From the Laboratoire de Chimie-Physique
Macromoléculaire, UMR 7568 CNRS, ENSIC-INPL, 54000 Nancy, France
and the § Institut de Biologie Moléculaire et
Cellulaire, UPR 9021 CNRS, 67000 Strasbourg, France
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ABSTRACT |
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The antigenic activity of a 19-mer peptide
corresponding to the major antigenic region of foot-and-mouth disease
virus and its retro-enantiomeric analogue was found to be completely
abolished when they were tested in a biosensor system in
trifluoroethanol. This suggests that the folding pattern, which is
The major immunogenic site of foot-and-mouth disease virus
(FMDV),1 which is contained
in the so-called G-H loop comprising amino acid residues 135-158 of
capsid protein VP1 (viral capsid protein 1), is located in a disordered region on the surface of the
particle (1). A single inoculum of a peptide corresponding to this
region usually elicits levels of neutralizing antibodies that protect guinea pigs against a severe challenge with the cognate virus (2). In
some instances, however, although the total reactivity of antibodies
evaluated in immunochemical tests such as enzyme-linked immunosorbent
assay is high, the level of neutralizing antibodies is low. This
important problem has generated numerous studies aimed at enhancing the
immunogenicity of this peptide with the hope of developing a peptide
construct that could be used successfully as a synthetic vaccine (3).
Recently, we have shown that antisera raised in rabbits against
peptides corresponding to the sequence 141-159 of two variants of
serotype A cross-reacted strongly with the corresponding retro-inverso
analogue peptides. Most importantly, the retro-inverso analogues, also
called retro all-D or retro-enantio peptides (4, 5), induced greater
and longer-lasting antibody titers than did the respective parent
peptides (6). Moreover, we showed with the FP variant analogue (which
contains Phe and Pro residues at positions 148 and 153, respectively)
that a single inoculation of the retro-inverso peptide elicited high
levels of neutralizing antibodies that persisted longer than those
induced against the corresponding L-peptide and protected guinea pigs challenged with the cognate virus (7). The enhanced immunogenic reactivities of retro-inverso analogues may result from their higher
resistance to proteolysis in biological fluids (7). However, this
property does not explain why in immunochemical assays, retro-inverso
peptides are often better recognized than the parent peptide by
anti-virus, anti-protein, or anti-peptide antibodies with equilibrium
affinity constants which can be increased by 10-100 in some instances
(7-9).
To better understand the structural basis for antigenic mimicry between
the L-peptide and its retro-inverso analogue, Carver et al.
(10) recently analyzed the L- and retro-inverso peptides 141-159 of
the SL variant (with Ser and Leu residues at positions 148 and 153) by
NMR spectroscopy and restrained molecular modelling. The NMR structures
of the peptides were determined in trifluoroethanol-d2 (TFE-d2) at different temperatures. As expected (because
the retro-inverso and L-peptides share inherently chiral secondary
structure elements) both peptides were found to wind in opposite
directions. A detailed analysis of the results showed the presence of
other different structural features between the peptides that exhibited
only gross structural similarity. Thus, the L- and retro-inverso
peptides maintain an approximately constant backbone conformation
within the conserved RGD triplet (residues 145-147), which represents the principal site for virus attachment. However, the backbone of the
RGD triplet is not equivalent in the L- and retro-inverso peptides. In
the L-peptide, the RGD motif is enclosed in a type IV Peptides--
Two natural (L-peptides) and two retro-inverso
peptides corresponding to the VP1 region 141-159 of FMDV (serotype A,
subtype 12, FP variant) were used in this study. The sequence of the
parent peptide is 141GSGVRGDFGSLAPRVARQL159.
This peptide contains the highly conserved RGD cell attachment site at
residues 145-147. An additional cysteine residue was added at the N
terminus of the peptides to allow their selective conjugation to the
BIAcore sensor chips. CD and NMR studies were performed with peptides
that contained no additional cysteine. The synthesis and purification
of the peptides were described previously (6, 7). The N terminus of the
L-peptides was acetylated, whereas their C terminus was left as -COOH.
