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J. Biol. Chem., Vol. 275, Issue 44, 34459-34464, November 3, 2000
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From the
Received for publication, June 16, 2000, and in revised form, July 27, 2000
Lysines in apolipoprotein (apo) E are key
factors in the binding of apoE to the low density lipoprotein
receptor, and high affinity binding requires that apoE be
associated with lipid. To gain insight into this effect, we examined
the microenvironments of the eight lysines in the 22-kDa fragment of
apoE3 (residues 1-191) in the lipid-free and lipid-associated states.
As shown by 1H,13C heteronuclear single
quantum coherence nuclear magnetic resonance, lysine resonances in the
lipid-free fragment were poorly resolved over a wide pH range, whereas
in apoE3·dimyristoyl phosphatidylcholine (DMPC) discs, the lysine
microenvironments and protein conformation were significantly altered.
Sequence-specific assignments of the lysine resonances in the spectrum
of the lipidated 22-kDa fragment were made. In the lipid-free protein,
six lysines could be resolved, and all had pKa
values above 10. In apoE3·DMPC complexes, however, all eight lysines
were resolved, and the pKa values were 9.2-11.1.
Lys-143 and Lys-146, both in the receptor binding region in helix 4, had unusually low pKa values of 9.5 and 9.2, respectively, likely as a result of local increases in positive
electrostatic potential with lipid association. Shift reagent
experiments with potassium ferricyanide showed that Lys-143 and Lys-146
were much more accessible to the ferricyanide anion in the apoE3·DMPC
complex than in the lipid-free state. The angle of the nonpolar face of
helix 4 is smaller than the angles of helices 1, 2, and 3, suggesting
that helix 4 cannot penetrate as deeply into the DMPC acyl chains at
the edge of the complex and that its polar face protrudes from the edge
of the disc. This increased exposure and the greater positive
electrostatic potential created by interaction with DMPC may explain
why lipid association is required for high affinity binding of apoE to
the low density lipoprotein receptor.
Human apolipoprotein
(apo)1 E serves a critical
function in cholesterol and lipoprotein metabolism by modulating the
lipolysis and clearance of plasma lipoproteins and the production of
very low density lipoprotein triglyceride (5). Humans (6, 7) and mice
(8-10) lacking apoE cannot clear remnant lipoproteins from the plasma
and are at increased risk for atherosclerosis. ApoE is a high affinity
ligand for the low density lipoprotein receptor (LDLR) family and for
cell surface heparan sulfate proteoglycans (1, 4). Defective binding of
apoE to receptors causes cholesterol-rich lipoprotein particles to
accumulate in the plasma and is the mechanism of type III
hyperlipoproteinemia, a genetic disorder characterized by elevated
plasma cholesterol and triglyceride levels and accelerated coronary
artery disease (11).
ApoE is a single polypeptide chain of 299 amino acids
(Mr = 34,200) (1) consisting of two
independently folded functional domains (2, 3). The C-terminal domain
contains the major lipid binding region. The N-terminal domain exists
in the lipid-free state as a four-helix bundle of amphipathic
The binding of apoE to the LDLR is thought to involve ionic interaction
between acidic residues in the ligand binding domain of the LDLR and a
cluster of basic residues in the receptor binding region of apoE.
Replacing these basic residues with neutral residues reduces receptor
binding affinity (12). High resolution x-ray crystallography of the
22-kDa N-terminal thrombolytic fragment of apoE in the lipid-free state
has provided detailed information about molecular features of the
The receptor binding region of lipid-associated apoE has not been
examined directly, and the conformational change it undergoes upon
binding to lipid is poorly understood. Neither x-ray crystallography nor multidimensional nuclear magnetic resonance (NMR) can be
applied readily to plasma lipoprotein particles to solve protein
structure. Lipoprotein particles are too big for sufficient resolution
by NMR, and lipoprotein crystals of adequate quality have not been obtained. In this study, we used multidimensional NMR to characterize the microenvironments of the eight lysines in the 22-kDa fragment of
apoE3 in the lipid-free and lipid-associated states.
