The Structure of Human Apolipoprotein A-IV as Revealed by Stable Isotope-assisted Cross-linking,Molecular Dynamics,and Small Angle X-ray Scattering

Apolipoprotein (apo)A-IV plays important roles in dietary lipid and glucose metabolism, and knowledge of its structure is required to fully understand the molecular basis of these functions. However, typical of the entire class of exchangeable apolipoproteins, its dynamic nature and affinity for lipid has posed challenges to traditional high resolution structural approaches. We previously reported an x-ray crystal structure of a dimeric truncation mutant of apoA-IV, which showed a unique helix-swapping molecular interface. Unfortunately, the structures of the N and C termini that are important for lipid binding were not visualized. To build a more complete model, we used chemical cross-linking to derive distance constraints across the full-length protein. The approach was enhanced with stable isotope labeling to overcome ambiguities in determining molecular span of the cross-links given the remarkable similarities in the monomeric and dimeric apoA-IV structures. Using 51 distance constraints, we created a starting model for full-length monomeric apoA-IV and then subjected it to two modeling approaches: (i) molecular dynamics simulations and (ii) fitting to small angle x-ray scattering data. This resulted in the most detailed models yet for lipid-free monomeric or dimeric apoA-IV. Importantly, these models were of sufficient detail to direct the experimental identification of new functional residues that participate in a “clasp” mechanism to modulate apoA-IV lipid affinity. The isotope-assisted cross-linking approach should prove useful for further study of this family of apolipoproteins in both the lipid-free and -bound states.     

A three-dimensional homology model of lipid-free apolipoprotein A-IV using cross-linking and mass spectrometry

Human apolipoprotein A-IV (apoA-IV) is a 46-kDa exchangeable plasma protein with many proposed functions. It is involved in chylomicron assembly and secretion, protection from atherosclerosis through a variety of mechanisms, and inhibition of food intake. There is little structural basis for these proposed functions due to the lack of a solved three-dimensional structure of the protein by x-ray crystallography or NMR. Based on previous studies, we hypothesized that lipid-free apoA-IV exists in a helical bundle, like other apolipoprotein family members and that regions near the N and C termini may interact. Utilizing a homobifunctional lysine cross-linking agent, we identified 21 intramolecular cross-links by mass spectrometry. These cross-links were used to constrain the building of a sequence threaded homology model using the I-TASSER server. Our results indicate that lipid-free apoA-IV does indeed exist as a complex helical bundle with the N and C termini in close proximity. This first structural model of lipid-free apoA-IV should prove useful for designing studies aimed at understanding how apoA-IV interacts with lipids and possibly with unknown protein partners.     

Specific sequences in the N and C termini of apolipoprotein A-IV modulate its conformation and lipid association

Apolipoprotein (apoA-IV) is a 376-residue exchangeable apolipoprotein that may play a number of important roles in lipid metabolism, including chylomicron assembly, reverse cholesterol transport, and appetite regulation. In vivo, apoA-IV exists in both lipid-poor and lipid-associated forms, and the balance between these states may determine its function. We examined the structural elements that modulate apoA-IV lipid binding by producing a series of deletion mutants and determining their ability to interact with phospholipid liposomes. We found that the deletion of residues 333-343 strongly increased the lipid association rate versus native apoA-IV. Additional mutagenesis revealed that two phenylalanine residues at positions 334 and 335 mediated this lipid binding inhibitory effect. We also observed that residues 11-20 in the N terminus were required for the enhanced lipid affinity induced by deletion of the C-terminal sequence. We propose a structural model in which these sequences can modulate the conformation and lipid affinity of apoA-IV.     

