(Received for publication, July 26, 1995; and in revised form, November 2, 1995)
From the
The synthetic lipid-associating peptide, LAP-20
(VSSLLSSLKEYWSSLKESFS), activates lecithin-cholesterol acyltransferase
(LCAT) despite its lack of sequence homology to apolipoprotein A-I, the
primary in vivo activator of LCAT. Using SDS and
dodecylphosphocholine (DPC) to model the lipoprotein environment, the
structural features responsible for LAP-20's ability to activate
LCAT were studied by optical and two-dimensional H NMR
spectroscopy. A large blue shift in the intrinsic fluorescence of
LAP-20 with the addition of detergent suggested that the peptide formed
a complex with the micelles. Analysis of the CD data shows that LAP-20
lacks well defined structure in aqueous solution but adopts helical,
ordered conformations upon the addition of SDS or DPC. The helical
nature of the peptides in the presence of both lipids was confirmed by
upfield H
NMR secondary shifts relative to random coil
values. Average structures for both peptides in aqueous solutions
containing SDS and DPC were generated using distance geometry methods
from 329 (SDS) and 309 (DPC) nuclear Overhauser effect-based distance
restraints. The backbone (N, C
, C=O) RMSD from
the average structure of an ensemble of 17 out of 20 calculated
structures was 0.41 ± 0.15 Å for LAP-20 in SDS and 0.41
± 0.12 Å for an ensemble of 20 out of 20 calculated
structures for LAP-20 in DPC. In the presence of SDS, the distance
geometry and simulated annealing calculations show that LAP-20 adopts a
well defined class A amphipathic helix with distinct hydrophobic and
hydrophilic faces. A similar structure was obtained for LAP-20 in the
presence of DPC, suggesting that both detergents may be used
interchangeably to model the lipoprotein environment. Conformational
features of the calculated structures for LAP-20 are discussed relative
to models for apolipoprotein A-I activation of LCAT.
In human plasma, free and esterified cholesterol circulate as
constituents of lipoproteins. The primary enzyme responsible for
modulating plasma levels of cholesterol is lecithin-cholesterol
acyltransferase (LCAT; EC
2.3.1.43)()(1, 2) , a 60-kDa glycoprotein
that catalyzes transesterification of a fatty acid from the sn-2 position of lecithin (phosphatidylcholine) to
cholesterol. The key in vivo metabolic activator of LCAT is
apoA-I(3) , a protein also believed to play an important role
in the biogenesis of high density lipoprotein particles, which serve as
acceptors of peripheral cell-associated free cholesterol in the reverse
cholesterol transport pathway(4, 5) . High plasma high
density lipoprotein-cholesterol levels have been inversely correlated
with the risk of developing coronary artery disease(6) .
A
prominent feature of apoA-I and the other exchangeable apolipoproteins
is repeating amino acid motifs of 11 or 22 residues, which, based upon
predictions from primary sequences, may adopt amphipathic helical
structures when associated with lipid(7) . Such a motif is
characterized by polar and nonpolar amino acid residues aligned on
opposing faces of the long axis of an -helix. The positively and
negatively charged amino acid residues are distributed in the
polar-nonpolar interface and along the center of the polar face,
respectively. The lipid-associating properties of the apolipoproteins
are believed to result from hydrophobic interactions between the
nonpolar amino acid side chains and the phospholipid acyl
chains(8) . The model also allows for ionic interactions
between (i) the positively charged protein side chains in the interface
with the negatively charged phosphate groups of the phospholipid and
(ii) the negatively charged protein side chains in the hydrophobic face
with the positively charged quaternary amines of the phospholipid.
In order to more precisely define secondary structural features
responsible for LCAT activation, synthetic model peptides have been
studied. Synthetic fragments of apoA-I designed to localize the
position of the major LCAT activating region indicate that residues
143-185 and 121-164, which both contain two predicted
amphipathic regions(9, 10) , activate LCAT 24 and 30%,
respectively, of the rate of native apoA-I. De novo peptides
have shown that charge distribution plays a role in the peptides'
affinity for lipid and ability to activate
LCAT(11, 12) . For example, one acidic 30-residue
peptide, GALA, was observed to activate LCAT almost as effectively as
apoA-I(13) . Studies using de novo amphipathic
peptides of various lengths suggest that 10-12 residues are
enough for lipid association (14) but that longer sequences are
necessary for LCAT activation(15) . The critical role of the
primary sequence is illustrated by the inability of a 16-residue
peptide, LAP-16, to associate with DMPC or activate LCAT, while the
addition of four residues to the amino terminus of LAP-16 (LAP-20)
results in a dramatic increase in lipid association and LCAT
activation(16) . ApoA-I mimetic peptides have also illustrated
that proline punctuation between contiguous pairs of predicted
amphipathic -helices increases lipid affinity and LCAT
activation(17) , although the absence of such a residue in
LAP-20 and GALA suggests that intramolecular interaction between
proline and LCAT is likely not involved in LCAT activation.
