From the Architecture et Fonction des
Macromolécules Biologiques (AFMB), UMR 6098, CNRS et
Universités d'Aix-Marseille I et II, 31 ch. Joseph Aiguier,
13402 Marseille, Cedex 20, France and the § Institute of
Physiology, University of Hohenheim, Garbenstrasse 30, 70593 Stuttgart,
Germany, and the ¶ Laboratoire d'Ingénierie des
Systèmes Macromoléculaires, UPR 9027 CNRS, IFR1, 31 ch. Joseph Aiguier, 13402 Marseille Cedex 20, France
Received for publication, January 25, 2001, and in revised form, March 26, 2001
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ABSTRACT |
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Pheromone-binding proteins (PBPs), located in the
sensillum lymph of pheromone-responsive antennal hairs, are thought to
transport the hydrophobic pheromones to the chemosensory membranes of
olfactory neurons. It is currently unclear what role PBPs may play in
the recognition and discrimination of species-specific pheromones. We
have investigated the binding properties and specificity of PBPs from
Mamestra brassicae (MbraPBP1), Antheraea
polyphemus (ApolPBP1), Bombyx mori (BmorPBP), and a
hexa-mutant of MbraPBP1 (Mbra1-M6), mutated at residues of the internal
cavity to mimic that of BmorPBP, using the fluorescence probe
1-aminoanthracene (AMA). AMA binds to MbraPBP1 and ApolPBP1, however,
no binding was observed with either BmorPBP or Mbra1-M6. The latter
result indicates that relatively limited modifications to the PBP
cavity actually interfere with AMA binding, suggesting that AMA binds in the internal cavity. Several pheromones are able to displace AMA
from the MbraPBP1- and ApolPBP1-binding sites, without, however, any
evidence of specificity for their physiologically relevant pheromones.
Moreover, some fatty acids are also able to compete with AMA binding.
These findings bring into doubt the currently held belief that all PBPs
are specifically tuned to distinct pheromonal compounds.
In the context of molecular recognition, the perception of
pheromones by male moths is a system that shows both high specificity and significant affinity, resulting in mating within the same species
and with the response being elicited by relatively low concentrations
of pheromone. In a generally recognized scheme, the hydrophobic
pheromone molecules enter the antennal sensilla (sensory hairs) through
pores in the cuticle and traverse the aqueous sensillum lymph
transported by low molecular mass (15-17 kDa) pheromone-binding
proteins (PBPs),1 finally
reaching receptors located in the dendritic membranes of the olfactory
neurons, where they are recognized either free or in association with
the PBP (1-5).
PBPs are present in the sensillum lymph at millimolar concentrations
and were originally identified by their ability to bind radiolabeled
compounds of the pheromone blend (1) and by their N-terminal sequence
(6). The primary structures of PBPs from various moth species,
established from molecular cloning (7-14), have indicated diversity
among PBPs and their classification into a large family of insect
pheromone/odorant-binding proteins, which also includes two classes of
general odorant-binding proteins 1 and 2 (10, 11, 15),
antennal-binding protein Xs (11, 14, 16), and antennal-binding proteins
(14). Insect PBPs and OBPs are Although molecular cloning has rapidly increased the information
available on moth PBPs primary structures, cloned genes have not
greatly increased our knowledge of their binding affinities and
specificities. Heterologously expressed PBPs from Antheraea polyphemus (ApolPBP1), Antheraea pernyi (AperPBP1 and
-2), and Lymantria dispar (LdispPBP1) have already been used
to develop quantitative binding assays with photoactivable derivatives
of the pheromone or radioactive pheromones (8, 21-24). However, synthesis of photoactivable or radioactive pheromones is difficult and
limits the candidate compounds tested, usually to the main components
of pheromone blends identified in behavioral studies.
In the present study, we have examined by fluorescence the binding
affinity of 1-aminoanthracene (AMA) to recombinant PBPs of A. polyphemus (ApolPBP1), Mamestra brassicae (MbraPBP1),
Bombyx mori PBP (BmorPBP), and a mutant of MbraPBP1
(Mbra1-M6) mutated at residues in the internal cavity to mimic the
cavity found in B. mori PBP. The results show that AMA binds
with high affinity to ApolPBP1 and MbraPBP1, but neither BmorPBP nor
Mbra1-M6. The comparison of the binding behavior of MbraPBP1 and
Mbra1-M6 indicates that AMA binds in the internal cavity of PBPs. The
fluorescence of bound AMA was used to follow and quantify the
binding of several pheromonal compounds, taking advantage of the
ability of pheromones to displace AMA from the binding site. The
dissociation constants estimated demonstrate a high affinity of
MbraPBP1 and ApolPBP1 both for pheromones and fatty acids. The binding
data reveal that the interaction of pheromones with binding sites of
the two PBPs examined by AMA fluorescence shows little specificity.
