BACKGROUND
the sex pheromone detection by male moths  is highly sensitive and capable of detecting volatile hydrocarbons at low concentrations, as well as discriminating among stereoisomers and pheromone blends  <cit> .
female moths produce and emit volatile sex pheromones from pheromone glands located in distal segments of the abdomen, and perception can cause conspecific males to be attracted from a distance of 100 m  <cit> .
the specificity of male reception and response to female sex pheromones is mediated by olfactory response neurons  located in antennal trichoid sensilla.
detection of female pheromones can induce physiological or behavioral changes in males following downstream signal transduction events mediated by the orn  <cit> .
the first step of pheromone detection is the transport of the hydrophobic pheromone across the hydrophobic lymph of the trichoid sensillum.
pheromone binding proteins  are a subgroup of the odorant binding protein  family that have been reported in many species of lepidoptera  <cit> , and are hypothesized to be essential in environmental detection of sex pheromones by male moths  <cit> .
pbps are small, water-soluble, extracellular proteins that are found in the lymph surrounding the dendrites of pheromone-sensitive orns  <cit> .
pbps bind short to medium chain length fatty acid derivatives that enter the sensillum lymph through cuticular pores, and function as chaperones that transport pheromones to the sex pheromone receptors localized on the dendritic membranes of orns.
pbps have six highly conserved cysteine residues that help stabilize the globular protein and the internal hydrophobic binding pocket  <cit> .
nerve impulses are generated by orns following the delivery of sex pheromones by the pbps are mediated by transmembrane odorant receptors  located on the surface of neuron dendrite.
insect ors dimerize with a highly conserved co-receptor termed the odorant receptor co-receptor  to form a ligand-gated ion channel  <cit> .
pheromones are released from carrier pbps when lower ph microenvironments are encountered at the neuron surface, where selectivity of pbps may be enhanced  <cit> .
pheromone ligand release by pbps and subsequent binding to the orco + orx complex will cause an electrical impulse to be generated  <cit> .
changes in the structure or function of these sexual communication pathway components form a hypothetical basis for the evolution of novel mechanisms of male attraction, and may be involved in establishment of sexual isolation between recently diverged species.
species from the genus ostrinia are models for the study of male sex pheromone detection systems.
different sex pheromone components are produced in the pheromone gland of females in the genus ostrinia , but different ratios of e-11- and z-11-tetradecenyl acetate  predominate among most species.
one exception is found with females of the asian corn borer , o. furnacalis , that produce and emit pheromone components that are synthesized with the double bond at the twelfth carbonyl carbon  <cit> ; e-12- and z-12-tetradecenyl acetate .
mating between o. furnacalis and the closely related european corn borer , o. nubilalis , occurs in the laboratory, but behavioral isolation and differences in response to sex pheromone may likely prevent high degrees of hybridization under natural conditions.
the gene coding sequence of five pbps were sequenced for both o. furnacalis and o. nubilalis, and with the exception of pbp <dig>  the amino acid sequences between orthologs are highly conserved  <cit> .
a significant overrepresentation of non-synonymous mutations predicted between pbp <dig> orthologs suggested a potential role in pheromone binding specificity between e11- and z11-14:oac, and e12- and z12-14:oac  <cit> .
in this study, we expressed and compared binding affinities of the five o. furnacalis pbp paralogs for synthetic female o. furnacalis and o. nubilalis pheromones, and mutagenized four amino acid residues predicted to coordinate e12- and z12-14:oac pheromones within the hydrophobic binding pocket.
the data presented in this study are important for understanding the functional residues that are involved in effecter binding by ostrinia pbp <dig>  but does not attempt to address the evolutionary changes that impart divergent function between o. furacalis compared to o. nubilalis pbps.
regardless, the data presented here are important for understanding the residues involved in general sex pheromone binding, but additional research is required to investigate any residues involved in the selectivity of ofurpbp <dig> compared to onpbp <dig>
materials
rna extraction and cdna synthesis
a laboratory colony of o. furnacalis was maintained at the institute of plant protection, chinese academy of agricultural sciences.
pupae were allowed to emerge in a netted cage at room temperature, and moths were fed a 10% honey solution.
antennae were dissected from three day old male moths, immediately frozen in liquid nitrogen and stored at −80 °c prior to use.
total rna was extracted from o. furnacalis antennae using trizol™ reagent  according to manufacturer instructions.
rna was then reverse transcribed into 1st strand cdnas in a reaction primed with an oligodt <dig> oligonucleotide, and synthesized with avian myeloblastosis virus  reverse transcriptase  at 40 °c for 1 h. the reaction was stopped by incubation at 95 °c for 5 min and then cooling on ice for 20 min.
the cdna sequences for five asian corn borer pbp genes were previously reported by allen and wanner , and were used to design primer pairs that amplified the in frame mature gene coding sequence for each gene .
each primer incorporated a novel 5-prime overhang with palindromic sites for cleavage by bamhi  or hindiii , which allowed for subsequent directional cloning into the restriction endonuclease sites of the multicloning site in the pet30a expression vector .
briefly, pcr primers for the amplification of mature ofurpbp1-ofurpbp <dig> coding sequences were designed using primer <dig>  <cit>  with a minimum length of  <dig> nucleotides and a predicted melting temperature  of ≥58 °c.
the 5-prime extensions that encompassed the bamhi preceding the n-terminal cds and hindiii site following the c-terminal coding region were inserted manually, the details of the primers was shown in additional file 1: s <dig>  the subsequent pcr reactions used 5 pmol of each primer used  <dig>  μl of cdna template diluted 1: <dig> with the high fidelity polymerase .
thermal cycling was performed on a biometra t-gradient thermocycler  under the following conditions: 94 °c for 2 min 30 s,  <dig> cycles of 94 °c for 30 s, 58 °c for 30 s, and 72 °c for 1 min, and 72 °c for 10 min.
an aliquot of each pcr product was separated on a  <dig> % agarose gel containing  <dig>  μg ml− <dig> ethidium bromides and visualized under uv illumination.
pcr products were cloned into the pgem-t vector , and single clones were obtained after selection on lb agar plates containing 50 μg ml− <dig> ampicillin.
for each ofurpbp clone, plasmid dna isolated and sequenced from the t <dig> primer binding site at beijing sinogenomax co., ltd  on an abi 3730xl dna analyzer .
electropherogram data were compared to cdna sequences of respective pbps in genbank accessions gu <dig> to gu <dig> previously published by  <cit>  using vector nti software .
independent multiple sequence alignments were made using ofurpbp <dig> to ofurpbp <dig> sequences generated in this study, o. furnacalis , o. nubilalis e-strain , and o. nubilalis z-strain  using the clustal w algorithm .
prokaryotic expression and purification
the pgem plasmid inserts were excised using bamhi and hindiii double digestion, excised from a 1% agarose gel and fragments purified using nucleic acid purification kit .
the bamhi and hindiii digestion products for ofurpbp <dig> to ofurpbp <dig> were then ligated individually into bamhi and hindiii cut and dephosphorylated pet30a vector by incubation with t <dig> ligase  for 4 h at 16 °c.
recombinant pet30a vectors containing inserts derived from the five pheromone binding proteins  were used to transform bl <dig> escherichia coli cells by electroporation, and cells allowed to recover in soc medium for 1 h at 37 °c.
transformed cells were selected by spread plating on lb agar plates containing 25 μg ml− <dig> kanamycin sulfates as described by the manufacturer.
