(Received for publication, February 7, 1997, and in revised form, April 11, 1997)
From the Lipid and Lipoprotein Research Group and
§ Medical Research Council Group in Protein Structure and
Function, Department of Biochemistry, University of Alberta,
Edmonton, Alberta T6G 2S2, Canada
Apolipophorin III (apoLp-III) from the insect
Manduca sexta is a 166-residue (Mr
18,340) member of the exchangeable apolipoprotein class that functions
to stabilize lipid-enriched plasma lipoproteins. In the present
study, we present the secondary structure and global fold
of recombinant apoLp-III derived from three-dimensional
heteronuclear NMR spectroscopy experiments. Five discrete
-helical segments (21-30 residues in length) with well defined
boundaries were characterized by four NMR parameters: medium range
nuclear Overhauser enhancement contacts between proton pairs, chemical
shift index, coupling constants, and amide proton exchange rates. An
antiparallel arrangement of helical segments has been obtained based on
the long range interhelical nuclear Overhauser enhancement contacts.
The NMR solution structure reveals a globular, up and down helix bundle organization similar to that of Locusta migratoria
apoLp-III (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesenberg,
G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991)
Biochemistry 30, 603-608). However, a short helix
(comprised of 5 amino acids) has been identified in the region between
helix 3 and helix 4. This helix is postulated to play a role in lipid
surface recognition and/or initiation of binding. Our results also
indicate the existence of buried polar and charged residues in the
helix bundle, providing a structural basis for the relatively low
stability of apoLp-III in its lipid-free state. It is suggested that
the intrinsic low stability of lipid-free apoLp-III may be important in
terms of its ability to undergo a reversible, lipid binding-induced,
conformational change. This study underscores the striking resemblance
in molecular architecture between insect apoLp-III and the N-terminal
domain of human apolipoprotein E. The potential for application of NMR techniques to studies of the exchangeable apolipoproteins, possibly in
their biologically active, lipid-associated state, has broad implications in terms of our understanding of the molecular basis of
their physiological functions.
Plasma lipoproteins are stable noncovalent assemblies of lipids and specialized proteins termed apolipoproteins. The apolipoproteins can be broadly categorized as exchangeable or nonexchangeable. Whereas nonexchangeable apolipoproteins are integral, nontransferable components, the exchangeable apolipoproteins stabilize lipoproteins through reversible binding as a function of particle surface defects or exposed hydrophobic regions. Specific exchangeable apolipoproteins are known to regulate lipoprotein metabolism through modulation of lipid metabolic enzymes or by functioning as ligands for cell surface receptors. To date, approximately 11 distinct human plasma-exchangeable apolipoproteins have been isolated and characterized (1). The capacity to form a stable interaction with lipid surfaces represents a common function shared by all exchangeable apolipoproteins and is essential for the expression of their alternate functions. Plasma levels of certain exchangeable apolipoproteins are correlated with several human diseases including dyslipidemia and cardiovascular disease (2). For example, overexpression of apolipoprotein E (apoE)1 in transgenic mice results in a decrease in plasma cholesterol (3), whereas apoE knockout mice exhibit massive accumulation of remnant lipoproteins, leading to premature atherosclerosis (4, 5).
Structural studies on exchangeable apolipoproteins indicate that the
amphipathic helix provides an essential structural motif for
lipoprotein binding (6). A major advance in our understanding of
exchangeable apolipoprotein structure comes from studies using x-ray
crystallography to determine the three-dimensional structures of the
22-kDa N-terminal domain of human apoE (7) and apolipophorin III
(apoLp-III) from the insect Locusta migratoria (8). These structures, which were determined in the lipid-free state, share a
similar molecular architecture comprised of a bundle of elongated amphipathic
helices. Whereas the N-terminal domain of human apoE
exists as a four-helix bundle, L. migratoria apoLp-III is organized as a five-helix bundle. The helices orient their hydrophobic faces toward the center of the bundle while their hydrophilic faces
interact with the solvent. It has been postulated that association with
a lipid surface triggers a dramatic conformational change wherein the
loop regions between
-helical segments function as hinges about
which the molecule opens to expose its hydrophobic interior, which then
becomes available for lipid binding (8, 9). To date, no lipid-bound
structure of an exchangeable apolipoprotein is available.
