The Spatial Organization of Apolipoprotein A-I on the Edge of Discoidal High Density Lipoprotein Particles
A MASS SPECTROMETRY STUDY*
W. Sean Davidson
and
George M. Hilliard ¶
From the
Department of Pathology and Laboratory
Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0529 and the
¶Department of Molecular Sciences and Center of
Excellence in Genomics and Bioinformatics, University of Tennessee Health
Science Center, Memphis, Tennessee 38163
Received for publication, March 18, 2003
, and in revised form, April 28, 2003.
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ABSTRACT
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The three-dimensional structure of human apoA-I on nascent, discoidal HDL
particles has been debated extensively over the past 25 years. Recent evidence
has demonstrated that the
-helical domains of apoA-I are arranged in a
belt-like orientation with the long axis of the helices perpendicular to the
phospholipid acyl chains on the disc edge. However, experimental information
on the spatial relationships between apoA-I molecules on the disc is lacking.
To address this issue, we have taken advantage of recent advances in mass
spectrometry technology combined with cleavable cross-linking chemistry to
derive a set of distance constraints suitable for testing apoA-I structural
models. We generated highly homogeneous, reconstituted HDL particles
containing two molecules of apoA-I. These were treated with a thiol-cleavable
cross-linking agent, which covalently joined Lys residues in close proximity
within or between molecules of apoA-I in the disc. The cross-linked discs were
then exhaustively trypsinized to generate a discrete population of peptides.
The resulting peptides were analyzed by liquid chromatography/mass
spectrometry before and after cleavage of the cross-links, and resulting peaks
were identified based on the theoretical tryptic cleavage of apoA-I. We
identified at least 8 intramolecular and 7 intermolecular cross-links in the
particle. The distance constraints are used to analyze three current models of
apoA-I structure. The results strongly support the presence of the salt-bridge
interactions that were predicted to occur in the "double belt"
model of apoA-I, but a helical hairpin model containing the same salt-bridge
docking interface is also consistent with the data.
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INTRODUCTION
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High plasma levels of high density lipoprotein
(HDL)1 are widely
thought to be protective against human cardiovascular disease. Apolipoprotein
(apo)A-I, a 243-amino acid, 28-kDa protein is a key mediator of HDL function.
It is required for lecithin:cholesterol acyl transferase-mediated maturation
of HDL and may be a major ligand by which cholesteryl esters are delivered to
the liver via the scavenger receptor type B class 1 receptor
(1). In addition, the recent
discovery of the importance of the ATP binding cassette protein in lipid
transport
(24)
indicates that an important apoA-I to cell surface interaction may occur
during HDL formation and reverse cholesterol transport. One of the major
obstacles to a better understanding of these interactions has been the paucity
of detailed structural information for apoA-I in its various states of lipid
association.
Homogeneous discoidal forms of HDL are easily reconstituted from purified
protein and lipids in vitro
(5), and these reconstituted
HDL (rHDL) analogs have been used heavily for structural studies. The discs
likely exist as a phospholipid/cholesterol bilayer surrounded at its edges by
the hydrophobic regions of the amphipathic helices of apoA-I (for a review see
Ref. 6). Segrest et
al. and others (7,
8) proposed in the late 1970s
that the
-helices of apoA-I were arranged around the disc circumference
with the long axis of the helices perpendicular to the acyl chains. This
became known as the "belt" or "bicycle wheel" model.
Alternatively, other investigators theorized that the 22-amino acid helical
repeats could traverse the bilayer edge with the helices parallel to the acyl
chains (9). This "picket
fence" model was challenged by the first successful x-ray crystal
structure of a lipid-free fragment of apoA-I by Borhani et al.
(10). The crystal structure
showed a tetramer of highly
-helical apoA-I molecules arranged in a
ring-shaped complex, with no evidence of hairpin turns. Borhani et
al. hypothesized that the ring motif in the crystal structure could be
applied to the case of lipid-bound apoA-I on a disc. Since then, a belt-like
orientation for the helices of apoA-I has been supported by polarized infrared
spectroscopy experiments performed by Koppaka et al.
(11). In addition, we
published a series of studies in which various single tryptophan mutants of
apoA-I were analyzed in discoidal HDL particles containing phospholipids with
quenching groups at various positions along the acyl chain. The results
clearly showed that all eight 22-amino acid helices in apoA-I were oriented
perpendicular to the phospholipid acyl chains
(12,
13).
With the question of apoA-I helical orientation addressed, attention has
focused on determining spatial relationships between two molecules of apoA-I
on a disc. Segrest et al.