With respect to the native sequence, the N terminus
(Gly141) of the retro-inverso peptides was carboxamidated,
and a 2-substituted malonic acid derivative was substituted for the
native C-terminal L residue (7). Because the malonic acid derivative
was introduced as a race mate during the synthesis, two
diastereoisomers were generated. They were separated by HPLC, and only
the most antigenically active diastereoisomer, which was the most
slowly eluted (6), was considered in this study. The purity of the L-
and retro-inverso peptides was greater than 90% as checked by
analytical HPLC. Peptide identity was verified by matrix-assisted laser
desorption/ionization mass spectrometry using a protein time-of-flight
apparatus (Bruker GMBH, Bremen, Germany) and gave the expected results
for all peptides (data not shown). Control experiments were carried out
with the L- and retro-inverso peptides of sequence IRGERA. These
peptides, as well as the anti-IRGERA monoclonal antibody 4x11, were
described previously (8, 9).
Antibodies--
Anti-peptide antibodies used in this study were
described elsewhere (6). They were raised either in guinea pigs
immunized against the L- and retro-inverso peptides covalently
conjugated to keyhole limpet hemocyanin in the presence of aluminum
hydroxide as adjuvant or in rabbits immunized with peptides coupled to
small unilamellar liposomes containing monophosphoryl lipid A as
adjuvant (6).
Assay in TFE--
Changes in the ability of peptides to bind
antibodies in the presence of increasing concentrations of TFE were
measured using the Pharmacia BIAcore biosensor instrument (Pharmacia
Biosensor, AB, Uppsala, Sweden). The conventional immobilization
technique was used to couple the L- and the retro-inverso peptides to
the sensor chips through their N-terminal thiol reactive groups (9). 267 and 185 pg of L- and retro-inverso peptides were respectively immobilized per mm2 (corresponding to 267 and 185 RU).
After the peptide immobilization, the remaining reactive groups on the
sensor surface were deactivated by a pulse with cysteine. The antibody
preparation was then injected at a constant flow rate of 5 µl/min
during 4 min at 25 °C, and the response expressed in RU was
measured. After dissociating the bound antibody from the peptide
surface in the presence of HCl (20 µl; flow rate, 5 µl/min) and
washing, 20 µl of TFE in HEPES buffer (12.5%, v/v) were injected at
a constant flow rate of 5 µl/min during 4 min at 25 °C. The
peptide surface was then washed by injecting 10 µl of HEPES buffer
(without TFE) at a constant flow rate of 5 µl/min for 2 min at
25 °C, and then the antibody preparation in HEPES (20 µl) was
injected as described above, and the RU was measured. The same series
of steps was repeated for each TFE concentration (i.e. 25, 50, 75, and 100%). As a control, between each TFE step the peptide
surface was regenerated (dissociation of the bound antibody) and
washed, and the antibody was injected in HEPES without TFE. By
measuring the RU between each step, we thus checked that the TFE
treatment did not irreversibly alter the peptide surface reactivity.
CD Measurements--
They were performed at room temperature on
a Jasco J-710 spectropolarimeter flushed with nitrogen. Spectra were
recorded in a wavelength interval of 250-190 nm using a 1-mm path
length rectangular cell. Each spectrum was the average of five scans
taken at a scan rate of 50 nm/s with a spectral bandwidth of 1 nm and
was corrected for base line using Jasco software. TFE titration was
carried out by dissolving the lyophilized peptide trifluoroactetate
salts in the appropriate solvent: (i) 0.1 mM sodium
phosphate buffer, pH 5.7, and varying concentrations of TFE or (ii)
100% TFE. Typically, the peptide concentration was 0.2 mM.
