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Gunde Ziegelberger - One of the best experts on this subject based on the ideXlab platform.

  • Identification of PLCβ and PKC in pheromone receptor neurons of Antheraea polyphemus
    Neuroreport, 2000
    Co-Authors: Rosario Maida, Alexei Redkozubov, Gunde Ziegelberger
    Abstract:

    Two proteins of the IP3 transduction pathway were identified by Western blots in homogenates of isolated pheromone-sensitive sensilla of the silkmoth Antheraea polyphemus. A 110 kDa protein was recognized by an antiserum raised against the Drosophila phospholipase C beta (PLC beta p121) and a 80kDa protein was labelled by an antiserum against a synthetic peptide of a conserved region of protein kinase C (PKC). Incubation of homogenized sensory hairs with the main sex pheromone component, (E,Z) 6-11 hexadecadienyl acetate, resulted in a 6-fold increase in the activity of PKC compared to controls without pheromone. In contrast, incubation with pheromone did not affect the activity of protein kinase A (PKA). Activation of PKC by the membrane permeable dioctanoylglycerol led to excitation of the pheromone-sensitive receptor neurons. These data support the current concept that pheromone perception of moths is mediated by the IP3 transduction pathway.

  • three pheromone binding proteins in olfactory sensilla of the two silkmoth species Antheraea polyphemus and Antheraea pernyi
    FEBS Journal, 2000
    Co-Authors: Rosario Maida, Jurgen Krieger, Therese Gebauer, Ute Lange, Gunde Ziegelberger
    Abstract:

    Females of the sibling silkmoth species Antheraea polyphemus and A. pernyi use the same three sex pheromone components in different ratios to attract conspecific males. Accordingly, the sensory hairs on the antennae of males contain three receptor cells sensitive to each of the pheromone components. In agreement with the number of pheromones used, three different pheromone-binding proteins (PBPs) could be identified in pheromone-sensitive hairs of both species by combining biochemical and molecular cloning techniques. MALDI-TOF MS of sensillum lymph droplets from pheromone-sensitive sensilla trichodea of male A. polyphemus revealed the presence of three major peaks with m/z of 15702, 15752 and 15780 and two minor peaks of m/z 15963 and 15983. In Western blots with four antisera raised against different silkmoth odorant-binding proteins, immunoreactivity was found only with an anti-(Apol PBP) serum. Free-flow IEF, ion-exchange chromatography and Western blot analyses revealed at least three anti-(Apol PBP) immunoreactive proteins with pI values between 4.4 and 4.7. N-Terminal sequencing of these three proteins revealed two proteins (Apol PBP1a and Apol PBP1b) identical in the first 49 amino acids to the already known PBP (Apol PBP1) [Raming, K., Krieger, J. & Breer, H. (1989) FEBS Lett.256, 2215–2218] and a new PBP having only 57% identity with this amino-acid region. Screening of antennal cDNA libraries with an oligonucleotide probe corresponding to the N-terminal end of the new A. polyphemus PBP, led to the discovery of full length clones encoding this protein in A. polyphemus (Apol PBP3) and in A. pernyi (Aper PBP3). By screening the antennal cDNA library of A. polyphemus with a digoxigenin-labelled A. pernyi PBP2 cDNA [Krieger, J., Raming, K. & Breer, H. (1991) Biochim. Biophys. Acta1088, 277–284] a homologous PBP (Apol PBP2) was cloned. Binding studies with the two main pheromone components of A. polyphemus and A. pernyi, the (E,Z)-6,11-hexadecadienyl acetate (AC1) and the (E,Z)-6,11-hexadecadienal (ALD), revealed that in A. polyphemus both Apol PBP1a and the new Apol PBP3 bound the 3H-labelled acetate, whereas no binding of the 3H-labelled aldehyde was found. In A. pernyi two PBPs from sensory hair homogenates showed binding affinity for the AC1 (Aper PBP1) and the ALD (Aper PBP2), respectively.