Pheromones and Analogues
Pheromones and analogues were purchased from Chemtech B.V. (The
Netherlands) and Sigma, and stored as specified by the manufacturers. AMA was from Fluka. All alcoholic solutions were freshly prepared.
Subcloning in pET22b(+) Vector and Expression
Escherichia coli strain XL1-Blue was used for DNA
subcloning and propagation of the recombinant plasmid. The
MbraPBP1 gene was amplified by PCR using pQE30/PBP1 as
template (25) with the following primers: Mbra1-MscI
5'-CGAGTAAAGAACTGATCACG-3'; Mbra1-NotI
5'-CCGGGCGGCCGCCTACACGGCCGTCATGATCTC-3'. The amplified PCR fragment was
purified and digested with MscI and NotI before being cloned between the same restriction sites of the pET22b(+) vector
(Novagen). Recombinant MbraPBP1 was produced by growing E. coli BL21(DE3) transformed with the recombinant pET22b(+)/MbraPBP1 plasmid at 37 °C in LB medium supplemented with 50 µg/ml
carbenicillin. Cultures were induced with 0.5 mM
isopropyl-1-thio- The gene of ApolPBP1 was amplified, subcloned, and expressed
in a similar way. PCR was performed using the primers:
ApolPBP1-MscI 5'-CTTCGCCAGAGATCATGAAGAAT-3' and
ApolPBP1-XhoI 5'-AGACACTCGAGCAATCTAAACTTCAGCTA-3'. An
ApolPBP1 cDNA clone (Apo-3 (7)) was used as template.
The fragment obtained by PCR was purified, digested by XhoI,
and cloned into the MscI and XhoI restriction
sites of the pET22b(+) vector. The recombinant pET22b(+)/ApolPBP1
plasmid was transformed into E. coli BL21(DE3) and
expression was performed following the protocol of Wojtasek and Leal
(26) by growing the cells without induction at 28 °C in LB medium
supplemented with 50 µg/ml carbenicillin until
A600 reaches a value >2.5. The gene of
BmorPBP was amplified, subcloned, and expressed as described by
Wojtasek and Leal (26).
Site-directed Mutagenesis of MbraPBP1
Six residues of MbraPBP1, Ile-5, Met-8, Ala-56,
Leu-61, Ile-62, and Met-68 were replaced by Met, Leu, Ser, Met, Leu,
and Leu, respectively, by PCR (Fig. 1). 5' and 3' regions of the
cDNA of MbraPBP1 were amplified separately using pET22b(+)/MbraPBP1
as template and the following mutated primers (mutations are
underlined, NruI site is in italic and encoded amino acids
are indicated between parentheses).
5' Region--
M6-NruI,
5'-CCGGTCGCGAGTAAAGAACTGATG(M5) ACGAAACTG(L8)AGTAGTGG-CTTCACGAAA-3';
M6-int-reverse,
5'-AGCCTTCCCGTGGTGGAG(L68)CTTCTGGTCTTCGCCCAG(L62)-CAT(M61)GTCCAGCTTGTTACT(S56)CATACACATCACCAT-3'.
3' Region--
M6-int-forward,
5'-ATGGTGATGTGTATGAGT(S56)AACAAGCTGGACATG(M61)CTG(L62)-GGCGAAGACCAGAAGCTC(L68) CACCACGGGAAGGCT-3'.
Mbra1-NotI, 5'-CCGGGCGGCCGCCTACACGGCCGTCATGATCTC-3'.
After amplification and purification, the PCR fragments were used
to reconstruct a single and continuous cDNA using
M6-NruI and Mbra1-NotI primers. After
purification and digestion by NruI and NotI, the
mutated cDNA was cloned between the MscI and
NotI restriction sites of the pET22b(+) vector (as described
above) and the final construct was referred as pET22b(+)/Mbra1-M6. The mutant, Mbra1-M6, was produced by growing E. coli BL21(DE3)
transformed by the recombinant pET22b(+)/Mbra1-M6 plasmid at 37 °C
in LB medium supplemented with 50 µg/ml carbenicillin. When
A600 reached the value of 0.8, the cultures were
induced with 0.1 mM
isopropyl-1-thio- Purification Procedure
The purification of recombinant MbraPBP1 and Mbra1-M6 mutant was
initiated by centrifugation harvesting of 0.5-4 liters of induced
cultures after 20-24 h of induction. The periplasmic proteins were
released by osmotic shock as described in the pET system manual.
