(Received for publication, October 11, 1996, and in revised form, February 28, 1997)
From the Center for Molecular and Vascular Biology
and the
Laboratory of Chemical and Biological Dynamics,
University of Leuven, B-3000 Leuven, Belgium
The Arg123-Tyr166
central and Ala190-Gln243 carboxyl-terminal
pairs of helices of apoA-I were substituted with the pair of helices of apoA-II, resulting in the
apoA-I((Arg123-Tyr166),
A-II(Ser12-Ala75))
and
apoA-I(
(Ala190-Gln243),
A-II(Ser12-Gln77))
chimeras, respectively. The structures of these chimeras in aqueous solution and in reconstituted high density lipoproteins (rHDL)
and the lecithin:cholesterol acyltransferase (LCAT) activation properties of the rHDL were studied. Recombinant human apoA-I and the
chimeras were expressed in Escherichia coli and purified from the periplasmic space. Binding of the apolipoproteins with palmitoyloleoylphosphatidylcholine was associated with a
similar shift of Trp fluorescence maxima from 337 to 332 nm, from 339 to 334 nm, and from 337 to 333 nm, respectively. All rHDL had a Stokes
radius of 4.8 nm and contained 2 apolipoprotein molecules/particle. Circular dichroism measurements revealed eight
-helices per apoA-I and per chimera molecule. The catalytic efficiencies of LCAT activation were 1.5 ± 0.33 (mean ± S.D.; n = 3),
0.054 ± 0.009 (p < 0.001 versus
apoA-I), and 1.3 ± 0.32 (p = not significant
versus apoA-I) nmol of cholesteryl
ester/h/µM, respectively. The lower LCAT activity of the
central domain chimera was due to a 27-fold reduced
Vmax with unaltered Km.
Binding of radiolabeled LCAT to rHDL of apoA-I and
apoA-I(
(Arg123-Tyr166),
A-II(Ser12-Ala75))
was very similar. In conclusion, although substitution of the Arg123-Tyr166 central or
Ala190-Gln243 carboxyl-terminal pair of
helices of apoA-I with the pair of helices of apoA-II yields chimeras
with structure similar to that of native apoA-I, exchange of the
central domain (but not the carboxyl-terminal domain) of apoA-I reduces
the rate of LCAT activity that is independent of binding to rHDL.
ApoA-I is synthesized as a prepropeptide, cotranslationally
cleaved to pro-apoA-I, and, upon secretion, processed to mature 243-amino acid apoA-I (1). It is folded into amphipathic -helices with hydrophilic and hydrophobic surfaces (2-4), as demonstrated with
complexes of phospholipids with apoA-I or model peptides (5-11).
ApoA-I, when associated with phospholipids in discoidal complexes
(12-14), contains eight putative amphipathic
-helices oriented
around the edge of the discs, parallel to the acyl chains of the
phospholipids, with their hydrophobic surface toward the lipid core and
their hydrophilic surface toward the aqueous phase. The first
amino-terminal domain (residues 44-63) has the lowest helical
structure probability, whereas the second
-helix (residues 69-85)
is not involved in a pair (12-14). The six carboxyl-terminal
-helical structures most likely form pairs of antiparallel
-helices stabilized by protein-protein interactions. A minimum
length of 17-20 amino acids (five to six helical turns) appears to be
required for effective phospholipid binding and
LCAT1 activation (12-14).
The structures in apoA-I involved in phospholipid binding and/or LCAT
activation remain largely unidentified. Reported differences in LCAT
activity of apoA-I and deletion mutants may result from altered folding
and/or organization of these molecules in rHDL rather than from
deletion of a functional domain (15, 16). Therefore, in this study, the
apoA-I((Arg123-Tyr166),
A-II(Ser12-Ala75))
and
apoA-I(
(Ala190-Gln243),
A-II(Ser12-Gln77))
chimeras were produced, in which the
Arg123-Tyr166 central or
Ala190-Gln243 carboxyl-terminal pair of
-helices of apoA-I was deleted (
) and substituted (
) with the
pair of
-helices of apoA-II. The average structural properties in
solution and in reconstituted high density lipoprotein particles of the
two chimeras were found to be unaltered, but the central domain chimera
had a markedly reduced LCAT activity.