The results were expressed as mean residue ellipticity [ NMR Experiments--
For the NMR experiments, 3 mM
peptide was dissolved in 500 µl of phosphate buffer with 10%
D2O, pH 5.7 (noncorrected). All NMR experiments were
recorded at 285 K on a 400 MHz DRX-Bruker spectrometer. The following
homonuclear two-dimensional experiments were recorded:
double-quantum-filtered J-correlated spectroscopy and total
correlation spectroscopy with a mixing time ( Structure Calculation--
Intensitiy of peaks was extracted
from the NOE spectra using XEASY software (12), and the interproton
distances were calculated taking the distance of 1.78 Å between the
two Pro153 Antigenic Activity of the L-peptide and Its Retro-inverso Analogue
in Increasing Concentrations of TFE--
Changes in the ability of
peptides (corresponding to the sequence of the FP variant) to bind
antibodies were measured using a biosensor instrument based on surface
plasmon resonance. The peptides were coupled to the sensor chips
through their N-terminal thiol reactive groups. After immobilization of
peptide and deactivation of any remaining reactive groups on the sensor
surface by a pulse with cysteine, the antibody preparation was injected
at a constant flow rate of 5 µl/min during 4 min at 25 °C, and the
response expressed in RU was measured. After dissociating the bound
antibody from the peptide surface in the presence of HCl and washing,
TFE in HEPES buffer (12.5%, v/v) was injected at a constant flow rate of 5 µl/min during 4 min at 25 °C. The peptide surface was then washed by injecting HEPES buffer (without TFE) at a constant flow rate
of 5 µl/min during 2 min at 25 °C; the antibody preparation in
HEPES (20 µl) was then injected as described above, and the RU was
measured. The same series of steps was repeated for each TFE
concentration (i.e. 25, 50, 75, and 100%). Control
experiments performed to check that TFE treatment did not irreversibly
alter the peptide surface reactivity are described under "Materials and Methods." This protocol allowed us to study the effect of TFE on
the antigenicity of the peptides without affecting the reactivity of
antibodies that remained in the standard HEPES buffer, pH 7.4.
The data presented in Fig. 1 illustrate
the reactivity of guinea pig and rabbit antibodies raised against the
parent and retro-inverso peptides 141-159 with the L-peptide and its
retroenantiomer as a function of TFE concentration. A slight decrease
in antibody binding was observed when the peptides were in 12.5% TFE,
and a sharp drop of binding was found when peptides were in 25-50% TFE. In general, guinea pig (Fig. 1, a and b) and
rabbit (Fig. 1, c and d) antibodies did not react
or reacted very weakly with peptides in 75 and 100% TFE. A control
experiment with the hexapeptide of sequence IRGERA, known to remain
unordered in 100% TFE (15), was performed in parallel. Using a
monoclonal antibody induced against the L-hexapeptide (9) and the
protocol described above, no change in antibody binding was found when
the parent and retro-inverso IRGERA peptides were in HEPES or in HEPES
containing 12.5-100% TFE (Fig. 1e). These results indicate
that our procedure does not affect the reactivity of the antibody
itself and that changes in antibody binding are only observed when the
structure of the peptide becomes an
Fig. 2 shows that when the parent and
retro-inverso FMDV peptides were in 50% TFE, the time required to
restore the structure recognized by antibodies was between 5 and 7 min
in HEPES buffer at a constant flow rate of 5 µl/min. It is possible
that when the dextran-linked parent and retro-inverso FMDV peptides are exposed to TFE, due to the local peptide density on the dextran matrix
and the tendency of this FMDV peptide to form an amphipathic
In summary, these results indicate very clearly that antibodies from
immunized animals do not bind the L- and retro-inverso peptides
141-149 when they adopt an Circular Dichroism Analysis--
The conformational features of
both the L- and the retro-inverso peptides were investigated in
solution by CD spectroscopy. An overlay of the CD spectra of the L- and
retro-inverso peptides (0.2 mM) in 0.1 M
phosphate buffer solution at pH 5.7 and in TFE is shown in Fig.
3a. As expected, both peptides
exhibited almost symmetrical CD curves in phosphate buffer solution as
well as in TFE. In aqueous solution, the spectra showed only a strong Cotton effect at 197 nm ([
The results presented above strongly suggest that the folding pattern
in the NMR and Molecular Modeling Analysis--
NMR data for both L- and
retro-inverso peptides were collected on a DRX-400 spectrometer
(Bruker) in aqueous solution (phosphate buffer, pH 5.7). A summary of
NOE connectivities observed in the NMR spectra used for structure
calculations of both peptides is shown in Fig.