positive clones were cultured, and plasmid dna isolated and inserts sequenced using the t <dig> primer as described above.
positive pet30a clones containing correct insert dna from each of the pbp sequences were incubated separately in fresh liquid lb medium containing 25 μg ml− <dig> kanamycin sulfate.
cultures were shaken at 37 °c until the optical density  of each culture reached to  <dig> – <dig> , where od was measured on a shimadzu spectrophotometer .
protein expression was induced in each culture by the addition of isopropyl-β-d-1-thiogalactopyranoside  to a final concentration of  <dig>  mm, followed by incubation on a 150 rpm shaker at 37 °c for 6 h. the cells were harvested by centrifugation at 7500×g for 15 min at 4 °c, chilled on ice, and treated with ultrasonic disruptor.
after centrifugation, all five proteins were highly present in inclusion bodies.
the protein pellets were suspended using previously published methods  <cit> .
proteins were purified by application of the lysate to ni–nta columns and purified as specified by the manufacturer , followed by digestion of eluted proteins with enterokinase to remove the his tag.
protein preparations were desalted by dialysis, quantified by means of the bradford method  <cit> , and purity verified by sds-page.
pheromone-binding affinity
initial measures of the binding affinity of the fluorescent ligand n-phenyl-1-naphthylamine  to recombinant ofurpbp <dig>  ofurpbp <dig>  ofurpbp <dig>  ofurpbp <dig> and ofurpbp <dig> was determined by titrating to final concentrations of  <dig> – <dig>  µm 1-npn with 2 µm of each protein in  <dig>  ml 50 mm tris–hcl, ph  <dig>  and ph  <dig> .
the 1-npn probe was excited at 337 nm, and emission spectra were recorded between  <dig> and 450 nm on a shimadzu fluorescence spectrophotometer .
each trial was run in triplicate, and scatchard plots of the ratio of bound to unbound ligand were created using microsoft excel  <dig>  competitive binding assays were performed with each of the ofurpbps using 1-npn as the probe.
assay reactions were comprised of 2 µm of each ofurpbp diluted individually in  <dig>  ml 20 mm tris–hcl, ph  <dig> , which were then titrated with  <dig>  mm increments sex pheromone diluted in methanol to a final concentrations of 1–6 µm.
the synthetic standards that correspond to the sex pheromones of o. furnacalis, o. nubilalis and other lepidopteran species were purchased from sigma-aldrich .
excitation and emission spectra were recorded from triplicate assays as described above.
the binding affinity of each purified ofurpbp to the different sex pheromones was measured by calculation of binding constants via competitive fluorescence-binding assay.
in brief, relative fluorescent intensities were analyzed by graphpad prism  <dig> software  and calculation was done using the equation: ki = /, where  is the free concentration of 1-npn and k1-npn is the dissociation constant of the complex protein/1-npn  <cit> .
sex pheromone binding affinity with different pbps was analyzed using one-way anova in sas  <dig>  .
molecular modeling and ligand docking
the amino acids of ofurpbp <dig> were submitted to swiss-model tool to automate homology-modeling  <cit> .
the sequence of ofurpbp <dig> was compared to all the known protein sequences and that had a high similarity with the target sequence on rcsb protein data bank  .
the ofurpbp <dig> used atrapbp <dig> for a homology model  <cit> .
the main pheromone components of o. furnacalis, z12- and e12-14:oac were used respectively in molecular docking with the simulated model of ofurpbp <dig> based on the advanced docking program cdocker  <cit> .
molecular dynamics scheme based on charmm was used to dock ligands into a binding site in the cdocker program.
total interaction , van der waals , and electrostatic interaction energies  were calculated for all ofurpbp <dig> residues predicted to be involved in the binding of intraspecific sex pheromone components.
site-directed mutagenesis
four amino acids of ofurpbp <dig> with some of the most negative estimated e
total values were mutated using quick-change lightning site-directed mutagenesis kit ; ile to asn at position  <dig> , m <dig> phe to ser at position  <dig> , m <dig> ile to asn at position  <dig> , and m <dig> leu to arg at position  <dig> .
the sequence of each primers used to alter the aforementioned sites by nucleotide mismatch are shown in additional file 3: s <dig>  after initial denaturation at 94 °c for 5 min, the subsequent pcr followed with  <dig> cycles of 94 °c for 30 s, 56 °c for 30 s, and final extension at 72 °c for 1 min.
changes within site-directed mutant fragments were verified by cloning into the pgem-t easy vector  by sanger sequencing.
all mutated ofurpbps proteins were expressed and used within in vitro binding assays as described above.
RESULTS
rna extraction and cdna synthesis
five amplified fragments predicted to contain the mature ofurpbp <dig> to ofurpbp <dig> gene coding sequences were successfully amplified by rt-pcr , cloned into the pgem vector, and sanger sequenced.
multiple nucleotide sequence alignments of these cloned ofurpbp sequences showed 100% similarity with homologous regions of genbank accessions for o. furnacalis pbp <dig> to pbp <dig> previously identified by allen and wanner  <cit> .
one exception was predicted in a single nucleotide polymorphism at position  <dig> of ofurpbp <dig> which changed a guanine  in gu <dig>  to an adenosine  in our cloned ofurpbp <dig> sequence.
this snp on ofurpbp <dig> produced a synonymous mutation, such that the amino acid sequence was unchanged and the same as that reported by allen and wanner  <cit> .
prokaryotic expression and purification
five mature ofurpbp gene coding sequences were successfully excised from pgem, and re-inserted into to the pet30a expression vector.
end sequencing of inserts from the t <dig> primer binding site flanking the pet30a vector multicloning site confirmed the correct orientation and sequence of each ofurpbp insert .
each of the iptg induced cultures derived from e. coli pet30a-ofurpbp transformants showed high expression of a his-tagged fusion protein in the approximately 16–19 kda size range , and subsequent purification of each on ni–nta columns successfully removed visible endogenous e. coli proteins .
digestion of purified ofurpbps fusion proteins with enterokinase  resulted in decreased estimated molecular weights, which were less than the 15–17 kda predicted for full-length lepidopteran pbps containing leader peptides .
this was expected since the cloned fragments were devoid of leader peptides and represent fully functional processed peptides.
after analysis, most of the proteins were expressed in inclusion bodies.
bradford assays estimated the concentration of each purified ofurpbp protein at ~ <dig> to 2 μg ml− <dig>  and extracts were used for the further experiments in this study.fig.
<dig> sds-page analysis of recombinant ostrinia furnacalis pheromone binding proteins  induced from e. coli clones carrying in frame insertions of ofurpbp <dig>  ofurpbp <dig>  ofurpbp <dig>  ofurpbp <dig>  or ofurpbp <dig> coding sequences in the pet30a expression vector.
replicate gel lanes correspond to  cell culture prior to addition of isopropyl-β-d-1-thiogalactopyranoside ,  iptg induced cells at o.d.
