From the
Protein Engineering Network Centres of
Excellence and Department of Biochemistry, University of Alberta, Edmonton,
Alberta T6G 2S2, Canada and the ¶Lipid Biology in
Health and Disease Research Group, Children's Hospital Oakland Research
Institute, Oakland, California 94609
Received for publication, February 19, 2003 , and in revised form, April 16, 2003.
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ABSTRACT |
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INTRODUCTION |
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Biophysical studies reveal that apoE is comprised of two structural domains, a 22-kDa N-terminal (NT) domain and a 10-kDa C-terminal domain (4, 5). The N- and C-terminal domains are connected by a flexible, unstructured, region encompassing amino acids 191216 that is susceptible to proteolytic cleavage. Studies conducted with isolated domains reveal that the NT domain contains amino acids responsible for binding to the LDL receptor (6). Several lines of evidence have led to a consensus that localizes the receptor-binding site to residues 136150 (7). This region of the protein is rich in basic amino acids, and their proposed role in receptor interactions is consistent with studies demonstrating loss of receptor binding following chemical modification of lysine and arginine residues (8, 9). In the absence of lipid, the isolated NT domain is not recognized by the LDL receptor. On the other hand, complexation with phospholipids results in a particle that binds efficiently to the LDL receptor (6). These data indicate that a lipid binding-induced conformational adaptation of apoE, which can be mimicked by the isolated NT domain, is an essential feature of apoE function as a ligand for receptor-mediated endocytosis of plasma lipoproteins.
X-ray crystallography of lipid-free apoE3-NT has yielded high resolution
structures (10,
11). This domain exists as an
elongated globular four-helix bundle. Each -helix segment is
amphipathic, orienting its hydrophobic face toward the center of the bundle.
The structure of lipid-free apoE3-NT provides a useful starting point for
development of models of how the helix bundle alters its structure upon
interaction with lipid surfaces to adopt a receptor-active conformation.
Weisgraber et al.
(12) studied the surface
properties of apoE3-NT at the air/water interface on a monolayer balance.
These investigators concluded that the protein spreads on the surface to
occupy a volume greater than can be accounted for by the globular helix bundle
conformation. More recently, NMR spectroscopy studies have provided evidence
for a major conformational change in the NT domain upon interaction with lipid
(13). In 1994, Weisgraber
(7) proposed an "open
conformation" model in which the loop segment that connects helix 2 and
helix 3 in the bundle functions as a hinge, about which the protein opens to
expose a continuous hydrophobic surface. Raussens et al.
(14) investigated the
structural organization of apoE3-NT in dimyristoylphosphatidylcholine (DMPC)
complexes by infrared spectroscopy. These investigators presented a model
wherein apoE-NT adopts an open conformation, circumscribing the perimeter of
the disc bilayer with its helical axes aligned perpendicular to the fatty acyl
chains of DMPC, to adopt a receptor active conformation
(6,
15). Support for this model
has been obtained from studies employing fluorescence resonance energy
transfer to evaluate distance relationships between specific sites in the
protein as a function of lipid binding
(16,
17). Lu et al.
(18) provided evidence for the
conformational opening model by demonstrating that apoE-NT dependent
transformation of DMPC bilayer vesicles into disc complexes is abolished when
helical segments in the bundle conformation are tethered by disulfide bond
engineering.
Using an alternate approach Raussens et al.