Given recent advances in the application of NMR spectroscopy to protein solution structure determination, this technique should be applicable to studies of exchangeable apolipoproteins. It has been shown by others that NMR techniques can be used to determine three-dimensional structures of proteins up to 30 kDa (10). Whereas most exchangeable apolipoproteins meet this size limitation, their tendency to self-associate in the absence of lipids hinders the application of NMR methods. Several investigators have used two-dimensional 1H NMR to study apolipoprotein peptide fragments in the range of 15-36 amino acids (11-14). To date, however, no NMR solution structures of intact exchangeable apolipoproteins have been reported.
NMR structure determination is attractive because it can be used to study the conformations as well as the dynamic changes of exchangeable apolipoproteins in both lipid-free and lipid-bound states. In considering possible candidate apolipoproteins for solution structure determination by NMR, we found that apoLp-III (Mr 18,300) meets several essential criteria. Although the x-ray crystal structure of L. migratoria apoLp-III has been solved (8), there are no reports of expression of this protein in bacteria, possibly due to the presence of complex carbohydrate moieties (15), which may be important for protein stability in the lipid-free state (16). On the other hand, the homologous apoLp-III from the sphinx moth, Manduca sexta, lacks covalent oligosaccharide moieties (17), is monomeric over a broad concentration range (18), and has been overexpressed in bacteria (19). Although the sequence identity between these two apoLp-IIIs is <30%, the two proteins are functionally indistinguishable (20).
The goal of our research is to understand the molecular basis of
reversible conformational adaptations of the exchangeable apolipoproteins upon lipid binding. As the first step, we report here
the solution secondary structure and global fold of lipid-free apoLp-III derived from three-dimensional heteronuclear NMR experiments. To our knowledge, this represents the first solution structure determination of an intact exchangeable apolipoprotein. Furthermore, it
represents one of the largest -helical proteins whose structure has
been solved using NMR techniques (21). The NMR structure of M. sexta apoLp-III provides new insight into the mechanism of
apoLp-III lipid surface recognition/binding and provides a structural
rationale for the reversibility of its interactions with lipoprotein
surfaces.
Bacterial expression of recombinant apoLp-III was
carried out as described by Ryan et al. (19). For isotopic
labeling experiments, a saturated overnight cell culture (grown in 2 × yeast tryptone medium at 37 °C) was diluted 1:100 (v/v) in M9
minimal medium containing 0.1% 15NH4Cl
(Isotec, Miamisburg, OH) and/or 0.15%
D-[6-13C]glucose (Isotec) as the sole
nitrogen and/or carbon source with 50 µg of ampicillin/ml and
cultured at 30 °C. The initial A600 values
for cell cultures upon dilution into minimal media were typically
0.06-0.09. After 4-5 h, when the A600 reached
0.7-0.9, protein expression was induced by the addition of
isopropyl--D-thiogalactopyranoside (1 mM
final concentration). As reported by Ryan et al. (19), significant amounts of recombinant apoLp-III specifically accumulate in
the culture medium following induction. Recombinant apoLp-III is
recovered from the cell culture by pelleting the cells and collecting
the supernatant. Upon concentration to <50 ml, the culture supernatant
was dialyzed against deionized H2O for 48 h and
lyophilized. Protein was dissolved in water and subjected to high
pressure liquid chromatography on a preparative Zorbax C8
reversed phase column. The purity of the sample was assessed by
SDS-polyacrylamide gel electrophoresis, and the relative isotope enrichment was characterized by electrospray ionization mass
spectrometry. Using the protocol described above, 70-100 mg of
13C,15N double-labeled or 120-150 mg of
15N single-labeled apoLp-III was obtained per liter of cell
culture with >99% purity and an isotope enrichment of >95%.