LAP-20 is unique in that, while it has no sequence homology with apoA-I, it activates transacylation by LCAT to 65% of that of apoA-I in the presence of DMPC and cholesterol(16) . Because the latter value is greater than twice the value obtained with the most potent synthetic peptide from apoA-I, we chose to study in greater detail the conformational changes that occur upon the association of LAP-20 with lipid.
Sodium dodecyl sulfate and dodecylphosphocholine, agents
commonly used to model membranes(18, 19) , were used
to model the lipoprotein environment(20, 21) . The
amount of helical structure adopted by LAP-20 in the absence and in the
presence of increasing concentrations of SDS and DPC was estimated by
circular dichroism spectroscopy and H NMR secondary
shift analysis. Two-dimensional
H NMR studies were
performed in the presence of perdeuterated SDS
(SDS-d
) and perdeuterated DPC
(DPC-d
) at a molar ratio of peptide to detergent
of 1:40. Using distance restraints obtained from NOESY data, average
structures for LAP-20 in SDS and DPC were obtained using distance
geometry/simulated annealing methods. A comparison was made between the
structures calculated for LAP-20 in the presence of SDS and DPC to
determine if the different lipid head groups have any significant
structural effects. The biological significance of the major structural
features observed for LAP-20 in the model lipid environments is
discussed in relation to models of LCAT activation.
NMR experiments were run on a Bruker AMX spectrometer
operating at a proton resonance frequency of 600.13 MHz as reported
earlier(20) . Standard phase-sensitive (TPPI) two-dimensional
NOESY(23) , TOCSY(24, 25) , and DQF-COSY (26) spectra were recorded at 25 °C. Water suppression in
the TOCSY and NOESY experiments was by WATERGATE (27) employing
a 3-9-19 pulse sequence(28) . NOESY data were recorded using
mixing times of 75, 100, 150, and 225 ms. A 75-ms mixing time and
2.5-ms trim pulse were used in the MLEV-17 spinlocking sequence of the
TOCSY experiments. Prior to Fourier transformation, the data were
zero-filled to generate a 2K 2K matrix and apodized by a cosine
function in D2 and a sine function in D1. A fifth-order polynomial
function was applied to base-line correct all processed spectra in both
dimensions.
The CD spectra from a titration of an aqueous solution of LAP-20 with lipid are shown in Fig. 1. In the absence of detergent the LAP-20 spectrum shows a negative band around 200 nm and a very weak band near 220 nm, which characterize peptides that lack a well defined secondary structure(32) . Spectra obtained without lipid did not change over the pH range 3-11 and, as observed by Pownall et al.(16) , over the temperature range of 10-50 °C. The addition of SDS or DPC to LAP-20 effected changes in the CD spectra, which suggested an increase in ordered secondary structure. Above a peptide:detergent ratio of 1:4 (SDS) and 1:6 (DPC), no further changes were observed in the CD spectra, suggesting that LAP-20 was completely associated with lipid in a micelle-bound state(20, 33) . The lower molar ratio of SDS required to obtain such a condition may reflect a marginally greater affinity of LAP-20 for the negatively charged SDS than the zwitterionic DPC(31) . In both lipid complexes the CD spectra of LAP-20 possess a double minimum at 222 and 208-210 nm and a substantial maximum at 191-193 nm. Such features are indicative of a helical conformation (34) and were observed for LAP-20 in complexes with DMPC(16, 29) .
Figure 1: Circular dichroism spectra of LAP-20 at 25 °C, pH 5.0, in aqueous solution and at various molar ratios of peptide:detergent. a, no detergent; b, 1:2.4 DPC (dashed line); c, 1:1 SDS; d, 1:40 DPC (dashed line); e, 1:40 SDS.