(19) studied a fragment
derived from human apoE. A 58-residue peptide encompassing the receptor
binding region of apoE was generated by CNBr cleavage of recombinant
apoE3-(1183), purified and characterized. Far UV CD spectroscopy of the
peptide showed that it is unstructured in aqueous solution. Importantly,
however, apoE3-(126183) efficiently transforms anionic phospholipid
vesicles into LDL receptor competent, peptide-lipid complexes. Analysis of
these complexes by electron microscopy revealed disc-shaped particles with an
average diameter of 13 ± 3 nm. Subsequently, the structure of this
receptor-active apoE peptide was determined by NMR experiments conducted in
the presence of the lipid mimetic cosolvent trifluoroethanol (TFE)
(20). In 50% TFE,
apoE-(126183) forms a continuous amphipathic -helix over
residues Thr130Glu179. To extend these findings
and investigate the structural organization of apoE-(126183) in the
presence of lipid, we have determined the structure of apoE-(126183) in
complex with the micelleforming single acyl chain phospholipid
dodecylphosphocholine (DPC). Electrostatic and geometric features of the
apoE-(126183) DPC-bound structure suggest that apoE binds to the LDL
receptor by interacting with more than one of its ligand binding repeats. The
results are discussed in terms of structural determinants responsible for apoE
conformational adaptability and binding to the LDL receptor family.
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EXPERIMENTAL PROCEDURES |
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NMR SpectroscopyNMR experiments were performed on 2
mM 15N-labeled apoE-(126183) in the presence of
34 mM DPC-d38,in500 µl of 10% H2O/90%
D2O, pH 6.0, containing 0.01% (w/v) NaN3 and 2
mM 2,2-dimethyl-2-silapentane-5-sulfonate as an internal chemical
shift reference. NMR experiments were carried out at 25 °C on Varian INOVA
500 MHz and Unity 600 MHz NMR spectrometers. Data were processed using NMRPIPE
(21) and analyzed using
NMRVIEW (22). Complete
1H and 15N spectral assignments of apoE-(126183)
were obtained using gradient-enhanced three-dimensional 15N-edited
TOCSY and NOESY (
mix, 75 ms) experiments to identify spin
systems and inter-residue connectivities as described by Wüthrich
(23). Confirmation of
side-chain assignment was obtained through the use of three-dimensional HNHB
and two-dimensional natural abundance 13CHSQC spectra.
15N T1, T2, and heteronuclear NOE relaxation data were recorded at 25 °C on both 500 and 600 MHz spectrometers using the enhanced sensitivity gradient pulse sequences developed by Farrow et al. (24). The T1 relaxation decay was sampled at 11 time points on each spectrometer: 11.1, 111, 222, 333, 444, 555, 666, 777, 888, 999, and 1110 ms. The T2 relaxation decay was sampled at different 11 time points: 16.6, 33.2, 49.8, 66.4, 83.0, 99.7, 116.3, 132.9, 149.5, 166.1, and 182.7 ms on the 500 MHz spectrometer and 16.5, 33.1, 49.6, 66.2, 82.7, 99.3, 115.8, 132.4, 148.9, 165.4, and 182.0 ms on the 600 MHz spectrometer. The exponential decay curves for T1 and T2 peak intensities were fit using the in-house program Xcrvfit (inhouse written program; www.pence.ualberta.ca/software). 1H-15N NOE values were obtained from the ratio of the peak intensity from proton-saturated and unsaturated spectra. Reduced spectral density mapping was carried out as described in Farrow et al. (25).
Structure CalculationAn ensemble of 147
apoE-(126183) structures was computed from the distance and dihedral
angle restraints available (Table
I) starting with an extended chain using a simulated annealing
protocol (26,
27) in X-PLOR version 3.851
(28). Inter-proton distance
restraints were derived from three-dimensional 15N-edited NOESY
experiments recorded with a mix of 75 and 120 ms. Distances
were calibrated according to Slupsky and Sykes
(29).
backbone dihedral
angles were calculated based on measured
3JHN-H
coupling constants in an HNHA
experiment (30) and the
Karplus equation (31).
dihedral angle restraints were obtained from the ratio of the
dN
(i,i)/d
N(i1,i)
in the three-dimensional 15N-edited NOESY spectrum
(32). The statistical values
for the 50 lowest energy structures are presented in
Table I. Families of structures
were extracted from the ensemble of structures using the program NMRCLUST
(33). For clustering, the
backbone heavy atoms of residues 134149 of the 50 structures were
superimposed, and clustering was based on residues 133177.