Materials--
DMPC was purchased from Avanti Polar Lipids
(Pelham, AL), and stock solutions were stored in chloroform/methanol
(2/1) under nitrogen at
Bacteriological media were obtained from Difco Laboratories. The
prokaryotic expression vector pET21 and the competent Escherichia coli strain BL21 (DE3) were from Novagen (Madison, WI). Competent E. coli strain DH5 Preparation of ApoE Variants--
Site-directed mutagenesis by
overlap extension polymerase chain reaction (16) was performed to
introduce lysine to glutamine point mutations (AAA
To express protein (12), a glycerol stock of the E. coli was
streaked on a Luria-Bertani medium plate containing 100 µg/ml ampicillin and incubated at 37 °C overnight. A single colony from the plate was cultured at 37 °C in a G24 environmental incubator shaker (New Brunswick Scientific, Edison, NJ) to an absorbance (660 nm)
of 0.4-0.6. Bacteria were collected by centrifugation (3,000 × g for 10 min at 4 °C), and the pellet was used to
inoculate 6 liters of Luria-Bertani medium containing 100 µg/ml
ampicillin. When the absorbance (660 nm) reached 0.4-0.6, 0.4 mM isopropyl- Preparation of ApoE 22-kDa·DMPC Complexes--
The 22-kDa
fragment of apoE3 was combined with DMPC and isolated by gel filtration
chromatography as follows. DMPC (40 mg) was dried from
chloroform/methanol solution under nitrogen to a thin film on the walls
of a 15-ml glass conical centrifuge tube (Corex). The residue in the
tube was redissolved in 1-2 ml of benzene, shell frozen with dry ice,
and lyophilized. The lipid was incubated with 5 ml of buffer (150 mM NaCl, 1 mM disodium EDTA, 10 mM
Tris-HCl, pH 7.6) for at least 30 min at room temperature and then
sonicated for 45 min at 24 °C in a Branson 350 sonicator fitted with
a tapered tip (5 min on, 2 min off). The mixture was then centrifuged
at low speed to remove titanium released from the sonicator tip. The
slightly translucent solution of DMPC vesicles was kept at room
temperature. A 10-mg aliquot of the 22-kDa fragment was dissolved in
0.1 M NH4HCO3, pH 8.1, to a final
concentration of 2 mg/ml and pipetted into a plastic tube.
Intermolecular disulfide bonds were reduced by adding
Reductive Methylation of the 22-kDa Fragment--
The
13C label was introduced into the DMPC-associated 22-kDa
fragment by reductive methylation of lysines with
[13C]formaldehyde as described elsewhere (18-20). The
reducing agent (sodium cyanoborohydride) was maintained at 5-10-fold
molar excess over the lysines, whereas the molar ratio of formaldehyde
to lysine was kept at 10/1. This allowed maximal labeling of the
lysines, which was reproducible to ± 3%. The level of
incorporation of label was monitored by doping the
[13C]formaldehyde with [14C]formaldehyde
and determining the 14C specific activity of the mixture.
The degree of labeling estimated from 13C incorporation
(NMR) agreed closely with the value from 14C incorporation
determined by liquid scintillation counting. This labeling procedure
did not cause apoprotein degradation, as determined by
SDS-polyacrylamide gel electrophoresis. No labeling of lipid occurred
because no 14C radioactivity could be extracted into
chloroform/methanol.
NMR Measurements--
1H,13C
heteronuclear single quantum coherence (HSQC) two-dimensional NMR
spectra of the 13C-labeled apoE3 22-kDa·DMPC complexes
were obtained with a Bruker DMX400 wide bore spectrometer equipped with
a SGI 02 computer and a 5-mm inverse broad band probe. The spectra were
correlated by using double INEPT transfer (21) and gradient pulses for coherence selection. The temperature for the two-dimensional NMR spectra was set at 310 K. The two-dimensional
1H,13C HSQC spectra were recorded with carbon
decoupling during acquisition. The time proportional phase increment
method (22) was used to obtain phase-sensitive spectra. Chemical shifts
and line widths for lipid-protein complexes were measured as described
elsewhere (18, 23-25). The pseudocontact shifts observed when
K3FeCN6 was added to the aqueous phase were
used to explore the exposure of (13CH3)2 lysines to the aqueous
medium (18). The chemical shifts of
Analytic Methods--
Protein concentrations were determined by
the Lowry procedure (26). Phospholipid content was monitored by
phosphorus analysis (27). Negative staining electron microscopy (28)
was used to measure the size of the apoE3 22-kDa·DMPC discs.
