Proline

Progress in
Lipid Research
Progress in Lipid Research 43 (2004) 350–380
www.elsevier.com/locate/plipres
Review
Contributions of domain structure and lipid interaction to

the functionality of exchangeable human apolipoproteins
Hiroyuki Saito, Sissel Lund-Katz, Michael C. Phillips *
Lipid Research Group, The Children’s Hospital of Philadelphia, Abramson Research Center, Suite 1102,
3615 Civic Center Boulevard, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4318, USA
Abstract
Exchangeable apolipoproteins function in lipid transport as structural components of lipoprotein par-

ticles, 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 a-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 apo-
lipoprotein 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.
Ó 2004 Elsevier Ltd. All rights reserved.
Abbreviations: ABCA1, ATP-binding cassette transporter A1; ANS, 8-anilino-1-naphthalenesulfonic acid; apo,
apolipoprotein; CD, circular dichroism; CE, cholesteryl ester; DMPC, 1,2-dimyristoyl phosphatidylcholine; FRET,
fluorescence resonance energy transfer; FC, free cholesterol; HDL, high density lipoprotein; HSPG, heparan sulfate
proteoglycan; LCAT, lecithin: cholesterol acyltransferase; LDL, low density lipoprotein; LDLR, LDL receptor; LRP,
LDL receptor-related protein; PL, phospholipid; SR-BI, scavenger receptor class B type I; VLDL, very low density
lipoprotein.
*
Corresponding author. Tel.: +1-215-590-0587; fax: +1-215-590-0583.
E-mail address: phillipsmi@email.chop.edu (M.C. Phillips).
0163-7827/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.plipres.2004.05.002
H. Saito et al. / Progress in Lipid Research 43 (2004) 350–380 351
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
2. Structures of apoA-I and apoE in the lipid-free state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352

2.1. Primary and secondary structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
2.2. Tertiary structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
2.3. Quaternary structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
3. Interaction of apoA-I and apoE with lipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358

3.1. Molecular mechanism of lipid-binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
3.2. Lipid-bound conformation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
4. Other members of the exchangeable apolipoprotein family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362

4.1. ApoA-II . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................................ 363
4.2. ApoA-IV. . . . . . . . . . . . . . . . . . . . . . . . . . . ........................................ 363
4.3. ApoA-V . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................................ 364
4.4. ApoC-I, C-II and C-III . . . . . . . . . . . . . . . . ........................................ 364
5. Structure–function relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366

5.1. ApoE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
5.2. ApoA-I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
6. Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
1. Introduction
The exchangeable apolipoproteins (apo) are part of a multigene family [1] and members of
the A, C and E classes of apolipoproteins play critical roles in lipoprotein metabolism. In
particular, they are structural components of lipoprotein particles and their ability to interact
with lipids is central to this function. ApoA, C and E regulate the transport of lipids into and
out of cells by acting as cofactors for plasma enzymes and ligands for cell-surface receptors.
The structures of human apoA-I and apoE have been studied the most extensively but many
aspects of their structures are poorly defined. This lack of molecular detail has limited un-
derstanding of the structure–function relationships of these proteins. Recently, we have shown
that apoA-I and apoE adopt similar conformations, as might be expected for members of the
same gene family. This new understanding of tertiary structure is providing important insights
into the molecular basis for the biological functions of apoA-I and apoE. The aim of this
review is to summarize recent advances in this area. Additional background information can
be obtained from some excellent reviews on apoA-I and apoE that have been published in the
last several years [2–7].
352 H. Saito et al. / Progress in Lipid Research 43 (2004) 350–380
2. Structures of apoA-I and apoE in the lipid-free state
2.1. Primary and secondary structure
Exchangeable apolipoproteins (apoA, C and E) have the same genomic structure and are
members of multigene family that probably evolved from a common ancestral gene [1]. The last
exon codes for a primary structure of 11- and 22-amino acid tandem repeats that span residues
44–243 in apoA-I and 62–299 in apoE (Fig. 1(a)) [1,8]. Each of these repeats has the periodicity of
an amphipathic a-helix and these helices are often separated by a proline residue [8,9]. The am-
phipathic a-helices have been classified into several distinct classes according to the distribution of
charged residues around the axis of the helix [10]. The class A helix is a major lipid-binding motif
of exchangeable apolipoproteins and is characterized by the location of basic residues near the
hydrophilic/hydrophobic interface and acidic residues clustered at the center of the polar face
(Fig. 1(b)) [11]. Class G* and class Y helices have also been identified in the exchangeable apo-
lipoproteins and these types of amphipathic a-helix are proposed to have reduced lipid affinity
[12]. The class G* helix is distinguished by a random radial arrangement of positive and negative
amino acid residues on the polar face, while the class Y helix is characterized by the presence of
three clusters of basic amino acids in the polar face forming a Y pattern (Fig. 1(b)) [11].
Fig. 1. (a) Distribution of amphipathic a-helices in the human exchangeable apolipoproteins,apoA-I, apoA-IV and
apoE. The letter ‘‘P’’ below the rectangles indicates positions of all proline residues. (b) Amphipathic helix classes found
in the exchangeable apolipoproteins. Classification is based on the distribution of charged residues (see Section 2.1).
These figures were adapted from Segrest et al. [8].
Human apoA-I is a 243 amino acid protein (molecular mass ¼ 28.1 kDa) in which exon 3
encodes an N-terminal domain (residues 1–43) and the remaining region coded by exon 4 is
predicted to contain eight 22-mer and two 11-mer amphipathic a-helices with most of the helices
being punctuated by prolines (Fig. 1(a)) [1,3]. Comparison of sequences amongst mammals in-
dicates that the N-terminal region of apoA-I is highly conserved while the central and C-terminal
regions show conservative substitutions between species [5]. Despite changes in the primary
structure of apoA-I amongst different species, the secondary structure appears to be more con-
served [13]. Studies of synthetic peptides corresponding to each of the 22-residue amphipathic
segments of human apoA-I have shown that the first (residues 44–65) and last (residues 220–241)
repeat helices have the greatest lipid affinity [14]. The C-terminal region of human apoA-I is very
hydrophobic as revealed by hydropathy analysis of the amino acid sequence (Fig. 2(a)) [15].
Furthermore, recent studies using specific mutations in the C-terminal region [16] and site-directed
spin-label electron paramagnetic resonance spectroscopy [17] indicate that there is a stable helical
structure in the extreme C-terminus of apoA-I. Interestingly, the N-terminal region (residues 1–43)
also contain a very hydrophobic segment centered around residue 18 (Fig. 2(a)), suggesting that
this region also has significant lipid binding ability [18,19].
Human apoE is a single polypeptide chain of 299 amino acid residues with a molecular mass of
34.2 kDa [20]. There is a high degree of sequence conservation across species with exceptions at
the N- and C-termini: homology begins in the vicinity of residue 26 in the human sequence and
continues to approximately residue 288 [2]. Human apoE exists in three major isoforms, apoE2,
apoE3 and apoE4, each differing by cysteine and arginine at positions 112 and 158. ApoE3, the
most common form, contains cysteine and arginine at these positions, respectively, whereas
apoE2 contains cysteine and apoE4 contains arginine at both sites [21]. Physical and biochemical
analyses have demonstrated that there are two independently folded domains in apoE: a 22-kDa
N-terminal domain (residues 1–191) and a 10-kDa C-terminal domain (residues 216–299) [22,23]
(cf. Section 2.2). The N-terminal domain contains the low density lipoprotein (LDL) receptor-
binding region (cf. Section 5) and the C-terminal domain has a high affinity for lipid and is re-
sponsible for lipoprotein binding [2]. Secondary structure predictions indicate that both domains
are highly helical and the N-terminal and the C-terminal domains contain predominantly class G*
and class A amphipathic helices, respectively (Fig. 1(a)) [8]. The hydropathy plot (Fig. 2(b)) shows
that the extreme C-terminus as well as part of the linker region (residues 192–215) are relatively
hydrophobic.
2.2. Tertiary structure
High-resolution structures for intact human apolipoproteins in the lipid-free state are not
available to date. ApoE is perhaps the best defined because the crystal structure for the 22-kDa
N-terminal fragment was solved by X-ray crystallography over a decade ago (Fig. 3) [24]. This
domain forms a globular bundle of four elongated a-helices in which the helices are arranged in an
antiparallel manner with their hydrophobic faces oriented towards the interior of the bundle. This
structure shares the same basic architecture as the five-helix bundle of insect apolipophorin III
[25]. The N-terminal fragments of all three apoE isoforms adopt such a four-helix bundle motif,
but subtle differences in the side-chain conformations and salt-bridge arrangements of the iso-
forms affect their functions and characteristics [26–28]. Studies of guanidine [29] and thermal [30]
2
(a): apoA-I
1
-1
-2
-3
0 50 100 150 200 250
2
Nonpolar
(b): apoE3
1
-1
-2
Polar
-3
0 50 100 150 200 250 300
2
(c): apoA-IV
1
-1
-2
-3
0 50 100 150 200 250 300 350