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RESULTS |
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Structure of ApoE-(126183)Fig. 1 displays a two-dimensional 1H-15N HSQC NMR spectrum of DPC-bound apoE-(126183) obtained at 600 MHz. Despite the presence of broader peaks than those observed in 50% TFE (20), most resonances were well resolved. Three pairs of resonances overlap (Glu132 with Arg172, Leu148 with Leu159, and Asp153 with Arg167) and other resonances partially overlap (Ala138 with Leu155 and Arg178, and Arg142, Asp151, and Gln163). In addition, three residues, including the two N-terminal residues, Leu126, Gly127 and the residue preceding the C-terminal proline, Gly182, are missing.
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The secondary structure of apoE-(126183) was determined using NMR
spectroscopy based upon NOE connectivities, the H NMR
chemical shift index (CSI)
(34,
35) and the ratio of
dN
/d
N NOEs
(32). A summary of these data
is illustrated in Fig. 2.
Helical secondary structure was defined as previously described
(20).
Fig. 2 shows that DPC-bound
apoE-(126183) is composed of a single
-helix spanning the
sequence from Glu131 to Glu179, with the first five and
last four residues unstructured. Fig.
2 also shows evidence of the simultaneous presence of unambiguous
d
N(i,i+2) and d
N(i,i+4) NOEs for residues
Leu133Val135,
Ala152Asp154, and
Gln156Arg158. According to Wüthrich
(23),
d
N(i,i+4) is characteristic of an
-helix whereas
d
N(i,i+2) is characteristic of a 310-helix. The
simultaneous presence of both NOEs for the same residue may reflect a degree
of internal flexibility for these residues.
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An amide proton secondary shift plot
(Fig. 3) shows a periodicity of
34 residues from Thr130 to Arg178, typical of a
curved amphipathic -helix
(36,
37). In general, hydrophobic
residues tend to have a downfield deviation for this type of structure.
ApoE-(126183) deviates somewhat from the ideal situation. For example
Arg134, Arg145, and Gln156 show unexpected
downfield deviations characteristic of hydrophobic residues, while
Val135, Ala160, and Val161 show upfield
deviations. In a helical wheel representation of this region of the peptide
(data not shown) Arg134, Arg145, and Gln156
are located on the hydrophobic face of the helix, close to the interface
between the polar and nonpolar sides of the amphipathic helix. Since Arg and
Gln have relatively long side-chain structures, the C
and C
(as
well as C
for Arg) acyl groups may be considered as apolar motifs,
maintaining hydrophobic interactions with other nonpolar residues in this
region while the polar terminus of the side-chain is capable of protruding
beyond the hydrophobic face of the helix. Ala160 is located near
the polar-nonpolar interface, close to the polar side. This could explain the
small upfield deviation observed for this residue. Val161 is
located on the polar face of the helix and on the predicted convex side of the
curvature, explaining the unexpected upfield deviation observed for this
hydrophobic amino acid, as the amide proton secondary deviation is related to
the hydrogen bond length. Two regions of the peptide, around
Ala152Asp153 and
Gln163Gly169, show a 34 residue
periodicity but with a lesser intensity and a non-significant amide shift
deviation. These regions might indicate differences in helix curvature around
these amino acids since secondary structural data indicate these residues are
helical. Indeed, previously it was shown with model peptides that a lack of
amide proton secondary shift 34-residue periodicity is not due to the
absence of a helical conformation but, rather, comes from a less curved
structure (37).
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The three-dimensional structure of apoE-(126183) was calculated from
NOE and dihedral restraints as described under "Experimental
Procedures." An ensemble of 147 structures was computed, none of which
contained distance restraint violations greater than 0.2 Å or dihedral
angle violations greater than 2°. The 50 structures with the lowest
calculated total energy were selected for further consideration. According to
PROCHECK-NMR (38), 99.7% of
the non-glycine residues have (,
) angles in the most favored or
the additionally allowed regions of the Ramachandran plot for these 50
structures (data not shown). The average structure, resulting from
superposition of the 50 lowest energy structures, is a long curved helix
spanning residues Glu131 to Arg178, according to a DSSP
analysis (39). The hydrophobic
amino acids are, for the most part, oriented toward the interior of the
curvature as expected from the amide secondary chemical shift behavior for an
amphipathic helix, and as expected for a helix bound to the surface of a lipid
micelle.