Impact of self-association on function of apolipoprotein A-I

Self-association is an inherent property of the lipid-free forms of several exchangeable apolipoproteins, including apolipoprotein A-I (apoA-I), the main protein component of high density lipoproteins (HDL) and an established antiatherogenic factor. Monomeric lipid-free apoA-I is believed to be the biologically active species, but abnormal conditions, such as specific natural mutations or oxidation, produce an altered state of self-association that may contribute to apoA-I dysfunction. Replacement of the tryptophans of apoA-I with phenylalanines (ΔW-apoA-I) leads to unusually large and stable self-associated species. We took advantage of this unique solution property of ΔW-apoA-I to analyze the role of self-association in determining the structure and lipid-binding properties of apoA-I as well as ATP-binding cassette A1 (ABCA1)-mediated cellular lipid release, a relevant pathway in atherosclerosis. Monomeric ΔW-apoA-I and wild-type apoA-I activated ABCA1-mediated cellular lipid release with similar efficiencies, whereas the efficiency of high order self-associated species was reduced to less than 50%. Analysis of specific self-associated subclasses revealed that different factors influence the rate of HDL formation in vitro and ABCA1-mediated lipid release efficiency. The α-helix-forming ability of apoA-I is the main determinant of in vitro lipid solubilization rates, whereas loss of cellular lipid release efficiency is mainly caused by reduced structural flexibility by formation of stable quaternary interactions. Thus, stabilization of self-associated species impairs apoA-I biological activity through an ABCA1-mediated mechanism. These results afford mechanistic insights into the ABCA1 reaction and suggest self-association as a functional feature of apoA-I. Physiologic mechanisms may alter the native self-association state and contribute to apoA-I dysfunction.     

Apolipoprotein A-IV is a novel substrate for matrix metalloproteinases

Screening of matrix metalloproteinase (MMP)-14 substrates in human plasma using a proteomics approach previously identified apolipoprotein A-IV (apoA-IV) as a novel substrate for MMP-14. Here, we show that among the tested MMPs, purified apoA-IV is most susceptible to cleavage by MMP-7, and that apoA-IV in plasma can be cleaved more efficiently by MMP-7 than MMP-14. Purified recombinant apoA-IV (44-kDa) was cleaved by MMP-7 into several fragments of 41, 32, 29, 27, 24, 22 and 19 kDa. N-terminal sequencing of the fragments identified two internal cleavage sites for MMP-7 in the apoA-IV sequence, between Glu(185) and Leu(186), and between Glu(262) and Leu(263). The cleavage of lipid-bound apoA-IV by MMP-7 was less efficient than that of lipid-free apoA-IV. Further, MMP-7-mediated cleavage of apoA-IV resulted in a rapid loss of its intrinsic anti-oxidant activity. Based on the fact that apoA-IV plays important roles in lipid metabolism and possesses anti-oxidant activity, we suggest that cleavage of lipid-free apoA-IV by MMP-7 has pathological implications in the development of hyperlipidemia and atherosclerosis.     

Structure and function of the apoA-IV T347S and Q360H common variants

Human apolipoprotein A-IV (apoA-IV) is involved in chylomicron assembly and secretion, and in reverse cholesterol transport. Several apoA-IV isoforms exist, the most common in Caucasian populations being apoA-IV-1a (T347S) and apoA-IV-2 (Q360H). The objective of the present study was to investigate the impact of these common aminoacid substitutions on the ability of apoA-IV to bind lipids, to promote cell cholesterol efflux via ABCA1, and to maintain endothelial homeostasis. Recombinant forms of wild-type apoA-IV, apoA-IV Q360H, and apoA-IV T347S were produced in Escherichia coli. ApoA-IV Q360H and apoA-IV T347S showed a slightly higher α-helical content compared to wild-type apoA-IV, and associated with phospholipids faster than wild-type apoA-IV. The capacity to promote ABCA1-mediated cholesterol efflux was significantly greater for the apoA-IV T347S than the other apoA-IV isoforms. No differences were observed in the ability of apoA-IV isoforms to inhibit the production of VCAM-1 and IL-6 in TNFα-stimulated endothelial cells. In conclusion, the apoA-IV T347S common variant has increased lipid binding properties and cholesterol efflux capacity, while the apoA-IV Q360H variant has only slightly increased lipid binding properties. The two common aminoacid substitutions have no effect on the ability of apoA-IV to maintain endothelial homeostasis.     