14C radioactivity was assessed by standard liquid
scintillation procedures. Polyacrylamide gel electrophoresis (8-25%
gradient) in either the presence or absence of SDS was performed using
a Pharmacia Phast Electrophoresis System to monitor the purity of the
apoE3 22-kDa fragment or determine the size of DMPC discs.
Circular dichroism spectra were obtained on a Jasco J600
spectropolarimeter equipped with a temperature-controlling device and
interfaced with a computer. The The apoE3 22-kDa fragment (residues 1-191) rather than the
full-length apoE3 was employed to evaluate the microenvironments of the
lysines because there are only 8 lysines in the fragment compared with
12 in the intact molecule, simplifying the resolution and assignment of
the resonances in the NMR spectrum. It is important to note that the
apoE3 22-kDa fragment adopts a receptor-active conformation when
complexed with DMPC and binds to the LDLR with an affinity similar to
that of full-length apoE3 (30). The structural motif adopted by the
apoE3 22-kDa fragment in the lipid-free state is the four-helix bundle
(31), and it has been demonstrated that the bundle undergoes a
conformational change in which it opens, exposing the hydrophobic faces
of the helices when binding to DMPC (14). The x-ray structure of the
lipid-free apoE3-22-kDa molecule (Fig.
1) shows that the receptor binding region
in helix 4 contains a cluster of basic amino acids including Lys-143
and Lys-146 located on the polar face.
To gain insight into the conformational change that gives rise to high
affinity binding of apoE to the LDLR, we used multidimensional 1H,13C HSQC NMR to examine individual lysines
in the 22-kDa fragment of apoE3 in the lipid-free state and in a
complex with DMPC, as applied by Zhang et al. (32) to
calmodulin. In our previous 13C NMR studies of apoE (20)
and amphipathic The spectrum of the 22-kDa fragment of apoE3 in the lipid-free state
showed a well resolved N-terminal
Effects of Lipid Interaction on the Lysine Microenvironments in
Apolipoprotein E*
§,,,,
, and
Joseph Stokes Jr. Research Institute, The
Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
19104-4318, the ¶ Department of Biochemistry, MCP Hahnemann
University, Philadelphia Pennsylvania 19129, and the Gladstone
Institute of Cardiovascular Diseases, Cardiovascular Research
Institute, and Department of Pathology, University of California,
San Francisco, California 94141
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices and contains the LDLR binding region (amino acids 136-150
in helix 4), which coincides with a heparin binding site (2).
-helices (see Fig. 1). However, apoE must be lipidated to bind with
high affinity to the LDLR (13). When the 22-kDa fragment of apoE is
complexed with dimyristoyl phosphatidylcholine (DMPC) as a simple model
of lipidation, the amphipathic -helices open, exposing their
hydrophobic faces (2, 14). This conformational change is associated
with receptor binding activity.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Its purity was assayed by thin
layer chromatography on Silica Gel G plates (Analtech, Newark, DE) in
chloroform/methanol/water (65/25/4, v/v). Lipids were visualized by
spraying developed thin layer plates with a 50% sulfuric acid solution
and charring at 200 °C for 15 min; 100-µg quantities gave a single
spot by charring. D2O (Cambridge Isotope Laboratories,
Andover, MA) was routinely deoxygenated and stored under nitrogen.
[13C]Formaldehyde (99% isotopic enrichment) as a 20%
solution in water was also obtained from Cambridge Isotope
Laboratories. [14C]Formaldehyde (40-60 Ci/mol) in
distilled water was purchased from PerkinElmer Life Sciences.
