 |
INTRODUCTION |
Plant nonspecific lipid transfer proteins
(ns-LTPs)1 were first
isolated from spinach leaves and named based on their ability to
mediate in vitro the transfer of phospholipids between
membranes (1). ns-LTPs are widely distributed and form a superfamily of
related proteins subdivided into two families: the type 1 ns-LTPs (ns-LTP1) and the type 2 ns-LTPs (ns-LTP2) (see Refs. 2 and 3 for
review). Both families are multigenic, and more than 150 sequences of
plant ns-LTPs are listed in data bases. Only a limited number of
proteins have been isolated from plant, and in vitro lipid
transfer or binding has been demonstrated for an even more limited
number of proteins.
The biological functions of ns-LTP1 have not yet been clearly
determined, the most favored hypothesis being a role in the transport
of cutin monomers (4, 5) or in plant defense mechanisms (6-8) for
ns-LTP1. ns-LTP2 gene expression has been reported in the Zinnia
elegans cell differentiation process (9, 10), in barley and rice
developing seeds (11, 12), under abiotic stress conditions in barley
roots (12), or during nodulation in Vigna unguiculata root
hairs (13). However, there is no biological evidence of their function
in these different contexts. The recent discovery that some ns-LTPs are
pan-allergens of plant-derived foods has brought new interest for their
study. Most of the ns-LTP allergens identified so far belong to the
ns-LTP1 family (14-17), whereas ns-LTP2 has been reported only as a
potent allergen of the pollen of Brassica rapa (18).
The three-dimensional structure of four cereal ns-LTP1s has been
determined, i.e. wheat (19), barley (20), maize (21, 22),
and rice (23, 24). In addition, seven structures of plant ns-LTP1 in
complex with ligands have been determined, including those of maize
ns-LTP1 with palmitate (21) or palmitoyl-lyso-phosphatidylcholine (22),
barley ns-LTP1 with palmitoyl CoA (25) or palmitate (26), and wheat
ns-LTP1 with di-myristoyl-phosphatidylglycerol (27),
lyso-myristoyl-phosphatidylcholine (28), or prostaglandin B2 (29). All
of these data showed that ns-LTP1 are compact single domain proteins
whose fold is stabilized by four disulfide bonds. They are
characterized by a four-
-helix bundle and a C-terminal region with
no regular secondary structure. The most interesting feature of ns-LTP1
structure is the tunnel-like hydrophobic cavity that runs through the
molecule and appears as a potential site for lipid binding. Although
plant ns-LTP1s exhibit very similar global folds, the shape and size of
this hydrophobic cavity vary considerably depending on the protein
and/or on the ligand. This clearly indicates a high plasticity of the
cavity that is able to accommodate a variety of hydrophobic molecules.
In contrast, an antifungal protein extracted from onion seeds that
showed a structure similar to those of ns-LTP1 except that the internal cavity, obstructed by several aromatic side chains, is unable to
transfer lipids (30).
ns-LTP1s have been studied more extensively than ns-LTP2s that are
distinct in terms of primary sequence with less than 30% homology
(Fig. 1), size (7 kDa versus 9 kDa), and lipid transfer efficiency. In wheat, both ns-LTP1 and ns-LTP2
have been biochemically characterized, and ns-LTP2 exhibits a higher
lipid transfer activity than ns-LTP1 (31). They share the same
8-cysteine skeleton, but their disulfide bond assignment has been shown
to be different (32). Because the wheat ns-LTP2 exhibits a lipid
transfer activity, one can hypothesize the presence of an hydrophobic
cavity. However, whether ns-LTP2s have a fold that is similar to or
different from that of ns-LTP1 remained to be elucidated at the
initiation of the present work. This paper presents the refined
solution structure of a recombinant wheat ns-LTP2 liganded with a C16
lyso-phospholipid. In the final stages of preparation of this
manuscript, the structure of a rice ns-LTP2 was reported (33) (Protein
Data Bank entry 1L6H).

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 1.
Primary sequence alignment of various ns-LTP1
and ns-LTP2. The proteins are detailed by the Swiss-Prot accession
number or the Protein Data Bank accession number when available. The
disulfide bridges are shown with black lines and numbered
according to wheat ns-LTPs.
|
|
 |
EXPERIMENTAL PROCEDURES |
Production/Purification--
The Pichia
pastoris transformant GS115-Tdltp18-tr5.2 (34) expressing a wheat
ns-LTP2 was used for the production of the 15N-labeled
protein. Production was carried out in an Applikon fermentor (400 ml of
culture) with 99.4% 15N-labeled ammonium sulfate
(Eurisotop) as nitrogen source. Labeled (15NH4)2SO2 (0.9% w/v)
was added from the very beginning of the biomass production phase and
during the induction phase at 0, 24, and 48 h (0.27, 0.27, and
0.36 w/v, respectively). Production of ns-LTP2 was induced by methanol
after 22 h of culture and lasted for 77 h. The amount of
secreted protein was estimated by densitometer analysis of SDS gels,
using purified ns-LTP2 as a standard. Folding and accumulation of the
recombinant protein was also directly monitored by 15N HSQC
NMR (34). A nonlabeled sample was also produced as described earlier
(35).