Periplasmic proteins, obtained from induced cultures of BL21(DE3)
(pET22b(+)/MbraPBP1) were submitted to a 100% ammonium sulfate
precipitation, dialyzed overnight against 50 mM Tris-HCl, 50 mM NaCl, pH 8.0, and purified by anion exchange on a
ResourceQ column (Amersham Pharmacia Biotech, Äkta FPLC)
pre-equilibrated in the same buffer. Elution was carried out with a
linear gradient of 0-1 M NaCl in 50 mM Tris,
pH 8.0. Fractions (1 ml each) were collected and analyzed by SDS-PAGE.
Fractions containing the MbraPBP1 were pooled and concentrated by a
10-kDa Microsep (Filtron), and loaded onto a Superdex 200 gel
filtration column (Amersham Pharmacia Biotech) pre-equilibrated with 50 mM Tris, 150 mM NaCl, pH 8.0; the elution was
carried out at 0.25 ml/min. Purification of Mbra1-M6 followed the same
procedure, with the exception of the ammonium sulfate precipitation
step. The periplasmic fraction was directly dialyzed overnight against
50 mM Tris-HCl, 25 mM NaCl, pH 8.0, before
purification by anion exchange on a ResourceQ column followed by gel
filtration (see above). A second anion exchange on a ResourceQ column
was performed with a linear gradient of 0-1 M NaCl in 10 mM Tris, pH 8.0. Fractions (0.5 ml each) were analyzed by
SDS-PAGE. Both proteins were shown to be >95% pure in SDS and native
PAGE. The integrity of the hexamutant was checked by mass spectrometry and N-terminal sequencing.
Purification of the recombinant ApolPBP1 and BmorPBP proteins was
performed by adjusting the periplasmic fraction to 10 mM Tris, pH 8.0, and loading it onto a 20-ml DEAE HR16/10 column (Tyopearl
650S, TOSOH). Proteins were eluted with a linear gradient of 0-300
mM NaCl in 10 mM Tris, pH 8.0. The collected
fractions (1 ml) were analyzed by SDS-PAGE. Fractions containing PBP
were pooled, concentrated in a Centriprep-10 (Amicon), and separated on
a Superdex 75 gel filtration column (Amersham Pharmacia Biotech) pre-equilibrated with 10 mM Tris, 150 mM NaCl,
pH 8.0. PBP containing fractions (1 ml each) were determined by
SDS-PAGE, pooled, and desalted by dialysis against 20 mM
Tris, pH 8.0. Purified ApolPBP1 and BmorPBP protein was shown to be
>95% pure in SDS and native PAGE.
Mass Spectrometry and Circular Dichroism Analysis
Mass analysis of recombinant PBPs was performed with a
Voyager-DE RP spectrometer (PerSeptive Biosystems). Samples (0.7 µl containing 15 pmoles) were mixed with an equal volume of sinapinic acid
matrix solution and spotted on the target, then dried at room
temperature for 10 min. The mass standard was apomyoglobin.
The CD spectra were measured on a CD6 spectropolarimeter (Jobin Yvon).
Spectra were recorded in 10 mM Na phosphate, pH 7.0, at
20 °C between 178 and 260 nm, with a 30-s averaging.
Fluorescence Assays
In the case of MbraPBP1, BmorPBP, and Mbra1-M6, the fluorescence
spectra were recorded on a Jobin-Yvon FL 3-21 spectrofluorimeter at
20 °C using a front face fluorescence accessory. Slit widths of 1 and 10 nm were used for excitation and emission, respectively. The
spectra were processed with DataMax software. In the case of ApolPBP1,
the spectra were recorded in a right angle configuration on a
PerkinElmer LS 50B spectrofluorimeter using a 1-cm light path
fluorimeter quartz cuvette. Slit width of 5 nm were used for both
excitation and emission.
Intrinsic Fluorescence of PBPs
The interaction of AMA and bombykol with PBPs was monitored by
following the quenching of the intrinsic protein fluorescence (excitation at 280 nm and emission 290-420 nm, slits as described above). Spectra were recorded with 1 µM protein in 20 mM Tris buffer, pH 8.0, 0.3% methanol and under the same
conditions in the presence of different concentrations of AMA (0-10
µM).
Fluorescence Emission Spectra Using AMA
The effects of solvents, such as methanol, ethanol, and dimethyl
sulfoxide on AMA binding have previously been tested with rat OBP (27).