(14) recently published a
computer model referred to as the "double belt" model for a
reconstituted HDL particle 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 forming an almost continuous helix that wraps around the perimeter
of the phospholipid disc in an anti-parallel orientation. Computer analysis of
the model predicted a particular registry between the monomers resulting in
the greatest potential for salt bridge connections between the two molecules.
An alternative belt-like model, initially suggested by Brouillette
(15), predicts that apoA-I
molecules are arranged in a hairpin orientation. In this model, about half of
the molecule interacts with one leaflet, there is a turn, and the other half
runs anti-parallel to the first on the opposing leaflet. This idea was
supported by fluorescence energy transfer experiments performed by Tricerri
et al. (16). The
model preserves the potential for stabilizing salt bridge interactions between
the same residues that were proposed for the double belt, although these must
occur intramolecularly in the hairpin model. We have proposed a third model
termed the "Z" belt orientation
(13). This arrangement is
similar to the hairpin except that, instead of traversing back along itself,
the molecule proceeds in the same direction on the opposing leaflet, giving
the potential for interlocking interactions between the molecules.
Although traditional spectroscopic methods such as fluorescence and
circular dichroism have proven useful for studying the generalities of apoA-I
structure in rHDL, data from these approaches are not suitable for high
resolution modeling. Nuclear magnetic resonance (NMR) and x-ray
crystallography data would be very useful, but these techniques have not yet
been successfully applied to native rHDL particles. However, Bennett et
al. (17) recently
reported an elegant study that demonstrated the power of combining high
precision mass spectrometry/peptide analysis with cross-linking chemistry to
identify sites of interaction between two protein molecules. Young et
al. (18) used a similar
approach in combination with a sequence threading technique to generate a
structure of monomeric human fibroblast growth factor that matched well with
the known NMR structure of the protein. In this work, we report the successful
adaptation of this approach to the problem the spatial relationships of two
molecules of apoA-I on the edge of a discoidal HDL particle. The results
provide the most comprehensive determinations of distance constraints within
an rHDL particle and strongly confirm the presence of the salt bridge
interactions predicted by Segrest et al.
(14) present in both the
double belt and hairpin models.
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EXPERIMENTAL PROCEDURES
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ApoA-I PurificationPurified human plasma apoA-I was
obtained from human HDL (1.21 < density > 1.062 g/ml) isolated as
reported (19). Briefly, HDL
was freeze-dried and extracted with chloroform/methanol. The pellet was
suspended in 10 mM Tris HCl with 6 M urea and applied to
a Q-Sepharose column (XK 2.6/40, Amersham Biosciences) pre-equilibrated and
eluted at 4 ml/min at room temperature in the same buffer. Fractions
containing apoA-I as determined by SDS-PAGE electrophoresis were dialyzed into
5 mM ammonium bicarbonate buffer and freeze-dried. Proteins were
solubilized in 3 M guanidine for 1 h and then dialyzed into
standard phosphate buffer (SPB) (20 mM sodium phosphate, 0.15
M NaCl, pH. 7.8) prior to use in reconstitution experiments.
Preparation of rHDL ParticlesReconstituted HDL (rHDL)
particles were prepared using 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC)
(Avanti Polar Lipids, Alabaster, AL) at lipid to protein molar ratios of 90:1
according to the method of Jonas
(5). Lipids were dried under
nitrogen and resuspended in SPB. Deoxycholate (Fisher, deoxycholate: lipid,
1.3:1, w/w) was added and incubated at 37 °C for 1.5 h with mild vortexing
every 15 min. The protein was added and incubated a 37 °C for 1 h. The
cholate was removed by dialysis against SPB (5 changes of 2 liters for at
least 4 h each at 4 °C). The particles were analyzed on a non-denaturing,
native polyacrylamide Phast gel (Amersham Biosciences, Piscataway, NJ)
(20). Before cross-linking,
the particles were passed down a Superdex 200 gel filtration column (Amersham
Biosciences) to remove unreacted protein and lipid. Fractions corresponding to
the 96-Å diameter complex were pooled and concentrated by filtration.
The phosphorus method of Sokolof and Rothblat
(21) and the Markwell
modification of the Lowry assay
(22) determined the final
phospholipid and protein concentrations, respectively. The atomic phosphorus
standard was obtained from Sigma (St, Louis, MO).