The protein was purified from culture supernatant by a single-step
procedure (36) using expanded bed chromatography (Streamline SP-XL;
Amersham Biosciences), and 650 mg of protein were obtained. The column
was equilibrated in 30 mM sodium acetate, pH 5.5, and the
proteins were eluted with 1 M NaCl. The purified protein
was dialyzed against H2O, lyophilized, and analyzed by
SDS-PAGE, MALDI-TOF, and HSQC. MALDI-TOF mass spectrometry experiments
were conducted on BiFlex III mass spectrometer (Bruker Daltonics,
Bremen, Germany). 100 pmol of the protein were solubilized in 10 µl
of water containing 0.1% trifluoroacetic acid. A 0.5-µl sample of
this solution was mixed with 0.5 µl of matrix (a saturated solution
of
-cyano-4-hydroxycinnamic acid in water/acetonitrile 50/50 v/v)
and deposited on the target (dried droplet preparation). The spectra
were recorded on the linear positive mode using the Pulsed Ion
Extraction. For the tryptic digestion analysis, the protein was reduced
by dithiothreitol and alkylated by iodoacetamide prior to digestion.
The protein was digested for 4 h by trypsin (modified, sequencing
grade from Roche Molecular Biochemicals) in 25 mM ammonium
carbonate buffer (pH 8) at an enzyme/protein ratio of 1/20 (w/w).
Proteolysis was stopped by adding diluted trifluoroacetic acid. The
digest mixture was analyzed by MALDI-TOF mass spectrometry in the
positive reflector mode.
Mass spectrometry analysis revealed the heterogeneity of the purified
protein, 15% being glycosylated. The sugar moiety, assumed to be
mannose, were located in the first 29-residue fragment. The protein was
thus further purified using reverse phase chromatography (Source 15 RPC/Akta Purifier 10; Amersham Biosciences), for a final amount of 300 mg of protein.
NMR and Fluorescence ns-LTP2/Lipid
Screening--
Preliminary experiments revealed that NMR spectra could
be significantly improved when ns-LTP2 interacts with a lipid. To find
the best conditions for the study, several ligands and physicochemical conditions were screened by fluorimetry as well as by NMR. The interaction with L-
-palmitoylphosphatidylglycerol
(LPG), L-
-palmitoylphosphatidylcholine, dimyristoylphosphatidylglycerol, dimyristoylphosphatidylcholine, and
cholesterol was tested by fluorimetry and NMR. Lipids from Avanti polar
were prepared in solution in ethanol at a concentration of 10 mM or in small unilamellar vesicle obtained by sonication in H2O.
The fluorescence experiments were performed on a ISS instrument
equipped with Hamatsu detector at 294 K in a 150-µl volume, excitation was set at 275 nm (300 W), and the spectra were recorded between 285 and 400 nm. Increasing amounts of ligand (0.1-100 equivalent) were added to a solution of ns-LTP2 (30 µM).
NOESY spectra were recorded for 12 h on a Bruker 600 MHz
spectrometer at 300 K on a 2.7 mM ns-LTP2 solution in 60 mM phosphate buffer containing 1 eq of lipid. HSQC spectra
were recorded for 30 min on a Bruker 400 MHz on a 1.1 mM
ns-LTP2 solution, 10% D2O. The impact of several
parameters was investigated: temperature (300-315 K), pH (3.45-6.5),
ionic strength (0-100 mM NaCl and 0-60 mM
phosphate buffer), and lipid concentration (0-1.5 eq).
Sequence Alignment and Model Prediction--
Primary sequence
alignments were performed with the following software: CLUSTAL W (37)
and Psi-Blast (38). Early three-dimensional models of construction and
three-dimensional comparison were performed using a web metaserver
(bioserv.cbs.cnrs.fr; Ref. 39) with the following threading methods:
3DPSSM (40) and TITO (41).
