Putative ligand binding sites of two functionally characterized bark beetle odorant receptors

Abstract Background Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. Results We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. Conclusions The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.

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Putative ligand binding sites of two functionally characterized bark beetle odorant receptors

10.1101/2020.03.07.980797 ◽

2020 ◽

Cited By ~ 4

Author(s):

Jothi K. Yuvaraj ◽

Rebecca E. Roberts ◽

Yonathan Sonntag ◽

Xiaoqing Hou ◽

Ewald Grosse-Wilde ◽

...

Keyword(s):

Bark Beetle ◽

Pest Control ◽

Binding Sites ◽

Functional Characterization ◽

Biological Significance ◽

Odorant Receptors ◽

Spruce Bark ◽

Ligand Binding Sites ◽

The Common

AbstractBark beetle behavior is to a large extent mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control during outbreaks. Such an approach requires information on the function of receptors and their interactions with ligands. Hence, we annotated 73 ORs from an antennal transcriptome of the spruce bark beetle Ips typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are selective for single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. We use homology modeling and molecular docking to predict their binding sites. The importance of residues Tyr84 and Thr205 in ItypOR46 in the activation by ipsenol is experimentally supported, and hydrogen bonding appears key in pheromone binding. The biological significance of the characterized ORs positions them as prime targets for pest control and use in biosensors to detect bark beetle infestations.

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Functional evolution of a bark beetle odorant receptor clade detecting monoterpenoids of different ecological origins

10.1101/2020.12.28.424525 ◽

2020 ◽

Author(s):

Xiao-Qing Hou ◽

Jothi Kumar Yuvaraj ◽

Rebecca E. Roberts ◽

C. Rikard Unelius ◽

Christer Löfstedt ◽

...

Keyword(s):

Bark Beetle ◽

Odorant Receptor ◽

Behavioral Responses ◽

Ips Typographus ◽

Odorant Receptors ◽

Functional Evolution ◽

Host Trees ◽

Spruce Bark ◽

Species Specific ◽

Insight Into

AbstractInsects detect odors using an array of odorant receptors (ORs), which may expand through gene duplication. How specificities evolve and new functions arise in related ORs within a species remain poorly investigated. We addressed this question by functionally characterizing ORs from the Eurasian spruce bark beetle Ips typographus, in which antennal detection and behavioral responses to pheromones, volatiles from host and non-host trees, and fungal symbionts are well described. In contrast, knowledge of OR function is restricted to two receptors detecting the pheromone compounds (S)-(–)-ipsenol (ItypOR46) and (R)-(–)-ipsdienol (ItypOR49). These receptors belong to a species-specific OR-lineage comprising seven ItypORs. To gain insight into the functional evolution of related ORs, we characterized the five remaining ORs in this clade, using Xenopus oocytes. Two receptors responded primarily to the host tree monoterpenes (+)-3-carene (ItypOR25) and p-cymene (ItypOR27). Two receptors responded to oxygenated monoterpenoids mainly produced by the beetle-associated fungi, with ItypOR23 specific for (+)-trans-(1R,4S)-4-thujanol, and ItypOR29 responding to (+)-isopinocamphone and similar ketones. ItypOR28 responded to the pheromone E-myrcenol from the competitor Ips duplicatus. Overall, the OR responses match well with those of previously characterized olfactory sensory neuron classes except that neurons detecting E-myrcenol have not been identified. Our results showing shared OR-specificity for monoterpenoid compounds is in line with ‘Sensory Drive’ predicting that evolutionarily related ORs are likely to detect structurally similar odorants. However, the variation in functional groups among OR ligands and their diverse ecological origins suggest that neofunctionalization has also taken place during the evolution of this OR-lineage.

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Mapping of ligand binding sites provides insight into the function of factor H-related protein 5

Molecular Immunology ◽

10.1016/j.molimm.2017.06.094 ◽

2017 ◽

Vol 89 ◽

pp. 145

Author(s):

Alexandra Papp ◽

Marcell Cserhalmi ◽

Ádám I. Csincsi ◽

Barbara Uzonyi ◽

David Ermert ◽

...

Keyword(s):

Ligand Binding ◽

Binding Sites ◽

Factor H ◽

Related Protein ◽

Ligand Binding Sites ◽

Insight Into

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Quantifying the cooperative subunit action in a multimeric membrane receptor

Scientific Reports ◽

10.1038/srep20974 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 6

Author(s):

Nisa Wongsamitkul ◽

Vasilica Nache ◽

Thomas Eick ◽

Sabine Hummert ◽

Eckhard Schulz ◽

...