Cross-linking, Reduction, and Generation of Tryptic
PeptidesDSP (dithiobis(succinimidyl propionate)) (Pierce) was
weighed out and dissolved in ice-cold Me2SO to a concentration of
6.5 mg/ml and used within 5 min. A 7:1 molar ratio of DSP to apoA-I protein
was added to a solution containing human apoA-I/POPC discs in SPB on ice at a
concentration between 0.5 and 1.0 mg/ml. The reaction was incubated at 4
°C for 24 h with periodic vortexing. The reaction was quenched by adding a
stock of 1 M Tris, pH.7.8, to a final Tris concentration of 100
mM. The samples were dialyzed into 5 mM ammonium
bicarbonate to remove any unreacted cross-linker and were lyophilized to
dryness. The lipids were extracted with chloroform/methanol, and the protein
fraction was solubilized in SPB with 3 M guanidine HCl. In some
experiments, the monomeric protein was separated from the dimeric protein
after cross-linking by passage down a Superdex 200 column equilibrated in the
same buffer. Fractions corresponding to the dimer and monomer were collected,
concentrated, and dialyzed into SPB. Each cross-linked sample was split in two
equal fractions. One was labeled "reduced" and incubated with 25
mM dithiothreitol (DTT) from a stock solution in water for 2 h at
37 °C. The other fraction was labeled "x-linked" and treated
identically except receiving the same volume of water instead of DTT. Both
samples were dialyzed against 5 mM ammonium bicarbonate (in
separate containers). 5% (weight of trypsin to apoA-I) sequencing grade
trypsin (Promega) was allowed to digest the protein at 37 °C for 2 h. The
samples were lyophilized to dryness in a microcentrifuge tube in 100-µg
aliquots. The samples were stored at -20 °C until used.
Mass SpectrometryLiquid chromatography mass spectrometry
(LCMS) experiments were carried out on a Sciex QSTAR DE (quadrupole
time-of-flight (TOF)) mass spectrometer fitted with an atmospheric
electrospray ionizer controlled by using Analyst QS 1.1 software (Applied
Biosystems). The spectrometer was programmed for TOF-MS scans from 100 to 2800
atomic mass units at a 1.0-s accumulation time. An Agilent 1100 capillary HPLC
with an Agilent ZORBAX SB-C18 0.5-mm x 15-cm reverse phase column at a
flow rate of 7.5 µl per min was used to separate tryptic peptides prior to
introduction into the mass spectrometer by complex peptide gradient
chromatography. Lyophilized samples were solubilized in mobile phase A
(distilled water with 0.1% trifluoroacetic acid) and eluted with a
0100% gradient of mobile phase B (95% acetonitrile in water with 0.085%
trifluoroacetic acid). 160 pmol of protein was injected per run. The mass
spectra were internally calibrated by a three-point linear method based on the
monoisotopic masses of the following peptides derived from apoA-I:
154160 (mass of 780.4242 Da), 161171 (1300.6412 Da), and
6277 (1931.9265 Da). These peptides were used because they exhibited
prominent peaks in all samples and covered much of the expected mass
range.
Data AnalysisUsing the Analyst QS software, individual mass
spectra were generated for each peak in the total ion chromatograph (TIC) for
each sample. Masses that exhibited an intensity of at least 25 detector counts
were recorded and identified in terms of ion type (M+H, M+2H, etc.).
Monoisotopic masses were determined by averaging the ion series for each mass.
The resulting list of masses was analyzed by the software GPMAW (ChemSW, Inc.)
to assign a putative amino acid sequence identity as either an unmodified
peptide of apoA-I or one or more peptides containing one or more DSP
modifications (see Table I). To
identify cross-links occurring between two peptides, a spreadsheet was used to
sum the masses of all possible combinations of peptides and one or more
intervening cross-links. The resulting data base of theoretical cross-linked
peptide masses was then searched for a given experimental mass. Mass identity
assignments were made using the following criteria: 1) An assignment was made
only if the experimental mass matched the theoretical mass within 40 ppm. This
cutoff was sufficient to identify all single cleavage peptides in control
experiments using unmodified apoA-I that had been completely trypsinized. 2)
All peptides present in a cross-link must contain a Lys residue (not at the C
terminus). 3) It was assumed that trypsin does not cleave on C-terminal side
of a modified Lys residue
(17). This feature of trypsin
was advantageous because it reduced the complexity of the peptide mixture in
the cross-linked sample. 4) Putative cross-links were assigned to masses
present in the cross-linked sample but only if they completely disappeared
when the sample was reduced with DTT. 5) Identities were assigned for putative
intermolecular or intramolecular cross-links only if all peptide
components were recovered with the appropriate number of reduced cross-links
on eligible Lys residues after DTT reduction. 6) It was assumed that trypsin
fully cleaved the protein at every opportunity, with no partials.