  • Redox‐Shift of the Pheromone‐Binding Protein in the Silkmoth Antheraea polyphemus
    European journal of biochemistry, 1995
    Co-Authors: Gunde Ziegelberger
    Abstract:

    In pheromone-sensitive hairs of the male silkmoth Antheraea polyphemus, two electrophoretically distinct pheromone-binding proteins (PBPs) are present. They indicate no amino acid sequence diversity according to peptide mapping, but differ in their redox state, as shown by free-sulfhydryl-group-specific cleavage at cysteine residues with 2-nitro-5-thiocyanobenzoic acid. In kinetic studies, the pheromone was initially bound mainly by the reduced PBP but later by the oxidized PBP, where all six cysteine residues form disulfide bonds. This redox shift was observed only in the homogenate of isolated olfactory hairs, where proteins of the sensillum lymph and receptive dendrites are present. In control experiments with purified binding proteins, the proportion of pheromone bound to the oxidized PBP did not increase with increasing incubation time, suggesting that disulfide formation does not occur spontaneously but is mediated by the sensory hairs, possibly by interaction with the receptor cell membrane. These data suggest that arriving hydrophobic pheromone molecules are first bound by the reduced PBP and transported through the aqueous sensillum lymph towards the receptor molecules of the dendritic membrane. The oxidized complex might not be able to activate further receptors and, thus, effectively deactivate the pheromone molecules within the sensillum lymph.

  • redox shift of the pheromone binding protein in the silkmoth Antheraea polyphemus
    FEBS Journal, 1995
    Co-Authors: Gunde Ziegelberger
    Abstract:

    In pheromone-sensitive hairs of the male silkmoth Antheraea polyphemus, two electrophoretically distinct pheromone-binding proteins (PBPs) are present. They indicate no amino acid sequence diversity according to peptide mapping, but differ in their redox state, as shown by free-sulfhydryl-group-specific cleavage at cysteine residues with 2-nitro-5-thiocyanobenzoic acid. In kinetic studies, the pheromone was initially bound mainly by the reduced PBP but later by the oxidized PBP, where all six cysteine residues form disulfide bonds. This redox shift was observed only in the homogenate of isolated olfactory hairs, where proteins of the sensillum lymph and receptive dendrites are present. In control experiments with purified binding proteins, the proportion of pheromone bound to the oxidized PBP did not increase with increasing incubation time, suggesting that disulfide formation does not occur spontaneously but is mediated by the sensory hairs, possibly by interaction with the receptor cell membrane. These data suggest that arriving hydrophobic pheromone molecules are first bound by the reduced PBP and transported through the aqueous sensillum lymph towards the receptor molecules of the dendritic membrane. The oxidized complex might not be able to activate further receptors and, thus, effectively deactivate the pheromone molecules within the sensillum lymph.

  • Esterase activity in the olfactory sensilla of the silkmoth Antheraea polyphemus.
    Neuroreport, 1995
    Co-Authors: Rosario Maida, Gunde Ziegelberger, Karl-ernst Kaissling
    Abstract:

    We studied in individual males of Antheraea polyphemus the activity of the sensillar esterase, a pheromone-degrading enzyme present in the sensillum lymph surrounding the olfactory receptor cells. In parallel, receptor potentials from single pheromone-sensitive sensilla trichodea were recorded. Our screening revealed a large variability of the enzyme activity in individuals with similar electrophysiological responses. In some moths the sensillar esterase was not detectable, i.e. present with 100-fold less activity. However, such variable esterase activity showed no correlation to the time course of the receptor potential. Thus, enzymatic pheromone degradation does not seem to be involved in the rapid pheromone inactivation at the end of the stimulus, but rather serves as the final pheromone sequestration step.

Richard G. Vogt - One of the best experts on this subject based on the ideXlab platform.

  • expression of snmp 1 in olfactory neurons and sensilla of male and female antennae of the silkmoth Antheraea polyphemus
    Cell and Tissue Research, 2001
    Co-Authors: Matthew E Rogers, Rudolf Alexander Steinbrecht, Richard G. Vogt
    Abstract:

    SNMP-1 (sensory neuron membrane protein 1) is an olfactory-specific membrane-bound protein which is homologous with the CD36 receptor family. Previous light level immunocytochemical studies suggested that SNMP-1 was localized in the dendrites and distal cell body of sex-pheromone-specific olfactory receptor neurons (ORN); these studies further suggested SNMP-1 was expressed in only one of two to three neurons in male-specific pheromone-sensitive trichoid sensilla. To better understand the expression and localization of SNMP-1, an immunocytochemical study was performed using electron microscopy to visualize the distribution of SNMP-1 among the neurons of several classes of olfactory sensilla of both male and female antennae of the silkmoth Antheraea polyphemus. SNMP-1 antigenicity was primarily restricted to the receptive dendritic membranes of ORNs of all sensilla types examined and was observed in cytosolic granules, but not plasma membranes, of the cell soma. Mean labeling densities ranged from 1 to 16 gold particles per micrometer of dendrite circumference; dendrites of trichoid and intermediate sensilla showed significantly higher labeling densities than those of basiconic sensilla. Larger dendrites of trichoid sensilla showed significantly higher mean labeling densities (13–16/µm) than smaller diameter dendrites (3–7/µm). Immunofluorescence studies using baculovirus expressed SNMP-1 and multiphoton photon laser scanning microscopy (MPLSM) indicated that rSNMP-1, which was post-translationally processed to the in vivo molecular weight, was inserted into the plasma membrane in a topography presenting extracellular epitopes. These studies suggest SNMP-1 is a common feature of the ORNs, is asymmetrically expressed among functionally distinct neurons, and possesses a topography which permits interaction with components of the extracellular sensillum lymph.