NMR Spectroscopy--
All of the NMR samples were prepared by
dissolving the 15N-labeled and unlabeled recombinant wheat
ns-LTP2 in H2O (10% D2O), at a concentration
of 2.8 mM in presence of 70 mM phosphate at pH
3.5 and 1 mM NaN3. 1.5 eq of
L-
-palmitoylphosphatidylglycerol (1-palmitoyl-2-Hydroxy-sn-glycero-3-[phospho-rac-(1-glycero)] sodium salt named LPG) was added to all of the protein preparations. Optimum phospholipid concentration was determined by a step by step
titration monitored by 15N HSQC. An improvement of the
spectrum was observed with increasing lipid concentration from 0 to 1.2 eq, with further lipid addition having no effect on the protein
spectrum. The NMR spectra were recorded on a Bruker AMX 600 spectrometer, operating at 599.94 MHz, equipped with a triple resonance
inverse probe with a field gradient unit on the z axis. All
of the data processing was performed with the version 4 of the
Gifa software (42).
On the 15N-labeled sample, a three-dimensional HSQC-TOCSY
(45-ms mixing time, 80-h acquisition 305.2 K), a three-dimensional HSQC-NOESY (200-ms mixing time, 64-h acquisition 305.2 K), and a
three-dimensional HSQC-NOESY (200-ms mixing time, 82-h acquisition, 295.2 K) were performed. Three-dimensional experiments were processed by linear prediction along the proton and 15N indirect axes
to obtain at least 256 complex points along each axis. Two
two-dimensional NOESY (200-ms mixing time, 18.5-h acquisition, 295.2 and 305.2 K) were also performed on this sample.
On the unlabeled sample, COSY, NOESY (200-ms mixing time), and TOCSY
experiments were performed at the temperatures of 295.2, 310, and 323 K. Additional experiments were also performed in pure D2O.
Three NOESY experiments (
µ = 50, 100, and 200 ms) and
a COSY experiment were performed on the unlabeled protein liganded with
a perdeuterated dodecylphosphocholine lipid (DPC) as ligand and
confirmed the assignment. Deuterium exchange experiments were performed
by following the evolution of the TOCSY spectrum on a 3.3 mM sample in D2O (60-ms mixing time, pH 3.3, 1 eq of LPG). To follow the most labile amide protons, the lyophilized protein was dissolved in D2O at a temperature of 273 K. The
exchange was then monitored by several experiments, slowly raising the temperature. The first spectrum was acquired at 273 K, starting directly after mixture, and a second one was acquired at the same temperature starting 3.5 h after the mixture. Then several spectra were acquired while raising the temperature to follow less labile protons: at 295.2 K 7 h after initial mixture and at 310 K 21 h after mixture. The sample was then left at room temperature for 4 days, and a final experiment was acquired at 295.2 K. A natural
abundance 13C HSQC experiment was performed in
D2O to confirm methylene assignment. Rotating frame
NOE-edited spectroscopy experiments were performed at 295.2 K (50 and
100 ms) on the unlabeled protein liganded with LPG and with DPC.
Diffusion ordered spectroscopy (43) was performed at 295.2 K (diffusion
duration, 200 ms; gradient duration, 1.5 ms, with varying intensities
from 0.5 to 47 G/cm, 1-h acquisition). Diffusion ordered spectroscopy
experiments were performed on the unlabeled sample at 290 K, and the
stimulated echo-longitudinal eddy current delay sequence (44, 45) was
used with a WATERGATE filter applied for water suppression. A set of
small monomeric globular proteins was measured under the same
conditions to determine the molecular mass
calibration.2 Several
31P NMR experiments were performed at 295.2 K on a AMX 400 Bruker spectrometer on a 2 mM sample (pH 3.5, 80 mM NaH2PO4 with 2.5 mM
of LPG). Two 31P exchange spectroscopy two-dimensional
experiments (mixing time of 0.2 and 2 s, respectively) did not
present any evidence of a bound/free equilibrium. All of the NMR data
sets have been deposited in the NMRb data bank
(nmrb.cbs.cnrs.fr; NMRb number ltpt2a).
NMR Spectra Assignment--
The assignment of the wheat ns-LTP2
complexed with the phospholipid was performed from the set of
three-dimensional (HSQC-NOESY and HSQC-TOCSY) and two-dimensional
experiments (COSY, TOCSY, and NOESY) using the sequential assignment
strategy (47) with the help of the Rescue software for the amino acid
typing step (48). The assignment module of the Gifa program
was used for this purpose (49).
In the HSQC experiment, all of the amide peaks could be found. All of
the side chain labile hydrogens from Lys, Asn, Arg, and Gln residues
were also assigned. Additional peaks belonging to the glycosylated
proteins were sorted out in the HSQC spectrum from their reduced
intensity. On the basis of sequential dNN(i,i+1) and d
N(i,i+1) all of the amino acids have been
found, and 98% of all the nonlabile 1H chemical shifts
have been assigned. The natural abundance 13C HSQC spectrum
was used to help the assignment of side chain methylenes.
Stereospecific assignment of
protons of methylene groups was
performed by examination of the NOE intensity and coupling constant patterns.