Anionic contaminants were removed from the formaldehyde by passage
through a small Dowex 1-chloride column (15). NaCNBH3
(Aldrich) was recrystallized from methylene chloride before use
(15). K3FeCN6 was from Aldrich; other salts and
reagents were analytical grade.
and nucleotides were from Life
Technologies, Inc. Pfu DNA polymerase was obtained from
Stratagene (La Jolla, CA). Restriction enzymes were purchased from New
England Biolabs (Beverly, MA).
Isopropyl-
-D-galactopyranoside, -mercaptoethanol, aprotinin, and ampicillin were from Sigma. Ultrapure guanidine HCl was
from ICN Pharmaceuticals (Costa Mesa, CA). Oligonucleotides and DNA
purification kits were from Oligos Etc. (Wilsonville, OR) and Qiagen
(Chatsworth, CA), respectively.
CAA/AAG CAG)
in human apoE cDNA. The template pET-21a(+) apoE3 22-kDa was made
by inserting the human apoE3 22-kDa cDNA into the pET-21a(+)
plasmid (17). Reactions were carried out with pfu DNA
polymerase. Plasmid DNA was prepared with a Qiagen plasmid maxi kit,
and DNA gel extractions were done with a Qiagen QIAEX II gel extraction
kit. The mutation, sequence, and cDNA orientation were confirmed by
restriction enzyme analysis and double-stranded DNA sequencing. The
expression host, E. coli BL21 (DE3), is deficient in some
proteases and has the T7 RNA polymerase gene under the control of the
lacUV5 promoter. Upon induction with
isopropyl--D-galactopyranoside, the lac
repressor is inactivated, leading to T7 RNA polymerase synthesis and to induction of the target gene in the plasmid.
-D-galactopyranoside was added,
and the bacteria were cultured for 2 h. This culture was
centrifuged at 3,500 × g for 20 min at 4 °C. The
pellet was resuspended in 90 ml of ice-cold buffer (150 mM
NaCl, 20 mM Na2HPO4, 25 mM EDTA, pH 8.0, 0.1% -mercaptoethanol, 1% aprotinin,
2 mM phenylmethylsulfonyl fluoride) and sonicated on ice to
lyse the bacteria (6-10 min, 50% pulse cycle). The bacterial debris
was removed by centrifugation (40,000 × g, 20 min at
4 °C), and the supernatant containing the apoE was dialyzed
overnight against 2.5 mM Tris, 2.0 mM EDTA, pH
8.0, at 4 °C. DMPC (650 mg) was dispersed in 45 ml of ice-cold 10 mM Tris, pH 8.0, sonicated on ice (cycles of 5.0 min on,
2.0 min off) until translucent. The DMPC small unilamellar vesicle
preparation was then mixed with the supernatant containing the
expressed apoE at a DMPC/protein ratio of 3.75/1 (w/w). KBr was added
to the DMPC/protein mixture to increase the density to 1.21 g/ml, and
the solution was centrifuged at 302,000 × g for 48-72
h at 15 °C. The top lipid layer was removed from the tubes, dialyzed
against 2.5 mM EDTA, pH 8.0, at 4 °C overnight and
lyophilized. This preparation of apoE·DMPC complexes was delipidated, and the apoE pellet was dissolved in 6 M guanidine HCl, pH
7.2, containing 1% -mercaptoethanol. The apoE was isolated by gel filtration chromatography on a Sephacryl S-300 column. Analysis by
SDS-polyacrylamide gel electrophoresis showed that the apoE was 
95% pure. If necessary, the apoE was purified further by ion exchange
or heparin-Sepharose chromatography.
-mercaptoethanol (0.5 µl/100 µg of protein) and incubating the
mixture for 30 min at room temperature. DMPC vesicles were added, and
the mixture was recycled three times through the gel-liquid crystal
transition temperature of DMPC (23.5 °C) by warming to ~30 °C
and cooling to ~15 °C. The apoE3 22-kDa·DMPC complexes were
separated from uncomplexed protein and lipid by gel filtration
chromatography. The complex (10 mg of apoE3 22-kDa in a 5-ml volume)
was applied to a calibrated Superdex 200 Prepgrade column with a fast
protein liquid chromatography system. Fractions (1 ml each) were
collected, and the absorbance of each fraction was monitored at 280 nm
to locate the protein peak and estimate the particle diameter.