Small-angle X-ray Scattering of Apolipoprotein A-IV Reveals the Importance of Its Termini for Structural Stability

ApoA-IV is an amphipathic protein that can emulsify lipids and has been linked to protective roles against cardiovascular disease and obesity. We previously reported an x-ray crystal structure of apoA-IV that was truncated at its N and C termini. Here, we have extended this work by demonstrating that self-associated states of apoA-IV are stable and can be structurally studied using small-angle x-ray scattering. Both the full-length monomeric and dimeric forms of apoA-IV were examined, with the dimer showing an elongated rod core with two nodes at opposing ends. The monomer is roughly half the length of the dimer with a single node. Small-angle x-ray scattering visualization of several deletion mutants revealed that removal of both termini can have substantial conformational effects throughout the molecule. Additionally, the F334A point mutation, which we previously showed increases apoA-IV lipid binding, also exhibited large conformational effects on the entire dimer. Merging this study''s low-resolution structural information with the crystal structure provides insight on the conformation of apoA-IV as a monomer and as a dimer and further defines that a clasp mechanism may control lipid binding and, ultimately, protein function.     

Modulation of apolipoprotein A-IV lipid binding by an interaction between the N and C termini

Apolipoprotein A-IV (apoA-IV) is a 376-amino acid exchangeable apolipoprotein made in the small intestine of humans. Although it has many proposed roles in vascular disease, satiety, and chylomicron metabolism, there is no known structural basis for these functions. The ability to associate with lipids may be a key step in apoA-IV functionality. We recently identified a single amino acid, Phe(334), which seems to inhibit the lipid binding capability of apoA-IV. We also found that an intact N terminus was necessary for increased lipid binding of Phe(334) mutants. Here, we identify Trp(12) and Phe(15) as the N-terminal amino acids required for the fast lipid binding seen with the F334A mutant. Furthermore, we found that individual disruption of putative amphipathic alpha-helices 3-11 had little effect on lipid binding, suggesting that the N terminus of apoA-IV may be the operational site for initial lipid binding. We also provide three independent pieces of experimental evidence supporting a direct intramolecular interaction between sequences near amino acids 12/15 and 334. This interaction could represent a unique "switch" mechanism by which apoA-IV changes lipid avidity in vivo.     

Apolipophorin III: a lipid-triggered molecular switch

Apolipophorin III (apoLp-III) is a low molecular weight exchangeable apolipoprotein that plays an important role in the enhanced neutral lipid transport during insect flight. The protein exists in lipid-free and lipid-bound states. The lipid-bound state is the active form of the protein and occurs when apoLp-III associates with lipid-enriched lipophorins. ApoLp-III is well characterized in two evolutionally divergent species: Locusta migratoria and Manduca sexta. The two apolipoproteins interact in a similar manner with model phospholipid vesicles, and transform them into discoidal particles. Their low intrinsic stability in the lipid-free state likely facilitates interaction with lipid surfaces. Low solution pH also favors lipid binding interaction through increased exposure of hydrophobic surfaces on apoLp-III. While secondary structure is maintained under acidic conditions, apoLp-III tertiary structure is altered, adopting molten globule-like characteristics. In studies of apoLp-III interaction with natural lipoproteins, we found that apoLp-III is readily displaced from the surface of L. migratoria low-density lipophorin by recombinant apoLp-III proteins from either L. migratoria or M. sexta. Thus, despite important differences between these two apoLp-IIIs (amino acid sequence, presence of carbohydrate), their functional similarity is striking. This similarity is also illustrated by the recently published NMR solution structure of M. sexta apoLp-III wherein its molecular architecture closely parallels that of L. migratoria apoLp-III.     

A complete backbone spectral assignment of lipid-free human apolipoprotein E (apoE)

Apolipoprotein E is an exchangeable apolipoprotein that plays an important role in lipid/lipoprotein metabolism and cardiovascular diseases. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer’s disease, cognitive function, immunoregulation, cell signaling and infectious diseases. Although the X-ray crystal structure of the apoE N-terminal domain was solved in 1991, there is no structure available for the apoE C-terminal domain and full-length apoE. Here we report a complete NMR backbone spectral assignment of lipid-free human apoE.     