The helical content of LAP-20 in the absence and presence of SDS and DPC was estimated by deconvoluting the CD spectra using convex constraint analysis(35) , and the results are presented in Table 1. While the absolute percentages of secondary structure obtained by such analyses will vary depending on the weighting of secondary structures contributing to the spectra used in the basis set, the convex constraint analysis results do illustrate trends. An increase in helical secondary structure of LAP-20 is observed as the lipid concentration is increased, with essentially identical values obtained at a peptide:lipid ratio of 1:40. Similar observations were made upon the titration of other peptides with SDS (20, 33, 36) and DPC (21, 37) . Because the calculated mean molar ellipticity values are sensitive to other factors(38, 39) , the CD data were also analyzed using two parameters, R1 and R2, which are independent of inaccuracies in determined peptide concentrations as well as those caused by small shifts in wavelength(40) . Such parameters, defined and tabulated in Table 1, also follow a trend characteristic of an increase in helical secondary structure as the lipid concentration is increased.
Fig. 2illustrates the H-H
regions
of 5 mM LAP-20 solutions in the presence of
SDS-d
and DPC-d
. All of the
proton resonances for LAP-20 in both detergents could be assigned to
one unique species using TOCSY spectra to identify spin systems, NOESY
spectra to obtain interresidue connectivities and to distinguish
between degenerate spin systems, and DQF-COSY spectra to confirm side
chain resonance assignments(44) . The proton chemical shifts
for LAP-20 in the presence of 40-fold molar excess
SDS-d
and DPC-d
are
summarized in Table 2and Table 3, respectively. Unique
proton assignments, and the preparation of clear LAP-20 solutions with
similar NMR spectral features at 5 and 20 mM, implied that the
peptide was tightly associated with the detergent micelles in a single
conformation.
Figure 2:
H-H
regions of
the 600-MHz NOESY spectra (
= 150 ms) of LAP-20
(5 mM) in the presence of 200 mM SDS-d
(pH 5.0) and 200 mM DPC-d
(pH 4.0) at 25 °C. The constructs
follow the sequential connectivities, with the
H
-H
cross-peaks
labeled.
Using random coil proton chemical shifts determined
for peptides in unstructured conformations(44) , it is possible
to calculate chemical shift changes that occur when a peptide or
protein adopts an ordered conformation. With respect to random coil
chemical shift values, the H resonances move upfield in
an
-helical conformation and downfield in a
-sheet
conformation(44, 45) . H
secondary
shifts (
H
) were calculated by subtracting the
measured H
chemical shifts of LAP-20 in detergent
complexes ( Table 2and Table 3) from the corresponding
random coil values obtained by
Wüthrich(44) . Most of the H
secondary shifts are positive, which suggests that LAP-20 adopts
a helical conformation in the presence of lipid. Furthermore, except
for Trp
, Lys
, and two residues at the
N-terminal, the difference in
H
for LAP-20 in the
presence of both lipids is less than 0.04 ppm, suggesting a similar
conformation in both lipid environments. A semiquantitative estimation
of this helical content is obtained by dividing the average H
secondary shift by 0.35, the average upfield H
shift observed in the amino acid residues of proteins in an
-helical conformation(46) . The results, tabulated in Table 1, agree with the CD data and show that LAP-20 is highly
helical in the presence of both detergents. A more detailed examination
shows that the H
secondary shifts of residues toward
the N and C termini approach 0 in both lipids, suggesting some fraying
at both ends.
Figure 3: Summary of the sequential and medium range NOEs observed for LAP-20 in an aqueous solution of deuterated SDS and DPC at a mixing time of 150 ms. Classification of NOE intensities into strong, medium, and weak is indicated by the height of the bars.
Figure 4:
Conformational ensembles of 17 and 20 out
of 20 calculated structures for LAP-20 in SDS (A) and DPC (B), respectively. The backbone atoms
(N-C-C=O) of residues 3-18
have been superimposed on the average structure. C, The
backbone of the average structures for ensembles A and B have been
replaced by a ribbon and superimposed on residues 3-18. Black, SDS; gray, DPC.
Figure 5:
Plots of the mean pairwise RMSDs to the
mean structure for each residue of LAP-20 in SDS (solid) and
in DPC (dashed). Squares, backbone atoms
(N-C-C=O); circles, all
atoms. The plots were generated by moving a window of three residues
along the sequence and plotting the mean pairwise RMSD (Å) over
the central residue.