Superposition of the final 50 structures along the length of the helix revealed a rather poor convergence. Since the peptide is bound to DPC micelles, at first glance this did not appear to make sense. A relaxation analysis (discussed below) revealed that residues 134149 are quite rigid whereas residues 150178 appear to show slightly increased internal motions in addition to increased slow motions. Superposition of 15 of the most similar 50 structures over residues 134149 reveals a backbone RMSD of 0.55 ± 0.11 Å and a heavy atom RMSD of 1.4 ± 0.11 Å (Fig. 4a). As may be observed, while the entire helix remains intact, the relative position of residues 150178 with respect to residues 134149 changes. Because of this, we tried to extract conformationally related structure subfamilies using the program NMRCLUST (33). For clustering, the backbone heavy atoms of residues 134149 of the 50 structures were superimposed, and clustering was based on residues 133177. Nine subfamilies were obtained with four structures considered as outliers. Among the subfamilies, the first three contain more than 50% of the structures. Fig. 4b shows the representative structures of the three most populated subfamilies in complex with a modeled DPC micelle composed of 54 DPC molecules (40). While the DPC micelle is only a model and is slightly bigger than what we would expect based on the overall correlation time, the complex gives a conceptual idea of how this peptide may adopt different conformations when bound to DPC. It suggests that the C-terminal portion of the peptide (residues 150183) has a lower affinity for the micelle than residues 134149. As will be discussed later, this has important implications for the interaction of apoE-(126183) with the receptor. The structures in each subfamily are well defined over the entire helical length, with RMSDs about the mean coordinate positions of 2.45 Å for subfamily 1, 2.04 Å for subfamily 2, and 1.94 Å for subfamily 3 for backbone atoms of residues 137173. However, for the most stable helical region of the peptide (residues 134149, see below), the RMSDs about the mean coordinate positions for backbone atoms are as follows: 0.73 Å for subfamily 1, 0.71 Å for subfamily 2, and 0.66 Å for subfamily 3. This shows that the structure is well defined locally. As observed previously in TFE, the structure subfamilies display differing degrees of helix curvature.
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Relaxation Measurements of ApoE-(126183)To gain
insight into the motions of DPC-bound apoE-(126183), longitudinal
(T1) and transverse (T2)
15N NMR relaxation times as well as 1H-15N
heteronuclear NOEs were measured. The R1
(1/T1) and R2
(1/T2) relaxation rates as well as the heteronuclear NOEs
at field strengths of 500 and 600 MHz are shown in
Fig. 5. Of the 58 residues in
apoE-(126183), the three missing and twelve overlapping resonances were
not used in the backbone dynamics analysis. In general terms,
apoE-(126183) bound to an isotropic DPC micelle of 50 molecules/
micelle (aggregation number for DPC is 5060, Ref.
41) should result in an
overall rotational correlation time of
13 ns, characteristic of an
isotropically tumbling molecule of
26 kDa. Consistent with this concept,
the average rotational correlation time for DPC bound apoE-(126183) was
determined to be
12.5 ns.
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Fig. 5 reveals the measured
relaxation characteristics of apoE-(126183) bound to DPC micelles.