Position
Fig. 2. Hydropathy plots of the amino acid sequences of human apoA-I, apoE3 and apoA-IV. Hydropathy was cal-
culated as described by Kyte and Doolittle [15] using a sliding window of nine residues.
denaturation demonstrated that the N-terminal fragments of the apoE isoforms differ in stability
(apoE4 < apoE3 < apoE2), indicating that replacing cysteine residues by arginine results in a
cumulative decrease in hydrophobicity and stability of the helix bundle (Table 1). The stabilities of
the N-terminal fragments are rather similar to that of other globular proteins [29]. The stability of
the C-terminal fragment is lower and similar to that of other apolipoproteins such as apoA-I
(Table 1): the structural organization of this domain is not well characterized. Recent 8-anilino-1-
naphthalenesulfonic acid (ANS) fluorescence measurements have suggested that the C-terminal
Fig. 3. Ribbon model of the structure of the 22-kDa fragment of human apoE3. Four of the five helices are arranged in
an antiparallel four-helix bundle. Reproduced with permission from [2].
domain of apoE forms a more solvent-exposed, less organized structure (Table 1) [31]. A model
has been proposed in which the helices in the C-terminal domain form an inter-molecular coiled-
coil helix structure [32].
A variety of studies using deletion and site-directed mutagenesis techniques have provided
important insights into the lipid-free structure of apoA-I (for reviews, see [4,5]). Proteolysis
analysis [33] and deletion mutagenesis studies [34,35] have suggested that the lipid-free apoA-I
molecule is organized into two structural domains; the N-terminal and central parts form a helix
bundle whereas the C-terminal helices form a separate, less organized structure (Fig. 4). The helix
bundle organization in the N-terminal domain is also supported by fluorescence studies of single
tryptophan mutants of human [36] and chicken apoA-I [37]. The crystal structure of N-terminal
truncated D1–43 apoA-I revealed a-helices arranged in a linear fashion rather than in a bundle but
this structure is postulated to represent more closely a lipid-bound conformation of apoA-I [38].
Such a helix bundle motif of the N-terminal domain in apoA-I is basically similar to apoE except
for the N-terminal helix bundle being less organized and less stable than that in apoE. As shown
Table 1
Parameters characterizing the domains of apoA-I and apoE isoforms
Apolipoprotein Average hydropathy per Tm b (°C) D1=2 c (M) ANS fluorescence
residuea ()kcal/mol) intensityd
apoE2 0.69 52 1.0, 2.7 (biphasic) 1.2
apoE3 0.71 46 1.0, 2.4 (biphasic) 1.2
apoE4 0.73 45 1.6 (biphasic but 1.4
overlapping)
apoE2 22 kDa 0.70 63 2.8 0.5
apoE3 22 kDa 0.74 57 2.5 0.4
apoE4 22 kDa 0.78 50 1.6 0.4
apoE 10 kDa 0.68 56 1.1 1.3
apoA-I 0.80 60 1.0 1.0
D190–243 apoA-I 0.93 56 1.0 0.6
a
Calculated as described by Kyte and Doolittle [15]. A more negative hydropathy is more polar.
b
Midpoint of thermal denaturation determined by far-UV CD measurements [29,35].
c
Midpoint of guanidine denaturation determined by far-UV CD measurements [29,39].
d
Values are ratios to wild-type apoA-I. Estimated error is within 0.1.
Fig. 4. Model of the two-step lipid binding mechanism of apoA-I on a spherical particle.In the lipid-free state, apoA-I
is organized into two structural domains in which the N-terminal domain forms a helix bundle whereas the C-terminal
domain forms a separate, less organized structure. Initial lipid binding occurs through amphipathic a-helices in the
C-terminal domain accompanied by an increase in a-helicity probably in the region including residues 187–220. Sub-
sequently, the helix bundle in the N-terminal domain undergoes a conformational opening, converting hydrophobic
helix–helix interactions to helix–lipid interactions. Reproduced with permission from [35].
in Table 1, guanidine-induced denaturation curves of apoE indicate that the N-terminal and C-
terminal domains unfold independently [29], whereas guanidine-induced denaturation of apoA-I
is monophasic [39]. In addition, a thermal unfolding study has demonstrated that the apoA-I
molecule exhibits a loosely folded, molten globular-like structure (i.e., the a-helices do not occupy
fixed positions with respect to one another and are not organized into a unique tertiary structure)
[40]. The fact that the N-terminal helix bundle domains in apoA-I and apoE contain different sorts
of amphipathic helix in which the helices are class G* in apoE and class A in apoA-I [8], may
underlie the differences in stability. The difference in charged group distribution (Fig. 1(b)) may
affect the burial of polar groups which is known to affect helix bundle stability [41]. In addition,
the proline punctuation is much more regular in apoA-I than in apoE (Fig. 1(a)), presumably
preventing the helices in the bundle from becoming as long and highly organized as they are in
apoE (Fig. 3).
The tertiary arrangement of the C-terminal domain in apoA-I is not understood well. An
electron paramagnetic resonance spectroscopic study indicated that the helices in the C-terminal
domain form a compact antiparallel alignment with residues 188–205 existing as a flexible loop
[17], whereas fluorescence resonance energy transfer (FRET) measurements suggest an extended
conformation [42,43]. Regardless of its exact conformation, the C-terminal domain appears to be
relatively disorganized because it contains an exposed hydrophobic surface that is accessible to
ANS binding (see the difference in ANS fluorescence between WT and D190–243 apoA-I in Table 1)
[35]. In addition, an interaction between the N- and C-terminal domains in the apoA-I molecule is
suggested in which the N- and C-termini interact with each other to maintain the overall structure
of the protein molecule [42,44].
The concept of domain interaction was introduced to account for the influence of the poly-
morphism at position 112 in the N-terminal domain of apoE on the lipid-binding properties of the
C-terminal domain [45]. In apoE4, the N- and C-terminal domains interact differently than they
do in the other isoforms: Arg-112 causes a rearrangement of the Arg-61 side chain in the
N-terminal domain of apoE4, allowing it to interact with Glu-255 in the C-terminal domain
[28,46]. This domain interaction in human apoE4 leads to a less organized structure in the
C-terminal domain and preferential association with very low density lipoprotein (VLDL) rather
than high density lipoprotein (HDL) which is contrary to the behavior of apoE3 [2]. The domain
interaction in apoE4 has been suggested to contribute to the accelerated catabolism of this iso-
form and, consequently, the increased cholesterol and LDL levels in the plasma of individuals
with this genotype [47]. A recent FRET study indicates that the N- and C-terminal domains of
lipid-free apoE3 are in a spatially proximate orientation with respect to each other, probably
through weak hydrophobic interaction [48].
2.3. Quaternary structure
Lipid-free apoA-I and apoE have a propensity to self-associate in aqueous solution because of
hydrophobic interaction between amphipathic a-helices in different molecules. Lipid-free apoA-I
tends to form oligomers at concentrations >0.1 mg/ml [49], but the oligomerization is less efficient
for deletion mutants lacking the C-terminal region [34], indicating that the self-association of
apoA-I is mediated by hydrophobic interaction between the C-terminal helices. Similarly, apoE
exists as a tetramer in the lipid-free state [50] and this tetramerization is thought to be mediated by
the C-terminal domain because the 22-kDa fragment is monomeric while the 10-kDa fragment is
tetrameric in aqueous solution [23]. Sedimentation equilibrium and C-terminal truncation anal-
yses indicate that the extreme C-terminal residues 267–299, which are shown to be very hydro-
phobic by hydropathy analysis (Fig. 2(b)), are responsible for the self-association of apoE [51].
Thus it can be inferred that self-association of apoA-I and apoE promotes stabilization of the
potential a-helical segments of the C-terminal domain that are less organized in the monomeric
form. Interestingly, apoE4 having a more solvent-exposed, less organized structure of the
C-terminal domain [31] shows a greater propensity to self-associate compared to apoE3 [52].
3. Interaction of apoA-I and apoE with lipids