LAP-20 was shown to associate with both SDS and DPC by
optical spectroscopy and displayed optical properties similar to those
observed for LAP-20 in the presence of DMPC(16, 29) ,
a lipid in which LAP-20 activates LCAT, suggesting that the
peptide's conformation is similar in all three lipid
environments. Previously, Pownall et al.(16) confirmed a physical association of LAP-20 with DMPC
discoidal complexes using a variety of methods, including
ultracentrifugation in a density gradient and size exclusion
chromatography. Our H NMR data indicate that LAP-20
associates with SDS and DPC, and all the evidence suggests it
associates as a monomer. While the unique set of proton resonance
linewidths for LAP-20 in the presence of detergent are sharp enough to
allow unambiguous assignments, they are still too broad to obtain
H
-H
coupling constants, indicating
the peptide is part of a large molecular weight micellar
complex(19, 20, 46) .
The CD data,
H secondary shifts (Table 1), and the pattern of
the interresidue NOEs ( Fig. 2and Fig. 3) (44, 47) suggest that LAP-20 adopts a helical
conformation when associated with SDS or DPC. Detailed
three-dimensional structures generated for LAP-20 in the presence of
SDS and DPC, using distance geometry calculations, verify such
structures as illustrated by the superimposed backbone atoms of the
calculated structures in Fig. 4, A (SDS) and B (DPC). Fig. 4C, which overlays ribbons drawn
through the backbone atoms of the mean LAP-20 structure of the
ensembles in Fig. 4, A and B, illustrates the
formation of a similar, well defined helical structure over the length
of the molecule in the presence of both detergents, apart from some
dynamic fraying at the termini. Because the different head groups on
SDS and DPC have little effect on the structure adopted by LAP-20 in
the micelle, we suggest that both detergents may be equally suited to
model the lipoprotein environment.
Fig. 6illustrates the
superposition of the side chains for the ensemble of calculated
structures shown for the backbone atoms of LAP-20 in SDS from Fig. 4A, while Fig. 7illustrates a stereo view
of the mean orientation of the side chains for the ensemble of
calculated structures for the backbone atoms of LAP-20 in DPC from Fig. 4B. In both lipid environments, the side chains
are clustered in three distinct regions: hydrophobic, hydrophilic, and
interfacial. Trp is fixed in the interfacial region with
the hydrophobic six-membered ring oriented toward the hydrophobic face
and the polarizable imino group intruding into the hydrophilic face.
Such an orientation (which generated a 21-nm blue shift in the maximum
Trp fluorescence) is predicted to be energetically favored (48, 49, 50, 51) and is similar to
the orientation observed for the Trp residue in a proposed lipid
binding domain of
apoC-I(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 20) .
Glu
and Glu
, which are negatively charged in
the SDS solution at pH 5.0, lie along the center of the hydrophilic
face. The two positively charged residues, Lys
and
Lys
(dark gray), are located in the
polar-nonpolar interface. Such an overall orientation of the peptide
side chains for LAP-20 in the presence of SDS and DPC fits the
definition of a class A amphipathic helix (7, 51) and
was predicted for LAP-20 from the primary structure(16) .
Figure 6:
Superposition of the side chains of the
ensemble of calculated structures for LAP-20 in SDS shown in Fig. 4A. The hydrophilic amino acids (Ser,
Ser
, Ser
, Ser
, Glu
,
Tyr
, Ser
, Ser
, Glu
,
Ser
, and Ser
) are shown in boldface
type, the positively charged amino acids (Lys
and
Lys
) are shown in dark gray, and the remaining
side chains are shown in light gray. Except for the N-terminal
valine, the side chains are segregated into distinct hydrophobic and
hydrophilic domains. One-letter amino acid codes are
used.
Figure 7:
Stereo
view of the side chains of the calculated structure for LAP-20 in DPC
that most closely superimposes on the average structure of the ensemble
in Fig. 4B. Ser, Ser
,
Ser
, Ser
, Glu
, Tyr
,
Ser
, Ser
, Glu
, Ser
,
and Ser
are shown in boldface type, the
positively charged amino acids (Lys
and Lys
)
are shown in dark gray, and the remaining side chains are
shown in light gray. If the lysine side chains truly
snorkeled, their positively charged termini should have been observed
to extend fully into the hydrophilic face.
The nonpolar side chains that extend from the hydrophobic face of
LAP-20 presumably interact with the hydrophobic acyl chains of the
lipid. On the other hand, the polar and charged side chains, located at
the polar-nonpolar interface or the hydrophilic face, presumably
interact with the aqueous milieu that includes the negatively charged
head groups of SDS or the zwitterionic head groups of DPC. Because the
LAP-20 structures are similar regardless of the head group of the
detergent, hydrophobic interactions between the nonpolar face of the
peptide and the hydrophobic interior of the micelle are likely the
primary force stabilizing the helix(50, 52) . The
surface occupied by the hydrophobic face, as estimated by looking down
the long axis of the helix, is a pie-shaped wedge that occupies
30% of the total area. This proportion of peptide, which
presumably penetrates into the micelle surface, is considered optimal
for enzyme activation(9) .