Typically, in a rigid globular protein, the R1 and
R2 relaxation rates as well as the heteronuclear
1H-15N-NOE remain somewhat constant throughout the
sequence except for the flexible ends and flexible loop regions. Generally,
flexibility is expressed in these relaxation parameters as increased
R1 and decreased R2 relaxation rates
as well as decreased heteronuclear 1H-15N-NOE. For
apoE-(126183) bound to DPC micelles, residues 126133 at the N
terminus of the peptide as well as residue 182 at the C terminus of the
peptide appear rather flexible. This is expected since secondary structural
information indicates that the peptide forms a contiguous -helix
starting from residue 131 and ending at residue 179
(Fig. 2), with little secondary
structural characteristics outside these regions. Interestingly, residues
134149 exhibit the lowest R1, and the highest
R2 relaxation rates in addition to the highest
1H-15N-NOE. Starting at residue 150, the relaxation
rates start to vary indicating changes in flexibility. These changes are
exaggerated in the R2/R1 ratio. This
ratio reflects the overall rotational correlation time, assuming the value of
R2 in not dominated by exchange
(42,
43). Due to a lack of
significant field dependence on R2, no significant
exchange contributions can explain the relaxation characteristics. Notably,
changes in the relaxation rates appear to coincide with the appearance of
aspartic or glutamic acid residues in the sequence. At the N terminus,
residues Glu131 and Glu132 act to break up the
long-chain hydrophobic amphipathic character of residues 134149.
Asp151, Asp153, and Asp154 appear to
accomplish a similar task after residue 149. A small long-chain hydrophobic
patch of amino acids (Leu159Val161) appears to
increase the R2/R1 ratio slightly,
which is subsequently decreased again due to a string of polar and glycine
residues (Tyr162, Gln163, Gly165). Residues
Glu168 and Glu171 appear to decrease
R2/R1 again followed by a slight
increase involving residues Leu174 and Ile177. Finally,
Glu178 terminates the helix.
The time scales of motions present may be represented by the calculation of
the reduced spectral density functions.
Fig. 6 illustrates the reduced
spectral density functions J(0), J(N), and
J(0.87
H) that describe the motion of the HN bonds
derived from the relaxation parameters R1,
R2, and the 1H-15N NOE. While the
spectral density function at zero frequency, J (0), is sensitive to motions on
all time scales, the high frequency spectral density functions
J(
N) and J(0.87
H) are sensitive to fast
internal motions on the time scales 1/
N and
1/
H (44).
Fig. 6 illustrates the relative
inflexibility of residues 134149. These residues exhibit a
J(0.87
H) consistently lower than 7.5 ps/rad, which indicates
a lack of internal flexibility. Not surprisingly, residues 126130 and
residue 182 exhibit J(0.87
H) spectral densities greater than
15 ps/rad indicating marked flexibility. Interestingly, residues 150180
exhibit J(0.87
H) spectral densities higher than for residues
134149. At 500 MHz, these spectral densities are higher than 7.5
ps/rad. It appears that residues 150154 experience slightly elevated
J(0.87
H), which decreases for residues 156160.
Between residues 164 and 180, J(0.87
H) increases even more.
J(0) data follows the opposite trend for both 500 and 600 MHz data. Residues
134149 exhibit J(0) values of between 4.6 and 5.3 ns/rad. Residues
150162 exhibit J(0) values between 5.0 and 5.2. Finally, between
residues 164 and 180, J(0) values are between 3.5 and 5.2
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DISCUSSION |
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Comparison of ApoE-(126183) in DPC and TFEApoE-(126183) bound to DPC forms a curved amphipathic helix from residue Glu131 to Arg178, in agreement with the structure of this peptide in 50% TFE (20). As with the TFE structure, the helix content of the DPC-bound peptide determined by NMR (83%) is higher than that calculated on the basis of far UV CD spectroscopy (70%). While the secondary structure content of apoE-(126183) is similar, differences exist with respect to the dynamic behavior of the molecule in TFE versus DPC. In TFE, apoE-(126183) exhibits more rigidity in the region between residues 149 and 159. Outside this region, residues 131143 and 163176 display a plasticity of motion such that the further away from the central region, the greater the movement. This type of dynamic behavior is consistent with a helix tumbling in solution.