Keyword(s):

Ion Channels ◽

Ligand Binding ◽

Cyclic Nucleotide ◽

Binding Sites ◽

Membrane Receptor ◽

Membrane Receptors ◽

Cooperative Action ◽

The Individual ◽

Insight Into

Abstract In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels.

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Mapping of ligand binding sites gives further insight into FHR-4 function

Molecular Immunology ◽

10.1016/j.molimm.2007.06.196 ◽

2007 ◽

Vol 44 (16) ◽

pp. 3984

Author(s):

Mario Hebecker ◽

Michael Mihlan ◽

Peter F. Zipfel ◽

Mihály Józsi

Keyword(s):

Ligand Binding ◽

Binding Sites ◽

Ligand Binding Sites ◽

Insight Into

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The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding

Biochemical Journal ◽

10.1042/bj20051982 ◽

2006 ◽

Vol 396 (3) ◽

pp. 469-477 ◽

Cited By ~ 27

Author(s):

Wei-I Chou ◽

Tun-Wen Pai ◽

Shi-Hwei Liu ◽

Bor-Kai Hsiung ◽

Margaret D.-T. Chang

Keyword(s):

Ligand Binding ◽

Binding Sites ◽

Catalytic Domain ◽

Rhizopus Oryzae ◽

Structural Information ◽

Molecular Model ◽

Site Directed Mutagenesis ◽

Carbohydrate Binding ◽

Carbohydrate Binding Module ◽

Ligand Binding Sites

The starch-hydrolysing enzyme GA (glucoamylase) from Rhizopus oryzae is a commonly used glycoside hydrolase in industry. It consists of a C-terminal catalytic domain and an N-terminal starch-binding domain, which belong to the CBM21 (carbohydrate-binding module, family 21). In the present study, a molecular model of CBM21 from R. oryzae GA (RoGACBM21) was constructed according to PSSC (progressive secondary structure correlation), modified structure-based sequence alignment, and site-directed mutagenesis was used to identify and characterize potential ligand-binding sites. Our model suggests that RoGACBM21 contains two ligand-binding sites, with Tyr32 and Tyr67 grouped into site I, and Trp47, Tyr83 and Tyr93 grouped into site II. The involvement of these aromatic residues has been validated using chemical modification, UV difference spectroscopy studies, and both qualitative and quantitative binding assays on a series of RoGACBM21 mutants. Our results further reveal that binding sites I and II play distinct roles in ligand binding, the former not only is involved in binding insoluble starch, but also facilitates the binding of RoGACBM21 to long-chain soluble polysaccharides, whereas the latter serves as the major binding site mediating the binding of both soluble polysaccharide and insoluble ligands. In the present study we have for the first time demonstrated that the key ligand-binding residues of RoGACBM21 can be identified and characterized by a combination of novel bioinformatics methodologies in the absence of resolved three-dimensional structural information.

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Bioinformatic and biochemical analysis of the key binding sites of the pheromone binding protein of Cyrtotrachelus buqueti Guerin-Meneville (Coleoptera: Curculionidea)

PeerJ ◽

10.7717/peerj.7818 ◽

2019 ◽

Vol 7 ◽

pp. e7818 ◽

Cited By ~ 3

Author(s):

Hua Yang ◽

Yan-Lin Liu ◽

Yuan-Yuan Tao ◽

Wei Yang ◽

Chun-Ping Yang ◽

...