Distinguishing Intraversus Intermolecular
Cross-linksIntra-versus intermolecular cross-links were
assigned by comparing the intensity of a given mass between spectra generated
from dimeric and monomeric forms of apoA-I isolated from a cross-linked rHDL
particle by gel filtration. The maximal detector count intensities for the
entire ion series for a given mass were summed for the dimeric and monomeric
cross-linked samples, respectively
(23). The ratio of the
dimeric/monomeric intensities was used to determine if a mass was
substantially less prevalent in the monomeric versus the dimeric
cross-linked protein. For a particular mass, an intensity ratio below 1.7 was
identified as a putative intramolecular cross-link. By contrast, an
intermolecular cross-link was proposed for ratios higher than 1.7. The
appearance of small amounts of intermolecular cross-links in the monomeric
sample was due to slight contamination of the dimeric form in the sample (see
Fig. 3).

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FIG. 3. Gel filtration separation of the DSP cross-linked rHDL sample into
monomeric and dimeric species. The apoA-I/POPC rHDL shown in
Fig. 2A was
cross-linked with DSP as under "Experimental Procedures." After
quenching the cross-linking reaction and removing unreacted cross-linker by
dialysis, the cross-linked apoA-I was delipidated by chloroform:methanol
extraction. The delipidated protein was applied to a Superdex 200 gel
filtration column. Fractions containing the monomeric and dimeric forms were
combined and concentrated by ultrafiltration. Shown is a denaturing
825% gradient SDS Phast gel stained with Coomassie Blue. Lane
1 shows the crosslinked rHDL particle prior to separation. Lane
2 shows the isolated monomeric form of the cross-linked protein. Lane
3 shows the isolated dimeric form of the cross-linked protein.
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FIG. 2. Characterization and cross-linking of a 96-Å apoA-I discoidal rHDL
particle. A, an 825% gradient native polyacrylamide Phast
gel. Lane 1, a discoidal rHDL particle compared with a set of
standards with the indicated hydrodynamic diameters. B, a denaturing
825% gradient SDS Phast gel of the same particle shown in A. Lane
2, an unmodified rHDL disc. Lane 3, DSP cross-linked rHDL.
Lane 4, the same sample as in lane 3 that has been reduced
with DTT. All gels were stained with Coomassie Blue.
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RESULTS
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The ApproachThe case of purified apoA-I on a well-defined
rHDL particle with two molecules of apoA-I is essentially a homodimeric
non-covalent interaction similar to that studied by Bennett et al.
(17) using mass spectrometry.
ApoA-I contains 21 Lys residues that are generally evenly spread throughout
the molecule, making it a manageable candidate for this approach. The
homodimer is first incubated with the homobifunctional cross-linking reagent
DSP, which reacts with the
-amine group of lysine residues. Numerous
cross-links randomly form both intra- and intermolecularly depending on the
number Lys residues within the reagent's spacer arm length of 12 Å. In
addition, the cross-linker may bind to one Lys residue but fail to cross-link
to a second Lys residue before spontaneous hydrolysis of the cross-linker
(17). Once cross-linked,
trypsin is used to cleave after Arg and Lys residues to generate a population
of peptides, some of which are unmodified whereas others are cross-linked. An
aliquot of the peptide mixture is treated with the reducing agent DTT to
cleave the disulfide linkage within DSP
(Fig. 1) to liberate any joined
peptides. A second aliquot is left untreated. Both the cross-linked and
reduced peptide mixtures are then separated by reverse phase HPLC and analyzed
immediately upon elution by electrospray MS. Highly accurate mass spectra are
taken for each peak as they elute from the column. If a particular mass is
present in both chromatograms, it represents a peptide that was not modified
by DSP. However, masses appearing in the cross-linked chromatogram, but not in
the reduced chromatogram, indicate the presence of cross-linked peptides that
were cleaved by DTT. Masses appearing in the reduced chromatogram are due to
the newly freed peptides. The mass data are used to assign a sequence identity
to each peptide by comparing the experimentally derived peptide mass to a data
base of theoretical peptide masses generated from the known protein sequence
and the known cleavage sites of trypsin. Unmodified peptides exhibit masses
equal to the sum of the amino acids in the peptide sequence. The mass of a
cross-linked peptide complex is the sum of each component peptide mass plus an
intact cross-link (see Table
I). Similarly, the mass of peptides containing cleaved cross-links
is the sum of the peptide mass plus a reduced cross-link. After peptide
identification, one can deduce that a cross-link was formed between peptides X
and Y in the native dimer. If each peptide had a single Lys residue in the
sequence, then one can conclude that the two Lys residues were within about 12
Å in the native protein structure.
rHDL Particle Reconstitution, Characterization, and Cross-linking
OptimizationWe generated a simple discoidal reconstituted rHDL
particle with a diameter of 96 Å that is well known to contain two
molecules of apoA-I and about 160 molecules of POPC
(24,
25). This particle was
selected because it is stable, easily produced in high yield in
vitro, and has been used for computer simulation studies testing various
models for apoA-I structure
(14,
26,
27).