In that study, methanol was demonstrated to be less effective than
ethanol in displacing the bound AMA from the binding site. In our
study, the displacement of AMA by ethanol and methanol has been
determined by successively adding aliquots of solvent to the
AMA·MbraPBP1 complex solution. As with rat OBP, ethanol was found to
compete significantly with AMA bound to MbraPBP1, and methanol was
found to have a lesser effect (data not shown). For this reason,
methanol was used for dissolving AMA and its competitors in the
fluorescence titrations.
MbraPBP1, Mbra1-M6, and BmorPBP
All the fluorescence experiments were carried out in 20 mM Tris, pH 8.0, at 20 °C (excitation 298 nm, emission
400-575 nm). The binding affinity for AMA was titrated by adding to
the protein sample (1 µM) aliquots of a stock solution of
AMA (10 mM) solubilized in 100% methanol. The fluorescence
of AMA was recorded after stabilization of the signal (5-6 min). In
competition experiments, PBP (1 µM) was incubated in the
presence of AMA (5 µM) for 1 h at room temperature, the fluorescence signal of AMA was then monitored and the decrease in
fluorescence intensity upon addition of different test compounds was
recorded. The competition experiments were carried out at constant
methanol concentration (0.3%) and with a test compound concentration
between 0 and 10 µM.
ApolPBP1
Binding and competition experiments were carried out in 20 mM Tris, pH 8.0, at 20 °C (excitation 256 nm, emission
440-600 nm). All values reported were obtained from three independent measurements. The curves for binding of AMA to ApolPBP1 were obtained by titration of 2 µM protein with increasing
concentrations of chromophore dissolved in methanol. In competition
assays, we monitored the fluorescence signal of AMA (2 µM) equilibrated with ApolPBP1 (2 µM) upon
addition of increasing amounts of competitor. Fluorescence intensities
at the maximum of emission (487 nm) were determined for different
concentrations of competitor and were corrected before further data
analysis by the extent of AMA fluorescence decrease due to the methanol
present in the cuvette.
Data Analysis
The affinity of different compounds for MbraPBP1 and ApolPBP1
was estimated by plotting the decrease of intensity of AMA fluorescence at the emission maximum, calculated as (I Design of the Mutant of MbraPBP1
Residues were chosen using two criteria: first, they are localized
in the binding pocket defined in the three-dimensional structure of
B. mori PBP (17) and second, they are not conserved among
the PBPs. Six residues of MbraPBP1 were thus replaced by their
counterparts in B. mori PBP (Fig.
1). Three primers were designed to
introduce these mutations by PCR. The final DNA sequence, subcloned in
pET22b(+), was verified by automated DNA sequencing to check the
introduction of mutations and PCR fidelity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUDING REMARKS
REFERENCES
-helical proteins (17), thus
completely different from mammalian OBPs which belong to the lipocalin
superfamily and have a
-barrel fold (18-20).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUDING REMARKS
REFERENCES
-D-galactopyranoside, and the
temperature was decreased to 28 °C when A600
reached the value of 0.8.
-D-galactopyranoside, and the
temperature was decreased to 28 °C.
Imin)/(I0
Imin) against the competitor concentration;
I0 is the maximum of fluorescence intensity of
the complex AMA·MbraPBP1 and AMA·ApolPBP1, at 492 and 487 nm,
respectively, I is the fluorescence intensity after addition
of an aliquot of competitor, and Imin the
fluorescence intensity at saturating concentration of the competitor.
The IC50 values were estimated on the direct plot by
non-linear regression with equation corresponding to a single binding
site using Prism 3.02 (GraphPad software, Inc).
Kdiss values were calculated according to
Kdiss = [IC50]/(1 + [AMA]/KAMA), in which [AMA] = free AMA concentration and KAMA = dissociation constant
for MbraPBP1/AMA and ApolPBP1/AMA, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUDING REMARKS
REFERENCES
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Fig. 1.
Sequence alignment of the three moth PBPs
used in this study and of the MbraPBP1 hexa mutant, obtained with
MultAlin (42). Residues in black and white
boxes indicate identity and similarity, respectively.