Previous denaturation studies of
lipid-free apoLp-III, monitored by circular dichroism spectroscopy,
revealed a midpoint of guanidine HCl-induced denaturation of 0.35 M, corresponding to a
GDH2O of
1.29 kcal/mol (22). Consistent with these results, a time course study
of apoLp-III stability monitored by 1H/15N HSQC
experiments showed that apoLp-III in H2O at pH 7.0 was stable for <16 h. New peaks that correspond to an unfolded state of
the protein appeared at longer times (data not shown). Empirical stability optimization studies monitored by NMR resulted in the finding
that apoLp-III was stable for >2 weeks in 250 mM potassium phosphate, 0.5 mM sodium azide, pH 6.4-6.5. In this
buffer, the chemical shift dispersion in both the 1H and
15N dimensions was acceptable. Thus, for three-dimensional
15N-edited NOESY, 15N-edited TOCSY, and HNHA
experiments as well as two-dimensional 1H/15N
HSQC and 15N/1H HMQC-J experiments,
[15N]apoLp-III was dissolved in 250 mM
potassium phosphate, pH 6.5, 0.5 mM NaN3 in
95% H2O, 5% D2O (1.2 mM final
protein concentration). Likewise, this buffer and protein concentration
of [13C,15N]apoLp-III was employed for
three-dimensional CBCA(CO)NNH, HNCACB, HCCH-TOCSY, and simultaneous
15N- and 13C-edited NOESY experiments. All
samples contained a small amount of 2,2-dimethyl-2-silapentanesulfonic
acid as an internal standard for proton chemical shifts.
All NMR experiments were carried out at
30 °C on a Varian Unity 600 NMR spectrometer equipped with three
channels, a pulsed field gradient triple resonance probe with a z
gradient, and a gradient amplifier unit. Carrier positions used in the
various three-dimensional NMR spectra were as follows: 15N,
119.0 ppm; 13C/
, 43.0 ppm;
1H, 4.71 ppm. Two-dimensional
1H/15N HSQC spectra (23) were acquired with the
following number of complex points and acquisition times: F1
(15N) 128, 91.5 ms; F2 (1H) 512, 73 ms, 8-32
transients. HMQC-J (24) spectra were recorded with the following number
of complex points and acquisition times: F1 (15N) 300, 214.5 ms; F2 (1H) 512, 73 ms, 96 transients. The spectral
widths for both HSQC and HMQC-J were 1398.8 Hz in F1 (15N)
and 7000 Hz in the F2 (1H) dimension.
Triple resonance three-dimensional NMR spectra correlating backbone
amide with and
carbons were acquired on a uniformly 15N,13C-labeled apoLp-III sample in
H2O with the following numbers of complex points and
acquisition times: HNCACB (25, 26), F1 (
/
-13C) 64, 7.0 ms, F2 (15N) 28, 20 ms, F3 (1H) 512, 73 ms
(8-16 transients); CBCA(CO)NNH (26, 27) F1 (
/
-13C)
64, 7.0 ms, F2 (15N) 32, 22.9 ms, F3 (1H) 512, 73 ms (8-16 transients). Pulsed field gradient HCCH-TOCSY (28) spectra
were acquired on a double-labeled H2O sample with a mixing
time of 16 ms (12 transients). The acquisition parameters for
HCCH-TOCSY were: F1 (1H) 512, 64 ms; F2
(1H-13C) 128, 35.6 ms; F3 (13C) 40, 12.6 ms. Simultaneous 15N- and 13C-edited NOESY
spectra (29) were recorded with mixing times of 50 and 100 ms. The
acquisition parameters for these experiments were: F1 (1H)
128, 35.6 ms; F2 (13C,15N) 32, 10.1 ms; F3
(1H) 512, 73 ms.
15N-Edited NOESY (23, 30) and 15N-edited TOCSY (23, 31) experiments were recorded on a uniformly 15N-labeled sample of apoLp-III in H2O using the enhanced sensitivity pulsed field gradient method (32, 33). 15N-Edited NOESY was carried out at 75- and 150-ms mixing times with the following number of complex points and acquisition times: F1 (1HN) 512, 73 ms; F2 (1H) 128, 20 ms; F3 (15N) 32, 22.9 ms. The same number of complex points and acquisition times was also used for 15N-edited TOCSY with a mixing time of 59 ms.
For backbone assignment of apoLp-III, 15N-edited NOESY and
15N-edited TOCSY spectra were used mainly due to the
-helical nature of the protein. HNCACB and CBCA(CO)NNH spectra were
used to confirm the backbone atom assignment obtained from
15N-edited NOESY and 15N-edited TOCSY spectra
as well as to assign those residues that could not be assigned due to
missing NH-NH connectivities in 15N-edited NOESY spectra.
1H/15N HSQC spectra of [15N]Leu,
[15N]Lys, and [15N]Val specifically labeled
apoLp-III facilitated assignment using the amide-amide walking strategy
(34). Side-chain atoms were assigned using HCCH-TOCSY,
15N-edited NOESY, 15N-edited TOCSY, and
13C-edited NOESY spectra.