Under the experimental conditions and because of the blurring of the
NOE spectra caused by the exchange cross-peaks, the phospholipid signal
could not be unambiguously assigned, except for the C16-terminal methyl
protons. The complete assignment of the 1H and
15N chemical shifts of the wheat ns-LTP2 complexed with LPG
has been deposited in the BioMagResBank (number 4977).
NMR Structure Calculation and Analysis--
The two-dimensional
and three-dimensional NOESY experiments, performed at three different
temperatures (295.2, 310, and 323 K), have been used to extract all of
the distance constraints used for the structure reconstruction. Peak
intensities were calibrated against a set of reference peaks, using the
standard tools provided with the Gifa program (49).
Intensity levels were analyzed with a 1/r6 law,
and distance constraints were obtained from the intensities by
classifying in long, medium, and short range distances; pseudo-atom corrections were used. Additional constraints were obtained on the
angle by measuring 3J NH-H
couplings constants using the
Ludvigsen method (50) and on the
1 angle by considering
the NOESY peak intensity between HN,H
spins and
H
1,H
2 spins, when a stereospecific
assignment was available.
The dynamical annealing protocol (anneal.inp) of the
crystallography NMR (CNS) software (51) was used to generate the
protein structure from the set of constraints. A first set of
structures was obtained from the constraint list, without any
constraint on the cysteine linkage and with no phospholipid in the
topology. From this set of structures, the disulfide linkage could be
unambiguously assigned, because all of the sulfur atoms were located in
compatible distances. The observed disulfide bridges are:
Cys-25/Cys-60, Cys-10/Cys-24, Cys-2/Cys-34, and
Cys-36/Cys-67. H
-H
NOE contacts were further observed in the
NOESY map, which confirmed this assignment.
The model of the liganded protein was constructed from the same set of
constraints. Additional constraints were used, corresponding to the
observed connectivity of the C16 terminal CH3 of the fatty acid chain
with H
and H
of Tyr-44 and Tyr-47 and the middle of the lipidic
chain (C8) with H
of Phe-35 with a long distance constraint. No
additional constraint was used to force the exit of the phospholipid
from the protein core. The distance constraints corresponding to the
S-S bounds were then added to the constraint list. The dynamical
annealing protocol was used for the calculation of 250 structures, from
which the 10 structures with the lowest global energy were conserved.
The obtained set of structures was deposited to the Protein Data Bank
(code 1N89).
The Procheck (52) program was used to check the quality of the obtained
structures, as well as to compute the Ramachandran maps. The Voidoo
(53-55) was used to compute the volume of the inner cavity,
 |
RESULTS AND DISCUSSION |
Production and Purification--
The recombinant protein
preparations were obtained as previously described (34-36). The
N-terminal sequence (ACQASQLAVC) of the recombinant ns-LTP2, determined
by mass spectroscopy, is identical to those of the wheat-purified
ns-LTP2 (56), indicating that the recombinant protein was correctly
processed by the P. pastoris KEX2 protease.
The mass spectroscopy was performed on the 15N-labeled
protein. A 7055-Da average molecular mass was measured, confirming a 15N isotopic labeling over 95%. The mass spectrum also
revealed that approximately 15% of the protein has been glycosylated
with one to four C6 sugar moieties, assumed to be mannose (162 Da). This is consistent with the fact that P. pastoris adds
O-oligosaccharides composed solely of mannose residues.
Peptide mass fingerprinting of the digested protein revealed that only
the first tryptic fragment at the N terminus of the protein (fragment
1-29) is glycosylated.
N-Glycosylation requires the Asn-Xaa-Ser/Thr consensus
sequence, whereas O-glycosylation requires the presence of a
Ser or Thr residue. The wheat ns-LTP2 does not contain the
Asn-Xaa-Ser/Thr sequence in its first 29 residues; however, it does
contain four serine residues (at positions
5, 12, 16, and 21) and one
threonine residue (at position 27), residues that are accessible for
possible glycosylation. The presence of a proline residue in the
vicinity of a serine or threonine residue can enhance
O-mannosylation (57). Of the four serine residues available
for O-linkage, residue 21 is the only one close to a proline
residue. All of these results indicated that a small fraction of the
wheat ns-LTP2 is expressed as a O-glycosylated protein. To
our knowledge, no glycosylation has been reported for ns-LTP purified
from plant.
Physicochemical Context Screening and Ligand Choice--
Intensive
screening of the physicochemical solution conditions was performed to
find a set of conditions that provides NMR spectra of good quality.
Ligand nature and ionic strength conditions were found to be critical.