Subcloning, Expression, and Purification
After PCR amplification, gel purification, digestion, and precipitation, the MbraPBP1, hexamutant Mbra1-M6, BmorPBP, and ApolPBP1 cDNAs were subcloned into the pET22b(+) vector. This vector leads to expression of a pelB leader/PBP fusion protein in E. coli, which targets the recombinant proteins from the cytoplasm to the periplasm of the bacteria. Upon translocation, the pelB signal peptide is cut off and the PBP is released into the oxidative environment of the periplasm, favorable to the formation of disulfide bonds. This system has been successfully used for the expression of the pheromone-binding protein of Bombyx mori (26), which contains 3 disulfide bridges (28), and in the case of a chemosensory protein from M. brassicae.2
PBP1 and Hexamutant of M. brassicae
Using this system, we routinely obtained 2-3 mg of pure
recombinant MbraPBP1 and Mbra1-M6 per liter of culture. Two predominant bands at around 17 and 20 kDa (Fig.
2A, lane 1) are present in the
SDS-PAGE of whole cell lysates of BL21(DE3) transformed with pET22b(+)/MbraPBP1. The band at 20 kDa is absent in the periplasmic fraction (Fig. 2A, lane 2), and present in the cytoplasmic
fraction (data not shown) suggesting that it corresponded to the
MbraPBP1 with its signal peptide (16,168 Da for MbraPBP1 + 2,228 Da for the pelB signal peptide), whereas the band at around 17 kDa,
present only in the periplasmic fraction, probably corresponded to the recombinant MbraPBP1 after cleavage of the signal peptide. Pure MbraPBP1 and Mbra1-M6 were obtained after two or three steps of purification (see "Experimental Procedures") as shown on 15%
SDS-PAGE (Fig. 2A, lanes 3 and 4). N terminus
sequencing and mass spectrometry confirmed the identity of the
recombinant MbraPBP1 and the mutant Mbra1-M6.
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PBP1 of A. polyphemus and PBP of B. mori
In the case of ApolPBP1, the expression was slightly better than MbraPBP1 and up to 5 mg of pure PBP per liter of culture could be purified. Two bands at about 16 and 18 kDa (Fig. 2B, lane 1), likely corresponding to recombinant ApolPBP1 (15,783 Da) and the pelB/ApolPBP1 fusion protein (15,783 Da + 2,228 Da) can be observed in the SDS-PAGE of whole cell lysates of BL21(DE3) cells transformed with the pET22b(+)/ApolPBP1 construct. In contrast, only the 16-kDa band can be seen in the SDS-PAGE of the periplasmic fraction (Fig. 2B, lane 2). This size is identical to that of the purified ApolPBP1 (Fig. 2B, lane 3). The identity of the isolated recombinant ApolPBP1 protein was confirmed by mass spectrometry and Western blot analysis using an antiserum directed against the wild type protein. Results similar to those described in Wojtasek and Leal (26) were obtained for BmorPBP.
Circular Dichroism
Analytical methods such as CD have been used to define structural
features of insect PBPs. The CD spectra of the four PBPs were recorded
and showed that they are all well folded and have similar secondary
structures. The two minima in the spectra around 208 and 222 nm are
typical of a fold with a majority of -helical secondary structure,
in agreement with the crystal structure of B. mori PBP (17).
The
-helical content obtained by CD (55-70%) is in agreement with
the Psi-Pred secondary structure predictions (30), which indicates
61-66% helical content.
Fluorescence Binding Assays Fluorescence Emission of
AMA Upon Binding to PBPs
When excited at 256 or 298 nm, AMA in aqueous buffer shows a weak
fluorescence emission with a maximum at 563 nm. In a hydrophobic environment, such as the binding pocket of MbraPBP1 or ApolPBP1, there
is a blue shift of the fluorescence emission maximum and a large
increase of intensity (Fig.
3A), resulting from the
modification in the AMA environment. The fluorescence emission spectra
show a maximum at 492 and 487 nm for MbraPBP1 and ApolPBP1,
respectively (Fig. 3,B and C, inset). The
concentration dependence of AMA binding to MbraPBP1 and ApolPBP1 can be
described by a hyperbolic curve as expected for a one-site binding
model (Fig. 3, B and C), and Kdiss values of 4.5 and 0.95 µM,
respectively, were calculated. The affinity of ApolPBP1 for AMA is thus
greater than that of MbraPBP1.
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In the case of BmorPBP, no increase in fluorescence emission and no saturation are observed with increasing AMA concentration. Interestingly, the Mbra1-M6 mutant behaves like BmorPBP. In these cases, the fluorescence increase observed is not significantly greater than the experimental error, furthermore, the lack of saturation suggests either some nonspecific interaction between AMA and the proteins, not related to binding, or the formation of superstructures by AMA.