All DNA manipulations were carried out essentially as described by Maniatis et al. (17). Oligonucleotide-directed mutagenesis was performed by the gapped-duplex method of Kramer et al. (18) using the pMa/c vector system of Stanssens et al. (19). This system employs phasmid (i.e. phage/plasmid hybrid) vectors, allowing cloning, site-directed mutagenesis, and sequencing using the same vector without recloning. Oligonucleotides were obtained by custom synthesis (Pharmacia, Brussels, Belgium). DNA sequences were determined using a primer walking strategy on an ALF DNA sequencer (Pharmacia, Uppsala). Template DNA was purified by alkaline hydrolysis followed by a polyethylene glycol precipitation step. The sequencing reactions were carried out using T7 DNA polymerase (Pharmacia) and a fast denaturation protocol as described (20). The reaction products were sized on 6% Hydrolink Long Ranger gels (AT Biochem, Malvern, PA) containing 1 × Tris borate/EDTA buffer and run with 0.6 × Tris borate/EDTA buffer. The sequence was assembled using the IntelliGenetics Suite 5.4 program.
Expression and Purification of ApoA-I and the ApoA-I/ApoA-II ChimerasThe pMc-5-apoA-I transfection vector, encoding wild-type recombinant apoA-I, for expression in Escherichia coli under control of the tac promoter and the PhoA signal peptide sequence was constructed starting from plasmid pA1-3 (21), the pUC-19 vector (22), and the pMa/c mutagenesis vector (19). Plasmid pA1-3, obtained by insertion of the apoA-I cDNA into the PstI site of pBR322 (21), was a kind gift of Dr. L. Chan (Baylor College of Medicine, Houston, TX).
Deletion mutagenesis with the 5-deoxyoligonucleotide
dCAAGCGCTGGCGCAGCTCGTCGCTTAAGGGCTCCACCTTCT was performed on the
pMc-5-apoA-I transfection vector to delete the
Arg123-Tyr166 coding sequence, resulting in
the pMc-5-apoA-I(
(Arg123-Tyr166)) vector.
By substitution of the CCG codon for Pro121 with the CCC
codon for Pro and of the CTG codon for Leu122 with the TTA
codon for Leu, an additional AflII restriction site was
created. The Ser12-Ala75 segment of apoA-II
was amplified by polymerase chain reaction using the
5
-deoxynucleotide dCATAAGCATGCTGTCTCAGTACTTCCAGACC primer, containing
the apoA-II(Ser12-Thr17) sequence and the
SphI restriction site, and the 3
-deoxynucleotide dATCAAATGCATGGCAGGCTGTGTTCCAAG primer, containing the
apoA-II(Ile70-Ala75) sequence and the
NsiI restriction site. Thirty cycles were performed, consisting of 1 min of denaturation at 94 °C, 2 min of annealing at
52 °C, and 1.5 min of extension at 72 °C. The reaction product was digested with NsiI and SphI, blunt-ended
using T4 polymerase, and ligated in the AflII-treated and
blunt-ended pMc-5-apoA-I(
(Arg123-Tyr166))
vector, resulting in the
pMc-5-apoA-I(
(Arg123-Tyr166),
A-II(Ser12-Ala75))
vector for the expression of
apoA-I(
(Arg123-Tyr166),
A-II(Ser12-Ala75))
in E. coli. The
pMc-5-apoA-I(
(Ala190-Gln243),
A-II(Ser12-Gln77))
vector for the expression of
apoA-I(
(Ala190-Gln243),
A-II(Ser12-Gln77))
in E. coli was constructed as described (23).
Apolipoproteins were expressed in the periplasmic space of E. coli WK6 host cells as described (16, 23). Standard apoA-I was isolated from normolipemic human plasma as described previously (1). The purity of proteins was established by SDS gel electrophoresis (24) and immunoblotting (25).
Preparation of Discoidal Apolipoprotein-POPC-Cholesterol ComplexesComplexes of the apolipoproteins with POPC (Sigma) and cholesterol, at an apolipoprotein/POPC/cholesterol ratio of 1:3:0.15 (w/w/w), were prepared using the cholate dialysis procedure (11, 26). The mixture was incubated at 43 °C for 16 h, and cholate was removed by extensive dialysis. Complexes were isolated by gel filtration on a Superdex 200 HR column equilibrated with 20 mM Tris-HCl, pH 8.1, containing 0.15 M NaCl and 0.02 mg/ml sodium azide in a Waters fast performance liquid chromatography system. One-ml fractions were collected. The sizes of these complexes were estimated by comparison of their migration positions on native 8-25% gradient polyacrylamide gels with those of standard proteins: thyroglobulin (Stokes radius of 8.5 nm), apoferritin (6.1 nm), catalase (5.2 nm), and lactate dehydrogenase (4.1 nm) (Pharmacia).