4. The parent peptide 141-159 was found
to be very flexible, and the NMR data then corresponded to a
conformational averaging. However, the N and C termini exhibited
different behavior that were analyzed using the program PROMOTIF (23).
It was found that the N-terminal region (residues 141-149) was very
flexible and contained no particular local structural motif. On the
contrary, the C-terminal part (residues 150-159) appeared to be more
rigid, with two distorted
Pegna et al. (21) have reported the NMR structures of two
VP1 peptides 141-160 (variants FP and SL) in TFE. Both peptides exhibited a helical motif from residues 151-158 and a loop
encompassing the RGD motif (residues 145-147), whereas our results
obtained in aqueous solution are rather in favor of two
The retro-inverso peptide 141-159 was subjected to the same NMR study
in aqueous solution. Again, no permanent hydrogen bond was observed,
but the analysis of the torsional angles and above all the
C Final Comments--
Very few studies describing the structures of
a peptide and its retro-enantiomer have been reported so far (10, 24,
25). It has been generally observed that the structures of L-peptides and their respective retro-inverso analogues are similar but not identical. The best topochemical similarity was achieved with a cyclo
hairpin peptide and its retroenantiomer (24). Likewise, in the present
study, only the C-terminal regions of the FMDV peptides, covering the
residues 156-159, could be superposed (Fig. 7). Although the CO-NH bonds are in
opposite directions, the peptide backbones in this region assume
closely similar conformations, and the side chains adopt the same
orientation. This similarity may explain in part the cross-reaction of
antibodies with these two peptides. Another important finding is that
the retro-inverso analogue appears to be significantly more rigid than
the parent peptide in aqueous solution, particularly in the region
145-154, which is unstructured and mobile in the parent peptide (Fig.
5). This feature supports our previous observation that the
retro-inverso peptide 141-159 is better recognized than the parent
peptide by anti-VP1 and anti-virus antibodies (6, 7). On the other hand, it has been reported that -helix in trifluoroethanol (confirmed by CD measurement), does not
correspond to the biologically relevant conformation(s) recognized by
antibodies. The NMR structures of both peptides were thus determined in
aqueous solution. These studies showed that the two peptides exhibit
similar folding features, particularly in their C termini. This may
explain in part the cross-reactive properties of the two peptides in
aqueous solution. However, the retro-inverso analogue appears to be
more rigid than the parent peptide and contains five atypical
-turns. This feature may explain why retro-inverso foot-and-mouth
disease virus peptides are often better recognized than the parent
peptide by anti-virion antibodies.
INTRODUCTION
Top
Abstract
Introduction
References
-turn, whereas
in the retro-inverso analogue it is part of a loose left-handed helix.
Conformational variability is found about glycine 149 in both families
of structures. Finally, a helical region encompasses residues 152-159
of the L-peptide and residues 150-159 of the retro-inverso analogue.
In both cases, the helix is amphipathic but as indicated above, it is
right-handed in the parent peptide and left-handed in the retro-inverso
peptide analogue. Thus, a number of different structural features
described for the L- and retro-inverso peptide 141-159 are
particularly significant, and this observation does not support the
fact that the retro-inverso peptide displays similar or superior
cross-reactive antigenic activity compared with the L-peptide with
regard to anti-virus or anti-protein antibodies. As suggested by Carver
et al. (10), this discrepancy could be explained by assuming
that the structures of the peptides in TFE do not correspond to those
assumed by each peptide in aqueous solutions used to test their
antigenic reactivity or in vivo when their immunogenic
activity is investigated. In an attempt to rationalize our previous
biological findings and explain why the retro-inverso analogue mimics
the antigenic activity of the parent peptide, we decided to test
whether anti-virus and anti-peptide antibodies effectively react with
the peptides in TFE and determine the preferred conformers of the L-
and retro-inverso peptides in aqueous solution.