Fig. 2A shows a
non-denaturing PAGE analysis demonstrating the homogeneity of this particle.
Its diameter measured 96 ± 3 Å, and it contained a final POPC to
apoA-I ratio of 79 ± 4:1. To work out the conditions for cross-linking
the apoA-I molecules on the complex with DSP, pilot experiments were performed
in which the molar ratio of DSP to apoA-I was varied from 2.5:1 to 50:1 (data
not shown). Ratios above 10:1 were sufficient to cross-link about 97% of the
apoA-I to a dimeric form as visualized by SDS-PAGE. Higher ratios could drive
the reaction completely to dimers. We chose a ratio of 7:1 for further
experiments to minimize the chances of perturbing the particle structure and
to give an opportunity to study the monomeric form of the cross-linked apoA-I
(see below). Cross-linking at 4 °C was found to reduce the heterogeneity
of the dimeric band versus incubations at room temperature probably
by minimizing thermal motions within the particle.
Fig. 2B shows the
results of a typical cross-linking experiment under these conditions. ApoA-I
(28 kDa) in the rHDL particle was cross-linked to a dimer (56 kDa), which
could be mostly reduced back to a monomer by cleavage of the cross-link with
DTT. We consistently observed a small amount of trimer (84 kDa) in these
particles (Fig. 2, lane 3,
top band) of about 5% of the total staining. We believe that this arose
from a slight contamination of the 108-Å rHDL particles in our
preparation, a complex with three molecules of apoA-I
(24). A small percentage
(about 5%) of the apoA-I remained as a monomer under these conditions. The
appearance of the dimer upon DSP cross-linking was independent of the rHDL
concentration between 0.5 to 5.0 mg/ml (data not shown), indicating that the
cross-links were formed within rHDL particles and not between two rHDL
particles (28). The presence
of the cross-links did not change the average helical content of apoA-I from
that of the unmodified form as measured by circular dichroism (both were about
75% helical), arguing that the cross-links did not significantly perturb the
structure of the protein. Furthermore, we found that the ratio of cross-linker
to apoA-I did not significantly affect the cross-links identifiable by MS
(data not shown).
To get a sense of which experimental mass values originate from
intermolecular versus intramolecular cross-links, a sample of
cross-linked rHDL particles (i.e. the sample in
Fig. 2, lane 3) was
delipidated and then separated into the component monomeric and dimeric
species by gel filtration chromatography (see "Discussion").
Fig. 3 shows that cross-linked
apoA-I was resolved into two fractions, which will hereafter be referred to as
the "cross-linked dimer" sample and the "cross-linked
monomer" sample, respectively. Despite our best efforts, there was a
slight contamination of <5% of the dimeric form in the cross-linked monomer
fraction.
Mass SpectrometryFour different delipidated apoA-I samples
were prepared from rHDL particle preparations. They were the unmodified
monomer, DSP cross-linked dimer, DSP cross-linked monomer, or cross-linked
then reduced by DTT (reduced monomer). Each was subjected to trypsin digestion
and the resulting peptides were analyzed by LCMS.
Fig. 4A shows the
total ion chromatograph (TIC) for the unmodified monomer. The TIC can be
thought of in much the same way as a UV trace for a typical HPLC chromatogram
except that, instead of an absorbance reading, the intensity value is
generated by the mass spectrometer as a summation of all ions striking the
detector during a scan at a particular elution time. The chromatogram shows
some 30 peaks that contained a total of 37 masses. All but five of these
masses were identifiable from the theoretical cleavage of human apoA-I. The
total apoA-I sequence coverage was 94% with all predicted peptides greater
than two amino acids identified. There was no evidence of partial cleavage
products generated by trypsin. The experimental monoisotopic mass values were
all within 40 ppm of the theoretical value.
Fig. 4B shows an
overlay of the TIC of the cross-linked dimer (blue) and the DTT
reduced monomer (red). It is clear that the peak pattern became more
complex in cross-linked versus unmodified protein. Numerous
differences between the cross-linked dimer and the reduced monomer
chromatograms are apparent, including several peaks that completely disappear
from the cross-linked dimer with new peaks appearing upon DTT treatment.

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FIG. 4. Total ion chromatographs (TIC) of untreated, cross-linked, and reduced
samples of apoA-I in a 96-Å rHDL disc. The peaks in each
chromatogram correspond to peptides eluting from a reverse phase HPLC column.
Each peak is a summation of the intensities of all ions striking the mass
spectrometer detector at the indicated elution time. There are typically from
one to five peptide masses present in each peak. A, TIC of an
unmodified apoA-I rHDL sample. B, overlays of the TIC of the
cross-linked dimer sample (blue) and the reduced monomer sample
(red). The same mass of protein was injected for each run. Peaks that
were unique to each chromatogram or exhibited a significant change in
intensity are indicated with the appropriately colored arrow. The
intensity is in detector counts per second.