The number of apolipoprotein molecules/rHDL particle was determined following cross-linking of apolipoproteins with bis(sulfosuccinimidyl) suberate (final concentration of 1 mM) for 6 h, as described previously (27), and separation of cross-linked proteins by SDS-PAGE on 10-15% gradient gels. The extent of oligomer formation was estimated by comparison with plasma cross-linked apoA-I (27). The levels of phospholipid and cholesterol were determined using commercial enzymatic kits (Biomérieux for phospholipid and Boehringer Mannheim for cholesterol), and the concentrations of apoA-I were determined according to Bradford (28).
Circular Dichroism SpectrometryCircular dichroism spectra
of lipid-free apolipoproteins in solution and of apolipoproteins in
discoidal apolipoprotein-POPC-cholesterol complexes were measured with
a Jasco J600 spectropolarimeter (Japan Spectroscopy, Tokyo) at
wavelengths between 200 and 250 nm, using 6 µM sample
solutions and a 1-mm path length cuvette. Backgrounds were measured at
250 nm for 5 min, followed by measurements of the -helical content
at 222 nm for 5 min. The fraction of
-helices in the secondary
structure of the apolipoproteins was estimated from the molar
ellipticities at 222 nm ([
]222 =
30,300
fH
2340, where fH is the
fraction of
-helical structure) (29).
Corrected steady-state fluorescence
spectra and intensities were measured as described previously (16).
Excitation was carried out at 295 nm, where the contribution of tyrosyl
fluorescence to the total intensity is minimal. Fluorescence was
measured with a 2-mm slit width in quartz cuvettes with optical path
lengths equal to 1 and 0.4 cm and wavelength resolutions of 7.2 and 3.6 nm for the emission and excitation wavelengths, respectively. Excitation was vertically polarized, and fluorescence was detected with
a polarizer at a 54° angle to reduce the influence of fluorescence depolarization and brownian motion on the detected intensity. The
spectra were corrected for the wavelength dependence of the emission
monochromator and the photomultiplier and for background intensities as
described previously (30). In the fluorescence-quenching experiments,
the spectra were recorded at increasing concentrations of KI. The
results were analyzed by the Stern-Volmer equation as modified by
Lehrer (31): F0/(F0 F) = (1/fa + 1/faKSV) × (1/[KI]), where
F0 and F are the fluorescence
intensities observed in the absence and presence of a given
concentration of KI, respectively, and fa is the
fraction of quenchable fluorescence (32). The Stern-Volmer constant
(KSV) is an index of the accessibility of
tryptophan residues to the aqueous phase.
The pUC-LCAT.10 plasmid, covering the entire coding region of human LCAT, was obtained from Dr. J. Mc Lean (Department of Cell Biology, Genentech Inc., San Francisco, CA). The LCAT cDNA was cloned into the pcDNA3 expression vector (Invitrogen, San Diego, CA), which contains the enhancer-promoter sequences from the immediate-early region of human cytomegalovirus and a neomycin resistance gene. After trypsinization, 8 × 106 293 cells were resuspended in 600 µl of Dulbecco's modified Eagle's medium (Gibco, Gent, Belgium) supplemented with 10% fetal bovine serum (Gibco) and transfected with 75 µg of pcDNA3-LCAT, linearized with XbaI, by electroporation. After electroporation, cells were plated at densities of 5 × 106, 106, 5 × 105, and 105/10-cm dish. One day after transfection, cells were placed on Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 600 µg/ml Geneticin (Gibco). Cell colonies, appearing 14 days after transfection, were picked, replated, and screened for LCAT expression by spectrophotometric assessment of butyryl ester of p-nitrophenol (33). Transfected 293 cells were cultured in serum-free Dulbecco's modified Eagle's medium for 48 h, and conditioned medium was collected and concentrated on a Centricon-10 filter (Amicon, Inc., Beverly, MA).