Fractions containing the complexes were pooled and dialyzed against
saline/EDTA before characterization and reductive methylation.
(13CH3)2 lysine and
(13CH3)2-terminal
amino residues of apoE3 22-kDa·DMPC complexes were determined as a
function of pH. Reductively methylated apoE3 22-kDa·DMPC complexes
(sample volume was typically 2.0 ml, 2.0 mg of apoE3 22-kDa/ml) were
introduced into a 5-mm NMR tube. The pH of the solution was adjusted
before each experiment by adding micromolar amounts of concentrated
NaOH or HCl. Two samples were used, one for titration from pH 10.0 to
12.5 and the other for titration from pH 5.5 to 10.0. At each pH, and
before each two-dimensional NMR experiment, a one-dimensional
13C spectrum was obtained with proton composite pulse
decoupling. The pKa values of the
13C-labeled dimethyl lysines were obtained by nonlinear
regression fitting of the chemical shifts at different pH values to the
Henderson-Hasselbalch equation with the GraphPad Prism computer program
(GraphPad Software). The sigmoidal equation is Y = (U + W × 10(X 
Xc))/(10(X Xc) + 1) where Y is the chemical shift, U is the lower
limit of the shift, W is the upper limit, X is
pH, and Xc is pKa.
-helical content of each apoE3
22-kDa·DMPC preparation was derived from the molar ellipticity at 222 nm by established procedures (20, 29).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (54K):
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Fig. 1.
Cross-section of the apoE3 22-kDa
four-helix bundle with the -helix backbones
displayed as strands. The x-ray structure was elucidated by Dong
et al. (31). The locations of seven lysines are indicated.
Lys-1 is located in an N-terminal segment (residues 1-22) for which
density is absent.
-helical peptides (33), methylation had no effect on
lipid binding affinity (20) or on the pKa values of
the lysines (15). Dimethylation of lysines in apoE does decrease
receptor binding activity (34) because some of the lysines are directly
involved in the receptor binding domain. However, this decrease does
not result from a structural perturbation of apoE induced by the
dimethylation or a significant change in the pKa
values of the lysines. Rather, it probably reflects the ~10%
reduction in ion pair energy caused by lysine dimethylation (35), the
reduced hydrogen bonding capability of dimethyl lysine, or both.
-amino group with a
13C chemical shift of 41.0 ppm at pH 10.0 (Fig.
2). The
(13CH3)2 resonances
from the eight lysines were relatively poorly resolved. Six resonances
with 13C chemical shifts of 43.1-43.9 ppm and
1H chemical shifts of 2.1-2.5 ppm were detected. The
existence of multiple (13CH3)2
lysine resonances reflects ordered structures because only a single
resonance is obtained when apoE is present in a disordered state in 8 M urea solution (20). The spectrum of the apoE3·DMPC complex showed striking differences in the lysine resonances, indicating significant changes in the lysine microenvironments and in
protein conformation (Fig. 2). The 13C resonances from the
DMPC molecules in the lipid-protein complex have been described before
(20). The apoE3 22-kDa·DMPC discs were homogeneous in size, with a
hydrodynamic diameter of ~16 nm as judged by their elution from a
calibrated Superdex 200 gel filtration column. Volumetric calculations
indicated that each disc contained about 750 DMPC molecules and seven
apoE3 22-kDa molecules.
View larger version (16K):
[in a new window]
Fig. 2.
Phase-sensitive
1H,13C HSQC spectra of the 22-kDa fragment
(residues 1-191) of human recombinant apoE3 in the lipid-free state
and complexed with DMPC. All of the lysines were converted to
(13CH3)2 lysine by reductive
methylation. The lipid-free 22-kDa fragment dissolved at 2.0 mg/ml in
0.02 M borate in D2O (pH 10.00), and the 3.2/1
w/w apoE3 22-kDa·DMPC discoidal complexes (2.2 mg of protein/ml) in
borate buffer (pH 10.00) are shown. The NMR spectra were recorded with
carbon decoupling during acquisition. Typically, each HSQC spectrum was
recorded under the following conditions: SW2 2400 Hz (6 ppm centered at
3.6 ppm), 2,000 data points in F2 and SW1 2000 Hz (20 ppm centered at
45 ppm), 256 experiments in F1, 48-128 scans. Data were zero filled
once in F1; a sinebell window shifted by 
/2 was applied in both
dimensions before Fourier transformation. Chemical shifts were
referenced to the resonance of 1,4-dioxane in D2O (
= 66.55 ppm) contained in an external capillary.