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A list of masses was accumulated from individual mass spectra for each peak
in all four chromatograms, and each was assigned an amino acid sequence
identity using the criteria described under "Experimental
Procedures." A summary of the identification process for masses observed
in the cross-linked dimer spectrum is shown in
Table II. Of the 110 masses
found, 56 were insensitive to DTT reduction. Most of these were identified as
peptides that either contained no Lys residues or contained Lys residues that
had escaped modification by DSP. We found evidence for at least some degree of
DSP modification for 19 of the 21 Lys in apoA-I. Lys28 and
Lys59 appeared to be completely inaccessible to the cross-linker,
suggesting that they may be buried within protein elements or lipid in the
native rHDL particle. By contrast, lysines 40, 118, 133, 140, 226, and 239
were among the most active sites of modification, because they were commonly
found with hydrolyzed cross-links or cross-links to numerous different
residues. Although DSP can modify the free amino group on Asp-1 of lipid-free
apoA-I, we saw no evidence that this residue was modified in the rHDL. We
noted that the peaks with the highest intensity tended to be identified as
those with hydrolyzed cross-links. This indicates that successful Lys-Lys
cross-links were a relatively rare event when compared with the case when a
cross-link forms at one site but cannot bind a second site before hydrolysis.
Table III lists the peptides
that were identified with reduced cross-links from the DTT-reduced monomer
sample. The agreement between the observed monoisotopic mass and the
theoretical masses illustrates the accuracy of the LCMS performed with this
instrumentation. Table IV lists
the identification of cross-links found in the cross-linked dimer sample and
the dimer/monomer ratio of intensity of each mass.
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TABLE III Identification of peptides with reduced cross-links after treatment of
the cross-linked dimer with DTT to cleave DSP cross-links
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DISCUSSION
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In the present study, we successfully used LCMS and a thiol-cleavable
cross-linker to derive the most comprehensive list of experimentally derived
distance constraints available to date for apoA-I in a discoidal rHDL
particle. Below, we discuss our interpretation of the MS data and then apply
the information to three recently proposed models for apoA-I on a disc
edge.
Data InterpretationThe random nature of the DSP
cross-linking reaction provides the potential for a wealth of structural
information, because many different Lys pairs residing within reach of the
cross-linker spacer can be identified. However, in proteins with even modest
numbers of Lys residues, the sheer number of potential combinations can
quickly dilute the abundance of any one particular linkage. Therefore, the
detection technique must be capable of accurately identifying small quantities
of cross-linked peptides within a complicated background of hundreds of
peptides that have large variations in abundance. For this reason, we chose
electrospray LCMS versus the more commonly used matrix-assisted laser
desorption ionization MS. The chromatography dimension of LCMS has the
advantage of separating large numbers of peptides into manageable units prior
to the analysis (23).
With over 110 experimentally derived masses in the cross-linked dimer
sample, we set up a stringent set of criteria to faithfully identify
cross-links (see "Experimental Procedures") and eliminate possible
misidentifications. The accuracy of the instrumentation allowed the rejection
of potential identifications if the theoretical mass differed from the
experimental mass by greater than 40 ppm. This translates to a maximal error
of about ± 0.20 Da for the largest peptide complex identified in this
study (4818 Da) or about ± 0.08 Da for a 2000-Da peptide. With this
degree of accuracy, most experimental masses had only one or perhaps two
suitable identification possibilities. In addition, putative intermolecular
cross-links were assigned only if both peptide components were
observed with a reduced cross-link after DTT reduction. Our philosophy was
that it was better to reject a "true" cross-link due to lack of
evidence than to include a misidentified cross-link in the model analysis.
Another important issue when studying homodimer interactions is determining
if experimentally observed cross-links occur inter- or intramolecularly. One
cannot distinguish between the two by studying the covalently linked dimer
alone (17) except in the case
of two identical Lys residues cross-linked together. Therefore, we separated
delipidated apoA-I from a cross-linked rHDL into the component monomeric and
dimeric species by gel filtration. By definition, the dimer contains at least
one pair of Lys resides cross-linked on two different molecules of apoA-I.