3.1. Molecular mechanism of lipid-binding
It is well established that the C-terminal domain is critical for lipid-binding of both apoA-I and
apoE molecules. In apoE, this domain is the 10-kDa fragment corresponding to residues 216–299
[2], and it contains three of the proposed 22-mer repeats [1]. Assuming from the homology in the
two domain structure of both molecules that the equivalent three C-terminal 22- mer repeats also
comprise the lipid-binding domain in human apoA-I, it follows that this domain spans residues
187–243 [1]. This notion about the domain boundary in the apoA-I molecule is consistent with
previous observations that the C-terminal 187–243 region is necessary for the initial lipid-binding
of apoA-I [34,53,54] and, also, with previous studies employing limited proteolysis of lipid-free
apoA-I that revealed major cleavage sites in the C-terminus particularly near residue 192 [33,53].
Upon binding of apoE to lipids, it has been proposed that the four-helix bundle in the
N-terminal domain undergoes a conformational reorganization to expose the hydrophobic faces
of its amphipathic helices for interaction with lipid molecules [2]. Molecular area measurements at
an air–water interface indicated that the N-terminal domain occupies a larger surface area than
can be accounted for by its globular four-helix bundle conformation, suggesting adoption of an
open conformation by the helix bundle [55]. Subsequent studies using infrared spectroscopy [56],
FRET [57], and interhelical disulfide mutants of the apoE N-terminal domain [58] confirmed that
the four-helix bundle undergoes conformational opening when apoE is complexed with
phospholipid (PL). Kinetic studies of 1,2-dimyristoyl phosphatidylcholine (DMPC) multilamellar
vesicle solubilization by the isoforms of apoE and their N- and C-terminal domains revealed that
the C-terminal fragment is much more reactive than the N-terminal fragment [59]. Furthermore,
the reaction rates of the N-terminal fragments of the isoforms appear to vary inversely with the
stabilities of these fragments [59–61]. In addition, the two domains in full-length apoE molecule
were shown to be not fully available to interact with lipid probably due to conformational re-
striction. This is consistent with the finding that the N- and C-terminal domains of lipid-free
apoE3 are in a spatially proximate orientation with respect to each other [48].
The flexible molten globular-like structure of apoA-I explains its lipid-binding properties [4].
The loose arrangement of a-helices allows rapid interaction of exposed hydrophobic regions of the
protein with lipid, leading to rapid solubilization of DMPC vesicles to form discoidal HDL
particles [59,62]. Based on the finding that the first and last 22-mer a-helices have the greatest lipid
affinity (cf. Section 2.1), a model of association of apoA-I with lipid was proposed in which the
two helices nearest the ends of the molecule bind to lipid first, facilitating binding of the middle
helical domains that have relatively low lipid affinity [14]. Combining this information with
structural studies of lipid-free apoA-I showing that the protein can adopt both globular helical
bundle and elongated rod-like conformations, Rogers et al. [63] proposed a multiple-step mech-
anism for apoA-I lipid-binding to form discoidal complexes. In their model, the initial binding to
lipid occurs through the C-terminal region in the elongated hairpin structure, followed by a
conformational switch of residues 1–43 which unmasks a latent lipid-binding domain comprising
residues 44–65.
Using a series of deletion mutants that progressively lacked different regions along the mole-
cule, we have shown recently that binding of apoA-I to lipids is modulated by reorganization of
the N-terminal helix bundle structure [35]. Given that the two-domain structure in apoA-I is like
that in apoE (Section 2.2), we proposed a two-step mechanism for binding of apoA-I to spherical
lipid particles (Fig. 4) [35]. In this model, apoA-I initially binds to a lipid surface through am-
phipathic a-helices in the C-terminal domain; this process is accompanied by an increase in
a-helicity in the region including residues 187–220. Subsequently, the helix bundle in the N-ter-
minal domain undergoes a conformational opening, converting hydrophobic helix–helix inter-
actions to helix–lipid interactions. In this step, the extra helicity in the N-terminal domain appears
to come from the residues 123–142 [64] as well as 1–43 [18,65,66]. Given the structural similarity of
apoA-I and other exchangeable apolipoproteins such as apoE that are part of the apolipoprotein
multigene family [1], the two-step lipid binding mechanism could be a general feature for lipid
interaction of exchangeable apolipoproteins composed of two structural domains.
The conformational transition from random coil to a-helix upon lipid-binding is thought to
provide the energetic source to drive the lipid interaction of apolipoproteins [67]. Calorimetric
measurements of binding of apoA-I [35,68,69], apoE [70], and apoA-I model peptides [71] to lipid
particles indicate that lipid-binding of these proteins is accompanied by a large exothermic heat,
consistent with the lipid-binding of apolipoproteins being enthalpically driven. We have shown
recently that the contribution of a-helix formation to the enthalpy of binding of apoA-I to egg PC
small unilamellar vesicles is )1.1 kcal/a-helical residue [66], in agreement with an early estimate
()1.3 kcal/a-helical residue) for plasma apolipoproteins using different membrane systems [67].
a-Helix formation in apoA-I also contributes to an increase in the favorable free energy of binding
to lipid ()42 cal/a-helical residue), leading to an approximately two-order of magnitude increase
in the affinity of binding (Fig. 5). This indicates that the transition of random coil to a-helix plays
a critical role in driving apoA-I to interact with a lipid surface. This phenomenon probably ex-
plains why many exchangeable apolipoproteins in the lipid-free state contain random coil
structure especially in the lipid-binding domain, such as the C-terminal domain of apoA-I and
apoE [2,4].
3.2. Lipid-bound conformation
ApoA-I is the major protein component of both nascent discoidal and circulating spherical HDL
particles [72]. HDL discs comprise a segment of PL bilayer surrounded at the edge by apoA-I
molecules. Two general models have been proposed for the organization of apoA-I molecules in
such discoidal HDL particles: a ‘‘picket fence’’ model in which the a-helix repeats of apoA-I are
arranged parallel to the PL acyl chains [73] and a ‘‘belt’’ model where a continuous series of
a-helices of apoA-I are aligned perpendicular to the acyl chains [74]. Since the determination of the
X-ray crystal structure of the N-terminal truncated D1–43 apoA-I molecule that consists of con-
tinuous amphipathic a-helices punctuated by kinks at the regularly spaced proline residues [38],
several pieces of experimental evidence have supported the conclusion that lipid-bound apoA-I
adopts a ‘‘belt-like’’ arrangement around the edge of discoidal complexes. Polarized internal re-
flection infrared spectroscopy [75], fluorescence parallax analysis [76,77], and FRET [78] studies
Fig. 5. Schematic representation describing the thermodynamics of binding of apoA-I to lipids. A hypothetical free
energy of binding for apoA-I that does not undergo a-helix formation upon lipid binding has a value of )9.7 kcal/mol
that corresponds to a Kd value of 120 lg/ml (4.3 lM). The increase in a-helix content when apoA-I binds to lipid
contributes )2.5 kcal/mol to the free energy of binding (possible regions forming a-helices are depicted by hatched
cylinders). This additional free energy leads to a reduction in Kd to 2 g/ml (0.06 lM) that is actually measured in the
binding of apoA-I to small unilamellar vesicles [66].
have confirmed that the orientation of most of the apoA-I helices is perpendicular to the PL acyl
chains. Based on the ‘‘belt’’ model, Segrest et al. [79] have proposed a ‘‘double belt’’ model for a
discoidal HDL complex containing two molecules of apoA-I. In this model, two ring-shaped
molecules of apoA-I are stacked on top of each other with both molecules in an anti-parallel
orientation, allowing the helix registry to maximize intermolecular salt-bridge interactions