Domain structure and lipid interaction in human apolipoproteins A-I and E,a general model

Detailed structural information on human exchangeable apolipoproteins (apo) is required to understand their functions in lipid transport. Using a series of deletion mutants that progressively lacked different regions along the molecule, we probed the structural organization of lipid-free human apoA-I and the role of different domains in lipid binding, making comparisons to apoE, which is a member of the same gene family and known to have two structural domains. Measurements of alpha-helix content by CD in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that deletion of the amino-terminal or central regions disrupts the tertiary organization, whereas deletion of the carboxyl terminus has no effect on stability and induces a more cooperative structure. These data are consistent with the lipid-free apoA-I molecule being organized into two structural domains similar to apoE; the amino-terminal and central parts form a helix bundle, whereas the carboxyl-terminal alpha-helices form a separate, less organized structure. The binding of the apoA-I variants to lipid emulsions is modulated by reorganization of the helix bundle structure, because the rate of release of heat on binding is inversely correlated with the stability of the helix bundle. Based on these observations, we propose that there is a two-step mechanism for lipid binding of apoA-I: apoA-I initially binds to a lipid surface through amphipathic alpha-helices in the carboxyl-terminal domain, followed by opening of the helix bundle in the amino-terminal domain. Because apoE behaves similarly, this mechanism is probably a general feature for lipid interaction of other exchangeable apolipoproteins, such as apoA-IV.     

The role of hydrophobic and negatively charged surface patches of lipid-free apolipoprotein A-I in lipid binding and ABCA1-mediated cholesterol efflux

Recent models of lipid-free apolipoprotein A-I, including a cross-link/homology model and an X-ray crystal structure have identified two potential functionally relevant “patches” on the protein surface. The first is a hydrophobic surface patch composed of leucine residues 42, 44, 46, and 47 and the second a negatively charged patch composed of glutamic acid residues 179, 191, and 198. To determine if these domains play a functional role, these surface patches were disrupted by site-directed mutagenesis and the bacterially expressed mutants were compared with respect to their ability to bind lipid and stimulate ABCA1-mediated cholesterol efflux. It was found that neither patch plays a significant functional role in the ability of apoA-I to accept cholesterol in an ABCA1-dependent manner, but that the hydrophobic patch did affect the ability of apoA-I to clear DMPC liposomes. Interestingly, contrary to previous predictions, disruption of the hydrophobic surface patch enhanced the lipid binding ability of apoA-I. The hydrophobic surface patch may be important to the structural stability of lipid-free apoA-I or may be a necessary permissive structural element for lipid binding.     

Interaction of an exchangeable apolipoprotein with phospholipid vesicles and lipoprotein particles. Role of leucines 32, 34, and 95 in Locusta migratoria apolipophorin III.

Apolipophorin III (apoLp-III) from Locusta migratoria is an exchangeable apolipoprotein that binds reversibly to lipid surfaces. In the lipid-free state this 164-residue protein exists as a bundle of five elongated amphipathic alpha-helices. Upon lipid binding, apoLp-III undergoes a significant conformational change, resulting in exposure of its hydrophobic interior to the lipid environment. On the basis of x-ray crystallographic data (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603-608), it was proposed that hydrophobic residues, present in loops that connect helices 1 and 2 (Leu-32 and Leu-34) and helices 3 and 4 (Leu-95), may function in initiation of lipid binding. To examine this hypothesis, mutant apoLp-IIIs were designed wherein the three Leu residues were replaced by Arg, individually or together. Circular dichroism spectroscopy and temperature and guanidine hydrochloride denaturation studies showed that the mutations did not cause major changes in secondary structure content or stability. In lipid binding assays, addition of apoLp-III to phospholipid vesicles caused a rapid clearance of vesicle turbidity due to transformation to discoidal complexes. L34R and L32R/L34R/L95R apoLp-IIIs displayed a much stronger interaction with lipid vesicles than wild-type apoLp-III. Furthermore, it was demonstrated that the mutant apoLp-IIIs retained their ability to bind to lipoprotein particles. However, in lipoprotein competition binding assays, the mutants displayed an impaired ability to initiate a binding interaction when compared with wild-type apoLp-III. The data indicate that the loops connecting helices 1 and 2 and helices 3 and 4 are critical regions in the protein, contributing to recognition of hydrophobic defects on lipoprotein surfaces by apoLp-III.     