However, because Lys residues are cross-linked randomly, it follows that a
small fraction of molecules, by chance, would contain the spectrum of
intramolecular cross-links but no intermolecular cross-links and thereby
remain a monomer. The dimeric form also contains the spectrum of
intramolecular cross-links but has the spectrum of intermolecular cross-links
as well. By comparing the peptide maps from both samples, one can distinguish
between the two types of cross-links. Because we had a small contaminant of
dimer from our isolated cross-linked monomer sample, we used the ratio of the
intensities of a given mass between the two samples as an index of their
abundance in each. By analyzing unmodified peptides, we found that the ratio
of the dimer/monomer intensity was between about 0.8 and 1.7; intramolecular
cross-links are expected to fall in this range. Intermolecular cross-links,
being more prevalent in the dimer sample, should give a ratio above 1.7. Note
that all four peptides containing intrapeptide cross-links, i.e. two
lysines cross-linked within the same tryptic peptide (and therefore must be
intramolecular), were correctly indicated to be intramolecular cross-links as
judged by the intensity ratio in Table
IV. Conversely, all three cases of cross-links occurring between
the same Lys residue (therefore must be intermolecular) were correctly
identified.
Superimposing the Cross-links on Belt Models of ApoA-I
Figs. 5,
6, and
7 depict two-dimensional
illustrations of three recently proposed models for apoA-I consistent with a
predominantly helical belt orientation. We wish to make clear that no attempt
was made to draw these models to any kind of molecular scale. Our purpose was
to qualitatively test the general fit of the cross-links to the models.
Detailed structural conclusions will await more sophisticated computer
modeling studies currently under way. Fig.
5 shows the double belt model in its LL 5/5 orientation as
proposed by Segrest et al. showing each Lys residue in its
approximate position in apoA-I. For each model, we took the list of
cross-links from Table IV and
superimposed them onto the models. If the Lys residues were located within
about one helix of each other as dictated by the model, they were drawn as an
appropriately colored line. If a cross-link failed to easily fit the model, it
was not drawn and the notation for the cross-link was underlined in the table
under the figure. Fig. 5 shows
that the majority of the cross-links fit the double belt model well. Because
they are so close in the primary sequence, the four intrapeptide cross-links
all fit the model. In fact, they fit all three models and therefore did not
offer significant information on the spatial orientation of the two molecules.
Three out of the four intramolecular cross-links also fit the double belt
model. Of interest is the intramolecular cross-link between Lys239
and Lys40. This could join the extreme C terminus with the globular
region at the N terminus within the same molecule of apoA-I. The
intramolecular cross-links involving Lys118 both fit, especially if
Lys133 is the other Lys residue participating rather than
Lys140. Similarly, all but two of the putative intermolecular
cross-links fit this model. Overall, of the 15 cross-links we identified, 12
were plausible in the double belt model.

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FIG. 5. Graphical representation of the compatibility of the observed
cross-links with the double belt model. Two molecules of apoA-I are
represented as either gray (molecule A) or white (molecule
B) as if they had been taken off of the edge of a three-dimensional rHDL disc
and laid flat on this printed page (or computer screen). The registry of the
helices is shown in the 5/5 anti-parallel orientation as modeled by Segrest
et al. (14). Residues
144 are predicted to exist in a globular conformation and are
represented as an octagon. Each amphipathic helical segment is
represented as a rectangle and is numbered according to the scheme of
Roberts et al. (29).
The 21 Lys residues are shown as numbered green dots in their
approximate location along each molecule. Intramolecular and intrapeptide
cross-links from Table IV are
shown in red. Putative intermolecular cross-links are shown in
blue. The Lys residues involved in each cross-link are listed on the
figure using the same color scheme. Cross-links that are not
consistent with the model are underlined. Dotted lines for some
cross-links reflect cases when two Lys residues are in such close proximity
that the cross-link may involve either residue.
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FIG. 6. Graphical representation of the compatibility of the various cross-links
with the parallel form of the "Z-belt" model. The layout of
the figure is as for Fig.
5.
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FIG. 7. Graphical representation of the compatibility of the observed
cross-links with the "head to head" (top) and "head
to tail" (bottom) hairpin model combinations. The layout of
the figure is as for Fig. 5.
Hairpins are shown with a break near the middle of the molecule in
helix 5 (13).
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|
Fig. 6 shows a model for
apoA-I termed the "Z-belt" (named because of its shape on the edge
of the disc), which we proposed to explain the presence of rHDL particles with
three molecules on the disc edge
(13). The model is attractive
from a symmetry standpoint, because all molecules, including a third, can
exist in the same conformation with helices in a belt orientation. Again, the
intrapeptide cross-links all fit the model. However only two of the
intramolecular and only one of the seven intermolecular cross-links were
consistent. Fig. 7 shows two
permutations of the helical hairpin in which the molecules are "head to
head" and "head to tail." Comparison of this figure with
Fig. 5 reveals that the hairpin
models are related to the double belt in that all helical interactions are
maintained with the same docking interface. The difference is that the helical
interactions are intramolecular for the hairpins instead of intermolecular for
the double belt. Despite this, all four intramolecular cross-links and four of
the seven intermolecular cross-links fit the head-to-head model. However, none
of the intermolecular links fit the head-to-tail version.