<dig> – <dig> ,  supernatant after ultrasonic treatment and centrifugation,  pellet after ultrasonic treatment and centrifugation,  ni–nta column purified proteins, and  enterokinase digested products with his-tags cleaved.
m corresponds to molecular weight marker.
a ofurpbp1; b ofurpbp2; c ofurpbp3; d ofurpbp4; e ofurpbp5
pheromone-binding affinity
the general affinity of the 1-npn reporter for each ofurpbp was measured , and a regression of these titrated values were plotted within scatchard plots and showed a linear relationship.
next, the binding specificity of  <dig> different ostrinia pheromone components were tested against each recombinant ofurpbp in competitive binding assays with 1-npn as the reporter .
the female sex pheromones, z12- and e12-14:oac are produced by o. furnacalis and z11- and e11-14:oac by o. nubilalis .
competitive binding assay results showed that z12- and e12-14:oac bound most strongly with ofurpbp <dig> , and also showed affinity for ofurpbp <dig> and ofurpbp <dig> .
results also showed that z12-14:oac bound ofurpbp <dig> weakly and e12-14:oac had no detectable binding to ofurpbp <dig>  in contrast, both z11- and e11-14:oac pheromones from o. nubilalis showed relatively high affinities for all five ofurpbps , with estimates of z11-14:oac binding to ofurpbp <dig> being the highest for all those that were tested .
additionally, the o. nubilalis pheromone e11-14:oac bound strongly to ofurpbp <dig> , and showed a significantly greater affinity compared to either z12- and e12-14:oac from o. furnacalis .
all five recombinant ofurpbps showed weak or lack of detectable binding to the interspecific lepidopteran sex pheromones, z9-14:oh and e11-14:oh.
however, under the ph  <dig> , none of the ofurpbps showed any binding to the sex pheromones that were tested .fig.
<dig> binding curves.
a binding curves and scatchard plots  for fluorescent probe 1-npn binding with purified ostrinia furnacalis pheromone binding proteins  at ph  <dig> .
competitive binding curves for different synthetic sex pheromone hydrocarbons  at ph  <dig>  to each of the ofurpbps, b ofurpbp <dig>  c ofurpbp <dig>  d ofurpbp <dig>  e ofurpbp <dig>  and f ofurpbp <dig>  the different sex pheromones tested are listed in additional file 2: s2
table  <dig> competitive ligand binding assay results among five recombinant native ostrinia furnacalis pbp orthologs  tested against six female sex pheromones found among species in the genus ostrinia
ic50
all assays performed at ph =  <dig> .
values reported as μm concentrations of each pheromone component
ic <dig> is the ligand concentration displacing 50% of the fluorescent reporter; ki is the binding constant calculated from the equation ki = /
mean ± se, data in the same column followed by the same letters were not significantly different  according to lsd test.
dashes indicate undetectable interations
3d modeling and docking
the ofurpbp <dig> was successfully modeling with atrapbp <dig> by using of swiss-model tool .
this figure visual displayed the 3-dimensional structure of ofurpbp <dig>  the qmean and gmqe were used to quality estimation, all the score reflected the expected accuracy of ofurpbp <dig> built with the alignment and template of atrapbp <dig>  based on the consideration of hydrophobicity at their binding site, ligand poses and consensus score programs were executed to evaluate binding pose affinities for the residues.
subsequently, optimal 3d binding conformations to the main sex pheromone components, z12- and e12-14:oac, were shown in fig.
3c, d. the interaction energies e
total, e
vdw, and e
ele between ofurpbp <dig> amino acids that interact with z12- or e12-14:oac were estimated .
results indicated that phe <dig> and leu <dig> interacted most strongly with z12-14:oac.
and phe <dig> and ile <dig> with e12-14:oac.fig.
<dig> the predicted 3d structure of ofurpbp <dig> of o. furnacalis and docking with sex pheromones.
a sequence alignment between ofurpbp <dig> and atrapbp <dig>  and the predicted α-helical secondary structure elements are shown along the top.
strictly identical residues are highlighted in white letters with a red background, and are framed in blue residues are chemically identical or similar.
residues with similar physico-chemical properties are shown in red letters.
the key amino acids used to mutated were marked by black triangle.
b the predicted 3d structure of ofurpbp <dig>  in silico predicted molecular docking between z12-14:oac  and e12-14:oac  with ofurpbp <dig> are shown with respective pheromone components in red interacting amino acids are in yellow
table  <dig> interaction energies between ofurpbp <dig> amino acid residues predicted to interact with z12- and e12-14:oac
e
total
e
vdw
e
eie
e
total
e
vdw
e
eie
phe12a
ile52a
leu94a
ile113a
e
total: total interaction energy; e
vdw: van der waals energy; e
ele: electrostatic interaction energy
aresidues chosen for site-directed mutagenesis
site-directed mutagenesis
based on the molecular modeling and docking results four amino acid residues with among the highest e
total were chosen , and in vitro mutagenesis altered ofurpbp <dig> mutants f12s, i52n, i94r, and i113n verified from sanger sequencing results .
subsequent competitive binding assays demonstrated that all ofurpbp <dig> mutants showed reduced affinities towards z11- and e11-14:oac  and z12- and e12-14:oac  compared to the wild-type ofurpbp <dig>  among the mutants, i113n and f12s had the least dramatic effect on affinity towards z12-14:oac .
surprisingly i113n showed a nearly  <dig> -fold reduction in z11-14:oac binding compared to the wildtype, and represented the greatest degree of change among all the assays.
all mutated ofurpbps showed the capacity to bind the sex pheromones components of other lepidopteran females, z9-14:oh and e11-14:oh , compared to the wildtype which failed to demonstrate this function.
in particular, i113n demonstrated the ability to bind z9:14-oh, which is a known antagonist of male response to e11- and z11-14:oac as well as e12- and z12-14:oac .table  <dig> competitive ligand binding assay results from four ostrinia furnacalis pbp <dig>  mutants generated by site-directed mutagenesis of ile <dig> to asn <dig> , phe <dig> to ser <dig> , ile <dig> to asn <dig> , and ile <dig> to arg <dig>
mutant and wildtype ofurpbp proteins were assayed against six female sex pheromones found among species in the genus ostrinia.
all assays performed at ph =  <dig> .
values reported as μm concentrations of each pheromone component
ic <dig> is the ligand concentration displacing 50% of the fluorescent reporter; ki is the binding constant calculated from the equation ki = /
mean ± se, date in the same column followed by the same letters were not significantly different  according to lsd test.
dashes indicate undetectable interations
discussion
we showed that the five recombinant ofurpbps have a range of binding affinities with synthetic o. furnacalis sex pheromones, and that ofurpbp <dig> and ofurpbp <dig> bind z12- and e12-14:oac with the greatest affinity .
these binding results might suggest that female o. furnacalis pheromones are bound strongly to male-antennal expressed ofurpbp <dig> and ofurpbp <dig> in vivo, and that these two chaperones could play a role in the specific transport of z12- and e12-14:oac within the sensillar lymph  <cit> .
the highly biased expression of ofurpbp <dig> and ofurpbp <dig> transcripts in the ostrinia male compared to female antennae has been used to suggest possible roles in the binding of female sex pheromones  <cit> , and the latter hypothesis was corroborated for the first time by our data .
the correlation between pbp expression level and molecular function was further corroborated by ofurpbp <dig> and ofurpbp <dig>  wherein both show female biased expression  and have weak or undetectable binding affinities towards e12- and z12-14:oac .
a difference in pheromone binding affinity has been previously shown for pbp <dig> from a. polyphemus and a. pernyi which were both bound strongly by e <dig> z-14:ac, but pbp <dig> and pbp <dig> from these species preferentially bound e <dig> z-16:ald and e <dig> z-14:ac, respectively  <cit> .