All spectra were processed on SUN workstations using NMRPipe software from Delaglio et al. (35). The spectral assignment was achieved using Pipp software (36). Postacquisition solvent subtraction was employed in spectra where NH protons were detected in the acquisition dimension (37). Typically, spectra were processed in the acquisition and indirect dimensions with 90°-shifted sine bell-squared apodization. For constant time 15N/13C evolution periods, mirror image linear prediction was used to double the time domain signals (38). A time domain deconvolution procedure (33) was used to minimize the signal from residual water for simultaneous 15N- and 13C-edited NOESY experiments.
torsion angles were obtained from either HNHA and/or HMQC-J
experiments. In HMQC-J experiments, few cross-peaks were split (<20),
indicating small coupling constants for most residues. Coupling
constants were obtained from the ratio of the peak intensity from the
diagonal to the
H-NH cross-peak in HNHA spectra for a given residue
(39). Amide proton exchange rates were estimated by the intensity of
the water peak in each residue strip of 15N-edited NOESY
spectra. For residues with no water peak in the strip, slow exchange
with solvent is concluded. In cases of weak intensity and medium or
strong intensity water peaks in the strips, medium and fast proton
exchange, respectively, is concluded.
For -helical proteins in the 150-amino
acid size range, the overall chemical shift dispersion is generally
poor, especially in the 1H dimension. In
1H/15N HSQC spectra of apoLp-III, the amide
proton chemical shift dispersion is <2.5 ppm, with significant
spectral overlap observed in the range of 7.5-8.5 ppm. By contrast,
the chemical shift dispersion in the 15N dimension is
relatively large, improving the overall cross-peak separation. Two
independent methods were used to obtain the assignment of apoLp-III.
One is through the amide-amide proton connectivities in the
three-dimensional 15N-edited NOESY together with the
information obtained from three-dimensional 15N-edited
TOCSY; the other is through the C
(i
1)-C
(i
1)-N(i)-NH(i) and
C
(i)-C
(i)-N(i)-NH(i)
connectivities in CBCA(CO)NNH and HNCACB experiments. In NOESY spectra
of an
-helical protein, strong/medium intensity dNN(i, i ± 1) NOEs can usually
be found (40). These dNN NOEs, which provide sequential
connectivities, were used for backbone atom assignment. In cases of
overlap in the NH-NH region, [15N]leucine-,
[15N]lysine-, and [15N]valine-specific
labeled apoLp-III 1H/15N HSQC spectra provided
useful additional information (34). For example, the
[15N]leucine-specific labeled apoLp-III
1H/15N HSQC spectrum gave 11 distinct
cross-peaks that served as the standard for the 11 leucine residues in
the three-dimensional 15N-edited NOESY spectra. Fig.
1, which shows strip plots extracted from the 150-ms
mixing time 15N-edited NOESY spectrum of apoLp-III
(residues Lys135-Val149), illustrates the
general quality of the spectra. The amide proton region (6.5-9.5 ppm)
clearly shows three amide-amide cross-peak patterns along the apoLp-III
sequence that involve dNN(i
1, i), diagonal peak and dNN(i,
i + 1) cross-peaks for residue i, indicating
these residues adopt an
-helical secondary structure. For example,
in the strip of residue Ile141, two amide-amide cross-peaks
can be observed other than the diagonal peak. Whereas the upfield peak
has the same chemical shift as the diagonal peak of residue
Lys140, the downfield peak has the same chemical shift as
the diagonal peak of residue Lys142. We assigned the
upfield peak as the dNN(Lys140,
Ile141) and the downfield peak as the
dNN(Ile141, Lys142), respectively.
It is worth noting that severe overlap was observed in several planes
of 15N-edited NOESY spectra due to chemical shift
degeneracy of backbone nitrogen atoms. For example, the nitrogen
chemical shift of residue Ala138 is degenerate with several
other residues (Fig. 1). Despite these problems, we managed to assign
~80% of the backbone NH and N atoms on the basis of sequential
NH-NH connectivities in 15N-edited NOESY spectra.