Fluorescence screening experiments were performed on a series of
phospholipids and fatty acids, highlighting a higher affinity for
negatively charged phospholipids. We finally selected LPG with an
average chain length (C16). The titration of ns-LTP2 by LPG, monitored
by NMR, shows that the ns-LTP2 becomes more structurally constrained in
the presence of this ligand. A narrowing and a spreading out of the
peaks are observed on the two-dimensional HSQC spectrum. This evolution
is stabilized around 1 eq of LPG. The addition of 70 mM
phosphate buffer was observed to also improve spectral quality (Fig.
2). On the other hand, we found that the protein could adapt a large range of pH (3.45-6.5) and temperatures (273 K to 323 K) without important modification of the HSQC spectra.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
HSQC spectrum of 15N-labeled
recombinant wheat ns-LTP2. The spectrum has been recorded at 295.2 K on a Bruker AMX 600 on a 2.8 mM sample complexed with 4.2 mM of L- -palmitoylphosphatidylglycerol in 70 mM phosphate buffer at pH 3.5. The assignment is indicated
by numbering the peaks with the identity of each residue. Unassigned
peaks are indicated with question marks.
|
|
Under these conditions, a diffusion ordered spectroscopy experiment was
used to determine the translational diffusion coefficient of the
protein in the experimental conditions, found at 117 µm2/s. Based on
a calibration performed on a series of small globular proteins measured
in the same conditions, this value confirms that the sample is
monomeric. This result is in good agreement with the relaxation study,
which has determined a rotational correlation time compatible with a
monomeric form (58).
Under these optimized conditions, the 1H-15N
HSQC spectrum of the 15N-labeled ns-LTP2 protein liganded
with LPG displays more peaks than expected, considering the number of
residues. A second step of purification designed to remove the
glycosylated proteins was performed on the sample, and a
1H-15N HSQC was recorded. This spectrum
displays a reduced number of peaks, but the peaks are still too
numerous (labeled with question marks in Fig. 2).
One-dimensional and exchange spectroscopy two-dimensional
31P spectra were recorded on this sample. Two phosphorous
lines can be observed in the one-dimensional spectrum, corresponding to the bound phospholipid and to the slight excess of free phospholipid in
solution. No additional peaks nor any exchange peak could be observed
in the exchange spectroscopy spectra, even at very long mixing time. To
observe eventual chemical exchange, a two-dimensional 1H
rotating frame NOE-edited spectroscopy experiment was performed on the
LPG-liganded protein. In this spectrum a strong exchange peak is
visible in the H
region, as well as several weaker peaks close to
the diagonal in the aliphatic and amide regions. The same peaks are
observed when the LPG ligand is replaced with a fully deuterated DPC
phospholipid. An exchange between the holo and the apo states of the
protein being excluded by the 31P experiments, this is an
indication that a conformational equilibrium between a major and a
minor form of the protein is taking place. This equilibrium is the
source of the additional peaks observed in the HSQC spectra. It was
taken into account during the assignment phase, and several peaks were
assigned to the minor form. However, no effort was done to fully assign
this minor form.
Assignments and Secondary Structure Elements--
The assignment
of the protein resonances was performed from the set of
15N-edited NOESY and TOCSY spectra. The sequential strategy
was used, aided by the 15N and natural abundance
13C HSQC spectra (Fig.
3).
The solvent protection experiments indicate that the secondary
structure is quite strongly established, because about 40% of the
amide protons remain unexchanged during the first hours of the
D2O exchange experiment at 1 °C, and eight remain
unexchanged after 5 days at room temperature. The solvent protection
patterns, as well as J-coupling and NOESY patterns, are indicative of a structure mostly helical. On the other hand, the secondary structure prediction program Jpred (59) anticipates two helical zones, ranging
from residues 22 to 29 and from residues 33 to 39.
Chemical assays as well as mass spectroscopy have shown that all eight
cysteines of the protein are engaged in disulfide bridges. We have not
been able to unambiguously assign the H
-H
NOE contacts characteristic of this structure because of the crowding of this spectral region. However, after the first run of structure generation, all of the obtained structures exhibited side chain proximities permitting the disulfide skeleton based on these prestructures to be
assigned. The disulfide bridges thus found are: Cys-25/Cys-60, Cys-10/Cys-24, Cys-2/Cys-34, and Cys-36/Cys-67. This is in agreement with the chemically determined assignment (32).
The 1H assignments of the LPG in the complexed form were
obtained from two-dimensional COSY homonuclear experiments on the free
LPG and by comparing the homonuclear spectra obtained from the
LPG-ns-LTP2 complex and from the DPC-ns-LTP2 complex. A few chemical shifts of the fatty acid chain were clearly identified: the
terminal methyl group (C16), its vicinal methylene (C15), as well as
the proximal methylene (C2); the glycerol moiety attached to the fatty
acid chains was also assigned. Some intermolecular NOE contacts were observed.