In addition to hydrophobic
interactions, -helical structures are stabilized by intramolecular
hydrogen bond formation between backbone amide and backbone carbonyl
groups four residues apart (5-7 kcal/bond). LAP-20, in a perfect
-helix, is predicted to form 16 backbone hydrogen bonds with a
H
O distance of 2.06 ± 0.16 Å
and a N-H
O bond angle of 155
± 11°(53) . The calculated structures for LAP-20 in
SDS and DPC show that the N-H
O
atoms are in a position to form 11 and 14 hydrogen bonds, respectively,
that meet the following conditions: (i) - (i + 4),
N-H
O bond angle between 120 and
180°, H
O distance < 3.0 Å.
Therefore, it is likely that the helical structure of LAP-20, in both
detergents, is stabilized by the formation of intramolecular hydrogen
bonds.
Segrest (7, 51) has proposed a stabilization
of amphipathic structures by a ``snorkeling'' of basic amino
acid side chains located at the polar-nonpolar interface. In such a
model the Lys and Arg residues are oriented with the side chains
aligned along the edge of the hydrophobic face, and the positively
charged ends are extended into the hydrophilic face. It is evident from Fig. 6and Fig. 7that, while the alkyl groups of
Lys and Lys
align along the edge of the
hydrophobic face, the positively charged ends extend into the
polar-nonpolar interface and not into the hydrophilic face, i.e. hydrophobic interactions dominate. We noted previously that
snorkeling was not extensive in apoC-I fragments bound to SDS, and
consequently, this study reinforces our contention that snorkeling is
likely not a general characteristic of amphipathic
helices(20) .
Intraresidue charge interactions, in the form
of salt bridges between oppositely charged side chains, may stabilize
-helical structures by up to 6 kcal/mol/salt
bridge(54, 55, 56) . While LAP-20 contains
two positively and two negatively charged side chains at pH 5.0, they
are either too close (1 residue) or too distant (>5 residues) to
stabilize the
-helix through intramolecular salt bridge formation.
It is therefore not surprising to find that the structure of LAP-20 at
pH 4.0 in DPC, where the Glu residues are protonated, does not differ
significantly from the structure obtained at pH 5.0 in SDS, where the
Glu residues are negatively charged. Furthermore, detailed structural
studies of the proposed lipid binding domains of apoC-I, which adopted
helical structures in the presence of SDS, showed that salt bridges did
not form(20) .
Features associated with peptides that activate LCAT are affinity for lipid (57) and helical secondary structure(58) . Our NOE-derived structures for LAP-20 confirm a helical conformation and show that ionic interactions (intermolecular salt bridges and snorkeling) play a minor role in stabilizing the lipid-bound state. We find, instead, that hydrophobic interactions between the nonpolar amino acid side chains and the phospholipid acyl chains (8) play the most important role in stabilizing the complex, followed by intermolecular backbone hydrogen bonding. The hydrophobic interactions are likely crucial for LCAT activation because LCAT is surface-active, i.e. it binds to the lipid/water interface(3) . Indeed, it has been suggested that the primary role of the amphipathic helix in activating LCAT is to disrupt the water-phospholipid interface and expose buried substrate to LCAT (15, 59) . Support for this hypothesis is the observation that LCAT does not require a co-factor to hydrolyze water soluble substrates such as the p-nitrophenyl esters of fatty acid(59) . However, while lipid binding is necessary for LCAT activation, many amphipathic peptides that bind to lipid do not activate LCAT(51) . Consequently, there must be one or more key topological features of these bound peptides responsible for their ability to effectively activate LCAT.
Segrest et al.(51) have proposed that the unique position of two negatively charged Glu residues, located in the nonpolar face of apoA-I regions 66-87 and 99-120, play a major role in the apoA-I activation of LCAT. In each of these two apoA-I 22-residue regions, the Glu is located at the 13th position. While a consensus 22-residue sequence containing a Glu at the 13th position is a poor activator of LCAT, a 44-residue dimer, obtained by linking two 22-mers together head-to-tail, activates LCAT as well as apoA-I(57) . Because the two Glu residues of LAP-20, a potent activator of LCAT, are located in the center of the hydrophilic face when bound to lipid, it is unlikely that negatively charged Glu residues in the nonpolar face are directly involved in the intermolecular activation of LCAT.