  • snmp 1 a novel membrane protein of olfactory neurons of the silk moth Antheraea polyphemus with homology to the cd36 family of membrane proteins
    Journal of Biological Chemistry, 1997
    Co-Authors: Matthew E Rogers, Michael R. Lerner, Ming Sun, Richard G. Vogt
    Abstract:

    While olfactory neurons of silk moths are well known for their exquisite sensitivity to sex pheromone odorants, molecular mechanisms underlying this sensitivity are poorly understood. In searching for proteins that might support olfactory mechanisms, we characterized the protein profile of olfactory neuron receptor membranes of the wild silk moth Antheraea polyphemus. We have purified and cloned a prominent 67-kDa protein which we have named Snmp-1 (sensory neuron membrane protein-1). Northern blot analysis suggests that Snmp-1 is uniquely expressed in antennal tissue; in situ hybridization and immunocytochemical analyses show that Snmp-1 is expressed in olfactory neurons and that the protein is localized to the cilia, dendrites, and somata but not the axons. Snmp-1 mRNA expression increases significantly 1–2 days before the end of adult development, coincident with the functional maturation of the olfactory system. Sequence analysis suggests Snmp-1 is homologous with the CD36 protein family, a phylogenetically diverse family of receptor-like membrane proteins. CD36 family proteins are characterized as having two transmembrane domains and interacting with proteinaceous ligands; Snmp-1 is the first member of this family identified in nervous tissue. These findings argue that Snmp-1 has an important role in olfaction; possible roles of Snmp-1 in odorant detection are discussed.

Jurgen Krieger - One of the best experts on this subject based on the ideXlab platform.

  • a receptor and binding protein interplay in the detection of a distinct pheromone component in the silkmoth Antheraea polyphemus
    International Journal of Biological Sciences, 2009
    Co-Authors: Maike Forstner, Heinz Breer, Jurgen Krieger
    Abstract:

    Male moths respond to conspecific female-released pheromones with remarkable sensitivity and specificity, due to highly specialized chemosensory neurons in their antennae. In Antheraea silkmoths, three types of sensory neurons have been described, each responsive to one of three pheromone components. Since also three different pheromone binding proteins (PBPs) have been identified, the antenna of Antheraea seems to provide a unique model system for detailed analyzes of the interplay between the various elements underlying pheromone reception. Efforts to identify pheromone receptors of Antheraea polyphemus have led to the identification of a candidate pheromone receptor (ApolOR1). This receptor was found predominantly expressed in male antennae, specifically in neurons located beneath pheromone-sensitive sensilla trichodea. The ApolOR1-expressing cells were found to be surrounded by supporting cells co-expressing all three ApolPBPs. The response spectrum of ApolOR1 was assessed by means of calcium imaging using HEK293-cells stably expressing the receptor. It was found that at nanomolar concentrations ApolOR1-cells responded to all three pheromones when the compounds were solubilized by DMSO and also when DMSO was substituted by one of the three PBPs. However, at picomolar concentrations, cells responded only in the presence of the subtype ApolPBP2 and the pheromone (E,Z)-6,11-hexadecadienal. These results are indicative of a specific interplay of a distinct pheromone component with an appropriate binding protein and its related receptor subtype, which may be considered as basis for the remarkable sensitivity and specificity of the pheromone detection system.