Sequence-specific assignments of the eight lysine resonances in the
spectrum of the lipidated 22-kDa fragment of apoE3 were made by
producing a series of recombinant variants in which each lysine was
individually mutated to glutamine. To ensure that these mutations did
not affect the properties of the apoE3 22-kDa fragment, we compared the
properties of the mutants and the wild-type molecule. As shown by
circular dichroism, the -helical content of each mutant protein in
the lipid-free state was ~55 ± 5%, which is similar to that of
the wild-type fragment (36, 37). When the mutants were complexed with
DMPC, their -helical content increased by 10-15%, in good
agreement with the increase exhibited by the wild-type protein.
Furthermore, the mutations did not affect the lipid binding capacity of
the protein, and the mutants interacted with DMPC to form complexes
identical in size and lipid-to-protein ratio to those formed with the
wild-type fragment.
Fig. 3 demonstrates how the resonance
from Lys-143 was assigned. Comparison of the two spectra reveals that
the resonance at 13C- = 43.30 ppm and
1H- = 2.26 ppm in the spectrum of the wild-type
protein is missing in the spectrum of the Lys-143 Gln mutant. Some
of the other lysine resonances are not at the same chemical shifts in
the two spectra in Fig. 3 because of a slight difference in pH.
However, when compared at the same pH, the chemical shifts of the
remaining seven lysine resonances (obtained from the pH titration
curves of the individual lysine resonances) were identical. This
finding confirms that mutation of the lysines does not change the
global conformation of the 22-kDa fragment of apoE3 and that the
lysines do not interact with one another. The other lysine resonances shown in the spectra of Fig. 3 were identified in similar fashion. In
each case, the lysine-to-glutamine mutation led to a loss of a single
resonance, as expected. Consistent with surface locations of the
lysines and high segmental motions, the 13C line widths of
all the lysine resonances were similar (20-30 Hz). For comparison, the
13C line width of the N(CH3)3
resonance from the DMPC polar group is about 10 Hz.
|
To characterize further the lysine microenvironments in apoE3
22-kDa·DMPC discs, we investigated the ionization behavior of the
lysines at different pH values. The pKa value for each lysine was obtained by monitoring the chemical shift as a function
of pH over a range of pH values (5.5-12.5). The titration curves were
fully reversible across the pH range studied. The pH dependence of the
13C chemical shifts for Lys-143, Lys-146, and Lys-157 is
shown in Fig. 4. The derived
pKa values for the eight lysines in apoE3
22-kDa·DMPC discs were 9.2-11.1, with Lys-143 and Lys-146 having
unusually low values of 9.5 and 9.2, respectively (Table I). The normal pKa
value for a fully hydrated, noninteracting lysine is 10.5. Because of
the loss of resolution of the lysine resonances in the spectrum of
lipid-free apoE3 22-kDa molecule (Fig. 2), pKa
values for all of the lysines could not be obtained. However, titration
curves showed that all of the lysines had a pKa
>10.0. Examination of the intensities of the resonances in the spectra
of the Lys-143 Gln and Lys-146 Gln variants, in the lipid-free
and DMPC-associated states, gave an estimated assignment for these two
lysines. The pKa values of these resonances were
10.1 and 10.4, respectively, in the lipid-free molecule. Thus,
comparison of the receptor binding domain in the 22-kDa fragment of
apoE3 in the lipid-free and lipidated states showed that the
pKa values of Lys-143 and Lys-146 decreased by at
least 0.6 and 1.2 pH units, respectively, upon binding to lipid. This
finding confirms that the microenvironment in the receptor binding
domain (amino acids 136-150) is altered by interaction with DMPC.