Kinetics of LCAT Activation by Discoidal Apolipoprotein-POPC-Cholesterol ComplexesLCAT activation by
discoidal apolipoprotein-POPC-cholesterol complexes was monitored by
incubation of the equivalent of 5-40 µM unesterified
cholesterol with a final concentration of 50 nM LCAT. The
reaction was carried out in 10 mM Tris-HCl, pH 8.0, containing 5 mM EDTA, 0.15 M NaCl, 4-16 mg/ml
delipidated bovine serum albumin, and 6 mM
-mercaptoethanol (LCAT buffer). After preincubation for 20 min at
37 °C, LCAT was added. The mixture was incubated at 37 °C for
10-180 min, after which the reaction was arrested by extraction of
cholesteryl esters with hexane/isopropyl alcohol (3:2, v/v) containing
cholesteryl heptadecanoate as an internal standard. Under these
reaction conditions, <20% of the cholesterol was esterified.
Generated cholesteryl esters were quantified by isocratic high
performance liquid chromatography on a reversed-phase ZORBAX ODS
column, eluted with acetonitrile/isopropyl alcohol (50:50, v/v), as
described previously (34). Initial activation rates were obtained from
plots of the concentration of generated cholesteryl esters
versus time, and the kinetic parameters Km (expressed in µM cholesterol) and
Vmax (expressed in nmol of cholesteryl esters
generated per h per µM of cholesterol (cf.
Table III)) were determined by linear regression analysis from
Lineweaver-Burk plots.
|
Recombinant LCAT was labeled with 125I by the IODOGEN method. Following the reaction, radiolabeled LCAT was separated from free 125I by passage over a Sephadex G-25M PD-10 gel filtration column (Pharmacia) pre-equilibrated with LCAT buffer. Specific activity was typically 225 cpm/ng of protein. Radiolabeled LCAT (final concentration of 3 µg/ml) was incubated with rHDL (final apolipoprotein concentrations ranging between 4.4 and 140 µg/ml) for 20 min, and free LCAT was separated from bound LCAT by filtration on a Centricon-100 filter (Amicon, Inc.).
Fig. 1 is a schematic representation of the
predicted amphipathic helical regions in apoA-I, apoA-II,
apoA-I((Arg123-Tyr166),
A-II(Ser12-Ala75)),
and
apoA-I(
(Ala190-Gln243),
A-II(Ser12-Gln77)).
The predicted number of amphipathic helices, according to Brasseur
et al. (12), was eight for apoA-I and for the two chimeras. Wild-type apoA-I and the apoA-I/apoA-II chimeras were expressed in the
periplasmic space of E. coli cells and purified to
homogeneity as evidenced by their migration as single bands on 10-15%
SDS-polyacrylamide gels (Fig. 2). The molecular masses
of the apolipoproteins, calculated from a plot of the logarithm of the
molecular masses of the standard proteins versus the
migration distance, were 28.3 kDa for apoA-I and 30.4 and 29.8 kDa for
the respective chimeras and thus were in agreement with those
calculated on the basis of the respective amino acid compositions.
Wild-type apoA-I migrated in the same position as plasma apoA-I. The
identity of each band was confirmed by immunoblot analysis using
polyclonal rabbit anti-human apoA-I antibodies (data not shown).
The -helical contents of the apolipoproteins in aqueous solution, as
determined by circular dichroism scanning, were 48% for apoA-I and 43 and 41% for the respective chimeras. The recovery of apolipoproteins
in discoidal apolipoprotein-POPC-cholesterol complexes after gel
filtration was 90% for wild-type apoA-I and the chimeras. The Stokes
radius of all rHDL was 4.8 nm (Fig. 3 and Table
I). Cross-linking of the apolipoprotein molecules within all the discoidal apolipoprotein-POPC-cholesterol particles revealed 2 apolipoprotein molecules/particle (Fig. 4 and Table I).
The phospholipid surface was calculated from the circumference of the
disc using the measured diameter minus 3 nm, i.e. 2 × radius of an
-helix, multiplied by a disc height of 3.8 nm. The
number of phospholipid molecules was calculated from the calculated
surface divided by 0.45 nm2, the surface area/condensed
phospholipid molecule. The calculated apolipoprotein/phospholipid molar
ratios were 1:90, 1:83, and 1:89, respectively, and were in agreement
with the ratios calculated on the basis of the composition of the
rHDL.
|
The -helical contents of the apoA-I proteins in discoidal
apolipoprotein-POPC-cholesterol complexes were 74% for apoA-I and 75 and 72% for the respective chimeras. The number of
-helices, calculated from the
-helical content determined by circular
dichroism scanning and from the protein length assuming a length of 22 amino acids/
-helical repeat (2, 12), was eight per apoA-I or per chimera molecule (Table I) and was thus in agreement with the predicted
values (12). The fraction of the phospholipid surface that was covered
by the
-helix was calculated as the total phospholipid surface in
nm2 divided by 4 nm2, the surface area of an
-helix, that contains 16 amino acids. The calculated fraction was
0.77 for apoA-I and the chimeras (Table I).