Fig. 2 shows C(i
1)-C
(i
1)-N(i)-NH(i) connectivities from a CBCA(CO)NNH
spectrum together with the corresponding
C
(i)-C
(i)-N(i)-NH(i)
and C
(i
1)-C
(i
1)-N(i)-NH(i) (in different sign) connectivities
identified by an HNCACB experiment for residues
Lys73-Thr86 of apoLp-III. An independent
assignment, obtained on the basis of these two spectra, was used to
confirm the assignment derived from 15N-edited NOESY and
15N-edited TOCSY spectra. For residues
Ala4-Gly5-Gly6-Asn7-Ala-8,
where no dNN connectivities were found, and residues
Ser33-Lys34-Asn35-Thr36-Gln37-Asp38-Phe39,
where significant overlap was observed for NH protons, assignment could
not be made on the basis of the 15N-edited NOESY spectrum
alone. In this case, HNCACB and CBCA(CO)NNH spectra were used to obtain
the assignment. Side-chain assignment was obtained by analysis of
HCCH-TOCSY, 15N-edited TOCSY, 15N-edited NOESY,
and 13C-edited NOESY spectra. The 15N-edited
TOCSY spectrum was not very useful in this regard, however, since it
generally showed the connectivities only up to
protons. Three-dimensional HCCH-TOCSY provided a powerful spectrum for side-chain assignment, although, in some cases, chemical shift overlap
was observed in some planes, creating ambiguity for some side-chain
assignments. In these cases, three-dimensional 15N-edited
NOESY and 13C-edited NOESY spectra were used to confirm the
assignment.
Fig. 3 shows a complete assignment of the
1H/15N HSQC spectrum of apoLp-III in 50 mM potassium phosphate, pH 7.0. The
1H/15N HSQC spectra of apoLp-III obtained in
our standard buffer condition (250 mM potassium phosphate,
pH 6.4-6.5) showed considerably more overlap in the center of the
spectrum, which makes it difficult to label the cross-peaks. Other than
the crowded central region, however, the overall chemical shift
dispersion is similar under both conditions. Five glycine residues in
apoLp-III only give rise to three cross-peaks in Fig. 3 due to fast
exchange of the amide protons of two glycines (Gly5 and
Gly6) at pH 7.0. At pH 6.5, however, five distinct
cross-peaks were observed for those five glycine residues. Cross-peaks
connected by dotted lines in the upper right
corner of Fig. 3 were derived from the side-chain amine groups of
Asn and Gln residues. Interestingly, several cross-peaks derived from
backbone N-H groups (such as Ser24, Ser128,
Ser66, and Ser29) were located in this region
of the spectrum. We have recently reported a strategy that results in
specific labeling of peptide backbone nitrogen atoms without side-chain
nitrogen atom labeling (34). The 1H/15N HSQC
spectrum of the backbone nitrogen specifically labeled apoLp-III sample
confirmed the assignment of these four Ser residues (data not
shown).
The Secondary Structure of ApoLp-III
Fig. 4
summarizes short and medium range NOE connectivities
(|i j|
4), amide exchange rates,
3J
N coupling constants, and
chemical shift index data for apoLp-III. The medium range (2
|i
j|
4) NOEs, especially d
N(i, i + 3) NOEs, together with
strong dNN(i, i + 1) and weak
d
N(i, i + 1) NOEs, indicate that
the peptide bonds adopt a folded structure (i.e.
helix),
due to the 3.6-residue repeats in the helical structure. The observed
slow amide proton exchange rates for specific residues suggest these
protons are hydrogen-bonded, which prevents proton exchange with the
solvent, again consistent with an
-helical structure. The chemical
shift index is a consensus of
proton and
and
carbon atom
chemical shift deviation from random coil values, which correlates with
specific secondary structure elements (41). In the case of apoLp-III,
the observed negative CSI for most residues in the protein is
consistent with
-helical secondary structure. Likewise, the observed
lack of consensus positive CSI for any residue indicates an absence of
structure in apoLp-III. Finally, the presence of
3J
N coupling constants <6.0 Hz
(shown by filled circles in Fig. 4) provides further
evidence of
helix structure for given residues. Whereas reliance on
a single parameter may cause ambiguity, the use of four independent NMR
measurements permits conclusive identification of secondary structure
elements within the protein sequence. The consensus NMR parameters for
apoLp-III shown in Fig. 4 clearly identify five discrete
-helical
regions (21-30 residues in length) with well defined boundaries. These
data are in accord with structural information on apoLp-III from
another insect, L. migratoria (8), despite the fact that
these proteins share <30% sequence identity. In M. sexta
apoLp-III, the NMR data also reveal a short helix, termed helix 3
,
located between helix 3 and helix 4 just beyond Pro95. This
helix includes residues Asp96-Glu100. The
existence of this short helix is confirmed not only by medium range
NOEs d
N(Glu99, Asp96),
d
N(Glu100, Val97),
dNN(Lys99, Val97), and
dNN(Glu100, Glu98), but also by
slower amide proton exchange rates, smaller
3J
N coupling constants, and
chemical shift index criteria.