Comparing the total cross-link fits between the double belt and the head to
head hairpin, both models allow 12 of the 15 cross-links. Surprisingly, they
only differed by one cross-link each. The Lys208-Lys45
intramolecular cross-link works in the hairpin but not in the double belt. By
contrast the intermolecular Lys195-Lys77 cross-link
works in the double belt but not in the hairpin. All the other cross-links fit
equally well to both models. The only cross-links that did not fit any of the
models are the intermolecular Lys208-Lys208 and the
Lys106-Lys106 connections. The reason for this is
unclear, but an interesting speculation is that these cross-links represent an
alternative registry of the double belt model. In
Fig. 5, if one slides molecule
A two helical positions to the right while keeping molecule B stationary (to a
4/4 orientation), both the Lys208-Lys208 and
Lys206-Lys206 cross-links will fit the model, although
all of the other cross-links that work in the 5/5 orientation will be broken.
This may support the idea of variable helical registry that has been proposed
by Li et al. (30),
allowing for the possibility that a fraction of the particles are in a
different registry. By contrast, we were unable to derive a permutation of the
hairpin models that could account for both of these cross-links.
From the model comparisons, it is clear that the cross-linking data
strongly suggest that apoA-I exists in at least one state that maintains the
salt-bridge docking interface proposed for the double belt model by Segrest
et al. (14). This
same interaction was also observed in an x-ray crystal structure of a
lipid-free oligomer of apoA-I
(10). Both the double belt and
the hairpin models maintain this orientation, whereas the Z-belt does not.
Thus, we feel that the Z-belt model can be safely ruled out. In terms of
distinguishing between the double belt and the hairpin, we observed only two
cross-links capable of distinguishing between the two models, the
Lys208-Lys45 (intramolecular) and the
Lys195-Lys77 (intermolecular). The fact that both of
these cross-links were present may argue for a mixture of the two models
existing in solution. Indeed, Segrest et al.
(14) have suggested that rHDL
particles containing three molecules of apoA-I may contain two present in the
double belt conformation with the third adopting a hairpin motif to be
accommodated on the disc edge. Because we did see a small amount of trimer
formation within our rHDL particle preparation (see
Fig. 2), one possibility is
that we observed cross-links originating from particles containing two
molecules of apoA-I both in the double belt conformation as well as some
particles containing three molecules of apoA-I with two in the double belt and
one in a hairpin. An alternative explanation is that both models may exist on
different particles in solution. The particular orientation adopted by apoA-I
may be determined by conformational factors at the time of initial lipid
binding. If the hairpin does exist, either in conjunction with the double belt
or alone on a particle, our data suggest that the head-to-head version is the
best possibility as no evidence was obtained for the head-to-tail version.
We believe that this work represents a major step forward in bridging the
gap between theoretical modeling and experimental evidence for apoA-I
structure in its lipid bound state. Future work will focus on identifying
additional distance constraints by using cross-linking agents with different
spacer arm lengths, proteases with different specificities, and by improving
our data analysis techniques to identify more complex cross-linked peptides.
Furthermore, the approach has nearly bound-less potential for applications in
lipoprotein structural biology. Other forms of rHDL such as smaller particles,
large ones containing three and four molecules of apoA-I, and even spherical
particles can be compared by this method to identify flexible regions within
apoA-I. In addition, because the masses that are derived from cross-links
within apoA-I are detectible even in the presence of other apolipoproteins
(18), the technique opens up
the exciting possibility of studying the structure of apoA-I and other
apolipoproteins in complex particles such as HDL isolated from human
plasma.
 |
FOOTNOTES
|
---|
* This work was supported in part by RO1 Grants HL62542 and HL67093 from
NHLBI, National Institutes of Health (to W. S. D.). The costs of publication
of this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
An Established Investigator of the American Heart Association. To whom
correspondence should be addressed: Dept. of Pathology and Laboratory
Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH
45267-0529. Tel.: 513-558-3707; Fax: 513-558-2289; E-mail:
Sean.Davidson{at}UC.edu.
1 The abbreviations used are: HDL, high density lipoprotein; apoA-I,
apolipoprotein A-I; DSP, dithiobis(succinimidyl propionate); DTT,
dithiothreitol; HPLC, high pressure liquid chromatography; MS, mass
spectroscopy; LCMS, liquid chromatography mass spectrometry; SPB, standard
phosphate buffer; TOF, time of flight; TIC, total ion chromatogram; POPC,
1-palmitoyl 2-oleoyl phosphatidylcholine; rHDL, reconstituted HDL; DTT,
dithiothreitol. 
 |
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