(Fig. 6(a)). In contrast to this extended belt-like conformation, a helical ‘‘hairpin’’ model for apoA-
I in discoidal HDL particles has been proposed [78,80]. The helical ‘‘hairpin’’ model offers an
explanation for the discrete particle size heterogeneity observed in reconstituted HDL particles; in
particular, it provides a model for discoidal reconstituted HDL particles containing three apoA-I
molecules [74]. A recent mass spectroscopic study indicated the presence of the salt-bridge inter-
actions between apoA-I molecules present in both the ‘‘double belt’’ and ‘‘hairpin’’ models [81].
Two models of the helix organization of apoE in discoidal particles have also been proposed:
the ‘‘picket fence’’ model [82] and the ‘‘belt’’ model [56]. A recent FRET analysis of the apoE
N-terminal domain in discoidal complexes suggests that the helix bundle opens to adopt a par-
tially extended conformation [83], consistent with the ‘‘belt’’ model. More recently, a fluorescence
parallax analysis study showed that the a-helices in both the N-terminal and the C-terminal
domains of apoE align perpendicular to the acyl chains of PL bilayers (Fig. 6(b)) [84]. It now
Fig. 6. (a) Double belt model for apoA-I structure at the edge of discoidal HDL complex. Two ring-shaped molecules
of apoA-I are stacked on top of each other with both molecules in an anti-parallel orientation, allowing the helix
registry to maximize intermolecular salt-bridge interactions. Only the charged residues at selected positions are ex-
plicitly displayed; basic residues are represented in blue, acidic residues in red, and prolines in green. Reproduced with
permisson from [79]. (b) Model of apoE in discoidal HDL complex depicting the locations of engineered tryptophan
residues on helix 4. Fluorescence from these amino acids was monitored to determine helix orientation. Two molecules
of apoE (blue and gray) of a total of about 4 molecules/particle are depicted in which the helical axes are orientated
perpendicular to the PL acyl chains. Reproduced with permission from [84].
seems clear that the perpendicular orientation of amphipathic helical domains with respect to PL
acyl chains in discoidal particles is a common feature of amphipathic exchangeable apolipopro-
teins.
Spherical HDL particles are quite heterogeneous in size and composition [72]. It is likely that
the apoA-I in spherical HDL is flexible with its conformation being governed by the size and core
lipid composition of the particles [85,86]. On the spherical surface, the amphipathic a-helices of
apolipoproteins are thought to be embedded between PL molecules with their hydrophobic faces
in contact with PL acyl chains [11]. In soluble apolipoproteins such as apoA-I and apoE con-
sisting of a number of different amphipathic helices, the conformational flexibility of the proteins
could allow some helices with low lipid affinity to be excluded from the particle surface [6,8,87].
Indeed, we demonstrated recently that the two domain structure in apoE leads to two different
lipid-bound conformations on lipoprotein-like spherical particles; the N-terminal four-helix
bundle can adopt either open or closed conformations, resulting from binding competition with
Fig. 7. Model of two possible conformations of apoE on spherical particle. This model proposes that at high apoE
surface concentration, the displacement of the N-terminal domain from the lipid surface by the C-terminal domain
causes the N-terminal four-helix bundle to adopt the closed conformation. At low surface coverage, the four-helix
bundle is open and all the a-helices are in contact with the lipid surface. Reproduced with permission from [70].
the C-terminal domain that has a high lipid affinity [70] (Fig. 7). Given the similar domain
structure of apoA-I and apoE [35], it is conceivable that the structural organizations of apoA-I on
discoidal and spherical particles are also different [66], such as with some helices in apoA-I being
out of contact with lipid on the spherical surface forming a so-called ‘‘hinge domain’’ [8,88]. It
seems likely that helices located around residue 100 are responsible for maintaining the plasticity
of the lipid-bound conformation of apoA-I, thereby allowing apoA-I to form discoidal HDL
particles of different size. Indeed, a recent FRET study suggested that apoA-I undergoes a con-
formational change as it adapts from a discoidal to a spherical surface [89]. The concept of
multiple lipid-bound conformations of apoA-I and apoE may provide new insights into how these
proteins modulate their functions (cf. Section 5).
4. Other members of the exchangeable apolipoprotein family
Compared to human apoA-I and apoE, the structure–function relationships of the other
members of the multigene family, apoA-II, apoA-IV, apoA-V and the apoCs have not been
examined as extensively. However, three-dimensional structures for some of these members of
the apolipoprotein family have been reported in recent years. The elucidation of these
structures has provided some insights into their biological functions and these are summarized
in this section.
4.1. ApoA-II
Human apoA-II is a major component of HDL particles and it is synthesized mainly in the
liver. Understanding of the contribution of apoA-II to lipid metabolism is much more limited
than that of apoA-I and the functions of apoA-II are still not entirely clear [90]. ApoA-II consists
of two 77 residue-long monomers that form a homodimer linked through a disulfide bridge and
the molecular mass of each monomer is 8.7 kDa [91]. Exon 4 of the apoA-II gene encodes for
residues 40–77 and this region of the protein is predicted to have only one 11-mer and one 22-mer
amphipathic a-helix: this can be compared to the two 11-mer and eight 22-mer helices encoded for
by exon 4 of apoA-I [1]. Three long a-helices make up most of the structure of each apoA-II
monomer [92]. The helices are largely punctuated at prolines, although not uniformly, unlike the
structure of apoA-I. Lipid-free apoA-II has been shown to have a hydrophobic core but is only
marginally stable under near-physiological conditions in plasma; this is consistent with a molten
globular-like state for lipid-free apoA-II [93]. A high-resolution crystal structure of lipid-free
human apoA-II has been published recently [92]. In this structure, each disulfide-bonded ho-
modimer contains three hydrophobic patches, including residues 6–29, 42–53 and 60–70 from
each chain. Two of these dimers associate to form a tetramer and these tetramers are held together
mostly by complementary interactions between their hydrophobic patches.
Interestingly, dimeric apoA-II binds well to PL and, in fact, with higher affinity than does
apoA-I probably because apoA-II is more hydrophobic [94]. Consistent with this enhanced
binding affinity, dimeric apoA-II can displace apoA-I from HDL particles [95–98]. This raises the
question of the role of the apoA-II cysteine 6-cysteine 6 disulfide bridge in human HDL structure
and function. It has been shown that reduction and carboxymethylation of the cysteine residues in
apoA-II to form monomeric apoA-II does not prevent binding to PL and formation of complexes
[99]. Experiments with synthetic peptides have suggested that there are at least two lipid-associ-
ating domains in apoA-II located at opposite ends of the molecule, between amino acids 12–31
and 40–77 [8]. The apoA-II dimer and monomer form discoidal complexes of similar size, with
twice as many of the latter molecules required per disc [100]. Removal of the disulfide bond in-
fluences the stability of the helical segments around the edge of a discoidal complex as seen by a
decrease in a-helix content of the monomeric protein. The discoidal particles containing the
monomeric form of apoA-II are somewhat more effective than particles containing either dimeric
apoA-II or apoA-I in removing cellular cholesterol [100]. Overall, reduction of the disulfide bridge
of apoA-II probably does not have a major effect in determination of HDL particle size in vivo.
The evolution of the cysteine 6–cysteine 6 disulfide bond in higher primates probably has not had
a major effect on the function of the apoA-II molecule.
4.2. ApoA-IV
ApoA-IV is a 46 kDa protein that was discovered in 1974 in rat plasma HDL [101]. In humans,