The five major helices in apoLp-III range between 21 and 30 residues
and, other than helix 3, are connected to one another by short
5-7-residue flexible loop segments. For example, helix 4 and helix 5 are connected by a seven-residue loop comprising amino acids 129-135.
Two d
N(i, i + 2) NOEs were found
between residue Glu131 and Lys135, indicating
turn-type structure in the loop. Helix 5 contains a proline at position
139 which, interestingly, does not break the helix. The C terminus of
helix 5 extends to residue 164 of the protein. Although residues 165 and 166 display d
N(i, i + 3) and
dNN(i, i + 2) NOEs, other NMR
parameters indicate these residues do not adopt a stable helical
secondary structure. These residues appear to be more flexible, as
judged by fast or medium amide proton exchange rates and the larger
3J
N coupling constants for these
residues. A summary of the secondary structure of M. sexta
apoLp-III and comparison with L. migratoria apoLp-III is
shown in Table I.
|
Whereas the
consensus NMR parameters shown in Fig. 4 identify secondary structure
elements, long range NOEs were used to characterize helix topology and
the tertiary structure of apoLp-III. These NOEs identify proton pairs
<5.0 Å apart in space, despite the fact that they may reside on amino
acids from distant regions of the primary sequence. Fig.
5 summarizes >300 interhelical long range NOEs derived
from 15N-edited NOESY spectra at 150-ms mixing time. The
helical wheel diagrams shown are based on the secondary structure of
apoLp-III identified by the consensus NMR parameters described above.
This depiction clearly demonstrates that each helix is amphipathic, with well segregated hydrophobic and hydrophilic surfaces. Analysis of
the pattern of long range NOEs indicates that the helices in apoLp-III
adopt an up and down, antiparallel orientation. For example, helix 1 contacts with both helix 4 and helix 5. We determined 46 interhelical
NOEs between helix 1 and helix 5 and another 46 between helix 1 and
helix 4. Long range NOEs were observed between residues 10 and 127, 20 and 153, and 29 and 159. These NOEs indicate a parallel orientation
between helix 1 and helix 5 and an antiparallel orientation between
helix 1 and helix 4. Interestingly, many more NOEs were found between
the N terminus of helix 1 and the C terminus of helix 4 than those
between the C terminus of helix 1 and the N terminus of helix 4, suggesting stronger interhelical contacts in one end of the two
helices. Helix 2 interacts with three helices, helix 3 (70 interhelical
NOEs), helix 4 (26 interhelical NOEs), and helix 5 (42 interhelical
NOEs). Helix 2 contacts both helix 3 and helix 5 in an antiparallel
manner, with contacts through the entire length of these three helices.
Helix 2 contacts helix 4 weakly in a parallel orientation since
interhelical NOEs were identified only between the two N termini and a
few residues in the center region of the two helices. Other than
contacting helix 2, helix 3 interacts with helix 4 in an antiparallel
arrangement. A total of 37 interhelical NOEs were found between helix 3 and helix 4, with NOEs observed along the entire length of the two helices.
The interhelical contacts among helices in apoLp-III are mainly from
hydrophobic residues such as leucine, valine, isoleucine, phenylalanine, and alanine. These hydrophobic contacts likely exert a
stabilizing force in terms of the globular fold of apoLp-III. However,
Fig. 5 also indicates that charged residues, such as Lys105
and Glu13, and several polar residues such as
Gln20, Asn40, Ser47,
Gln53, Ser58, Gln109,
Gln113, Ser119, Gln120, and
Gln156 are located within the hydrophobic core of the helix
bundle. These buried charged and polar residues, which are available
for contact with the other residues, may contribute to the low
intrinsic stability of this protein in the lipid-free state. Long range NOEs were identified between all residues in helix 3 and several residues in the N terminus of helix 2, indicating the existence of
interactions between helix 2 and helix 3
. These NOEs demonstrate that
helix 3
is in close proximity to the N terminus of helix 2, suggesting
the possible topology of helix 3
. Long range NOEs were also identified
in the loops that connect different helices. For example, NOE contacts
were found between residues in loop 1 and residues located in the N
termini of helix 2 and helix 4; from loop 2 to loop 4, the N terminus
of helix 5 and the C terminus of helix 3; and from loop 4 to loop 2. These long range NOEs allow us to identify the global fold and tertiary
structure of apoLp-III.