analogously, pbp <dig> and pbp <dig> from lymantria dispar male antennae respectively bind specifically to  and  enantiomers of the pheromone released by conspecific females  <cit> , which suggest that pbp gene family members can develop variant molecular functions and that our observed differences between ofurpbp <dig> and ofurpbp <dig> compared to ofurpbp <dig> and ofurpbp <dig> may not be surprising.
furthermore, the binding affinities of ofurpbp <dig>  ofurpbp <dig>  and ofurpbp <dig> to cognate pheromone components estimated in this study are comparable to analogous ki values estimated for helicoverpa armigera pbps  and h. assulta  to their corresponding sex pheromone  <cit> .
similarly, the binding affinities of the cockroach leucophaea maderae pbp, pbplma, to their sex pheromones 3-hydroxy-butan-2-on or butane- <dig> -diol were  <dig>  and  <dig>  μm, respectively  <cit> .
in contrast to predictions made by allan and wanner , ofurpbp <dig> did not show specific affinity towards z12- and e12-14:oac, but instead demonstrated a nearly equal affinity for z11- and e11-14:oac emitted by o. nubilalis females.
moreover, all ofurpbps showed a comparatively strong affinity towards z11- and e11-14:oac, including observation of strong binding by ofurpbp <dig> and ofurpbp <dig>  these ligand affinities are in direct opposition to species responses  <cit> , which might suggest that ofurpbps bind to z11- and e11-14:oac does not preclude a role in male sexual response, but only in potential for binding these components within the sensillar lymph.
transcripts encoding pbps have previously been found in non-pheromone sensitive female antennae and male sensilla  <cit> , which suggests that pbps may have evolved alternate roles such as host plant recognition  <cit> .
this notion was reinforced by evidence that compounds structurally similar to cognate pheromones can be bound by bombyx mori pbp <dig>  <cit> , and further by observations that of sex pheromones and plant volatiles can interact within the same peripheral sensory pathways  <cit> .
this altogether suggests that binding compounds or interspecific pheromone components to a pbp does not preclude subsequent interaction with the orco + orx complex resulting in sexual response.
grosse-wilde et al.
<cit>  showed that pheromone agonists bound to different pbp paralogs and lead to different responses by male sexually-responding orco − orx receptors, such that members of the pbp gene family may have evolved specific interactions at different orco − orx complexes.
the o. furnacalis or <dig>  was shown to specifically respond to z12-14:oac and e12-14:oac  <cit> .
the selectivity of pbp for delivery of these pheromone components to ofor <dig> was not investigated in this or any other previous studies, but may provide crucial information regarding the evolution of species-specific male sexual response.
regardless, our results are the first to demonstrate that male antennal expressed ofurpbp <dig> and  <dig> are capable of binding the intraspecific female pheromone components z12-14:oac and e21-14:oac.
a change in the 3-dimensional conformation of pbps occurs following a shift from neutral to acidic ph environments, and is associated with binding and release of cognate pheromone ligands.
crystal structures of pheromone-bound pbps indicated that the c-terminal amino acid sequence remains linear under neutral ph  <cit> , whereas this c-terminal region forms an alpha-helix in acidic conditions .
the more linear conformation with  <dig> alpha helices was used to predict the 3d structure of ofurpbp <dig> , wherein  <dig> residues within the hydrophobic binding pocket were predicted to interact with z12-14:oac and e21-14:oac .
this ph-dependent change is also accompanied by alteration of pbp 3-dimensional structure where salt bridges are formed between two asp residues on the c-terminal α <dig> helix and two corresponding deprotonated his residues in the pheromone binding pocket at low ph.
thus, the ph-dependent alpha-helix interactions with the pheromone binding pocket competes with sex pheromone ligands, and is a key component for pbp pheromone carrier and delivery functions.
specifically, neutral ph in sensillar lymph may place pbps into a competent pheromone-binding state, where these chaperones are capable of transporting hydrophobic pheromone hydrocarbons.
residues of ofurpbp <dig> potentially involved during the binding of intraspecific pheromones at neutral ph binding are important for understanding function within a species, and the e
total derived from cdocker predicted the  <dig> key amino acids which may interact with z12- and e12-14:oac ligands .
predictions from homology-based in silico structural modeling can be unreliable  <cit>  and translation into biological systems can be bolstered by experimental validation.
thus, site-directed mutagenizes of four residues with the highest e
total values were made in four separate mutant ofurpbps.
comparisons between wildtype and mutant ofurpbps clearly demonstrated the negative affects on binding affinities following change of native residues to those with different polar properties and suggest that phe <dig>  ile <dig>  ile <dig>  and ile <dig> are important for the efficient binding of cognate sex pheromones.
interestingly, after mutation, the ofurpbps showed weak binding affinity towards other lepidopteran female sex pheromone, z9-14:oh and e11-14:oh, that was not observed with the wild type ofurpbp <dig>  these differences may occur either through direct interaction with the bound hydrocarbons, or by stabilization of the hydrophobic binding pocket.
the affects of induced amino acid changes on the overall 3d structure and function of a protein remain largely unknown  <cit> .
this suggests that even single amino acid changes might alter pbp conformation in unforeseen ways, such that further research, such as using x-ray or nmr to analyze the structure of native and mutant binding proteins are likely required.
although our current study demonstrated the ability of ofurpbps to bind inter and intraspecific sex pheromones, further research is likely required to fully understand the structural, chemical, and environmental factors that may influence any specific interactions between pbps and their cognate sex pheromones.
CONCLUSIONS
our research provides crucial functional information regarding the specificity of pbps in an ostrinia moth species, which is a group that has emerged as a model for the study of sexual communication systems in lepidoptera.
this study provides the first biochemical evidence that ofurpbp <dig> and ofurpbp <dig> bind z12- and e12-14:oac with high affinity.
moreover, this current work demonstrates that despite prior evidence for directional selection and predicted divergent function between onpbp <dig> and ofurpbp <dig>  the latter shows a nearly equal affinity to o. furnacalis female z12- and e12-14:oac and o. nubilalis z11- and e11-14:oac.
the four amino acids, ser <dig>  asn <dig>  arg <dig>  and asn <dig> may play a role in binding of sex pheromone of pbp <dig>  but cannot be interpreted in the context of species-specific binding since we only mutagenized sites predicted to be important in protein interactions with pheromone ligands.
our results suggest that overall individual o. furnacalis pbps may be capable of equivalent binding of both intra- and inter-specific female sex pheromone components in vitro, and therefore could suggest that molecular discrimination in male response might reside within the behavioral pathway.
additional research is undoubtedly required to both dissect the molecular functions of pbps in concert with cognate ors with respect to ligand binding as well as the mechanisms that transmit neuronal signals which result in differential male responses to female emitted pheromones.
additional files
additional file 1: s <dig>  the primers used for the pbps expression.
the lists of primers used for pbps expression, and the digestion sites were underlined.
additional file 2: s <dig>  the message of sex pheromones used in binding assay.
the sex pheromones messages used in binding experiment, including the molecular weight, purity and company.
additional file 3: s <dig>  the primers used for site-directed mutants.
the lists of primers used for site-directed mutants, and the digestion sites were underlined.
additional file 4: s <dig>  the sequences of the wild type ofurpbp <dig> and the mutants.
the nucleotide sequences of wild type ofurpbp <dig> and the mutants .
tiantao zhang and yaqi sun contributed equally to this work
authors’ contributions
ttz, klh and zyw designed and conducted the research experiments.
ttz, kww and bsc interpreted the results and drafted the manuscript.
ttz and yqs are the principal investigator who designed the experiment, analyzed the data and critically reviewed the manuscript.
all authors read and approved the final manuscript.