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The exposure of the Lys-143 and Lys-146 side chains to the aqueous
phase was assessed by using K3Fe(CN)6 as a
paramagnetic shift reagent. The ferricyanide anion, a large ion
(diameter ~ 0.9 nm) that causes 13C resonances to
shift downfield, is expected to be attracted electrostatically to the
positively charged dimethyl lysines and to the
N(CH3)3 groups of the choline-containing
phospholipids. The changes in chemical shifts (
) are strongly
dependent on the distance of closest approach between the anion and the
13C atom of interest (18). Ferricyanide shifted the
resonances of Lys-143 and Lys-146 downfield more than the other lysine
resonances in apoE3 22-kDa·DMPC discs (Table
II). Also, the shifts in the resonances
of Lys-143 and Lys-146 were greater in DMPC complexes than in the
lipid-free state (0.19 and 0.25 versus 0.01 and 0.04 ppm,
respectively).
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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The current study presents the first characterization of the microenvironment within the LDLR binding region in a functionally active form of apoE. In particular, knowledge of the properties of the lysine side chains provides insight into how binding to lipid creates a functional LDLR binding site.
The NMR spectra in Fig. 2 demonstrated the major reorganization of the
four-helix bundle structure induced by binding to DMPC. As discussed
previously (2, 14, 36-39), it is apparent that the four-helix bundle
opens so that the nonpolar faces of the amphipathic -helix interact
with the acyl chains of the DMPC molecules. However, the organization
of the -helices in the discoidal complex with DMPC is not
established. Parallel (36) and perpendicular (37) orientations of the
helices relative to the DMPC acyl chains have been proposed. A
conundrum is that most of the basic residues in the LDLR binding domain
of the four-helix bundle (Fig. 1) are solvent-exposed and not involved
in salt bridges (2), but the lipid-free molecule does not bind to the
receptor with high affinity. Given the organization shown in Fig. 1,
one would expect that electrostatic interaction with acidic domains in
the LDLR would be possible. Although intermolecular effects may be
significant in the binding of apoE-containing lipoprotein particles to
the LDLR, the changes in the microenvironments of the LDLR binding domain revealed in this study strongly suggest that the intramolecular reorganization of the four-helix bundle structure induced by lipid is
critical for functionality.
The formation of apoE3 22-kDa·DMPC discs involves opening of the four-helix bundle to allow the nonpolar amino acid side chains to interact with the acyl chains of the DMPC molecules (36, 37). The opening is probably initiated at the flexible end, where there is a loop region near residue 80 (40). Helices 1 and 2 and helices 3 and 4 preferentially remain paired upon exposure of their hydrophobic faces (14, 39), at least in the initial stages of DMPC disc formation.
The current NMR data provide insights into how such a conformational change affects the microenvironments of the lysines in the 22-kDa fragment of apoE3.The local environments of the lysines in helices 2 and 3 (Fig. 1) were not greatly altered by the interaction with DMPC. In the lipid-free fragment, Lys-69, -72, -75, and -95 exhibited pKa values of 10.0-10.5 and ferricyanide-induced downfield shifts of 0.05-0.1 ppm; similar values were observed in apoE3 22-kDa·DMPC discs. Of these four lysines, Lys-69 had the highest pKa of 10.4 (Table I), perhaps because the intrahelical salt bridge with Glu-66 (2) is retained after the opening of the four-helix bundle.
In contrast to the relatively small DMPC-induced changes in the lysine environments in helices 2 and 3, the lysines in helix 4 were significantly affected by the reorganization of the four-helix bundle. The pKa values of Lys-143 and Lys-146 decreased by 0.6 and 1.2 pH units, respectively. Shift reagent experiments indicated that these lysines, which are three amino acids apart on the polar face of the helix, become much more accessible to the ferricyanide anion after binding of the 22-kDa fragment to DMPC. Lys-157, which is three turns along the helix from Lys-146, is not as accessible to the ferricyanide anion (Table II). Lys-157 had an unusually high pKa value of 11.1 (Table I), perhaps because the intrahelical salt bridges with Asp-153 and Asp-154 (2) are maintained after the four-helix bundle reorganizes upon interaction with DMPC.