When bound to DPC, however, apoE-(126183) behaves more isotropically as might be expected for a peptide bound to the surface of a micelle. R1, R2, and 1H-15N NOE have characteristics that, at first glance, appear to follow conventional relaxation behavior for a globular protein. Residues 134149 comprise a more rigid region while residues 150179 constitute a more flexible segment of the polypeptide. Between positions 134 and 149 there are six residues with aliphatic side chains (1 Val and 5 Leu). At residue 151, a series of aspartic acid residues (Asp151, Asp153, and Asp154) appear to disrupt binding of apoE-(126183) to the micelle. Between residues 159 and 161, the peptide is a bit less flexible yet increases dramatically in elasticity after Tyr162, presumably due to the presence of polar and acidic residues, including Glu168 and Glu171. Flexibility in this region may facilitate receptor interaction by permitting structural adaptation to constraints imposed by individual ligand binding repeats. PhD secondary structure prediction (49) analysis of apoE-(126183) yields results that are in agreement with the present NMR structure. PhD predicts two helical segments (Thr130-Glu168 and Arg171-Arg180). In the first predicted helix there is a small decrease in the reliability index for residues Leu149-Arg150. This region and the predicted helix interruption from residues 169 to 170, generally coincide with the present NMR-derived flexibility results.
Curvature and flexibility in this peptide likely facilitate interaction
with lipid surfaces such as lipoproteins or phospholipid disc complexes.
Consistent with these structural characteristics, intact apoE is known to bind
different sized lipoprotein particles, from small HDL to large chylomicron
remnants. Similar physical properties have also been observed for other
apolipoproteins. For example, NMR structures of apoC-I and apoC-II in a
lipid-mimicking environment revealed curved helical regions and/or linker
regions between helices with a loosely defined conformation that confers a
flexible curvature to the structures
(50,
51). The X-ray structure of an
N-terminal deletion mutant of apoA-I, (143)apoA-I, is comprised
of a series of curved amphipathic helices
(52) that, conceivably, could
circumscribe the perimeter of an HDL particle. These examples suggest that the
features observed for DPC-bound apoE-(126183) may represent typical
characteristics of exchangeable apolipoproteins.
Potential Implications of the ApoE-(126183) Structure on ApoE-LDL Receptor InteractionsThe structure of DPC-bound apoE-(126183) has implications with respect to apoE ability to bind to the LDL receptor. For example, Lys143 and Lys147 are known to display increased solvent accessibility in the presence of lipid, thereby increasing their electrostatic interaction potential with elements of the LDL receptor (13). This increase in solvent accessibility appears to be a consequence of helix curvature in this region induced by the presence of lipid. Likewise, structuring of residues 165179 in the presence of lipid most likely facilitates interaction with the LDL receptor since it is known Arg172 is required for optimal binding (47). In the case of DPC micelles it is evident that residues 134149 are anchored at the lipid interface while the region between residues 165 and 179 appears to bind less tightly. It is conceivable that the smaller size and higher radius of curvature of DPC micelles compared with natural lipoproteins affects the ability of apoE-(126183) to retain tight contact with the lipid surface. Although residues 165179 are not as tightly associated with the micelle surface, they are nonetheless helical, suggesting induction of helix in this region of the molecule may be a cooperative event triggered and/or maintained by lipid interaction of another region of the peptide (e.g. residues 134149).