Intrinsic Fluorescence Quenching of PBPs
AMA-- In order to check whether AMA binds within the internal cavity of the two PBPs, we measured the influence of AMA on the PBP intrinsic fluorescence. The intrinsic fluorescence spectra of MbraPBP1 decreases in intensity upon addition of AMA, the same behavior is observed with ApolPBP1 (data not shown). A dissociation constant of 6 ± 1.7 µM was obtained from intrinsic fluorescence quenching data for AMA binding to MbraPBP1 (compared with 4.5 µM from AMA measurements). The decrease of Trp fluorescence intensity may be due to a ligand induced quenching or to Förster resonance energy transfer (31), between the tryptophan (being the donor) and AMA (the acceptor). Upon excitation at 280 nm, the tryptophan fluorescence is quenched by AMA (acceptor quenched donor emission), however, in the case of Förster resonance energy transfer, the energy is transferred from the protein to AMA, and we should also observe an emission of fluorescence (donor sensitized acceptor emission) when AMA is bound to the protein. The excitation spectrum of bound AMA showed no evidence for tryptophan-sensitized fluorescence, which argues against a Förster resonance energy transfer mechanism, but rather for local environmental effects.
Bombykol-- To confirm the proper folding of BmorPBP, we have used bombykol as an intrinsic fluorescent probe, exploiting its conjugated double bond. Bombykol produced an increase in the intrinsic fluorescence of BmorPBP, and saturation was reached after addition of 5 equivalents. A very similar result was observed with Mbra1-M6 (Table I).
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AMA Displacement by Pheromones or Fatty Acids
To estimate the binding affinities of MbraPBP1 and ApolPBP1 for a variety of different ligands, we monitored the decrease of AMA fluorescence resulting from the ability of different pheromones and fatty acids to displace AMA, and determined the Kdiss values for the different compounds.
For MbraPBP1, the three specific M. brassicae pheromones, Z11-C16-aldehyde, alcohol, and acetate, exhibit very similar AMA displacement properties with Kdiss values of 0.29, 0.17, and 0.20 µM, respectively (Table II). However, these species-specific, and physiologically relevant, compounds are less efficient than bombykol, the pheromone specific to the moth B. mori (Kdiss = 0.13 µM; Table II; Fig. 4A). The best AMA competitor was found to be cetyl alcohol (C16-OH), the saturated equivalent of the Z11-C16-OH (Kdiss = 0.09 µM). Fatty acids also bind well to MbraPBP1, especially palmitic acid (C16-COOH) which exhibits a Kdiss (0.12 µM) very close to the Kdiss of bombykol. The Kdiss values determined for the compounds tested in competition with AMA binding to MbraPBP1 cluster in a narrow range, between 0.09 and 0.63 µM (7-fold), irrespective of their behavioral specificity (Table II).
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In the case of ApolPBP1, three types of test ligands were used in AMA
competition assays: the three species-specific pheromones, pheromones
from other species, and fatty acids. The Kdiss
values observed for all compounds were between 0.48 and 1.36 µM (Table II, Fig. 4B). The three components
of the pheromonal blend released by A. polyphemus females,
E6,Z11-C16-Ald, E6,Z11-C16-Ac, and E4,Z9-C14-Ac, display similar
Kdiss values of 0.50, 0.48, and 0.51 µM, respectively (Table II). The two former compounds are
the best AMA competitors of this series; however, like MbraPBP1, a low
Kdiss value, indicative for a high affinity to
ApolPBP1, has also been determined for bombykol (0.54 µM). Indeed this Kdiss value is
very close to that obtained for the components of the specific
pheromonal blend. As seen for MbraPBP1, fatty acids are also very
efficient in displacing AMA from ApolPBP1; Kdiss
values in this case ranged between 0.56 and 1.36 µM,
indicating an affinity of fatty acids to ApolPBP1 similar to that of
specific pheromones.
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DISCUSSION |
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Fluorescence of probes like AMA, 1-anilino-8-naphtalenesulfonic
acid, or 1,1'-bi(4-anilino)naphtalene-5,5'-disulfonic acid have already
been used to study the affinity constants of ligands to lipocalins,
such as rat OBP (32, 33), porcine OBP (34), apolipoprotein D,
insecticyanin, and bovine -lactoglobulin (35). Although fluorescence
spectroscopy has proved its worth in probing the binding of odorous
compounds to vertebrate OBPs, this technique has rarely been applied to
the study of the interaction between insect PBPs and pheromones.
Intrinsic fluorescence and 1-anilino-8-naphtalenesulfonic acid
fluorescence have previously been used, however, but only to check for
structural changes of B. mori PBP at different pH values and
for its binding to bombykol (26, 36). In the present study, we
successfully reproduced the effect of bombykol on the BmorPBP intrinsic
fluorescence. The behavior of Mbra1-M6, designed to mimic BmorPBP, with
AMA and bombykol used as fluorescence quenchers was found to be similar
to that of the authentic B. mori PBP. The intrinsic
fluorescence of MbraPBP1 and ApolPBP1 decreased upon increasing the
concentration of AMA, which indicates an interaction between AMA and
tryptophans of the protein.