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Binding Sites ◽

Dibutyl Phthalate ◽

Structural Model ◽

Site Directed Mutagenesis ◽

Economic Losses ◽

Directed Mutagenesis ◽

Wide Range ◽

Cyrtotrachelus Buqueti

The bamboo snout beetle Cyrtotrachelus buqueti is a widely distributed wood-boring pest found in China, and its larvae cause significant economic losses because this beetle targets a wide range of host plants. A potential pest management measure of this beetle involves regulating olfactory chemoreceptors. In the process of olfactory recognition, pheromone-binding proteins (PBPs) play an important role. Homology modeling and molecular docking were conducted in this study for the interaction between CbuqPBP1 and dibutyl phthalate to better understand the relationship between PBP structures and their ligands. Site-directed mutagenesis and binding experiments were combined to identify the binding sites of CbuqPBP1 and to explore its ligand-binding mechanism. The 3D structural model of CbuqPBP1 has six a-helices. Five of these a-helices adopt an antiparallel arrangement to form an internal ligand-binding pocket. When docking dibutyl phthalate within the active site of CbuqPBP1, a CH-π interaction between the benzene ring of dibutyl phthalate and Phe69 was observed, and a weak hydrogen bond formed between the ester carbonyl oxygen and His53. Thus, Phe69 and His53 are predicted to be important residues of CbuqPBP1 involved in ligand recognition. Site-directed mutagenesis and fluorescence assays with a His53Ala CbuqPBP1 mutant showed no affinity toward ligands. Mutation of Phe69 only affected binding of CbuqPBP1 to cedar camphor. Thus, His53 (Between α2 and α3) of CbuqPBP1 appears to be a key binding site residue, and Phe69 (Located at α3) is a very important binding site for particular ligand interactions.

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A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily

Journal of Biological Chemistry ◽

10.1074/jbc.m113.510743 ◽

2013 ◽

Vol 289 (3) ◽

pp. 1377-1387 ◽

Cited By ~ 8

Author(s):

Jagdeep Kaur ◽

Elena Olkhova ◽

Viveka Nand Malviya ◽

Ernst Grell ◽

Hartmut Michel

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Ligand Binding ◽

Isothermal Titration Calorimetry ◽

Functional Characterization ◽

Homology Model ◽

Docking Studies ◽

Site Directed Mutagenesis ◽

Titration Calorimetry ◽

Amino Acid Residues

Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed.

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Interference of pH buffer with Pb2+-peripheral domain interactions: obstacle or opportunity?

10.1101/2019.12.31.892232 ◽

2020 ◽

Author(s):

Sachin Katti ◽

Tatyana I. Igumenova

Keyword(s):

Binding Sites ◽

Metal Ion ◽

Functional Characterization ◽

Valuable Insight ◽

Cellular Environment ◽

Domain Interactions ◽

Ph Buffer ◽

Insight Into ◽

Peripheral Domain

ABSTRACTPb2+ is a xenobiotic metal ion that competes for Ca2+-binding sites in proteins. Using the peripheral Ca2+-sensing domains of Syt1, we show that the chelating pH buffer Bis-Tris enables identification and functional characterization of high-affinity Pb2+ sites that are likely to be targeted by bioavailable Pb2+.Significance to MetallomicsSyt1, a key regulator of Ca2+-evoked neurotransmitter release, is a putative molecular target of Pb2+. We demonstrate that the use of a chelating pH buffer Bis-Tris enables identification of Ca2+-binding sites that would be most susceptible to Pb2+ attack in the cellular environment. In addition, experiments conducted in Bis-Tris revealed the differences between the membrane-binding responses of two Ca2+-sensing domains of Syt1, C2A and C2B. This work advances the understanding of how Pb2+ interacts with multipartite Ca2+-binding sites, and illustrates that conducting the experiments under both chelating and non-chelating conditions could provide valuable insight into the mechanism of metallosensory proteins.

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Reversibly Sampling Conformations and Binding Modes Using Molecular Darting

10.26434/chemrxiv.12670676.v2 ◽

2020 ◽

Author(s):

Samuel C. Gill ◽

David Mobley

Keyword(s):

Monte Carlo ◽

Ligand Binding ◽

Binding Sites ◽

Degrees Of Freedom ◽

Dynamics Simulation ◽

Hiv Integrase ◽

Binding Modes ◽

System A ◽

Multiple Binding ◽

Internal Degrees Of Freedom

<div>Sampling multiple binding modes of a ligand in a single molecular dynamics simulation is difficult. A given ligand may have many internal degrees of freedom, along with many different ways it might orient itself a binding site or across several binding sites, all of which might be separated by large energy barriers. We have developed a novel Monte Carlo move called Molecular Darting (MolDarting) to reversibly sample between predefined binding modes of a ligand. Here, we couple this with nonequilibrium candidate Monte Carlo (NCMC) to improve acceptance of moves.</div><div>We apply this technique to a simple dipeptide system, a ligand binding to T4 Lysozyme L99A, and ligand binding to HIV integrase in order to test this new method. We observe significant increases in acceptance compared to uniformly sampling the internal, and rotational/translational degrees of freedom in these systems.</div>

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