Structural characteristics of the LDL receptor have been discussed in a
preceding study (20). Here, we
briefly outline features that are pertinent to this discussion. The presence
of highly conserved acidic residues within ligand binding modules of the
receptor has led to the hypothesis that ligand-receptor recognition may be due
to electrostatic interactions. Although ligand binding repeat 5 (LR5) was
demonstrated to be the most important
(53), experiments also suggest
a role for other repeats, located upstream of LR5
(53,
54). The well characterized
receptor binding region of apoE (136150) displays a highly positive
electrostatic surface potential and thus, may interact with LR5, the most
negatively charged ligand binding repeat. Residues 165178 become
structured in the presence of lipid and present another positively charged
surface that could interact with a second ligand binding repeat. Glutamic acid
residues punctuating the largely positive surface might be important for
proper orientation of the peptide at the surface of the receptor, interacting
with the few arginine and lysine residues present in almost every ligand
binding repeat. Once a ligand-binding repeat, presumably LR5, has recognized
the most basic region of apoE (residues 136150), adjacent repeat(s)
might interact with another region(s) of apoE (notably around
Arg172). This interaction may orient the apolipoprotein and, by
extension, the entire lipoprotein particle, in a favorable manner with respect
to the receptor. When bound to DPC or in TFE
(20), apoE-(126183)
adopts an elongated structure 70 Å in length. By the same token, a
single ligand-binding repeat has a maximum diameter of 25 Å. These
distances are consistent with the possibility that apoE interacts with more
than one repeat at the surface of the receptor in a process that would be
facilitated by the structural independence of adjoining ligand binding repeats
(55,
56). A recently reported
crystal structure of the entire extracytosolic domain of the human LDL
receptor provides support for this view
(57). It is known that, after
lipoprotein internalization, at endosomal pH (pH < 6), the LDL receptor
discharges its ligand before recycling to the cell surface. Because this
crystal structure was obtained at pH 5.3, it should represent the endosomal
conformation of the LDL receptor. The most remarkable feature of this
structure is that ligand binding repeats LR4 and LR5 are held in place through
interactions with the
-propeller motif of the receptor, while other
repeats seem to be fixed mainly by intermolecular crystal contacts. The
authors suggest that, at endosomal pH, following release of the lipoprotein
ligand, the
-propeller can serve as an alternative substrate for the two
ligand binding repeats, and this might be a pH-regulated, reversible process.
These conclusions favor a predominant role for LR4 and LR5 in ligand binding
activity of the receptor, in agreement with concepts presented here and
elsewhere (20).
Potential Molecular Mechanism Involved in Structuration of Residues
165178 The lipid bound structure of apoE-(126183)
raises questions about why helix 4 does not terminate at the same residue in
the presence and absence of lipid? In the crystal structure of apoE3-NT
domain, helix 4 ends at Ala164
(10). The amino acids around
this residue, Val-Tyr-Gln-Ala164-Gly-Ala correspond to a typical
Schellman C-capping terminal motif sequence
(Fig. 7), commonly found at the
end of -helices (Ref.
58; for a review see Ref.
59). Furthermore, analysis of
the x-ray coordinates of apoE3-NT (PDB code: 1LPE
[PDB]
; 10) provides evidence for
the presence of Schellman motif structural requirements
(Fig. 7, panel A).
Vadar analysis2
revealed the possibility of an H-bond between the NH group of
Gly165 and the CO group of Tyr162
(Fig. 7C)
corresponding to the C'C2 bond in a Schellman motif.
Although a C''C3 H-bond was not detected,
Ala166 (corresponding to C'' and the last residue observed in
the electron density), has a very high B factor suggesting its position and
orientation may not be accurately determined. The
angle value for the
Gly165 (corresponding to C')is133°, a positive value, as
expected for a left-handed amino acid conformation. The last criterion of a
Schellman motif, namely a hydrophobic interaction between C'' and
C3 (corresponding to Ala166 and Val161), is
more difficult to identify. We used the CSU program
(61) to identify potential
hydrophobic contacts in the vicinity of Ala166 and
Val161. Whereas the side chain of Ala166 is not oriented
correctly to promote hydrophobic interaction between these residues, CSU
analysis detected a potential hydrophobic interaction between the side chains
of Val161 and Leu93 (located at the beginning of helix
3; Fig. 7C). At the
same time, Leu93 makes hydrophobic contact with Ala166.
Thus, we suspect that the hydrophobic interaction required for stabilization
of the C capping motif is provided by a long-range interaction with
Leu93.