MATERIALS AND METHODS
] having
the units degrees·cm2·dmol
1 according to
the relation [
] =
/(100lcN), where
is the measured ellipticity, l is the
optical path length, c is the peptide concentration, and
N is the number of amino acid residues in the sequence.
m) of 70 ms
and NOE spectroscopy 32 with mixing times from 100 to 400 ms. Water
suppression was achieved using the WATERGATE sequence (11). Spectra
were processed on Silicon Graphics Indy workstation using the XWINNMR
1.2 software (Bruker GMBH). Distance restraints were derived from
cross-peak intensities of the NOE spectroscopy spectra with
m = 200 ms.
H protons as a reference. Distance restraints
were assigned as strong, medium, and weak and set at intervals of
1.8-2.5, 2.5-3.5, and 3.5-5.5 Å, respectively.
dihedral angles
were restrained within the
170,
10] and 10, 170 intervals for the
parent and retro-inverso peptides, respectively. DYANA-1.4 software
(13) was used to run the structure calculations: sets of 50 structures were initially calculated based on 37 and 41 inter-residues distance restraints for parent and retro-inverso peptides, respectively, following the torsion angle dynamics procedure. Refinement with restrained minimization was then performed on the structures having the
lowest energies, i.e. structures with target function
typically below 1. We had to introduce D-amino acids in the
DYANA library, changing signs of the z coordinates for all
H
and side chain atoms. The 2-isobutyl malonic acid
residue was also introduced in the library, from the Leu residue, which
was modified using the MOLMOL-2.5.1 software (14). Two series of simulations were run with R and S conformers of
2-isobutyl malonic acid. Final refinement was run only on the
S-2-isobutyl malonic acid because it led to less restraint violations.
RESULTS AND DISCUSSION
-helix in the presence of TFE.
Interestingly, in another study (16), it was shown in enzyme-linked
immunosorbent assay that when the native structure of peptides is an
-helix, certain monoclonal antibodies react better with the peptides
dried from TFE onto the solid phase than with the same peptides
adsorbed in aqueous solution.
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Fig. 1.
Antigenic activity of the parent and
retro-inverso (RI) peptide in TFE measured using the
biosensor instrument BIAcore. The successive steps of the assay
and the controls are described under "Materials and Methods." The
results reflect the effect of TFE on the antigenicity of peptides. The
antibodies used as probes were not in contact with the TFE solution
during the test. The peptides tested are described in the figure. The
antibodies tested were from guinea pig antisera (diluted 1:50) raised
against the L-peptide 141-159 (a) and RI-peptide 141-159
(b) and from rabbit antisera (diluted 1:20) raised against
the L-peptide 141-159 (c) and RI-peptide 141-159
(d). The mouse monoclonal antibody 4x11 directed against the
parent peptide IRGERA was tested as a control with the L- and
retro-inverso peptides IRGERA (e). The results are expressed
in RU.
-helix
in TFE (10), helix bundle structures are formed, and a strong secondary
structure stabilizing effect is exerted, explaining that several
minutes (and not milliseconds) are required to induce the folding to
unfolding transition in aqueous solution. Such a stabilizing effect has
been described by Mutter et al. (17) with template-assembled
synthetic peptides involving helical peptides.
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Fig. 2.
Restoration with time of the initial
peptide-antibody binding after peptide treatment with TFE. The
time needed to restore the initial level of antibody binding (0%
TFE) after incubating the immobilized L- and retro-inverso
peptides in the presence of TFE/HEPES (v/v; 4 min at a flow rate of 5 µl/min at 25 °C), and washing with a constant flow of TFE-free
HEPES (5 µl/min at 25 °C) was between 5 and 7 min for the L
(black bars) and retro-inverso (hatched bars)
peptides. The antibodies tested were from a guinea pig immunized
against the L-peptide 141-159.
-helical conformation. Furthermore the
antibody reactivity was completely restored after a few minutes (>5)
when the sensor chip-immobilized peptides were placed in a TFE-free buffer.