Binding of apoA-I and the chimeras to POPC was associated with a shift
of Trp fluorescence maxima to lower wavelengths from 337 to 332 nm for
apoA-I (both for plasma apoA-I and recombinant apoA-I), from 339 to 334 nm for the central domain chimera, and from 337 to 333 nm for the
carboxyl-terminal domain chimera (Table II), suggesting
that lipid binding is associated with a translocation of Trp residues
to a more apolar environment. The quenching parameters KSV and fa are summarized in
Table II. For totally exposed Trp residues, in the absence of
electrostatic or viscosity effects, KSV = 12 M1 and fa = 1, whereas for
totally protected Trp residues, KSV = 0 and
fa = 0. The quenchable fluorescence of all apolipoproteins in the respective rHDL represented, on average, 60% of
the total fluorescence (Table II).
|
Recombinant LCAT was obtained in serum-free conditioned medium of
transfected 293 cells. The homogeneity of the recombinant LCAT
preparation is illustrated in Fig. 5. LCAT activation by the discoidal apolipoprotein-POPC-cholesterol complexes obeyed Michaelis-Menten kinetics, as shown by linear Lineweaver-Burk plots of
the inverse of the initial activation rate
(1/V0) versus the inverse of the
cholesterol concentrations (1/[C]). The apparent kinetic
parameters Vmax and Km and
the Vmax/Km ratios for the
different apolipoprotein-POPC-cholesterol complexes are summarized in
Table III. Exchange of the
Arg123-Tyr166 paired helix of apoA-I with the
pair of helices of apoA-II reduced the LCAT activity of apoA-I 27-fold
due to a reduction of Vmax and not of
Km. In contrast, exchange of the
Ala190-Gln243 carboxyl-terminal domain helices
of apoA-I with the pair of helices of apoA-II had no effect on the LCAT
activity of apoA-I.
Fig. 6 illustrates the binding of radiolabeled LCAT to
rHDL of apoA-I and the
apoA-I((Arg123-Tyr166),
A-II(Ser12-Ala75))
chimera. Fifty % of maximal binding was obtained with 34 µg/ml apoA-I and 27 µg/ml
apoA-I(
(Arg123-Tyr166),
A-II(Ser12-Ala75)).
Reported differences in LCAT activity of apoA-I variants may be
due to defective interaction with phospholipids, structural changes in
rHDL, and/or deletion of functional domains. To further investigate the
role of the central and carboxyl-terminal domains of apoA-I in LCAT
activation, the
apoA-I((Arg123-Tyr166),
A-II(Ser12-Ala75))
and
apoA-I(
(Ala190-Gln243),
A-II(Ser12-Gln77))
chimeras were produced. The extent of in vitro phospholipid binding of these chimeras was similar to that of apoA-I, as
demonstrated by comparable disc formation after mixing the
apolipoproteins and phospholipids at equal weight ratios. This was
evidenced by a shift of the maximum Trp fluorescence to a shorter
wavelength and by a decreased accessibility of the Trp residues to
I
. The sizes of rHDL reconstituted with apoA-I and the
chimeras were identical: the respective rHDL contained 2 apolipoprotein molecules/particle, and circular dichroism scanning revealed eight
-helices per intact apoA-I molecule and per chimera molecule, in
agreement with the predicted numbers according to Brasseur et
al. (12). The calculated apolipoprotein/phospholipid molar ratios
of the different rHDL particles were very similar. Thus, substitution
of the Ala123-Tyr166 central or
Ala190-Gln243 carboxyl-terminal domain helices
of apoA-I with the pair of helices of apoA-II did not affect the size
and the composition of rHDL, and the conformation and helical
distribution in the different apolipoproteins in these particles were
very similar. Substitution of the carboxyl-terminal domain of apoA-I
with the helices of apoA-II did not reduce LCAT activity, but
substitution of the central domain resulted in a 27-fold reduction of
LCAT activity, suggesting that the
Ala123-Tyr166 segment is critical for LCAT
activation. Binding experiments revealed that the reduced LCAT activity
of the
apoA-I(
(Arg123-Tyr166),
A-II(Ser12-Ala75))
chimera was not due to reduced binding of LCAT to rHDL.