The three-dimensional structure of the B. mori PBP (17)
displays a buried cavity surrounded by the 6 helices constituting the
PBP fold. The sequences of MbraPBP1 and ApolPBP1 are, respectively, 44 and 67% identical to B. mori PBP (Fig. 1) and show no
insertions or deletions. Furthermore, the CD spectra indicate a very
similar overall fold for the four PBPs under study. It seems therefore reasonable to speculate on the three-dimensional structural properties of MbraPBP1 and ApolPBP1 based on those of the B. mori PBP.
In such a model, the two conserved tryptophans (at the same position in
the three sequences) have been localized relative to the cavity; Trp-37
is buried in the PBP and is an integral part of the cavity wall,
whereas Trp-127 is far from the cavity, and not fully exposed to the
solvent (Fig. 1). In this context, the decrease in intrinsic fluorescence in the presence of AMA for two PBPs is likely to be
mediated by the interaction between the fluorophore and Trp-37 as
suggested by the following arguments: first, upon binding to the
protein, the maximum emission wavelength of AMA is blue-shifted and the
quantum yield is substantially increased, indicating a significant
modification of the environment of AMA, which inside the protein
becomes protected from the solvent. Second, the intrinsic fluorescence
can be quenched only if the tryptophan is close enough to AMA (a few
Å) to interact electrostatically, and this excludes Trp-127. In the
three-dimensional model, AMA fits well into the cavity, positioned
about 5 Å from Trp-37 (Fig. 5). The
Kdiss of AMA for MbraPBP1 was found to be 6 ± 1.7 and 4.5 ± 0.3 µM by intrinsic fluorescence
and AMA fluorescence, respectively. Finally, the AMA binding assays
with MbraPBP1 and Mbra1-M6, where the first PBP binds AMA and its
variant not, indicate that mutations in the internal cavity have
impeded AMA binding, and, therefore, that AMA very likely binds to the
internal cavity in the native protein.
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With the aim of determining the specificity of MbraPBP1 and ApolPBP1, competitive binding experiments with several aliphatic ligands were performed. The data indicate that pheromones as well as fatty acids can displace AMA from its binding site. Furthermore, there was no obvious correlation between the presence, the position, and the number of double bonds, and Kdiss, nor were there any obvious effects of the functional group of the first carbon and the chain length (C14, -16, or 18). These results are in contrast with previous studies suggesting that pheromone discrimination might occur not only at a receptor level, but also in the first molecular step, i.e. binding of pheromones to PBPs (8, 21-24, 37-39). Concerning MbraPBP1, migration on native gels and autoradiography indicated that the antennal protein was able to bind the tritiated major pheromonal compound, Z11-C16-Ac (38). However, since this was the only compound tested, the result should be taken with caution. Furthermore, autoradiography studies with the recombinant PBP indicated an affinity not only for the Z11-C16-Ac, but also for the Z11-C16-TFMK (trifluoromethyl ketone), but not for the saturated C16-Ac (25). Indeed, the literature is punctuated with reports misgiving the specificity of PBPs. AperPBP1, expressed in insect cells, has been shown to bind three different compounds, E6,Z11-C16-acetate, -alcohol, and -aldehyde (22), while the PBP of A. polyphemus has been shown to bind compounds other than the specific pheromone, namely decyl-thio-trifluoropropanone, an inhibitor of pheromone receptor neurons of the moth (40). On this basis it has been suggested that decyl-thio-trifluoropropanone, like pheromones, could be carried through the sensillum lymph by PBP and compete with the pheromone-PBP complex at the receptor. Moreover, previous binding experiments of ApolPBP1 with two different photoactivable compounds, (E6,Z11)-[3H]hexadecadienyl diazoacetate and (E4,Z9)-[3H]tetradecadienyl diazoacetate, led to the proposition that two residues, Thr-44 and Asp-32, were part of the pheromones-binding site (41). However, based on the three-dimensional structure of B. mori PBP, these residues and the covalently modified regions do not belong to the binding cavity (17).