] =
1.08 × 104 and
1.04 × 104 for the L- and the retro-inverso peptides,
respectively). Although such a CD signature is often assigned to a
random-coil structure (18, 19), Siligardi et al. (20) argued
for an equilibrium between a limited number of secondary structures
that may include a left-handed extended helical conformation. In TFE,
both the isomeric L- and retro-inverso peptides presented the typical
pattern that can be assigned to the
-helical conformation, with a
trough at 222 nm and an extremum around 205 nm. However, in the case of
the retro-inverso peptide, the intensity of the Cotton effect at 222 nm
was stronger ([
] = +6.20 × 103 versus
4.49 × 103 calculated for the L-peptide). This
result is in good agreement with previous NMR investigations performed
in TFE-d2 on a related antigenic peptide (variant SL),
which revealed that the helical region present in the C-terminal half
(with respect to the parent sequence) of both peptides is more defined
in the retro-inverso isomer (10, 21). To further characterize the
structural behavior of the retro-inverso isomer in solution, an
H2O/TFE solvent titration was performed at room
temperature. The presence of a plateau between 25 and 75% TFE, clearly
deduced from the plot of
at 222 nm versus TFE
concentration (Fig. 3b), is indicative of a
folding-unfolding pathway between three stable conformational
components. It is worth noting that two plateaux and four
conformational states were observed by Siligardi et al. (20)
in TFE titration of the related L-peptide encompassing residues
141-160 (variant SL) with a free N terminus. From our data, it appears
that the retro-inverso isomer described in the present study shares
similar conformational features with the L-peptides corresponding to
variants SL and LP studied by Siligardi et al., which also
involved a three-state process and a higher helical content in 100%
TFE compared with the FP L-peptide (20, 22). However, during TFE
titration of the retro-inverso peptide, Siligardi et al.
observed two isodichroic points at 197 and 203 nm, which were not found
in our case. The finding that some major conformational changes already
occur when both the L- and retro-inverso peptides are in 5-25% TFE is
in good agreement with the drop in antibody binding observed when the
peptides were studied in this range of TFE concentration in mixed TFE
buffer solutions (Fig. 1).
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Fig. 3.
Overlay of the CD spectra of the L- and
retro-inverso peptides in 0.1 M phosphate buffer at pH 5.7 and in TFE. Mean residue ellipticity [ ] in
degrees·cm2·dmol
1 (a). Plot of
[
] (absolute values) at 222 nm versus the TFE
concentration (b).
-helix observed in TFE for both the L- and retro-inverso peptides does not in fact correspond to the biologically relevant conformation(s) recognized by antibodies. It was thus decided to
re-examine by NMR the structures of the L- and retro-inverso peptides
in aqueous solution and in the absence of TFE.
-turns, although no permanent hydrogen
bond was observed. The first
-turn involves residues 155-158 and is present in 81% of the structures obtained from the final run of DYANA
calculations (Fig. 5). The second one
involves residues 156-159 in 41% of the structures. Table
I summarizes angle values and
C
(i)
C
(i+3)
distances observed for these turns, and a superposition of the 10 structures with the best target functions is shown in Fig.
6a. The optimal fit of the
backbone atoms corresponds to the sequence 150-158 and yields to a
root mean square difference (r.m.s.d.) of 0.96 Å, whereas the 144-148 region (containing the RGD motif) gives an r.m.s.d. of 1.2 Å.
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Fig. 4.
Summary of nonambiguous observed
connectivities for the parent (a) and retro-inverso peptide
(b). For conventional reasons the numbering of
residues was maintained in L- and retro-inverso peptides regardless the
orientation of the peptide bonds. The strong, medium, or weak
intensities of the NOE cross-peaks are indicated by the
thickness of the lines.
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Fig. 5.
-Turns found in the parent and
retro-inverso peptides and frequency of occurrence. Only the
parent peptide sequence is reported and used as a reference. The
upper arrows (dotted) refer to the two
-turns
characterized in the parent peptide, and the lower arrows
(hatched) to the five
-turns found in the retro-inverso
peptide.
Angle values and C(i)C
(i+3) range of
-turns in
L-peptide structures
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Fig. 6.
Stereoviews showing ten superposed structures
coming out from DYANA calculations with the best target functions.
The L- and retro-inverso peptides present a very flexible N-terminal
region (residues 141-150). The best fit was found for the 151-159
region with an r.m.s.d. of 0.9 Å for the parent peptide (a,
blue) and 0.8 Å for the retro-inverso peptide
(b, red). C -C
bonds (thin lines)
were added to display the side chain orientations. This figure was
produced using the MOLMOL program (version 2.5.1) (14).