it is synthesized by the small intestine and secreted into the gastric lymphatics associated with
chylomicrons. It has been proposed to participate in numerous functions in vivo: these functions
include regulation of food intake [102], gastrointestinal motility [103], structural stabilization of
lipoprotein particles [104] and protection against lipid oxidation and atherosclerosis [105]. ApoA-
IV can also mimic certain roles of apoA-I such as in mediation of cholesterol efflux and activation
of lecithin: cholesterol acyltransferase (LCAT) [106,107]. The amino acid sequences of apoA-I,
apoA-IV and apoE are dominated by multiple 22-amino acid repeats that are predicted to form
amphipathic a-helices (Fig. 1) [1,108]. ApoA-IV contains at least 12 such domains, most of which
are punctuated by proline residues [1,108]. Lipid-free apoA-IV has been demonstrated to be less
stable than apoA-I in terms of its chemical denaturation characteristics [109]. Based on recent
studies in which our laboratory participated, it was concluded that apoA-IV exhibits one large
helix-bundle domain that may be similar to the N-terminal helical bundle domains of apoA-I and
apoE [110]. However, apoA-IV lacks the disordered and hydrophobic C-terminal lipid-binding
domain present in apoA-I and apoE. Indeed, apoA-IV contains a unique glutamine-rich domain
at the C-terminus that actually appears to inhibit the molecule from interacting with lipids and
promoting cholesterol efflux. Since there is no homolog of this sequence in any of the other ex-
changeable apolipoproteins, this domain may have evolved to allow apoA-IV to perform func-
tions that are distinct from other exchangeable apolipoproteins by suppressing its apoA-I-like
functions.
4.3. ApoA-V
Recently, a new family member of the exchangeable apolipoproteins has been identified [111].
Whereas genetic studies clearly demonstrate the important role of human apoA-V in plasma
triglyceride (triacylglycerol) metabolism [111–113], questions remain about the mechanism(s)
whereby this protein exerts its biological effects. The calculated molecular mass of the 366 amino
acid residues is 38.9 kDa and sequence analysis indicates that apoA-V is very hydrophobic [114].
Studies of the physicochemical properties of lipid-free apoA-V indicate that the protein is poorly
soluble between the pH limits of 3.5–9.0 at concentrations >0.1 mg/ml [115]. Like other apo-
lipoproteins such as apoA-I and apoA-IV, apoA-V contains a significant amount of a-helical
structure (32% a-helix, 33% b-sheet, 16% b-turn, and 18% random coil) [114,115]. However,
unlike apoA-I and apoA-IV, the a-helical regions in apoA-V are not composed of homologous
repeating 11- or 22-mer units but instead display considerable heterogeneity in both hydropho-
bicity and length. The observed differences in the hydrophobic properties and the unusual clus-
tering patterns of proline residues in apoA-V indicate that discrete independently folded domains
exerting distinct functional roles may exist within this protein. The association of apoA-V with
large HDL in mouse [111] suggests the presence of lipid-binding domains and, indeed, this has
been demonstrated by the ability of this protein to form discoidal complexes with DMPC [115].
As with other apolipoproteins, there is an increase in a-helix content when apoA-V interacts with
lipid [115]. It is not known which regions of apoA-V are responsible for lipid interaction and self-
association. Unlike apoA-I and apoA-IV that both have the ability to activate LCAT, apoA-V is
a poor activator of this enzyme. More studies will be required to understand the biological role of
this protein at the molecular level.
4.4. ApoC-I, C-II and C-III
The common ancestor of the apolipoprotein genes has been suggested to be very similar to the
apoC-I gene in structure and length. The present day apoC genes evolved through multiple du-
plications (for a review, see [1]). ApoC-I has both inhibitory and stimulatory effects on various
enzymes and lipid transfer proteins involved in lipoprotein metabolism. Thus, apoC-I deficiency
in HDL may modulate LCAT activity [116] and the protein can inhibit both phospholipase A2
[117] and hepatic lipase [118]. ApoC-I has also been shown to abolish cholesteryl ester transfer
protein activity in a concentration-dependent manner [119]. The majority of apoC-I is associated
with HDL in vivo with no detectable levels found in LDL. The apoE-mediated binding of
b-VLDL to the LDL receptor (LDLR) and LDL receptor-related protein (LRP) is inhibited by
apoC-I [120]. ApoC-I is the smallest exchangeable apolipoprotein (57 amino acid residues and a
molecular mass of 6.6 kDa) and it transfers among HDL, VLDL and chylomicron particles.
There appear to be at least two lipid-associating domains in apoC-I located between residues 1–31
and 32–57 [121]. Exon 4 encodes for only one 11-mer amphipathic a-helix in residues 40–57 and
the structure of apoC-I is believed to be comprised of two dynamic helices that are stabilized by
interhelical interactions and connected by a short linker region. The minimal folding unit in the
lipid-free state of this apolipoprotein comprises a helix-turn-helix motif formed of four 11-mer
sequence repeats [122,123]. The structures of two apoC-I fragments in ‘‘lipid-mimicking’’ sodium
dodecyl sulfate micelles have been solved by NMR [123]. A dramatic increase in a-helix content is
observed for both fragments and the three-dimensional structures correspond to the helix-turn-
helix motif with hydrophobic interactions as the stabilizing force.
ApoC-II is the major activator of lipoprotein lipase, a key enzyme in the regulation of tri-
glyceride levels in human serum [124]. It is a single polypeptide chain of 79 amino acid residues
and has a calculated molecular mass of 8.9 kDa. Exon 4 encodes for two 11-mer amphipathic
a-helices and the full-length apoC-II molecule contains amphipathic a-helical sequences that are
postulated to mediate binding to lipid surfaces and to account for the increase in a-helical content
on lipid binding [8]. The lipid-binding region of the apoC-II molecule is located towards the
N-terminus whereas the lipoprotein lipase-activating domain resides towards the C-terminus. It
seems that once apoC-II is bound to the surface of a lipid particle, formation of a high-affinity
complex with lipoprotein lipase is favored and activation occurs by interaction of the helical
apoC-II domain spanning residues 39–62 with the enzyme [125]. Lipid-free apoC-II is postulated
to exist as a compact but flexible intermediate in solution [126] and NMR spectroscopy has shown
that apoC-II in sodium dodecyl sulfate micelles contains three a-helical regions spanning residues
16–36, 50–56 and 63–77 [127].
ApoC-III is the most abundant C apolipoprotein in human plasma and it is synthesized mainly
in the liver and to a minor extent in the intestine. ApoC-III is the principal plasma inhibitor of
VLDL lipolysis; it acts both by direct inhibition of lipoprotein lipase activity and by interference
with lipoprotein association with cell-surface glycosaminoglycan matrix [128]. ApoC-III also
interferes with remnant lipoprotein clearance [128]. The mature apoC-III protein consists of 79
amino acid residues with a molecular mass of 8.8 kDa. Exon 4 encodes for one 11-mer and one
22-mer amphipathic a-helix [1]. No three-dimensional structure of apoC-III has been reported but
a model structure based on sequence alignment of apoC-III with apoA-II has been described [7].
The model structure exhibits a helix-turn-helix fold similar to that observed in apoC-I and apoC-
II. In addition, the apoC-III model reveals a cluster of C-terminal residues that are essential for
binding of apoC-III to PL. Recently, the lipid-interacting properties of the N-terminal domain of
apoC-III have been investigated [129]. Through the use of molecular modeling, it is predicted that
the fragment corresponding to residues 6–20 of apoC-III is obliquely oriented at the lipid-water
interface due to an asymmetric distribution of the hydrophobic residues along the a-helix axis. It
is hypothesized that this tilted peptide of apoC-III is responsible for its loose binding to lipo-
protein particles allowing it to exchange freely from the surface.
5. Structure–function relationships
Among the exchangeable apolipoproteins, the structure–function relationships of apoA-I and