 the sex pheromone detection by male moths is highly sensitive and capable of detecting volatile hydrocarbons at low concentrations, as well as discriminating among stereoisomers and pheromone blends. female moths produce and emit volatile sex pheromones from pheromone glands located in distal segments of the abdomen, and perception can cause conspecific males to be attracted from a distance of 100 m. the specificity of male reception and response to female sex pheromones is mediated by olfactory response neurons located in antennal trichoid sensilla. detection of female pheromones can induce physiological or behavioral changes in males following downstream signal transduction events mediated by the orn. the first step of pheromone detection is the transport of the hydrophobic pheromone across the hydrophobic lymph of the trichoid sensillum. pheromone binding proteins are a subgroup of the odorant binding protein family that have been reported in many species of lepidoptera, and are hypothesized to be essential in environmental detection of sex pheromones by male moths. pbps are small, water-soluble, extracellular proteins that are found in the lymph surrounding the dendrites of pheromone-sensitive orns. pbps bind short to medium chain length fatty acid derivatives that enter the sensillum lymph through cuticular pores, and function as chaperones that transport pheromones to the sex pheromone receptors localized on the dendritic membranes of orns. pbps have six highly conserved cysteine residues that help stabilize the globular protein and the internal hydrophobic binding pocket. nerve impulses are generated by orns following the delivery of sex pheromones by the pbps are mediated by transmembrane odorant receptors located on the surface of neuron dendrite. insect ors dimerize with a highly conserved co-receptor termed the odorant receptor co-receptor to form a ligand-gated ion channel. pheromones are released from carrier pbps when lower ph microenvironments are encountered at the neuron surface, where selectivity of pbps may be enhanced. pheromone ligand release by pbps and subsequent binding to the orco + orx complex will cause an electrical impulse to be generated. changes in the structure or function of these sexual communication pathway components form a hypothetical basis for the evolution of novel mechanisms of male attraction, and may be involved in establishment of sexual isolation between recently diverged species. species from the genus ostrinia are models for the study of male sex pheromone detection systems. different sex pheromone components are produced in the pheromone gland of females in the genus ostrinia, but different ratios of e-11- and z-11-tetradecenyl acetate predominate among most species. one exception is found with females of the asian corn borer, o. furnacalis, that produce and emit pheromone components that are synthesized with the double bond at the twelfth carbonyl carbon; e-12- and z-12-tetradecenyl acetate. mating between o. furnacalis and the closely related european corn borer, o. nubilalis, occurs in the laboratory, but behavioral isolation and differences in response to sex pheromone may likely prevent high degrees of hybridization under natural conditions. the gene coding sequence of five pbps were sequenced for both o. furnacalis and o. nubilalis, and with the exception of pbp the amino acid sequences between orthologs are highly conserved. a significant overrepresentation of non-synonymous mutations predicted between pbp orthologs suggested a potential role in pheromone binding specificity between e11- and z11-14:oac, and e12- and z12-14:oac. in this study, we expressed and compared binding affinities of the five o. furnacalis pbp paralogs for synthetic female o. furnacalis and o. nubilalis pheromones, and mutagenized four amino acid residues predicted to coordinate e12- and z12-14:oac pheromones within the hydrophobic binding pocket. the data presented in this study are important for understanding the functional residues that are involved in effecter binding by ostrinia pbp but does not attempt to address the evolutionary changes that impart divergent function between o. furacalis compared to o. nubilalis pbps. regardless, the data presented here are important for understanding the residues involved in general sex pheromone binding, but additional research is required to investigate any residues involved in the selectivity of ofurpbp compared to onpbp materials rna extraction and cdna synthesis a laboratory colony of o. furnacalis was maintained at the institute of plant protection, chinese academy of agricultural sciences. pupae were allowed to emerge in a netted cage at room temperature, and moths were fed a 10% honey solution. antennae were dissected from three day old male moths, immediately frozen in liquid nitrogen and stored at −80 °c prior to use. total rna was extracted from o. furnacalis antennae using trizol™ reagent according to manufacturer instructions. rna was then reverse transcribed into 1st strand cdnas in a reaction primed with an oligodt oligonucleotide, and synthesized with avian myeloblastosis virus reverse transcriptase at 40 °c for 1 h. the reaction was stopped by incubation at 95 °c for 5 min and then cooling on ice for 20 min. the cdna sequences for five asian corn borer pbp genes were previously reported by allen and wanner, and were used to design primer pairs that amplified the in frame mature gene coding sequence for each gene. each primer incorporated a novel 5-prime overhang with palindromic sites for cleavage by bamhi or hindiii, which allowed for subsequent directional cloning into the restriction endonuclease sites of the multicloning site in the pet30a expression vector. briefly, pcr primers for the amplification of mature ofurpbp1-ofurpbp coding sequences were designed using primer with a minimum length of nucleotides and a predicted melting temperature of ≥58 °c. the 5-prime extensions that encompassed the bamhi preceding the n-terminal cds and hindiii site following the c-terminal coding region were inserted manually, the details of the primers was shown in additional file 1: s the subsequent pcr reactions used 5 pmol of each primer used  μl of cdna template diluted 1: with the high fidelity polymerase. thermal cycling was performed on a biometra t-gradient thermocycler under the following conditions: 94 °c for 2 min 30 s, cycles of 94 °c for 30 s, 58 °c for 30 s, and 72 °c for 1 min, and 72 °c for 10 min. an aliquot of each pcr product was separated on a % agarose gel containing  μg ml− ethidium bromides and visualized under uv illumination. pcr products were cloned into the pgem-t vector, and single clones were obtained after selection on lb agar plates containing 50 μg ml− ampicillin. for each ofurpbp clone, plasmid dna isolated and sequenced from the t primer binding site at beijing sinogenomax co., ltd on an abi 3730xl dna analyzer. electropherogram data were compared to cdna sequences of respective pbps in genbank accessions gu to gu previously published by using vector nti software. independent multiple sequence alignments were made using ofurpbp to ofurpbp sequences generated in this study, o. furnacalis, o. nubilalis e-strain, and o. nubilalis z-strain using the clustal w algorithm. prokaryotic expression and purification the pgem plasmid inserts were excised using bamhi and hindiii double digestion, excised from a 1% agarose gel and fragments purified using nucleic acid purification kit. the bamhi and hindiii digestion products for ofurpbp to ofurpbp were then ligated individually into bamhi and hindiii cut and dephosphorylated pet30a vector by incubation with t ligase for 4 h at 16 °c. recombinant pet30a vectors containing inserts derived from the five pheromone binding proteins were used to transform bl escherichia coli cells by electroporation, and cells allowed to recover in soc medium for 1 h at 37 °c. transformed cells were selected by spread plating on lb agar plates containing 25 μg ml− kanamycin sulfates as described by the manufacturer. positive clones were cultured, and plasmid dna isolated and inserts sequenced using the t primer as described above. positive pet30a clones containing correct insert dna from each of the pbp sequences were incubated separately in fresh liquid lb medium containing 25 μg ml− kanamycin sulfate. cultures were shaken at 37 °c until the optical density of each culture reached to –, where od was measured on a shimadzu spectrophotometer. protein expression was induced in each culture by the addition of