Fig. 6
shows a cylinder diagram of the global fold and tertiary structure of
apoLp-III. This diagram clearly demonstrates a five-helix bundle
structure for lipid-free M. sexta apoLp-III that is similar
to the x-ray crystal structure of L. migratoria apoLp-III
(8) as well as the four-helix bundle architecture of the N-terminal
domain of human apoE (7). The short helix, helix 3, which connects
helix 3 and helix 4, orients almost perpendicular to helix 3 and helix
4 and is positioned at the end of the elongated helix bundle. In terms
of proposed helical repositioning upon lipid association (8), helix 3
may function in initiating conformational opening of the protein.
Interestingly, a similar short helix is present in the N-terminal
domain of apoE (7).
Previous NMR studies on exchangeable apolipoproteins have been limited to small fragments ranging from 15 to 36 residues using two-dimensional NMR techniques. For example, Rozek et al. (42) and Buchko et al. (12) reported NMR structures of two synthetic fragments of apolipoprotein C-I, whereas Lycksell et al. (11) investigated the structural properties of a 30-residue synthetic fragment of human apolipoprotein C-II (43). In a recent study, Wang et al. (13) determined the structure of a peptide fragment (residues 166-185) of apolipoprotein A-I and two fragments of apoE (14), which range from 14 to 18 residues, in complex with sodium dodecyl sulfate and dodecyl phosphocholine micelles. Using a similar approach, we have solved the structure of a 36-residue C-terminal fragment of M. sexta apoLp-III in the presence of SDS.2 The present study is unique in that we report the first NMR structure of an intact exchangeable apolipoprotein. We suggest that extension of this approach to the lipid-associated state of apolipoproteins employing model lipid surfaces such as dodecyl phosphocholine micelles, points to a potentially fruitful research direction.
Following an extensive empirical search for optimal conditions for NMR experiments, we found that apoLp-III is stable for >2 weeks in 250 mM phosphate buffer at pH 6.4-6.5. This is a critical step toward the practice of three-dimensional NMR studies of apoLp-III. It was known from previous denaturation studies that apoLp-III is relatively labile and susceptible to environmental perturbation (22). Indeed, the low stability of apoLp-III is a common property of most exchangeable apolipoproteins. For example, the midpoint of guanidine HCl-induced denaturation of the 10-kDa C-terminal domain of human apoE was found to be 0.7 M guanidine HCl (44); that for human apolipoprotein A-II was 0.6 M (45), and that for human apolipoprotein A-IV was found at 0.4 M (46). A potentially important factor contributing to the low stability of apoLp-III has been identified in the NMR structure of M. sexta apoLp-III. Although hydrophobic interactions are the major force contributing to stabilization of the helix bundle structure, Fig. 5 indicates that there are several charged residues, such as Lys105, Glu13, as well as several polar residues (e.g. Gln20, Asn40, Ser47, Gln53, Ser58, Gln109, Gln113, Ser119, Gln120, and Gln156) located in the interior of the helix bundle. We suggest that charged and polar residues localized in otherwise nonpolar regions of the molecule contribute to the low stability of apoLp-III. This hypothesis can be tested by site-specific mutagenesis experiments coupled with denaturation studies.
It is conceivable that the low stability of apoLp-III is required for its known reversible lipoprotein binding activity. Based on the crystal structure of L. migratoria apoLp-III, it was proposed that the helix bundle opens about putative hinge regions, exposing its hydrophobic interior, which interacts directly with lipid surfaces (8). This open conformation model is supported by the NMR structure of apoLp-III presented in this study. Whereas conformational opening of apoLp-III may disrupt certain interhelical contacts within the protein, this is more than compensated by subsequent replacement of helix-helix contacts with helix-lipid interactions. It is recognized that association with lipoprotein surfaces and consequent conformational opening of apoLp-III is induced by the content of surface monolayer-localized diacylglycerol. On the other hand, dissociation from lipoprotein surfaces and return to the helix bundle conformation results from depletion of lipoprotein diacylglycerol content (47-50). Thus, in essence, apoLp-III association/dissociation with lipoproteins is dictated by the lipid composition of the particle. The intrinsic reversibility of apoLp-III conformational changes permits the protein to function in multiple association/dissociation events during its lifetime in plasma.