apoE have been studied the most extensively. The high level of interest in these two proteins has
arisen because they both exhibit strong anti-atherogenic properties. The insights that under-
standing of the various structural domains in the apoA-I and apoE molecules provides into the
functionalities of these proteins are the focus of this section.
5.1. ApoE
The anti-atherogenic function of apoE arises, in large part, from its profound effect on plasma
lipoprotein levels. As a component of lipoprotein particles, apoE is a high-affinity ligand for the
LDLR and this interaction is followed by endocytosis of the lipoprotein particle [2,130]. This
process leads to hepatic clearance of apoE-containing lipoprotein particles and a reduction in
plasma cholesterol levels. A region in the center of the apoE molecule that is enriched in basic
residues is responsible for binding to acidic regions in the LDLR. Chemical modification and
mutagenesis studies with apoE have demonstrated that a cluster of arginine and lysine residues
located between residues 136–158 represents the binding site for the receptor [2]. The arginine
residue located in position 158 in the apoE3 isoform is mutated to cysteine in apoE2 and the loss
of a basic residue in the latter isoform leads to defective binding to the LDLR [2,130]. It seems
that maintenance of the appropriate positive charge at the C-terminal end of the receptor-binding
region is particularly critical for effective interaction with acidic residues on the LDLR [131].
Disruption of the amphipathic a-helix spanning residues 136–158 abolishes receptor binding,
indicating that this structural motif in apoE is critical for function [132]. As discussed in Section
2.2, the 22-kDa N-terminal domain of apoE adopts a four-helix bundle structure in the lipid-free
state (Fig. 3). The basic residues in the receptor-binding region are located on the polar face of the
fourth amphipathic a-helix in this bundle and they are exposed to the aqueous phase [2]. How-
ever, despite this apparent accessibility of the basic residues, apoE in the lipid-free state does not
bind to the LDLR. Interaction of apoE with PL is necessary to induce a conformational change
that promotes high affinity interaction with the receptor. Lipid-binding leads to opening of the
helix bundle (Section 3.1); this conformational change can increase exposure of lysines 143 and
146 to the aqueous phase [133] and induce helix formation by residues 165–179 [134]. These
changes increase the positive electrostatic potential in the receptor-binding domain of the apoE
molecule enhancing interaction with acidic elements of the LDLR. ApoE is also a ligand for
scavenger receptor class B type I (SR-BI) [135] and, as seen with the LDLR, lipidation of apoE
enhances binding to SR-BI [136]. Since the N-terminal helix bundle domain of apoE is recognized
by SR-BI [136,137], it is likely that opening of this bundle mediates the higher affinity binding of
lipidated apoE to SR-BI.
Since the conformation of an apoE molecule affects its ability to interact with a receptor such as
the LDLR, it is possible that not all apoE molecules on a given lipoprotein particle can function
as ligands for the receptor. This condition applies to VLDL isolated from hypertriglyceridemic
subjects where only a fraction of the apoE present on the surface mediates binding to the LDLR
[138]. This effect probably arises because apoE can exist in two conformations on the surface of
spherical lipid particles (cf. Section 3.2 and Fig. 7). When there is limited interfacial area available,
the N-terminal bundle is closed and not in contact with the particle surface. ApoE molecules in
this conformation are expected to bind poorly to the LDLR.
Besides being a ligand for the LDLR, apoE also interacts with other members of this receptor
family such as the VLDL receptor, apoE receptor 2, and LRP [139]. Interaction of apoE with the
latter receptor is required for hepatic catabolism of chylomicron remnants in the space of Disse
[130,140]. Heparan sulfate proteoglycans (HSPG) act in concert with LRP to complete the in-
teraction of chylomicron remnants with LRP [130]. ApoE in a lipid-poor state is bound to the
extracellular matrix of hepatocytes [141] where it is available for binding to lipids in chylomicron
remnant particles. This enrichment of the lipoprotein particle with apoE enhances binding to LRP
so that endocytosis can occur. It is likely that the two domains in the apoE molecule (Fig. 7) each
contribute to this process. Both domains of apoE contain a heparin-binding site [2], but the
N-terminal site plays a dominant role in the binding of apoE to heparin (Fig. 8) [142]. Thus, the
C-terminal lipid-binding domain of a lipid-free apoE molecule bound to HSPG on the surface of
hepatocytes is readily available for binding to chylomicron remnants. The cluster of positively
charged amino acid sidechains between residues 136–150 that is involved in binding to the LDLR
is involved in the heparin interaction. In the latter case, formation of the high affinity complex
involves juxtaposition of three arginine and two lysine residues of the apoE a-helix with several
complementary sulfate groups on the heparin molecule [143].
Besides modulating lipid transport, the ability of apoE to interact with members of the LDLR
family and with HSPGs can be significant for cell signalling events. Thus, apoE binding to LRP
activates cAMP-dependent protein kinase A and inhibits platelet-derived growth factor-stimu-
lated migration of smooth muscle cells [144]. On the other hand, inhibition of smooth muscle cell
proliferation by apoE is mediated by its binding to HSPG [145]. The anti-mitogenic effect of apoE
maps to its N-terminal domain and the Cox-2 (cyclooxgenase-2) gene is a target of the apoE
signaling [146].
ApoE is involved in the regulation of cholesterol transport in the central nervous system [139]
where it can contribute to neurodegenerative disorders [130,147]. For instance, apoE is associated
with neuritic amyloid plaques in brains of patients with Alzheimer’s disease. This plaque for-
mation seems to involve an interaction between apoE and b-amyloid peptide. The C-terminal
domain, but not the N-terminal domain, of apoE can complex efficiently with this peptide [148].
Interactions between the N- and C-terminal domains of human apoE (cf. Section 2.2) may
modulate the interaction with b-amyloid peptide because apoE3 and apoE4 behave differently in
this regard [147]. The acquisition of more insights into the biological consequences of the human
apoE4 domain interaction will be easier now that a mouse model for this has been created [149].
5.2. ApoA-I
The anti-atherogenic function of apoA-I arises primarily from its central role in reverse cho-
lesterol transport, the process whereby excess cholesterol is transported from cells in the periphery
back to the liver for eventual secretion from the body [150]. As the principal protein constituent of
(a)
0.6 lipid-free apoE
WT
K233E
ApoE bound (g/g dried weight of heparingel)