Structure Determination--
From the complete set of geometric
constraints extracted from the NMR spectra, a set of 10 structures has
been obtained. They do not display any important constraint violation,
and all of the residues are localized in the allowed regions of the
Ramachandran plot. The ensemble of the10 best structures present a root
mean square deviation computed on residues 2-67 of 0.9 Å for all of the heavy atoms and a root mean square deviation of 0.67 Å for the
backbone atoms. All of the statistics of the geometrical constraints and the structure reconstruction are given in Table
I. The set of solution structures of
ns-LTP2 liganded with LPG as obtained from this experimental work is
presented in Fig. 4.
View this table:
[in this window]
[in a new window]
|
Table I
NMR restraints and structural statistics of the ensemble calculated for
LPG-liganded wheat ns-LTP2 (10 structures)
|
|

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 4.
a, stereo view of the best 10 NMR
structures of wheat ns-LTP2. The phospholipid is not shown for clarity.
b, mean NMR structure, with the ensemble of the phospholipid
structure as observed in the 10 best protein generated structures. The
orientation is rotated by 90° along the OX axis with respect
to panel a. Basic residues around the phosphate group are
marked in green. c, schematic view of the
structure of ns-LTP2 with a CPK model of the liganded phospholipid. The
orientation is the same as in panel b.
|
|
The protein is observed as a globular protein with a diameter of about
30 Å. The structure is composed of five helices arranged in a
superhelix tertiary structure. Helix 1 is a 3-10 helix, encompassing residues 7-15. All of the other helices are
-helices. Helix 2 includes residues 22-31, helix 3 includes residues 34-40, helix 4 includes residues 44-49, and helix 5 includes residues 51-60. The
overall fold is a right superhelix. The localization of the helices is
in good agreement with the proton exchange experiment, which has shown
that there are five main zones in which the amide protons are protected
against solvent exchange: residues 14 and 15, residues 26-31, residues
38-41, residues 47-50, and residues 56-64. The LPG molecule
is found partly embedded in the structure of the protein, with the
superhelix structure of the protein coiled around the fatty acid chain,
and with the phosphate group and the external glycerol moiety
unstructured and located outside of the core of the protein.
Helices 1 and 2 are organized in a near anti-parallel conformation. The
contact between helices 1 and 2 is tightened by the Cys-10/Cys-24
disulfide bridge. They are linked by an extended strand from Ser-16 to
Gly-22. Lys-19 appears to be oriented toward the solvent and does not
display any NOE contact with other residue. It should be noted that in
a previous dynamic study, it has been observed that the H-N vector of
Lys-19 is highly mobile in the ns range (58). Helices 3-5 form a
square configuration. Helix 3 contains a characteristic Cys-34, Phe-35,
Cys-36 pattern, with Phe-35 buried into the structure and contributing
to the hydrophobic core of the structure. Cys-34 and Cys-36 are
respectively engaged in disulfide bridges with Cys-2 and Cys-67,
forming two diametrically opposed bonds relative to the helix axis. All
of the prolines are observed in trans conformation, as confirmed by the
H
(i)-H
-Pro(i + 1) contacts observed in
the NOESY spectra, for all of the proline residues (Pro-20, Pro-42,
Pro-51, and Pro-65).
Several additional secondary structure elements can be observed in the
structure. The C-terminal of helix 1 presents an unusual hydrogen bond
pattern, with the carboxyl moiety of Ser-12 being engaged with the HN
of Ala-18. This structure is found in the 10 structures generated, and
the HN of residue Ala-18 is found to be protected against solvent
exchange, thus confirming this result. A type-1
turn is observed
between helices 2 and 3, corresponding to residues Gln-31, Gln-32, and
Gly-33. A type-1
turn is observed between helices 3 and 4, corresponding to residues Asp-41, Pro-42, Thr-43, and Tyr-44, with the
acidic head of Asp-41 engaged in a hydrogen bond with the HN of Thr-43.
Three classic helix cappings can also be observed: the N-capping
of helix 2 with the OH of Ser-21 hydrogen-bonded to the HN of Glu-23;
the C-capping of helix 2 with the side chain of Gln-32 bonded to the CO
of Arg-29; and the N-capping of helix 5, with the OH of Ser-50 bonded
to the HN of His-52. Finally, a transient slat bridge between the amide moiety of Lys-40 and the C terminus of the backbone can be observed in
several structures of the NMR ensemble.
The protein presents an inner cavity, which has been measured at 341 Å3. All of the helices present hydrophobic side chains
directed toward the cavity. The phospholipid is found in this cavity.
Only one unique phospholipid position is observed in the cavity for all
10 retained structures. The fatty acid chain is completely embedded in
the protein structure. Its axis is aligned with the axis of the
tertiary superhelix and is orthogonal to the
-helix axes (Fig.