Compared with the crystal structure of L. migratoria
apoLp-III, the NMR structure of M. sexta apoLp-III reveals
an overall similar molecular architecture. One apparent difference,
however, is the presence of a short helix, helix 3, in M. sexta apoLp-III. Interestingly, a similar short helix was also
found in the x-ray crystal structure of human apoE N-terminal domain
(7). In human apoE, this short helix, which involves nine residues,
connects helix 1 and helix 2 and orients perpendicular to helix 1 and
helix 2. The short helix found in the NMR structure of M. sexta apoLp-III is in a similar orientation. It is worth noting
that helix 3
is a flexible helix displaying amide exchange rates that
are faster than those from the other helices.
The helix-short helix-helix organization in this molecule may comprise
an important structural element in apoLp-III in terms of initiating
interactions with lipoprotein surfaces. It is noteworthy that helix 3
is positioned where, in the open conformation model, it could initiate
contact with lipid surfaces. We have proposed a structural model to
describe the interactions between apoLp-III and the phospholipid
monolayer of insect lipophorins based on 31P NMR studies
designed to evaluate the mobility of phospholipids in different
lipophorin subspecies (48). In this model, we suggested that
diacylglycerol partitioning into the particle surface monolayer creates
a binding site for apoLp-III interaction. It is recognized that a
strong correlation exists between lipophorin diacylglycerol content and
apoLp-III binding, and recently we have provided direct experimental
evidence for surface localization of diacylglycerol in lipophorin
particles known to bind apoLp-III (49). We speculate that surface
diacylglycerol intercalates between phospholipids in the monolayer
surface, creating a gap between phospholipid head groups. This defect
is effectively repaired by apoLp-III binding.
An important question, however, is whether hydrophobic interactions are
responsible for initiation of binding or whether ionic interactions
localize apoLp-III at the particle surface, positioning the protein to
"respond" to surface defects created by diacylglycerol partitioning
into the surface monolayer. Soulages and Wells (51) have presented a
"hydrophobic sensor" hypothesis to describe the initiation of
interactions between exchangeable apolipoproteins and lipoproteins.
According to this hypothesis, a two-step sequential mechanism for
binding of apoLp-III to lipoprotein surfaces has been proposed. The
first step involves a recognition process (through exposed hydrophobic
amino acids) consisting of the adsorption of apoLp-III to a nascent
hydrophobic defect in the phospholipid bilayer caused by the presence
of diacylglycerol. This is followed by a conformational opening to
expose the protein interior. Soulages and Wells suggest (51) that in
L. migratoria apoLp-III, conserved leucines located in the
loops between helices are responsible for the initial association
process. An alternative proposal is that helix 3 may function in
initiation of stable lipoprotein binding. This concept is supported by
disulfide bond engineering studies, which indicate that apoLp-III
interactions with lipoproteins is oriented at the end of the molecule
containing helix 3
(52). Helix 3
contains five residues
(Asp96, Val97, Glu98,
Lys99, and Glu100), four of which possess
charged hydrophilic side chains. The suggestion that charge-charge
interactions between the phospholipid head groups and helix 3
provide
a relatively long range attraction, which localizes apoLp-III in close
proximity to the lipoprotein surface, is supported by studies with
model phospholipids (53). Once the recognition process is complete,
surface-exposed diacylglycerol could trigger opening of the helix
bundle, resulting in formation of a stable binding interaction. Judging
by the fact that the N-terminal domain of apoE contains a similar short
helix, an analogous binding mechanism may be suggested for human
exchangeable apolipoprotein-lipoprotein interactions. Although further
experiments are required to determine the precise mode of lipid
association of apoLp-III, it is likely that concepts developed with
this model protein may have important implications for
amphipathic-exchangeable apolipoproteins in general.
We thank Dr. Lewis Kay of the University of Toronto for providing us with the pulse sequences of three-dimensional heteronuclear NMR experiments carried out in this study. We thank the Protein Engineering Network of Centers of Excellence for the use of their computer and 600-MHz NMR spectrometer. Technical assistance from Dean Schieve is gratefully acknowledged.