0.4
0.2 K146E
0.0
(b)
0.6 DMPC discs
0.4 K233E
0.2 WT
K146E
0.0
0 100 200 300 400
Free protein (µg/ml)
Fig. 8. Contributions of different apoE domains to heparin binding. The heparin binding site in the N-terminal domain
of human apoE is located between residues 142–147 while the C-terminal site involves basic residues around lysine 233
[142]. The separate 22-kDa N-terminal and 10- kDa C-terminal fragments in the lipid-free state each bind to heparin,
indicating that both sites are functional in this situation. However, the data in this figure show that only the N-terminal
site is functional in either the lipid-free or the lipid-associated intact apoE molecule. Thus, disruption of the cluster of
heparin-binding basic charges by mutation of a lysine residue to glutamic acid greatly reduces binding when the mu-
tation is in the N-terminal site (K146E) but not when it is in the C-terminal site (K233E) (a). Similarly, in discoidal
complexes with DMPC, only the K146E mutation reduces binding relative to wild-type apoE (b). Surprisingly, en-
hanced heparin binding was observed in apoE (K233E)–DMPC complexes and this is probably due to subtle differences
in the open conformation of the N-terminal domain in the DMPC disc. The results are replotted from data in [142].
HDL, apoA-I mediates the following three steps in reverse cholesterol transport. (1) The for-
mation of nascent HDL particles from cellular free cholesterol (FC) and PL via the interaction
with the cell surface transporter, ATP-binding cassette transporter A1 (ABCA1). (2) The con-
version of FC in HDL particles to cholesteryl ester (CE) by acting as a cofactor for the HDL-
associated enzyme, LCAT. (3) The delivery of CE and FC from HDL to the liver by acting a
ligand for SR-BI located on the surface of hepatocytes. The conformational plasticity of apoA-I
(cf. Section 2) underlies its ability to perform these three quite distinct functions. Thus, apoA-I
can bind various amounts of lipid and form HDL particles of different size and shape (cf. Section
3.2), and its functionality is modulated by its physical state. Current understanding of how the
physical state and domain structure of apoA-I contribute to its functionality in reverse cholesterol
transport is outlined below.
ApoA-I molecules in a lipid-free or lipid-poor state are the preferred acceptor of FC and PL
released from cell plasma membranes by the action of ABCA1 [150,151]. ApoA-I in plasma is able
to cycle between lipid-rich and lipid-poor states [152]. Thus, lipid-free/poor apoA-I molecules
dissociate from HDL during remodeling of the particles by lipid transfer proteins and lipases to
become available to accept lipids from ABCA1. The amphipathic a-helices in apoA-I interact with
ABCA1 and this interaction is not very specific. Thus, ABCA1 can export cellular PL and FC to
different apolipoproteins and peptides [151] and experiments with apoA-I mutants indicate that
different combinations of apoA-I helices can mediate binding to ABCA1 [153]. PL and FC are
released simultaneously to apoA-I [154] and more than one type of HDL particle is created [155].
The larger of these nascent HDL particles are almost certainly discoidal structures (cf. Fig. 6). The
molecular mechanism by which apoA-I acquires lipid from ABCA1 is not defined yet, although
the lipid-binding capability of apoA-I is clearly needed for formation of stable nascent HDL
particles. Consistent with this, deletion of the strongly lipid-binding C-terminal domain of apoA-I
(cf. Sections 2 and 3) leads to a major reduction in ABCA1-mediated FC efflux to apoA-I
(Fig. 9(a)). In comparison, deletions in the helix bundle domain of apoA-I have relatively minor
effects on FC efflux.
The FC in nascent apoA-I-containing HDL particles formed in vivo by the action of ABCA1 is
converted to CE by LCAT. ApoA-I is required for activation of LCAT and its conformation in
the belt-like arrangement in discoidal HDL particles (cf. Fig. 6) is apparently optimal for this
process. Thus, discoidal HDL particles are more reactive with LCAT than the spherical CE-
containing HDL particles that discoidal HDL are converted into by the enzyme [156]. LCAT
binds with higher affinity to discoidal HDL and the initial binding is to lipid surface, followed by
interaction with apoA-I [157]. The LCAT/apoA-I protein/protein interaction decreases the rate
constant of dissociation of LCAT thereby activating the enzyme; this activation may involve
conformational changes in LCAT and/or apoA-I [158]. There has been a lot of interest in es-
tablishing which regions of the apoA-I molecule are involved in this activation. Fig. 9(b) shows
that deletion of either the C-terminal domain (residues 190–243) or certain helices in the helix
bundle domain (residues 139–170) abolishes the ability of apoA-I to activate LCAT. The removal
of the C-terminal domain eliminates the ability of apoA-I to bind to lipid with high affinity
(cf. Section 3.1). Consequently, since apoA-I has to be retained in the surface of the substrate
HDL particle to interact with LCAT, it is possible that the deletion indirectly attenuates the
ability of apoA-I to activate LCAT by reducing the apoA-I surface concentration. In contrast,
removal of residues 139–170 does not alter the lipid affinity of the apoA-I molecule so it is likely
that this region is involved directly in activating LCAT. Consistent with this idea, detailed mu-
tagenesis studies have established that the class A amphipathic a-helix spanning residues 143–164
in human apoA-I is critical for activation of LCAT [159]. The appropriate spatial organization of
specific amino acids in this helix is apparently important because proper alignment and orien-
tation of the helix is required for optimal activation. The fact that three arginine residues (R149,
R153 and R160) located in this helix are critical for activation supports a model in which these
three residues interact electrostatically with acidic residues in the LCAT molecule [160]. This
interaction may be responsible for the ability of apoA-I to retain LCAT on the HDL particle
surface. Given the importance of LCAT in the maturation of HDL particles in plasma, it is to be
expected that this process will be dependent on the presence of apoA-I molecules that can ef-
fectively activate the enzyme. Consistent with the concept, expression in mice of apoA-I mutants
ABCA1 (a)
3
T
6
5
3
6
3
-4
-6
16
24
24
W
-1
∆1
44
0-
3-
3-
44
22
∆
∆1
∆1
∆
∆
LCAT (b)
Relative activity
0
26
70
43
T
W
-1
2
9-
0-
44
9
∆1
∆1
∆
SR-BI (c)
0
43
66
43
3
T
-6
24
W
-2
-
-1
44
∆1
3-
44
90
22
∆
∆1
∆
Fig. 9. Contributions of various segments of the human apoA-I molecule to its interactions with ABCA1,LCAT and
SR-BI. The activities of human apoA-I mutants with the indicated deletions of N-terminal, central and C-terminal
residues are plotted relative to the activity of the WT protein. (a) ABCA1-mediated efflux of cholesterol from human
fibroblasts incubated for 4 h with 2 lg/ml of the apoA-I variants (C. Vedhachalam, S. Lund-Katz and M.C. Phillips –
unpublished observations). (b) Relative Vmax for formation of cholesteryl ester when reconstituted discoidal HDL
particles (dipalmitoyl PC/cholesterol/variant apoA-I) were incubated. (c) Relative binding to SR-BI of reconstituted