Lipid-free apoA-I structure - Origins of model diversity

Apolipoprotein A-I (apoA-I) is a prominent member of the exchangeable apolipoprotein class of proteins, capable of transitioning between lipid-bound and lipid-free states. It is the primary structural and functional protein of high density lipoprotein (HDL). Lipid-free apoA-I is critical to de novo HDL formation as it is the preferred substrate of the lipid transporter, ATP Binding Cassette Transporter A1 (ABCA1) Remaley et al. (2001) [1]. Lipid-free apoA-I is an important element in reverse cholesterol transport and comprehension of its structure is a core issue in our understanding of cholesterol metabolism. However, lipid-free apoA-I is highly conformationally dynamic making it a challenging subject for structural analysis. Over the past 20 years there have been significant advances in overcoming the dynamic nature of lipid-free apoA-I, which have resulted in a multitude of proposed conformational models.     

Interfacial exclusion pressure determines the ability of apolipoprotein A-IV truncation mutants to activate cholesterol ester transfer protein

We used a panel of recombinant human apolipoprotein (apo) A-IV truncation mutants, in which pairs of 22-mer alpha-helices were sequentially deleted along the primary sequence, to examine the impact of protein structure and interfacial activity on the ability of apoA-IV to activate cholesterol ester transfer protein. Circular dichroism and fluorescence spectroscopy revealed that the secondary structure, conformation, and molecular stability of recombinant human apoA-IV were identical to the native protein. However, deletion of any of the alpha-helical domains in apoA-IV disrupted its tertiary structure and impaired its molecular stability. Surprisingly, determination of the water/phospholipid interfacial exclusion pressure of the apoA-IV truncation mutants revealed that, for most, deletion of amphipathic alpha-helical domains increased their affinity for phospholipid monolayers. All of the truncation mutants activated the transfer of fluorescent-labeled cholesterol esters between high and low density lipoproteins at a rate higher than native apoA-IV. There was a strong positive correlation (r = 0.790, p = 0.002) between the rate constant for cholesterol ester transfer and interfacial exclusion pressure. We conclude that molecular interfacial exclusion pressure, rather than specific helical domains, determines the degree to which apoA-IV, and likely other apolipoproteins, facilitate cholesterol ester transfer protein-mediated lipid exchange.     

Interaction of apolipoprotein A-I in three different conformations with palmitoyl oleoyl phosphatidylcholine vesicles

Interactions of apolipoprotein A-I (apoA-I) with cell membranes appear to be important in the initial steps of reverse cholesterol transport. The objective of this work was to examine the effect of three distinct conformations of apoA-I (lipid-free and in 78 A or 96 A reconstituted high density lipoproteins, rHDL) on its ability to bind to, and abstract lipids from, palmitoyl oleoyl phosphatidylcholine membrane vesicles (small unilamellar vesicles, SUV, and giant unilamellar vesicles, GUV). The molecular interactions were observed by two-photon fluorescence microscopy, and the binding parameters were quantified by gel-permeation chromatography or isothermal titration microcalorimetry. Rearrangement of apoA-I-containing particles after exposure to SUVs was examined by native gel electrophoresis. The results indicate that lipid-free apoA-I binds reversibly, with high affinity, to the vesicles but does not abstract a significant amount of lipid nor perturb the vesicle structure. The 96 A rHDL, where all the amphipathic helices of apoA-I are saturated with lipid within the particles, do not bind to vesicles or perturb their structure. In contrast, the 78 A rHDL have a region of apoA-I, corresponding to a few amphipathic helical segments, which is available for external or internal phospholipid binding. These particles bind to vesicles with measurable affinity (lower than lipid-free apoA-I), abstract lipids from the membranes, and form particles of larger diameters, including 96 A rHDL. We conclude that the conformation of apoA-I regulates its binding affinity for phospholipid membranes and its ability to abstract lipids from the membranes.     

Structure-function studies of human apolipoprotein A-V: a regulator of plasma lipid homeostasis