-turns in
the 155-159 region. Therefore, it appears that TFE is promoting a helical structure in the 141-159 (160) sequence. On the other hand it
is notable that in good agreement with the results obtained in TFE by
Pegna et al. (21), we could not find any particular element
of secondary structure in the N-terminal part of the peptide (Figs. 5
and 6a).
(i)
C
(i+3)
distances revealed five atypical
-turns (Table
II). As in the parent peptide, the
C-terminal part of the retro-inverso peptide is folded by two
-turns
involving residues 156-159 (56% occurrence; Fig. 5) and residues
155-158 (30% occurrence). Two other
-turns are also present in the
central region of the peptide surrounding residues 150-151 and
152-153 with frequencies of 30 and 43%, respectively. Finally, the
RGD motif is also involved in a
-turn present in 10% of the
structures. Fig. 6b shows the optimal superposition of the
backbone atoms for the 10 structures with the lowest target functions.
The best fit corresponds to the sequence 150-158 and yields to an
r.m.s.d. of 0.8 Å. We also measured an r.m.s.d. of 0.9 Å on the
positions of the backbone atoms for the sequence 144-148, containing
the RGD motif. Carver et al. (10) found that in TFE, the
C-terminal part of the retro-inverso peptide (variant SL) as well as
the RGD motif were involved in helices. In contrast our results show that in aqueous solution, these two regions are involved in
-turns, illustrating again the helical promoter role of TFE. These
conformational changes could explain why the parent and retro-inverso
peptides are not recognized by antibodies when the peptides are in
TFE.
Angle values and C(i)C
(i+3) range of
-turns in
retro-inverso peptide structures
-turns in antigenic peptides are the
feature most commonly seen in peptide-antibody complex structures (26,
27). The three additional
-turns found in the retroenantio structure
(Fig. 5) represent putative important nucleation sites that may be
retained and further stabilized or rearranged into different structures
when the peptide interacts with the antibody paratope. As far as the
FMDV sequence studied in this work is concerned, our findings thus
suggest that the retro-inverso strategy has proven to be useful not
because it allowed the generation of a peptide whose surface closely
mimics the L-peptide or the cognate region in the virus, but rather
because it reduced the conformational space available to the peptide
when it binds to the corresponding antibody. In this respect, a
detailed comparison of the L- and retroenantiomeric peptide structures in contact with the paratope of specific monoclonal antibodies will
be of considerable interest.
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Fig. 7.
Stereoview showing best fit superpositions of
mean structure of parent (blue) and retro-inverso peptides
(red). Because the peptide bonds were inverted, only
the C were taken into consideration for superposition. Only the
heavy atoms from residues 155 to 159 are shown. The mean structures
were calculated using the MOLMOL program and minimized using the DYANA
program (13).
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ACKNOWLEDGEMENTS |
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We thank Dr. F. Brown (Plum Island Animal Disease Center, Greenport, NY) and Dr. J. Witz (UPR 9021 CNRS, Strasbourg, France) for helpful suggestions regarding the manuscript.
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FOOTNOTES |
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* This work was supported in part by a grant from Centre National de la Recherche Scientifique (Programme "Physique et Chimie du Vivant") and by European Community Grant FAIR 5-CT-3577.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is dedicated to Patrick Sodano.
The atomic coordinates and structure factors (codes 1bcv and 1bfw) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
¶ Recipient of a post-doctoral grant from SIDACTION (France).
To whom correspondence should be addressed: Institut de
Biologie Moléculaire et Cellulaire, UPR 9021 CNRS, 15 rue
Descartes, 67000 Strasbourg, France. Tel.: 33 388 41 70 27; Fax: 33 388 61 06 80; E-mail: smuller{at}ibmc.u-strasbg.fr.
The abbreviations used are: FMDV, foot-and-mouth disease virus; HPLC, high performance liquid chromatography; RU, resonance unit; TFE, trifluoroethanol; NOE, nuclear Overhauser enhancement; r.m.s.d., root mean square difference.
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