Our present results raise two important structural questions: 1) does
AMA bind to the same internal binding site as pheromones, the site
identified in the x-ray structure (17) of BmorPBP? 2) Do the various
recombinant PBPs have a three-dimensional structure identical to that
of the natural antennal PBPs? With regards to question 1, AMA binding
to both Mbra and Apol PBPs yields a significant decrease of intrinsic
fluorescence (see above), suggesting a close approach between AMA and a
tryptophan. Since, as deduced from the PBP structural model, the
external surface of both PBPs contains only a few hydrophobic residues
and no hydrophobic patches, it is not very likely that AMA binds at the
PBPs surface near the second tryptophan (Trp-127). Furthermore,
Mbra1-M6, in which only residues located within the cavity have been
changed to mimic the cavity of BmorPBP, loses its ability to bind AMA
thus resembling BmorPBP. With respect to the second question, due to
the lack of a direct comparison of three-dimensional structure or
binding data for natural and recombinant PBPs, no direct evidence for the correct folding of recombinant PBPs is available. However, several
lines of evidence suggest these proteins fold correctly. (i)
Recombinant PBPs are expressed in E. coli periplasm, in
conditions allowing disulfide bridge formation; (ii) no
post-translational modifications have been identified in natural PBPs,
therefore bacterial expression should be adequate for obtaining a
native fold; (iii) the CD spectra are all very similar and are in
agreement with the secondary structure predictions; (iv) BmorPBP
generated by the same procedure resulted in crystalline PBP leading to
the three-dimensional structure and binding bombykol (26).
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CONCLUDING REMARKS |
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Based on a fluorophore displacement assay, two recombinant PBPs
are shown not to display significant discriminatory capacity between
different pheromones, while two others do not bind the fluorophore AMA,
precluding displacement studies. The observed lack of discrimination
brings into question the hypothesis that PBP subtypes may be
specifically tuned to distinct species-specific pheromones. One way to
reconcile our data with previous results, is to postulate that among
the several PBPs found in a single moth species (often three different
PBPs), some might be nonspecific while others are more specific. Since
AMA is a bulky compound, very different from the insect pheromone
structures, it might be possible that PBPs able to bind AMA are rather
nonspecific. Among the three PBPs studied here, MbraPBP1 and ApolPBP1
would be nonspecific, while BmorPBP might be specific. Indeed current studies indicate that some other recombinant PBPs bind AMA and others
do not. While the existence of several PBPs, each specific for a
distinct component of the pheromonal blend, would be conceivable in a
molecular and evolutionary context, the co-existence of several nonspecific PBP subtypes retained throughout evolution is a challenging question. Alternatively, one could imagine that compounds may be
readily bound by the proteins without being able to activate them, thus
reflecting the situation of receptor proteins which are able to
interact with a large variety of pharmacological compounds, some of
which activate the receptor (agonists) whereas other do not activate
(antagonists), although the latter often display a higher binding affinity.
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ACKNOWLEDGEMENTS |
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We thank Gesa Dreesman for excellent technical assistance. We also thank Dr Jon Clardy for kindly providing us with the coordinates of the B. mori PBP before release.
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FOOTNOTES |
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* This work was supported in part by the Deutsche Forschungsgesellschaft, Provence Alpes Cote d'Azur region Grant 9811/2177, and European Union BIOTECH Structural Biology project BIO4-98-0420 OPTIM.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.
To whom correspondence may be addressed. Tel.:
33-491-16-45-12-01; Fax: 33-491-16-45-36; E-mail:
cambillau@afmb.cnrs-mrs.fr.
** To whom correspondence may be addressed. E-mail: tegoni@afmb.cnrs-mrs.fr.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M100713200
2 V. Campanacci, A. Mosbah, O. Bornet, R. Wechselberger, E. Jacquin-Joly, H. Darbon, C. Cambillau, and M. Tegoni, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: PBP, pheromone-binding protein; OBP, odorant-binding protein; Mbra, Mamestra brassicae; Apol, Antheraea polyphemus; Aper, Antheraea pernyi; PCR, polymerase chain reaction; AMA, 1-amino-anthracene; C16-OH, cetyl alcohol; Z11-C16-OH, (Z)-11-hexadecen-1-ol; C16-COOH, palmitic acid; Z9-C16-COOH, palmitoleic acid; Z9-C18-COOH, oleic acid; Z11-C16-Ald, (Z)-11-hexadecenal; E6, Z11-C16-Ald, (E6,Z11)-hexadecadienal; Z11-C16-Ac, (Z)-11-hexadecenyl-1-acetate; E6, Z11-C16-Ac, (E6,Z11)-hexadecadienyl-1-acetate; E4, Z9-C14-Ac, (E4,Z9)-tetradecadienyl-1-acetate; PAGE, polyacrylamide gel electrophoresis.
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