isopropyl-β-d-1-thiogalactopyranoside to a final concentration of  mm, followed by incubation on a 150 rpm shaker at 37 °c for 6 h. the cells were harvested by centrifugation at 7500×g for 15 min at 4 °c, chilled on ice, and treated with ultrasonic disruptor. after centrifugation, all five proteins were highly present in inclusion bodies. the protein pellets were suspended using previously published methods. proteins were purified by application of the lysate to ni–nta columns and purified as specified by the manufacturer, followed by digestion of eluted proteins with enterokinase to remove the his tag. protein preparations were desalted by dialysis, quantified by means of the bradford method, and purity verified by sds-page. pheromone-binding affinity initial measures of the binding affinity of the fluorescent ligand n-phenyl-1-naphthylamine to recombinant ofurpbp ofurpbp ofurpbp ofurpbp and ofurpbp was determined by titrating to final concentrations of –  µm 1-npn with 2 µm of each protein in  ml 50 mm tris–hcl, ph and ph. the 1-npn probe was excited at 337 nm, and emission spectra were recorded between and 450 nm on a shimadzu fluorescence spectrophotometer. each trial was run in triplicate, and scatchard plots of the ratio of bound to unbound ligand were created using microsoft excel competitive binding assays were performed with each of the ofurpbps using 1-npn as the probe. assay reactions were comprised of 2 µm of each ofurpbp diluted individually in  ml 20 mm tris–hcl, ph, which were then titrated with  mm increments sex pheromone diluted in methanol to a final concentrations of 1–6 µm. the synthetic standards that correspond to the sex pheromones of o. furnacalis, o. nubilalis and other lepidopteran species were purchased from sigma-aldrich. excitation and emission spectra were recorded from triplicate assays as described above. the binding affinity of each purified ofurpbp to the different sex pheromones was measured by calculation of binding constants via competitive fluorescence-binding assay. in brief, relative fluorescent intensities were analyzed by graphpad prism software and calculation was done using the equation: ki = /, where is the free concentration of 1-npn and k1-npn is the dissociation constant of the complex protein/1-npn. sex pheromone binding affinity with different pbps was analyzed using one-way anova in sas. molecular modeling and ligand docking the amino acids of ofurpbp were submitted to swiss-model tool to automate homology-modeling. the sequence of ofurpbp was compared to all the known protein sequences and that had a high similarity with the target sequence on rcsb protein data bank. the ofurpbp used atrapbp for a homology model. the main pheromone components of o. furnacalis, z12- and e12-14:oac were used respectively in molecular docking with the simulated model of ofurpbp based on the advanced docking program cdocker. molecular dynamics scheme based on charmm was used to dock ligands into a binding site in the cdocker program. total interaction, van der waals, and electrostatic interaction energies were calculated for all ofurpbp residues predicted to be involved in the binding of intraspecific sex pheromone components. site-directed mutagenesis four amino acids of ofurpbp with some of the most negative estimated e total values were mutated using quick-change lightning site-directed mutagenesis kit; ile to asn at position, m phe to ser at position, m ile to asn at position, and m leu to arg at position. the sequence of each primers used to alter the aforementioned sites by nucleotide mismatch are shown in additional file 3: s after initial denaturation at 94 °c for 5 min, the subsequent pcr followed with cycles of 94 °c for 30 s, 56 °c for 30 s, and final extension at 72 °c for 1 min. changes within site-directed mutant fragments were verified by cloning into the pgem-t easy vector by sanger sequencing. all mutated ofurpbps proteins were expressed and used within in vitro binding assays as described above.  rna extraction and cdna synthesis five amplified fragments predicted to contain the mature ofurpbp to ofurpbp gene coding sequences were successfully amplified by rt-pcr, cloned into the pgem vector, and sanger sequenced. multiple nucleotide sequence alignments of these cloned ofurpbp sequences showed 100% similarity with homologous regions of genbank accessions for o. furnacalis pbp to pbp previously identified by allen and wanner. one exception was predicted in a single nucleotide polymorphism at position of ofurpbp which changed a guanine in gu to an adenosine in our cloned ofurpbp sequence. this snp on ofurpbp produced a synonymous mutation, such that the amino acid sequence was unchanged and the same as that reported by allen and wanner. prokaryotic expression and purification five mature ofurpbp gene coding sequences were successfully excised from pgem, and re-inserted into to the pet30a expression vector. end sequencing of inserts from the t primer binding site flanking the pet30a vector multicloning site confirmed the correct orientation and sequence of each ofurpbp insert. each of the iptg induced cultures derived from e. coli pet30a-ofurpbp transformants showed high expression of a his-tagged fusion protein in the approximately 16–19 kda size range, and subsequent purification of each on ni–nta columns successfully removed visible endogenous e. coli proteins. digestion of purified ofurpbps fusion proteins with enterokinase resulted in decreased estimated molecular weights, which were less than the 15–17 kda predicted for full-length lepidopteran pbps containing leader peptides. this was expected since the cloned fragments were devoid of leader peptides and represent fully functional processed peptides. after analysis, most of the proteins were expressed in inclusion bodies. bradford assays estimated the concentration of each purified ofurpbp protein at ~ to 2 μg ml− and extracts were used for the further experiments in this study.fig.  sds-page analysis of recombinant ostrinia furnacalis pheromone binding proteins induced from e. coli clones carrying in frame insertions of ofurpbp ofurpbp ofurpbp ofurpbp or ofurpbp coding sequences in the pet30a expression vector. replicate gel lanes correspond to cell culture prior to addition of isopropyl-β-d-1-thiogalactopyranoside, iptg induced cells at o.d. –, supernatant after ultrasonic treatment and centrifugation, pellet after ultrasonic treatment and centrifugation, ni–nta column purified proteins, and enterokinase digested products with his-tags cleaved. m corresponds to molecular weight marker. a ofurpbp1; b ofurpbp2; c ofurpbp3; d ofurpbp4; e ofurpbp5 pheromone-binding affinity the general affinity of the 1-npn reporter for each ofurpbp was measured, and a regression of these titrated values were plotted within scatchard plots and showed a linear relationship. next, the binding specificity of different ostrinia pheromone components were tested against each recombinant ofurpbp in competitive binding assays with 1-npn as the reporter. the female sex pheromones, z12- and e12-14:oac are produced by o. furnacalis and z11- and e11-14:oac by o. nubilalis. competitive binding assay results showed that z12- and e12-14:oac bound most strongly with ofurpbp, and also showed affinity for ofurpbp and ofurpbp. results also showed that z12-14:oac bound ofurpbp weakly and e12-14:oac had no detectable binding to ofurpbp in contrast, both z11- and e11-14:oac pheromones from o. nubilalis showed relatively high affinities for all five ofurpbps, with estimates of z11-14:oac binding to ofurpbp being the highest for all those that were tested. additionally, the o. nubilalis pheromone e11-14:oac bound strongly to ofurpbp, and showed a significantly greater affinity compared to either z12- and e12-14:oac from o. furnacalis. all five recombinant ofurpbps showed weak or lack of detectable binding to the interspecific lepidopteran sex pheromones, z9-14:oh and e11-14:oh. however, under the ph, none of the ofurpbps showed any binding to the sex pheromones that were tested.fig.  binding curves. a binding curves and scatchard plots for fluorescent probe 1-npn binding with purified ostrinia furnacalis pheromone binding proteins at ph. competitive binding curves for different synthetic sex pheromone hydrocarbons at ph to each of the ofurpbps, b ofurpbp c ofurpbp d ofurpbp e ofurpbp and f ofurpbp the different sex pheromones tested are listed in additional file 2: s2 table  competitive ligand binding assay results among five recombinant native ostrinia furnacalis pbp orthologs tested against