Based on data obtained with synthetic peptides, it was concluded that the LCAT-activating domain of apoA-I resides in a 22-mer tandem repeat located between residues 66 and 121 (35). This was further supported by the finding that monoclonal antibodies directed against an epitope that spanned residues 95-121 inhibited the LCAT activation with apoA-I (36). Binding of antibodies to an epitope in the amino-terminal domain of apoA-I may, however, induce conformational changes in the central domain of apoA-I that may be responsible for the reduction of LCAT activity (37). Deletion of the Leu44-Leu126 amino-terminal domain of apoA-I indeed did not reduce its LCAT activity (16), suggesting that the amino-terminal domain of apoA-I is not critical for LCAT activation.
Using deletion mutants of apoA-I, Minnich et al. (38) found
that deletion of the Met148-Gly186 segment
resulted in decreased LCAT activity, whereas Sorci-Thomas et
al. (39) found that deletion of the
Pro143-Ala164 segment reduced LCAT activity.
In previous studies, the conformation of deletion mutants in their
respective rHDL was not investigated. However, it has been demonstrated
that the decreased LCAT activity of deletion mutants of apoA-I may be
due to differences in folding (decreased -helical content) and/or
organization of the apolipoproteins in rHDL (3 or 4 molecules/particle
as compared with 2 for intact apoA-I) rather than to the removal of
specific domains for LCAT activity (16). Indeed, it has been
demonstrated that rHDL discs containing wild-type apoA-I may have
discrete sizes, compositions (with 2, 3, or 4 protein
molecules/particle), and apoA-I conformations (with six, seven, or
eight
-helices/apoA-I molecule in contact with lipid) and that
differences in the apoA-I structure in these particles correlate with
their ability to activate LCAT (40).
Chimeras in which -helical segments of apoA-I are substituted with
helical segments of apoA-II, which does not activate LCAT, in such a
way that the average secondary structure of the apolipoprotein molecule
as well as the organization of the apolipoprotein molecules in rHDL are
not affected may be preferable reagents to address the function of a
particular structural domain in LCAT activation. Indeed, rHDL
containing apoA-II have a very low LCAT activity (41), and the addition
of apoA-II together with apoA-I to liposomes reduces LCAT activity by
70% (42). Thus, substitution of sequences in apoA-I that are critical
for the interaction with LCAT with sequences derived from apoA-II would
result in decreased LCAT activity. Substitution of the
carboxyl-terminal domain of apoA-I with helices of apoA-II was found
not to affect LCAT activity, but substitution of the central domain
resulted in a 25-fold reduction of LCAT activity, suggesting that the
central domain (but not the carboxyl-terminal domain) is essential for
LCAT activation.
The differences in LCAT activity were due to differences in apparent Vmax values, which reflect the activated enzyme concentration, and not to differences in apparent Km values, which reflect the affinity of LCAT for rHDL (43). Similar Km values are indeed in agreement with similar binding of LCAT to rHDL of apoA-I and the central domain chimera. Thus, the Ala123-Tyr166 segment of apoA-I appears to contain structures that are required for optimal phospholipid and cholesterol presentation to LCAT (44) that cannot be mimicked by the apoA-II segment. It is possible that substitution of the central domain affects the conformation of a hinged domain that is crucial for LCAT activation because antibodies that interfere with the mobility of a hinged domain in the central part of apoA-I inhibit LCAT activation (37). Previous data obtained with deletion mutants supported the existence of such a hinged domain overlapping either the Asn102-Lys140 or Ala124-His162 segment of apoA-I (16). Because conformational changes elsewhere in the molecule could not be excluded, these data were, however, not conclusive. The present study strongly suggests that this hinged domain most likely overlaps the Ala124-His162 domain of apoA-I.
In conclusion, substitution of the central or carboxyl-terminal pair of helices of apoA-I with the helices of apoA-II does not affect its average structure in rHDL. Substitution of the central domain (but not the carboxyl-terminal domain) results in a significant reduction of the rate of LCAT activation, although the binding of LCAT to rHDL is not reduced.
We thank G. Vanderheeren (Interdisciplinary Research Center, University of Leuven, Kortrijk, Belgium) for CD measurements.