To investigate structure and function relations of a new member of the exchangeable apolipoprotein family that modulates plasma lipid levels, recombinant human apolipoprotein (apo) A-V was produced in Escherichia coli and isolated by a combination of nickel chelation affinity chromatography and reversed-phase HPLC. Antibodies directed against apoA-V were generated and employed in immunoblotting experiments. Anti-apoA-V IgG gave a strong response against recombinant apoA-V from E. coli and human apoA-V expressed in transgenic mice, but did not recognize human apoA-I or apoA-IV. In neutral-pH buffers, at concentrations of >0.1 mg/mL, isolated lipid-free apoA-V is poorly soluble. By contrast, apoA-V is soluble in 50 mM sodium citrate (pH 3.0). Far-UV circular dichroism analysis and spectral deconvolution reveal that apoA-V possesses 32% alpha-helix, 33% beta-sheet, 16% beta-turn, and 18% random coil secondary structure conformers. Temperature-induced denaturation studies gave rise to a transition midpoint of 47.1 degrees C. Upon being cooled to ambient temperature from 85 degrees C, apoA-V failed to recover all of the negative ellipticity present in unheated apoA-V. ApoA-V interacts with bilayer vesicles of dimyristoylphosphatidylcholine to form discoidal complexes with diameters in the range of 15-20 nm. However, apoA-V was a poor activator of lecithin:cholesterol acyltransferase where the activity was 8.5 +/- 1.8% of that of apoA-I. Furthermore, apoA-V failed to support enhanced efflux of cholesterol from cAMP-treated J774 macrophages, although low levels of efflux were obtained from unstimulated cells. Taken together, the results demonstrate recombinant apoA-V possesses unique structural and functional characteristics, in keeping with its proposed role in the modulation of plasma lipid levels.     

Overexpression of apolipoprotein A-IV enhances lipid secretion in IPEC-1 cells by increasing chylomicron size

Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans.     

Structure of human apolipoprotein A-IV: a distinct domain architecture among exchangeable apolipoproteins with potential functional implications

Apolipoprotein A-IV (apoA-IV) is an exchangeable apolipoprotein that shares many functional similarities with related apolipoproteins such as apoE and apoA-I but has also been implicated as a circulating satiety factor. However, despite the fact that it contains many predicted amphipathic alpha-helical domains, relatively little is known about its tertiary structure. We hypothesized that apoA-IV exhibits a characteristic functional domain organization that has been proposed to define apoE and apoA-I. To test this, we created truncation mutants in a bacterial system that deleted amino acids from either the N- or C-terminal ends of human apoA-IV. We found that apoA-IV was less stable than apoA-I but was more highly organized in terms of its cooperativity of unfolding. Deletion of the extreme N and C termini of apoA-IV did not significantly affect the cooperativity of unfolding, but deletions past amino acid 333 on the C terminus or amino acid 61 on the N terminus had major destabilizing effects. Functionally, apoA-IV was less efficient than apoA-I at clearing multilamellar phospholipid liposomes and promoting ATP-binding cassette transporter A1-mediated cholesterol efflux. However, deletion of a C-terminal region of apoA-IV, which is devoid of predicted amphipathic alpha helices (amino acids 333-376) stimulated both of these activities dramatically. We conclude that the amphipathic alpha helices in apoA-IV form a single, large domain that may be similar to the N-terminal helical bundle domains of apoA-I and apoE but that apoA-IV lacks the C-terminal lipid-binding and cholesterol efflux-promoting domain present in these apolipoproteins. In fact, the C terminus of apoA-IV appears to reduce the ability of apoA-IV to interact with lipids and promote cholesterol efflux. This indicates that, although apoA-IV may have evolved from gene duplication events of ancestral apolipoproteins and shares the basic amphipathic helical building blocks, the overall localization of functional domains within the sequence is quite different from apoA-I and apoE.     

Contributions of domain structure and lipid interaction to the functionality of exchangeable human apolipoproteins

Exchangeable apolipoproteins function in lipid transport as structural components of lipoprotein particles, cofactors for enzymes and ligands for cell-surface receptors. Recent findings with apoA-I and apoE suggest that the tertiary structures of these two members of the human exchangeable apolipoprotein gene family are related. Characteristically, these proteins contain a series of proline-punctuated, 11- or 22-amino acid, amphipathic alpha-helical repeats that can adopt a helix bundle conformation in the lipid-free state. The amino- and carboxyl-terminal regions form separate domains with the latter being primarily responsible for lipid binding. Interaction with lipid induces changes in the conformation of the amino-terminal domain leading to alterations in function; for example, opening of the amino-terminal four-helix bundle in apolipoprotein E upon lipid binding is associated with enhanced receptor-binding activity. The concept of a two-domain structure for the larger exchangeable apolipoproteins is providing new molecular insights into how these apolipoproteins interact with lipids and other proteins, such as receptors. The ways in which structural changes induced by lipid interaction modulate the functionality of these apolipoproteins are reviewed.