discoidal HDL (palmitoyl-2-oleoyl PC/cholesteryl oleate/variant apoA-I) (S.T. Thuahnai, S. Lund-Katz and M.C.
Phillips – unpublished observations). 30 lg protein/ml of the HDL particles that were of similar size were incubated
with SR-BI-transfected COS-7 cells for 2 h at 37 °C and the binding was determined [135].
lacking central a-helices in the LCAT-activating domain leads to defective maturation of HDL
particles in vivo [161,162]. Other more C-terminally located a-helices in the helix bundle domain
also play a role in HDL maturation. Thus, the 22-mer repeats spanning residues 165–209 are
important in determining the size distribution of HDL particles [163].
There are several pathways by which the pool of LCAT-generated CE in mature HDL
particles can be delivered to the liver in the reverse cholesterol transport pathway. One such
route involves SR-BI-mediated uptake of the CE into hepatocytes [164,165]. The selective
uptake of CE from HDL occurs by a two-step process that involves (1) binding of HDL to
SR-BI and (2) diffusion of the CE molecules into the cell plasma membrane. ApoA-I is an
important ligand for SR-BI and the receptor can interact with multiple sites in the apoA-I
molecule [136,166]. A specific amino acid sequence in apoA-I is not responsible for the in-
teraction and the class A or class Y amphipathic a-helix (Fig. 1) is the recognition motif. The
data in Fig. 9(c) are consistent with the idea that the SR-BI/apoA-I interaction is not de-
pendent upon a specific site in apoA-I. Thus, deletions of a-helical segments in either the
helix-bundle domain or C-terminal lipid-binding domain have similar effects and decrease
discoidal HDL binding no more than 50%. This is in contrast to the situation with ABCA1
and LCAT where loss of certain helical segments in apoA-I essentially abolishes the activity
(cf. Fig. 9). Association with PL increases the amount of apoA-I binding to SR-BI [136] and
its conformation on discoidal HDL particles modulates the binding [167]. As a consequence,
larger discoidal HDL particles bind better than smaller particles and deliver more CE [168].
SR-BI also facilitates efflux of FC molecules from the cell plasma membrane to bound HDL
particles [150] and this process is sensitive to the conformation of apoA-I because large
discoidal HDL particles promote more efficient efflux than small particles [168]. This is in
contrast to efflux of cellular FC that occurs by simple aqueous diffusion [150] because, in this
case, changes in discoidal HDL particle size have little effect on efflux [169].
Naturally occurring mutations in apoA-I can affect its functionality [5]. As far as the reverse
cholesterol transport pathway is concerned, it is well established that point mutations in and
around the LCAT-activating region of the helix-bundle domain can reduce the ability of apoA-I
to activate the enzyme. Point mutations in the N-terminal helix bundle domain that interfere with
proper folding can also lead to a different dysfunctionality in the apoA-I molecule. Thus, small
N-terminal fragments of such apoA-I variants have been found in individuals with amyloidosis
[5]. The transition from a-helix to b-structure in these peptide fragments is associated with for-
mation of fibrils [170].
6. Summary and conclusions
It is established that the amphipathic a-helical repeats in the exchangeable apolipoproteins are
the key structural elements responsible for the functions of these molecules. However, the ways in
which these helices are organized to create tertiary structures are not well understood. Recently,
we have shown that two of the larger members of the family of exchangeable apolipoproteins,
apoA-I and apoE, share a common two-domain conformation. The relatively hydrophobic and
disordered C-terminal domain is responsible for binding to lipid. The multiple helices located
in the N-terminal and central regions adopt a helix-bundle conformation; this helix bundle is
relatively ordered and stable in apoE but disordered and unstable in apoA-I. The helix-helix
contacts in the bundle in the lipid-free state are replaced by helix-lipid interactions when binding
to lipid occurs. Thus, lipid binding causes the helix-bundle to open allowing specific a-helices
within this domain to be recognized. For example, the helix spanning residues 136–150 in human
apoE and the helix spanning residues 143–164 in apoA-I can bind to the LDLR and LCAT,
respectively. In comparison, not so much is known about the structures of apoA-IV and apoA-V
which are also larger members of this gene family. However, the lack of a hydrophobic C-terminal
domain in apoA-IV is apparently the reason that this protein binds weakly to lipoproteins.
Smaller members of the gene family, such as the apoC molecules, contain only one or two
amphipathic a-helices so they are unable to form an intramolecular helix-bundle domain.
It can be anticipated that in the future more will be learned about the nature of the helix-helix
interactions in the apolipoprotein molecules. It will be particularly important to generate such
information for these proteins in the lipid-bound state. Good progress is being made in defining
the helix organization in discoidal HDL particles where the ‘‘belt-model’’ is established. It remains
to be seen how the helices are arranged on spherical lipoprotein particles and what helix features
control the affinity for particles of different diameter. At this stage, it is difficult to predict how
changes of particular amino acids in an amphipathic a-helix affect the overall properties of the
helix. This information will be required to understand the effects of mutations on apolipoprotein
structure–function.
Acknowledgements
We are indebted to all our colleagues for their valuable contributions to the studies described
here from our laboratory. Our research reported here was supported by NIH Grants HL22633
and HL56083.
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Progress in

Contributions of domain structure and lipid interaction to

Exchangeable apolipoproteins function in lipid transport as structural components of lipoprotein par-

2. Structures of apoA-I and apoE in the lipid-free state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352

3. Interaction of apoA-I and apoE with lipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358

4. Other members of the exchangeable apolipoprotein family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362

5. Structure–function relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366

6. Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371

2. Structures of apoA-I and apoE in the lipid-free state

2.1. Primary and secondary structure

2.2. Tertiary structure

0 50 100 150 200 250 300 350

2.3. Quaternary structure

3. Interaction of apoA-I and apoE with lipids

3.1. Molecular mechanism of lipid-binding

3.2. Lipid-bound conformation

4. Other members of the exchangeable apolipoprotein family

4.4. ApoC-I, C-II and C-III

Among the exchangeable apolipoproteins, the structure–function relationships of apoA-I and

ApoE bound (g/g dried weight of heparingel)

6. Summary and conclusions

[32] Choy N, Raussens V, Narayanaswami V. Inter-molecular coiled-coil formation in human apolipoprotein E C-

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