4c). The terminal methyl group is positioned between the H1
and H4 helices. The fatty acid chain is inserted in the cavity
constituted by the hydrophobic residues (Leu-7, Leu-28, Phe-35, Tyr-38,
Tyr-44, Tyr-47, Ile-48, Ala-53, Leu-57, Val-64, and Pro-65). The chain
presents a turn near carbon 8 and exits the cavity in a cleft between
helices H4 and H5 and the C-terminal residue. The inner glycerol moiety
is located on a pocket on the surface of the protein. This pocket
presents a basic environment constituted by the Arg-49, Arg-54, and
His-66 side chains. These basic residues are observed in close
proximity with the phosphate moiety, equilibrating the phosphate charge
(Fig. 5). The cavity of ns-LTP2 is
asymmetric. The proximal entrance of the cavity, where the phosphate
group is found, presents several hydrophilic and basic groups: Arg-49,
Arg-54, Thr-58, and His 66. The distal opening of the cavity is
characterized with hydrophobic residues, such as Leu-7, Tyr-38, Tyr-44,
and Tyr-47.

View larger version (96K):
[in this window]
[in a new window]
|
Fig. 5.
CPK representation of the ns-LTP2
structure. Positively charged residues are colored blue
and negatively charged residues are colored red; the
phospholipid is represented in licorice mode. It can be seen how the
terminal glycerol group sticks out of the molecular surface and how the
basic residues surround the phosphate group (in yellow). The
orientation is similar to that in Fig. 4a.
|
|
This location is in good agreement with the following spectroscopic
observations: In the fatty acid chain only three unambiguous NOE
intermolecular contacts have been identified. Four NOE contacts connect
the LPG-terminal methyl groups (C16) with the Tyr-44 and Tyr-47
aromatic part. The other NOE connects the one of the methylenes of the
fatty acid chain with the H
of Phe-35. All of these residues are
found in the hydrophobic cavity. No NOE contacts were found between the
protein and LPG glycerol moiety. The chemical shifts of the
phospholipid glycerol moiety do not present much shift upon
complexation with the protein. On the other hand, the fatty acyl chain
chemical shift differences between isolated and liganded LPG are important.
The structure of the minor form present in solution has not been
studied, even though it appears to be structured. About one-third of
the amino acids seem to be involved in the conformation equilibrium; no
attempt was made to assign the residues involved in this equilibrium. Previous dynamic study (58) has shown that the major form is predominantly rigid, with S2 ranging from 0.8 to
0.9, except for Lys-19, exposed to the solvent.
ns-LTP2 versus ns-LTP1 Comparison--
The three-dimensional
structure is known for several ns-LTP1 from different species (19-29).
They are very similar among plant and consist in four
-helices
organized in a superhelix structure and connected by four disulfide
bridges. We compared the structure we present here with wheat ns-LTP1
(Protein Data Bank code 1gh1) (19) using the Visual
Molecular Dynamics software (46) (Fig. 6).

View larger version (62K):
[in this window]
[in a new window]
|
Fig. 6.
Structural comparison of wheat ns-LTP2
(red) with wheat ns-LTP1 (Protein Data Bank code 1gh1)
(blue). Orientation is the same as in Fig.
4a, only ns-LTP2 C-terminal and N-terminal are
labeled. Superposition of helices 1, 2, and 5 (ns-LPT2
numbering) with equivalent helices in ns-LTP1 can clearly been
observed. Helices 3 and 4 with no equivalent in ns-LTP1 are also
observed in front.
|
|
The best superposition is obtained when the H1, H2, and H5 (residues
3-16, 22-32, and 50-63) helices in ns-LTP2 are superimposed with the
H1, H2, and H4 helices (residues 5-8,10-19, 23-35, and 63-76) in
ns-LTP1. This gives a root mean square deviation of 1.88 Å (backbone
only). The global structure is similar and consists in an hydrophobic
cavity structure adapted to the lipid transport. Nevertheless, large
three-dimensional structural differences between ns-LTP1 and ns-LTP2
are observed in two regions: the H3 and H4 (respectively H3)
-helices and the C-terminal region. The last turn of the H2 helix is
also different in both structures.
The two short H3 and H4
-helices orthogonal to each other in ns-LTP2
are replaced by the longer H3 helix in ns-LTP1. ns-LTP1 H3 helix is
oriented differently and flanked by two large loops presenting no
regular secondary structure. This structural difference opens up the
cleft corresponding to the entrance of the cavity in which the ligand
is located. The C terminus of ns-LTP1 presents a large loop with no
secondary structure. The corresponding zone is deleted in ns-LTP2, thus
releasing access to the hydrophobic cavity. This deletion has globally
no impact on the position of the disulfide bridge involving the last
cysteine (Cys-67), which is conserved. As a consequence of these
structural changes, the wheat ns-LTP2 is smaller and more globular than
ns-LTP1.