six female sex pheromones found among species in the genus ostrinia ic50 all assays performed at ph = . values reported as μm concentrations of each pheromone component ic is the ligand concentration displacing 50% of the fluorescent reporter; ki is the binding constant calculated from the equation ki = / mean ± se, data in the same column followed by the same letters were not significantly different according to lsd test. dashes indicate undetectable interations 3d modeling and docking the ofurpbp was successfully modeling with atrapbp by using of swiss-model tool. this figure visual displayed the 3-dimensional structure of ofurpbp the qmean and gmqe were used to quality estimation, all the score reflected the expected accuracy of ofurpbp built with the alignment and template of atrapbp based on the consideration of hydrophobicity at their binding site, ligand poses and consensus score programs were executed to evaluate binding pose affinities for the residues. subsequently, optimal 3d binding conformations to the main sex pheromone components, z12- and e12-14:oac, were shown in fig. 3c, d. the interaction energies e total, e vdw, and e ele between ofurpbp amino acids that interact with z12- or e12-14:oac were estimated. results indicated that phe and leu interacted most strongly with z12-14:oac. and phe and ile with e12-14:oac.fig.  the predicted 3d structure of ofurpbp of o. furnacalis and docking with sex pheromones. a sequence alignment between ofurpbp and atrapbp and the predicted α-helical secondary structure elements are shown along the top. strictly identical residues are highlighted in white letters with a red background, and are framed in blue residues are chemically identical or similar. residues with similar physico-chemical properties are shown in red letters. the key amino acids used to mutated were marked by black triangle. b the predicted 3d structure of ofurpbp in silico predicted molecular docking between z12-14:oac and e12-14:oac with ofurpbp are shown with respective pheromone components in red interacting amino acids are in yellow table  interaction energies between ofurpbp amino acid residues predicted to interact with z12- and e12-14:oac e total e vdw e eie e total e vdw e eie phe12a ile52a leu94a ile113a e total: total interaction energy; e vdw: van der waals energy; e ele: electrostatic interaction energy aresidues chosen for site-directed mutagenesis site-directed mutagenesis based on the molecular modeling and docking results four amino acid residues with among the highest e total were chosen, and in vitro mutagenesis altered ofurpbp mutants f12s, i52n, i94r, and i113n verified from sanger sequencing results. subsequent competitive binding assays demonstrated that all ofurpbp mutants showed reduced affinities towards z11- and e11-14:oac and z12- and e12-14:oac compared to the wild-type ofurpbp among the mutants, i113n and f12s had the least dramatic effect on affinity towards z12-14:oac. surprisingly i113n showed a nearly -fold reduction in z11-14:oac binding compared to the wildtype, and represented the greatest degree of change among all the assays. all mutated ofurpbps showed the capacity to bind the sex pheromones components of other lepidopteran females, z9-14:oh and e11-14:oh, compared to the wildtype which failed to demonstrate this function. in particular, i113n demonstrated the ability to bind z9:14-oh, which is a known antagonist of male response to e11- and z11-14:oac as well as e12- and z12-14:oac.table  competitive ligand binding assay results from four ostrinia furnacalis pbp mutants generated by site-directed mutagenesis of ile to asn, phe to ser, ile to asn, and ile to arg mutant and wildtype ofurpbp proteins were assayed against six female sex pheromones found among species in the genus ostrinia. all assays performed at ph = . values reported as μm concentrations of each pheromone component ic is the ligand concentration displacing 50% of the fluorescent reporter; ki is the binding constant calculated from the equation ki = / mean ± se, date in the same column followed by the same letters were not significantly different according to lsd test. dashes indicate undetectable interations discussion we showed that the five recombinant ofurpbps have a range of binding affinities with synthetic o. furnacalis sex pheromones, and that ofurpbp and ofurpbp bind z12- and e12-14:oac with the greatest affinity. these binding results might suggest that female o. furnacalis pheromones are bound strongly to male-antennal expressed ofurpbp and ofurpbp in vivo, and that these two chaperones could play a role in the specific transport of z12- and e12-14:oac within the sensillar lymph. the highly biased expression of ofurpbp and ofurpbp transcripts in the ostrinia male compared to female antennae has been used to suggest possible roles in the binding of female sex pheromones, and the latter hypothesis was corroborated for the first time by our data. the correlation between pbp expression level and molecular function was further corroborated by ofurpbp and ofurpbp wherein both show female biased expression and have weak or undetectable binding affinities towards e12- and z12-14:oac. a difference in pheromone binding affinity has been previously shown for pbp from a. polyphemus and a. pernyi which were both bound strongly by e z-14:ac, but pbp and pbp from these species preferentially bound e z-16:ald and e z-14:ac, respectively. analogously, pbp and pbp from lymantria dispar male antennae respectively bind specifically to and enantiomers of the pheromone released by conspecific females, which suggest that pbp gene family members can develop variant molecular functions and that our observed differences between ofurpbp and ofurpbp compared to ofurpbp and ofurpbp may not be surprising. furthermore, the binding affinities of ofurpbp ofurpbp and ofurpbp to cognate pheromone components estimated in this study are comparable to analogous ki values estimated for helicoverpa armigera pbps and h. assulta to their corresponding sex pheromone. similarly, the binding affinities of the cockroach leucophaea maderae pbp, pbplma, to their sex pheromones 3-hydroxy-butan-2-on or butane- -diol were and  μm, respectively. in contrast to predictions made by allan and wanner, ofurpbp did not show specific affinity towards z12- and e12-14:oac, but instead demonstrated a nearly equal affinity for z11- and e11-14:oac emitted by o. nubilalis females. moreover, all ofurpbps showed a comparatively strong affinity towards z11- and e11-14:oac, including observation of strong binding by ofurpbp and ofurpbp these ligand affinities are in direct opposition to species responses, which might suggest that ofurpbps bind to z11- and e11-14:oac does not preclude a role in male sexual response, but only in potential for binding these components within the sensillar lymph. transcripts encoding pbps have previously been found in non-pheromone sensitive female antennae and male sensilla, which suggests that pbps may have evolved alternate roles such as host plant recognition. this notion was reinforced by evidence that compounds structurally similar to cognate pheromones can be bound by bombyx mori pbp, and further by observations that of sex pheromones and plant volatiles can interact within the same peripheral sensory pathways. this altogether suggests that binding compounds or interspecific pheromone components to a pbp does not preclude subsequent interaction with the orco + orx complex resulting in sexual response. grosse-wilde et al. showed that pheromone agonists bound to different pbp paralogs and lead to different responses by male sexually-responding orco − orx receptors, such that members of the pbp gene family may have evolved specific interactions at different orco − orx complexes. the o. furnacalis or was shown to specifically respond to z12-14:oac and e12-14:oac. the selectivity of pbp for delivery of these pheromone components to ofor was not investigated in this or any other previous studies, but may provide crucial information regarding the evolution of species-specific male sexual response. regardless, our results are the first to demonstrate that male antennal expressed ofurpbp and are capable of binding the intraspecific female pheromone components z12-14:oac and e21-14:oac. a change in the 3-dimensional conformation of pbps occurs following a shift from neutral to acidic ph environments, and is associated with binding and release of cognate pheromone ligands. crystal structures of pheromone-bound pbps indicated that the c-terminal amino