  • Species-specific pheromonal compounds induce distinct conformational changes of pheromone binding protein subtypes from Antheraea polyphemus.
    Invertebrate neuroscience : IN, 2002
    Co-Authors: Claudia Mohl, Heinz Breer, Jurgen Krieger
    Abstract:

    We have investigated the structural features of three pheromone binding protein (PBP) subtypes from Antheraea polyphemus and monitored possible changes induced upon interaction with the Antheraea pheromonal compounds 4E,9Z-14:Ac [(E4,Z9)-tetradecadienyl-1-acetate], 6E,11Z-16:Ac [(E6,Z11)-hexadecadienyl-1-acetate], and 6E,11Z-16:Al [(E6,Z11)-hexadecadienal]. Circular dichroism and second derivative UV-difference spectroscopy data demonstrate that the structure of subtype PBP1 significantly changes upon binding of 4E,9Z-14:Ac. The related 6E,11Z-16:Ac was less effective and 6E,11Z-16:Al showed only a small effect. In contrast, in subtype PBP2 pronounced structural changes were only induced by the 6E,11Z-16:Al, and the subtype PBP3 did not show any considerable changes in response to the pheromonal compounds. The UV-spectroscopic data suggest that histidine residues are likely to be involved in the ligand-induced structural changes of the proteins, and this notion was confirmed by site-directed mutagenesis experiments. These results demonstrate that appropriate ligands induce structural changes in PBPs and provide evidence for ligand specificity of these proteins.

  • probing a pheromone binding protein of the silkmoth Antheraea polyphemus by endogenous tryptophan fluorescence
    Insect Biochemistry and Molecular Biology, 2002
    Co-Authors: Stefanie Bette, Heinz Breer, Jurgen Krieger
    Abstract:

    One subtype of the pheromone binding proteins of the silkmoth Antheraea polyphemus (ApolPBP1) has been analysed exploiting the two endogenous tryptophan residues as fluorescent probe. The intrinsic fluorescence exhibited a rather narrow spectrum with a maximum at 336 nm. Site-directed mutagenesis experiments revealed that one of the tryptophan residues (Trp37) is located in a hydrophobic environment whereas Trp127 is more solvent exposed, as was predicted modeling the ApolPBP1 sequence on the proposed structure of the Bombyx mori pheromone binding protein. Monitoring the interaction of ApolPBP1 as well as its Trp mutants with the three species-specific pheromone compounds by recording the endogenous fluorescence emission revealed profound differences; whereas (E6,Z11)-hexadecadienal induced a dose-dependent quenching of the fluorescence, both (E6,Z11)-hexadecadienyl-1-acetate and (E4,Z9)-tetradecadienyl-1-acetate elicited an augmentation of the endogenous fluorescence. These data indicate that although ApolPBP1 can bind all three pheromones, there are substantial differences concerning their interaction with the protein, which may have important functional implications.

  • revisiting the specificity of mamestra brassicaeand Antheraea polyphemus pheromone binding proteins with a fluorescence binding assay
    Journal of Biological Chemistry, 2001
    Co-Authors: Valerie Campanacci, Heinz Breer, Jurgen Krieger, Stefanie Bette, James N Sturgis, Audrey Lartigue, Christian Cambillau, Mariella Tegoni
    Abstract:

    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.

  • revisiting the specificity of mamestra brassicae and Antheraea polyphemus pheromone binding proteins with a fluorescence binding assay
    Journal of Biological Chemistry, 2001
    Co-Authors: Valerie Campanacci, Heinz Breer, Jurgen Krieger, Stefanie Bette, James N Sturgis, Audrey Lartigue, Christian Cambillau, Mariella Tegoni
    Abstract:

    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.

Matthew E Rogers - One of the best experts on this subject based on the ideXlab platform.

  • expression of snmp 1 in olfactory neurons and sensilla of male and female antennae of the silkmoth Antheraea polyphemus
    Cell and Tissue Research, 2001
    Co-Authors: Matthew E Rogers, Rudolf Alexander Steinbrecht, Richard G. Vogt
    Abstract:

    SNMP-1 (sensory neuron membrane protein 1) is an olfactory-specific membrane-bound protein which is homologous with the CD36 receptor family. Previous light level immunocytochemical studies suggested that SNMP-1 was localized in the dendrites and distal cell body of sex-pheromone-specific olfactory receptor neurons (ORN); these studies further suggested SNMP-1 was expressed in only one of two to three neurons in male-specific pheromone-sensitive trichoid sensilla. To better understand the expression and localization of SNMP-1, an immunocytochemical study was performed using electron microscopy to visualize the distribution of SNMP-1 among the neurons of several classes of olfactory sensilla of both male and female antennae of the silkmoth Antheraea polyphemus. SNMP-1 antigenicity was primarily restricted to the receptive dendritic membranes of ORNs of all sensilla types examined and was observed in cytosolic granules, but not plasma membranes, of the cell soma. Mean labeling densities ranged from 1 to 16 gold particles per micrometer of dendrite circumference; dendrites of trichoid and intermediate sensilla showed significantly higher labeling densities than those of basiconic sensilla. Larger dendrites of trichoid sensilla showed significantly higher mean labeling densities (13–16/µm) than smaller diameter dendrites (3–7/µm). Immunofluorescence studies using baculovirus expressed SNMP-1 and multiphoton photon laser scanning microscopy (MPLSM) indicated that rSNMP-1, which was post-translationally processed to the in vivo molecular weight, was inserted into the plasma membrane in a topography presenting extracellular epitopes. These studies suggest SNMP-1 is a common feature of the ORNs, is asymmetrically expressed among functionally distinct neurons, and possesses a topography which permits interaction with components of the extracellular sensillum lymph.

  • snmp 1 a novel membrane protein of olfactory neurons of the silk moth Antheraea polyphemus with homology to the cd36 family of membrane proteins
    Journal of Biological Chemistry, 1997
    Co-Authors: Matthew E Rogers, Michael R. Lerner, Ming Sun, Richard G. Vogt
    Abstract:

    While olfactory neurons of silk moths are well known for their exquisite sensitivity to sex pheromone odorants, molecular mechanisms underlying this sensitivity are poorly understood. In searching for proteins that might support olfactory mechanisms, we characterized the protein profile of olfactory neuron receptor membranes of the wild silk moth Antheraea polyphemus. We have purified and cloned a prominent 67-kDa protein which we have named Snmp-1 (sensory neuron membrane protein-1). Northern blot analysis suggests that Snmp-1 is uniquely expressed in antennal tissue; in situ hybridization and immunocytochemical analyses show that Snmp-1 is expressed in olfactory neurons and that the protein is localized to the cilia, dendrites, and somata but not the axons. Snmp-1 mRNA expression increases significantly 1–2 days before the end of adult development, coincident with the functional maturation of the olfactory system. Sequence analysis suggests Snmp-1 is homologous with the CD36 protein family, a phylogenetically diverse family of receptor-like membrane proteins. CD36 family proteins are characterized as having two transmembrane domains and interacting with proteinaceous ligands; Snmp-1 is the first member of this family identified in nervous tissue. These findings argue that Snmp-1 has an important role in olfaction; possible roles of Snmp-1 in odorant detection are discussed.

Smita Mohanty - One of the best experts on this subject based on the ideXlab platform.

  • Pheromone Perception: Mechanism of the Reversible Coil-Helix Transition in Antheraea polyphemus Pheromone-Binding Protein 1.
    Biochemistry, 2019
    Co-Authors: Suman Mazumder, Bharat P. Chaudhary, Salik R. Dahal, Omar Al-danoon, Smita Mohanty
    Abstract:

    Pheromone-binding protein (PBP) in male moth antennae transports pheromone to the olfactory receptor neuron by undergoing a pH-dependent conformational switch, from PBPB at higher pH to PBPA at lower pH, associated with ligand binding and release, respectively. The characteristic feature of the dramatic protein switch is the pH-dependent reversible coil-helix transition of the C-terminus. In the PBPB conformation at pH >6.0, the C-terminus is exposed to the solvent as a coil while the ligand occupies the hydrophobic pocket. However, in the PBPA conformation at acidic pH, the C-terminus switches to a helix and releases the ligand by outcompeting it for the hydrophobic pocket. In Antheraea polyphemus PBP1 (ApolPBP1), the C-terminus (P129-V142) is composed predominantly of hydrophobic residues except for three strategically located acidic residues: Asp132, Glu137, and Glu141. Here, we report for the first time on the consequences of the mutation of one or more acidic residues in the pH-driven reversible coil-helix transition of the ApolPBP1 C-terminus through biophysical characterization. Mutation of any single acidic residue in the C-terminus to its neutral counterpart destabilizes the helix formation at lower pH; these mutants exist as a mixture of both conformations. However, mutation of the two terminal acidic residues together knocks out the protein switch and adversely affects both ligand binding and release functions. Thus, these mutant proteins remain in the open (PBPB) conformation at all pH levels.