All of the cysteine residues are involved in disulfide bridges, but the
wheat ns-LTP2 differs from ns-LTP1 in the way the connections are made
between the cysteines. ns-LTP1s exhibit a conserved CXC motif
located in helix 3, with X being an hydrophilic residue. The two
cysteines flanking this hydrophilic residue are linked to cysteines at
distal positions in a crossed scheme (as showed in Fig. 1). In the
wheat ns-LTP2, an hydrophobic residue (Phe-35) is present, and the two
flanking cysteines are involved in disulfide bridges in a noncrossed
scheme. The hydrophobic side chain of Phe-35 is displayed on the cavity
surface of the wheat ns-LTP2, whereas in ns-LTP1 the corresponding
hydrophilic residue is exposed to the solvent, because of a 180°
rotation of the entire helix 3 along its main axis.
This cysteine linkage pattern is not unique to ns-LTP2 and is observed
in the soy bean hydrophobic protein. Searching structural data bases
(40) for a protein presenting a similar structural arrangement to
ns-LTP2, the soy bean hydrophobic protein (Protein Data Bank code 1hyp)
is found as the best match. This protein shares some structural
similarity with ns-LTPs but does not transfer lipids. It presents an
analogous organization in terms of helix number and orientation, as
well as disulfide bridge topology. Helix 3 is involved in a noncrossed
disulfide bridge scheme and appears not to be amphiphilic when compared
with ns-LTP2. This could explain why soy bean hydrophobic protein does
not exhibit lipid transfer capability.
The presence of an hydrophobic cavity is a characteristic of ns-LTPs;
the hydrophobic ligands are bound in this cavity in a rather
nonspecific manner. No major differences have been observed between the
structure of the free and palmitate complexed maize (21) and barley
(25) ns-LTP1 and free and prostaglandin B2-liganded wheat ns-LTP1 (29),
whereas large conformational changes have been seen for barley ns-LTP1
when it complexes with palmitoyl CoA (25). Orientation of the lipid
within the hydrophobic cavity was found to be opposite in maize (21)
and barley (26) liganded structures, whereas wheat ns-LTP1 is able to
bind two monoacylated lipids insert head to tail in the hydrophobic
cavity (28).
Measured volumes of this hydrophobic cavity are highly variable in
ns-LTP1 (23), with typical values ranging from 150 to 580 Å3. However, barley ns-LTP1 exhibits a large volume change
upon palmitoyl-CoA binding, with a measured cavity volume increasing from 39 to 620 Å3. The volume observed for the wheat
ns-LTP2 appears to be in the same range, even though the protein is
smaller by 24 amino acids.
The orientation of the phospholipid main chain, observed in ns-LTP2, is
roughly orthogonal to the
-helix axes, and the chain runs from
helices H1 to H5. This is in contrast with most described ns-LTP1
cavities found with a main axis parallel to the
-helix axes.
Conclusion--
The present work presents the refined structure of
the wheat ns-LTP2 protein, liganded with
L-
-palmitoylphosphatidylglycerol, as determined by NMR
spectroscopy. The protein was observed as being composed of five
helices, structured as a right superhelix, surrounding the
phospholipid. This structure presents some homologies with other lipid
transfer proteins such as ns-LTP1; however, the phospholipid was found
in a quite different location than in most LTP1s. An hydrophobic cavity
was also observed, with a volume equivalent to the one found in ns-LTP1
but with a different geometry.
The ns-LTP2 protein structure also presents homology with the one soy
bean hydrophobic protein that does not exhibit any lipid transfer
activity. This permits us to devise a protein family encompassing
ns-LTP1 and ns-LTP2 but also soy bean hydrophobic protein and other
related vegetal proteins, based on structural homologies, rather than
on the function of primary sequence homologies.
Extensive ligand screening had to be undertaken to find a set of
conditions allowing structural study. This indicates that even if the
ns-LTP2 is able to adapt a large number of hydrophobic ligands in its
hydrophobic pocket, it certainly presents varying affinities
depending on the nature of the ligand. The better affinity for single
chain phospholipid, negatively charged, can be explained a
posteriori, by the size of the pocket and by the patch of
positively charged residues located around its entrance. Further
comparative studies on this protein and on homologous proteins will
have to be undertaken to improve the understanding on the phospholipid binding affinity and of the phospholipid transfer activity.
Finally it remains to be explained how the structural differences
observed in the plant LTP family might be related to the various
in vivo activities putatively assigned to its members. Comparative structural and dynamical studies on several isoforms of
wheat ns-LTPs are currently in progress in our group. This complementary work should permit us to draw stronger links between structural features and biological functions.