acid sequence remains linear under neutral ph, whereas this c-terminal region forms an alpha-helix in acidic conditions. the more linear conformation with alpha helices was used to predict the 3d structure of ofurpbp, wherein residues within the hydrophobic binding pocket were predicted to interact with z12-14:oac and e21-14:oac. this ph-dependent change is also accompanied by alteration of pbp 3-dimensional structure where salt bridges are formed between two asp residues on the c-terminal α helix and two corresponding deprotonated his residues in the pheromone binding pocket at low ph. thus, the ph-dependent alpha-helix interactions with the pheromone binding pocket competes with sex pheromone ligands, and is a key component for pbp pheromone carrier and delivery functions. specifically, neutral ph in sensillar lymph may place pbps into a competent pheromone-binding state, where these chaperones are capable of transporting hydrophobic pheromone hydrocarbons. residues of ofurpbp potentially involved during the binding of intraspecific pheromones at neutral ph binding are important for understanding function within a species, and the e total derived from cdocker predicted the key amino acids which may interact with z12- and e12-14:oac ligands. predictions from homology-based in silico structural modeling can be unreliable and translation into biological systems can be bolstered by experimental validation. thus, site-directed mutagenizes of four residues with the highest e total values were made in four separate mutant ofurpbps. comparisons between wildtype and mutant ofurpbps clearly demonstrated the negative affects on binding affinities following change of native residues to those with different polar properties and suggest that phe ile ile and ile are important for the efficient binding of cognate sex pheromones. interestingly, after mutation, the ofurpbps showed weak binding affinity towards other lepidopteran female sex pheromone, z9-14:oh and e11-14:oh, that was not observed with the wild type ofurpbp these differences may occur either through direct interaction with the bound hydrocarbons, or by stabilization of the hydrophobic binding pocket. the affects of induced amino acid changes on the overall 3d structure and function of a protein remain largely unknown. this suggests that even single amino acid changes might alter pbp conformation in unforeseen ways, such that further research, such as using x-ray or nmr to analyze the structure of native and mutant binding proteins are likely required. although our current study demonstrated the ability of ofurpbps to bind inter and intraspecific sex pheromones, further research is likely required to fully understand the structural, chemical, and environmental factors that may influence any specific interactions between pbps and their cognate sex pheromones.  our research provides crucial functional information regarding the specificity of pbps in an ostrinia moth species, which is a group that has emerged as a model for the study of sexual communication systems in lepidoptera. this study provides the first biochemical evidence that ofurpbp and ofurpbp bind z12- and e12-14:oac with high affinity. moreover, this current work demonstrates that despite prior evidence for directional selection and predicted divergent function between onpbp and ofurpbp the latter shows a nearly equal affinity to o. furnacalis female z12- and e12-14:oac and o. nubilalis z11- and e11-14:oac. the four amino acids, ser asn arg and asn may play a role in binding of sex pheromone of pbp but cannot be interpreted in the context of species-specific binding since we only mutagenized sites predicted to be important in protein interactions with pheromone ligands. our results suggest that overall individual o. furnacalis pbps may be capable of equivalent binding of both intra- and inter-specific female sex pheromone components in vitro, and therefore could suggest that molecular discrimination in male response might reside within the behavioral pathway. additional research is undoubtedly required to both dissect the molecular functions of pbps in concert with cognate ors with respect to ligand binding as well as the mechanisms that transmit neuronal signals which result in differential male responses to female emitted pheromones. additional files additional file 1: s the primers used for the pbps expression. the lists of primers used for pbps expression, and the digestion sites were underlined. additional file 2: s the message of sex pheromones used in binding assay. the sex pheromones messages used in binding experiment, including the molecular weight, purity and company. additional file 3: s the primers used for site-directed mutants. the lists of primers used for site-directed mutants, and the digestion sites were underlined. additional file 4: s the sequences of the wild type ofurpbp and the mutants. the nucleotide sequences of wild type ofurpbp and the mutants. tiantao zhang and yaqi sun contributed equally to this work authors’ contributions ttz, klh and zyw designed and conducted the research experiments. ttz, kww and bsc interpreted the results and drafted the manuscript. ttz and yqs are the principal investigator who designed the experiment, analyzed the data and critically reviewed the manuscript. all authors read and approved the final manuscript. 
these four amino acids may play a role in binding of sex pheromone, but this study does not address questions regarding specific response between males of o. furnacalis and o. nubilalis. directional selection was previously predicted between pbp orthologs of ostrinia furnacalis and ostrinia nubilalis were interpreted as being involved in sexual isolation. in vitro assays show that recombinant male ofurpbp bound o. furnacalis sex pheromones, z-12-tetradecenyl acetate and e-12-tetradecenyl acetate, as well as to ecb pheromones z11- and e11-14:oac. five pbps of o. furnacalis play important functions in ostrinia pheromones binding. additional studies are required determine the role, if any, pbps play in the evolution of sex pheromone communication. pheromone binding proteinbindingsex pheromonedockingmutanthttp://dx.doi.org/ /501100001809national natural science foundation of china31201753zhang tiantao special fund for agro-scientific research in the public interest201303026wang zhenying united states department of agriculture, agricultural research service3625-22000-017-00coates brad s. the iowa agriculture and home economics experiment station, ames, ia3543coates brad s. issue-copyright-statement© the author 2017
 pheromone binding proteins of male lepidoptera function in chemical communication, mate attraction and recognition. directional selection was previously predicted between pbp orthologs of ostrinia furnacalis and ostrinia nubilalis were interpreted as being involved in sexual isolation.  in vitro assays show that recombinant male ofurpbp bound o. furnacalis sex pheromones, z-12-tetradecenyl acetate and e-12-tetradecenyl acetate, as well as to ecb pheromones z11- and e11-14:oac. recombinant ofurpbp and ofurpbp bound e11- and z11-14:oac with greater affinity compared to z12- and e12-14:oac, and ofurpbp incapable of binding with e12-14:oac. in silico molecular docking predicted ofurpbp residues phe ile leu ile within a hydrophobic ligand-binding pocket and may participate in e12- and z12-14:oac binding. independent site-directed mutagenesis experiments demonstrated that ser asn arg and asn residues variants caused an approximately - to -fold reduction in ofurpbp affinity for z12- and e12-14:oac, and a - to -fold decrease in affinity towards e11- and z11-14:oac.  five pbps of o. furnacalis play important functions in ostrinia pheromones binding. these four amino acids may play a role in binding of sex pheromone, but this study does not address questions regarding specific response between males of o. furnacalis and o. nubilalis. additional studies are required determine the role, if any, pbps play in the evolution of sex pheromone communication. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords pheromone binding proteinbindingsex pheromonedockingmutanthttp://dx.doi.org/ /501100001809national natural science foundation of china31201753zhang tiantao special fund for agro-scientific research in the public interest201303026wang zhenying united states department of agriculture, agricultural research service3625-22000-017-00coates brad s. the iowa agriculture and home economics experiment station, ames, ia3543coates brad s. issue-copyright-statement© the author 2017 