqueous diffusion mechanism as well as by an apolipoprotein (apo)-mediated process. The aqueous diffusion mechanism is understood but the molecular mechanism of lipid-poor pre-beta-HDL-(apo-) mediated cholesterol removal is not known. Measurements of the initial rates of efflux of unesterified cholesterol and phospholipid from human fibroblasts to lipid-free, human apoA-I showed that both lipids are released from the cells during a 10-min incubation with apoA-I. The concentration-dependence of efflux of the lipids is the same (Km = 0.4 and 0.6 microg apoA-I/ml for cholesterol and phospholipid flux, respectively), suggesting a membrane microsolubilization process. A finite pool of about 1% of the plasma membrane cholesterol is accessible for release by solubilization; the limited size of this cholesterol pool is not due to a lack of availability of apoA-I, but rather to the restricted amount of phospholipid that is removed from the plasma membrane. Plasma membrane domains may be involved in membrane microsolubilization, but caveolar cholesterol seems not to be specifically accessed in this process. Membrane microsolubilization is the process by which pre-beta1-HDL removes cell cholesterol in the first step of reverse cholesterol transport. When apoA-I is present in the extracellular space, the relative contributions of cholesterol efflux by membrane microsolubilization and by aqueous diffusion are determined by the degree of lipidation of the apoA-I molecules.