What is the basis for the striking DMPC-induced changes in the
microenvironments of Lys-143 and Lys-146 (and, by inference, the
neighboring arginines)? The pKa of a lysine
decreases if the local environment becomes more hydrophobic. However,
this seems an unlikely explanation for the decreases in the
pKa values of Lys-143 and Lys-146 because they
became more accessible to the ferricyanide anion upon binding to DMPC.
Most likely these decreases resulted from increases in the local
positive electrostatic potential, which favors deprotonation of the
lysine 
-amino groups. One contribution to such an effect could
involve the interactions of Arg-147. In the lipid-free, four-helix
bundle this residue is involved in interhelical salt bridges with
Asp-107 and Asp-110 in helix 3 (2). If these and similar interactions
are disrupted by the interaction with DMPC, then the net positive
charge potential on the polar face of helix 4 could increase, thereby
decreasing the pKa values of Lys-143 and Lys-146.
This effect would be expected to be greater for Lys-146, as observed experimentally.
The amphipathic -helices in the four-helix bundle (Fig. 1) are all
of the G* type (41). In these helices, the acidic and basic residues
are distributed across the polar face, whereas the nonpolar face does
not contain any charged residues. One difference in helix 4 is that the
angle subtended by the nonpolar face is 100°, which is less than the
angles in helices 1, 2, and 3, which are in the range of 120-180°.
Consequently, the nonpolar face of helix 4 cannot penetrate as deeply
among the DMPC acyl chains at the edge of the discoidal complex. For
this reason, the polar face of helix 4 is expected to protrude at the
edge of the DMPC disc. This domain may be relatively sequestered in the
lipid-free state because of the close helix juxtaposition in the bundle
structure (Fig. 1).
The enhanced exposure of the polar face of helix 4 and the greater
positive electrostatic potential created by interaction with DMPC
probably explain why the apoE3 22-kDa·DMPC binds with high affinity
to the LDLR, whereas the lipid-free molecule does not. The fact that
binding is improved by greater exposure of the basic residues in helix
4 suggests that this helix has to fit into a complementary structure in
the LDLR. The LDLR binds apoE via tandemly repeated, structurally
independent modules that are cysteine-rich and contain an acidic region
that binds calcium (42). The modules have little recognizable secondary
structure, and the details of the ligand binding site are not known
although the presence of a patch of negative surface electrostatic
potential is consistent with ligand binding being mediated by
electrostatic complementarity of the LDLR and apoE (42). The
electrostatic interaction with acidic residues in the LDLR would be
improved by the DMPC-induced increase in positive electrostatic
potential around the domain containing Lys-143 and Lys-146. Hydrogen
bonding is an important component of the salt bridges formed between
the basic residues on apoE and the acidic residues on the LDLR (12). The ability of Lys-143 and Lys-146 to form ion pairs with acidic residues would be decreased because, as reflected in their lower pKa values, a greater fraction of their -amino
groups are deprotonated at neutral pH. Nonetheless, the lysine
-amino groups contribute significantly to the free energy of binding of apoE to the LDLR because replacement of these residues with neutral
amino acids decreases the binding significantly (12).
| |
ACKNOWLEDGEMENTS |
|---|
We thank Brian Auerbach and Barbara Engle for manuscript preparation and Stephen Ordway and Gary Howard for editorial assistance.
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FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grants HL56083 (to S. L.-K.) and HL41633 (to K. H. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Joseph Stokes Jr. Research Institute, The Children's Hospital of Philadelphia, Abramson Research Bldg., Suite 302, 3516 Civic Center Blvd., Philadelphia, PA 19104-4318. Tel.: 215-590-0588; Fax: 215-590-0583; E-mail: katzs@email.chop.edu.
Published, JBC Papers in Press, July 31, 2000, DOI 10.1074/jbc.M005265200
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ABBREVIATIONS |
|---|
The abbreviations used are: apo, apolipoprotein; LDLR, low density lipoprotein receptor; DMPC, 1,2-dimyristoyl phosphatidylcholine; HSQC, heteronuclear single quantum coherence.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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