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Other interactions seem to exist between the end of helix 4 and (a) residues connecting helices 2 and 3 (the 80s loop; Ref. 11) and (b) residues located at the end of helix 2 and the beginning of helix 3. Tyr162 forms many hydrophobic interactions through its aromatic ring atoms that orient between helices 2 and 3, locking the end of helix 4 in close proximity to the 80s loop (Fig. 7C). Val85 has a potential for hydrophobic contact with the side chain of Ala166 while Thr83 makes a main chain hydrogen bond with Ala164, as detected by Vadar and CSU. This H-bond serves to stabilize the CO group of Ala164 at the terminus of helix 4. Interestingly, most of the residues described here as involved in stabilizing interactions are conserved among different apoE sequences (Fig. 7B). The only non-conserved residue is Thr83. Insofar as Thr83 is substituted only by amino acids having smaller side-chains, these substitutions are not expected to sterically hinder H-bond formation between the backbone NH group at position 83 and Ala164.
The interaction of apoE-NT with lipid is believed to arise by opening the globular 4-helix bundle about a hinge region between helices 2 and 3 (7). It appears that helices 1 and 2 and helices 3 and 4 remain preferentially paired during the first stage of bundle opening (18) with possible subsequent reorganization of helix segments (1618). If this view is accurate, then the hinge region, implicated in the first stage of the opening, must include the 80s loop. Opening the bundle in this manner would disrupt interactions between 80s loop residues and the end of helix 4. Subsequent separation of helices 3 and 4 would abolish long-range hydrophobic interactions between Leu93 and Val161, which contribute to stabilization of the Schellman C capping motif. It has been observed that, upon disruption of such hydrophobic interactions (by the presence of a cofactor, for example), helix structure can extend despite the presence of a C-capping consensus sequence or a helix-breaking Gly residue (58).
In summary, we suggest that lipid binding-induced alteration of
interactions responsible for termination of helix 4 in lipid-free apoE
provides a molecular explanation for the apparent transition of residues
165178 from unstructured to -helix, thereby conferring LDL
receptor recognition properties to the protein. Reorganization of the hinge
region is likely to be an early event in apoE NT lipid binding, and one that
triggers structure formation within residues 165178. In addition to its
role in receptor recognition, structure formation in this region may be an
important adaptation that leads to a favorable orientation of the protein with
respect to the lipid surface. This proposal has specific advantages including
the following. 1) It accounts for the fact that the region of apoE around
Arg172 adopts a helical structure in the presence of DPC while it
is unstructured in absence of lipid. 2) It explains, at the molecular level,
how this region can transition from one structure to another (or more exactly
to the absence of structure); and 3) it explains why the lipid-bound
conformation is required for correct interaction between apoE and the LDL
receptor. The lipid surface effectively serves as a molecular switch to modify
stabilizing interactions (especially hydrophobic interactions) at one end of
the helix bundle, inducing a 15-residue conformational change from
unstructured to amphipathic
-helix that orients and aligns essential
elements of the receptor binding region of this protein.
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FOOTNOTES |
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A Postdoctoral Researcher of the National Funds for Scientific Research
(Belgium) and the recipient of an Alberta Heritage Foundation for Medical
Research (AHFMR) award. Present address: Structure and Function of Biological
Membranes, Free University of Brussels, Campus Plaine, CP 206/2, Blvd. du
Triomphe, B-1050 Brussels, Belgium.
|| To whom correspondence should be addressed. Tel.: 510-450-7645; Fax: 510-450-7910; E-mail: rryan{at}chori.org.
1 The abbreviations used are: apo, apolipoprotein; CSI, chemical shift index;
DMPC, dimyristoylphosphatidylcholine; DMPG, dimyristoylphosphatidylglycerol;
DPC, dodecylphosphocholine; HSQC, heteronuclear single quantum correlation;
LDL, low density lipoprotein; NOE, nuclear Overhauser effect; NOESY, nuclear
Overhauser effect enhancement spectroscopy; NT, N-terminal; RMSD, root mean
square deviation; TFE, trifluoroethanol; TOCSY, total correlation
spectroscopy.
2 Vadar,
www.pence.ualberta.ca/software/vadar.
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ACKNOWLEDGMENTS |
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REFERENCES |
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