  • Structural insights into the ligand binding and releasing mechanism of Antheraea polyphemus pheromone-binding protein 1: role of the C-terminal tail.
    Biokhimiia (Moscow Russia), 2013
    Co-Authors: Uma V. Katre, Suman Mazumder, Smita Mohanty
    Abstract:

    Pheromone-binding proteins (PBPs) in lepidopteran moths selectively transport the hydrophobic pheromone molecules across the sensillar lymph to trigger the neuronal response. Moth PBPs are known to bind ligand at physiological pH and release it at acidic pH while undergoing a conformational change. Two molecular switches are considered to play a role in this mechanism: (i) protonation of His70 and His95 situated at one end of binding pocket and (ii) switch of the unstructured C-terminus at the other end of the binding pocket to a helix that enters the pocket. We have reported previously the role of the histidine-driven switch in ligand release for Antheraea polyphemus PBP1 (ApolPBP1). Here we show that the C-terminus plays a role in the ligand release and binding mechanism of ApolPBP1. The C-terminus truncated mutants of ApolPBP1 (ApolPBP1ΔP129–V142 and ApolPBP1H70A/H95AΔP129–V142) exist only in the bound conformation at all pH levels, and they fail to undergo pH- or ligand-dependent conformational switch...

  • ligand binding turns moth pheromone binding protein into a ph sensor effect on the Antheraea polyphemus pbp1 conformation
    Journal of Biological Chemistry, 2009
    Co-Authors: Uma V. Katre, Suman Mazumder, Rabi K Prusti, Smita Mohanty
    Abstract:

    In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the “open” (bound) conformation at high pH but switches to the “closed” (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH-induced conformational switch of PBPs hold true only for the ligand-bound open conformation of the protein. Binding of various ligands to delipidated ApolPBP1 studied by solution NMR revealed that the protein in the closed conformation switches to the open conformation only at or above pH 6.0 with a protein to ligand stoichiometry of ∼1:1. Mutation of His70 and His95 to alanine drives the equilibrium toward the open conformation even at low pH for the ligand-bound protein by eliminating the histidine-dependent pH-induced conformational switch. Thus, the delipidated double mutant can bind ligand even at low pH in contrast to the wild type protein as revealed by fluorescence competitive displacement assay using 1-aminoanthracene and solution NMR.

  • the solution nmr structure of Antheraea polyphemus pbp provides new insight into pheromone recognition by pheromone binding proteins
    Journal of Molecular Biology, 2004
    Co-Authors: Smita Mohanty, Sergey Zubkov, Angela M Gronenborn
    Abstract:

    Pheromone-binding proteins (PBPs) located in the antennae of male moth species play an important role in olfaction. They are carrier proteins, believed to transport volatile hydrophobic pheromone molecules across the aqueous sensillar lymph to the membrane-bound G protein-coupled olfactory receptor proteins. The roles of PBPs in molecular recognition and the mechanisms of pheromone binding and release are poorly understood. Here, we report the NMR structure of a PBP from the giant silk moth Antheraea polyphemus. This is the first structure of a PBP with specific acetate-binding function in vivo. The protein consists of nine a-helices: a1a (residues 2 ‐ 5), a1b (8 ‐12), a1c (16 ‐23), a2 (27 ‐ 34), a3a (46 ‐ 52), a3b (54 ‐59), a4 (70 ‐79), a5 (84 ‐ 100) and a6 (107 ‐125), held together by three disulfide bridges: 19‐ 54, 50 ‐108 and 97 ‐117. A large hydrophobic cavity is located inside the protein, lined with side-chains from all nine helices. The acetate-binding site is located at the narrow end of the cavity formed by the helices a3b and a4. The pheromone can enter this cavity through an opening between the helix a1a, the C-terminal end of the helix a6, and the loop between a2 and a3a. We suggest that Trp37 may play an important role in the initial interaction with the ligand. Our analysis also shows that Asn53 plays the key role in recognition of acetate pheromones specifically, while Phe12, Phe36, Trp37, Phe76, and Phe118 are responsible for non-specific binding, and Leu8 and Ser9 may play a role in ligand chain length recognition.

  • pheromone binding protein from the silk moth Antheraea polyphemus (ApolPBP)
    2003
    Co-Authors: Smita Mohanty, Sergey Zubkov
    Abstract:

    Smita Mohantya,∗, Sergey Zubkova & Ramon Campos-Olivasb,c aDepartment of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, NY 11794, U.S.A.; bLaboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A.; cStructural and Computational Biology Program, Centro Nacional de Investigaciones Oncologicas, Madrid 28029, Spain