The key amino residues determining binding activities of the odorant

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Bioactive Constituents, Metabolites, and Functions

The key amino residues determining binding activities of the odorant binding protein AlucOBP22 to two host plant terpenoids of Apolygus lucorum Hangwei Liu, hongxia duan, Qi Wang, Yong Xiao, Qian Wang, Qiang Xiao, Liang Sun, and Yongjun Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05975 • Publication Date (Web): 03 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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The key amino residues determining binding activities of the odorant binding protein AlucOBP22

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to two host plant terpenoids of Apolygus lucorum

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Hangwei Liu, †, Hongxia Duan, †, Qi Wang, Yong Xiao, Qian Wang, Qiang Xiao, Liang

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Sun,, *, Yongjun Zhang, *

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 State

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Chinese Academy of Agricultural Sciences, Beijing, 100193, China

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

Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection,

Key Laboratory of Tea Quality and Safety Control, Key Laboratory of Biology, Genetics and

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Breeding of Special Economic Animals and Plants, Ministry of Agriculture, Tea Research

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Institute, Chinese Academy of Agricultural Sciences, Hangzhou, 310008, China

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 College

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†These

of Science, China Agricultural University, Beijing, 100193, China

authors contributed equally to this work.

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* Correspondence:

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Yongjun Zhang, E-mail: [email protected]

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Liang Sun, E-mail: [email protected]

18 19 20 21 22


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ABSTRACT: Odorant binding proteins (OBPs) are considered to be highly expressed at antennae

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sensillum lymph and play crucial roles in detection of insect host plant volatiles. The polyphagous

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mirid bug Apolygus lucorum is one of serious insect pests on many important agricultural crops

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which heavily rely on sophisticated olfaction to locate host plants. Previously, putative OBP genes

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and their tissue-related expression patterns in this pest species have been clarified. In this study,

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we characterized the ligand spectrum and the molecular binding mechanism of the

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antennae-biased AlucOBP22 to host plant volatiles of A. lucorum. Frist, the recombinant

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AlucOBP22 protein was constructed and purified, and its binding affinities to selected host plant

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volatiles were assessed. Two terpenoids, β-ionone and β-caryophyllene, could highly bind to

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AlucOBP22. Next, 3D model prediction indicated that AlucOBP22 employed six α-helices to

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form a typical pocket for ligand accommodation. Molecular docking analysis suggested both

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β-ionone and β-caryophyllene were located at AlucOBP22 pocket with some hydrophobic amino

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acid residues closely to the two chemicals, suggesting hydrophobic interactions might be crucial

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for ligand specific binding. Finally, site-directed mutagenesis combined with fluorescence binding

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assays revealed that mutants of five hydrophobic residues Leu5, Ile40, Met41, Val44 and Met45

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displayed significantly decreased or completely abolished binding affinities to the two ligands.

39

Our findings showed the specific binding characteristic of AlucOBP22 and suggested that

40

hydrophobic residues and their hydrophobic interactions were involved in AlucOBP22 binding to

41

terpenoids, which provided new insights into the molecular interaction mechanisms of hemipteran

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insect OBPs to host plant odors.

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KEYWORDS: Apolygus lucorum, AlucOBP22, plant terpenoids, binding feature, molecular

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docking, site-directed mutagenesis



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Insect miridae covers more than 1200 genera and 11000 described species, and some mirid

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species are of great economic importance for their progressively raised population size. The

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mirid bug Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae) is a destructive phytophagous

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pest of many crops including cotton, green beans, fruit trees and tea plants.1,

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control of this insect pest heavily relies on traditional chemical approaches, promoted the

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potential risk of residue, resistance and resurgence. The development of environmentally

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friendly pest managements could be one of the promising strategies.

INTRODUCTION

2

Nowadays,

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Previous studies have showed that A. lucorum can spatially and temporally switch host

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plants,3, 4 and in this host shift behavior olfaction of bugs plays important roles.5 Therefore, a

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better understanding of these bugs responding to their external plant released volatiles is the

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foundation for the development of effective management strategies. Insect antennae are

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important olfactory organs which contain numerous multiporous sensilla hairs housing odorant

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sensory neurons (OSNs).6, 7 The airborne chemicals initially enter the sensilla cavity through

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sensilla pores and are recognized and bound by odorant binding proteins (OBPs) and odorant

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receptors (ORs) and subsequently activate OSNs and finally trigger behavioral responses like

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foraging, egg-laying, mating and avoiding.8

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Generally, OBPs are predominately expressed in sensilla lymph of insects.9-13 Fluorescence

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competitive binding assays indicated that OBPs especially the antennae-biased expressed

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OBPs can bind to semiochemicals of both host plant released volatiles and insect produced sex

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pheromones.14-18 The convincing evidence of OBPs contribution to odorant detection are


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supported by RNA interference (RNAi) and CRISPR/Cas9 gene editing, which result into

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significant decreased sensitivities or specificities of odorant perceptions across insect different

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species. 19-25

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There is no doubt that OBPs play important roles in binding to odorant molecules,

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however, the detailed mechanism of OBPs specifically interact with odorants remains unclear.

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The OBP crystal structures have been reported in several species, and the modes of ligand

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binding and releasing are correspondingly proposed, such as pH-dependent conformational

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changes and so on.26-29 In addition, the difficulty obtained high-quality diffracting crystals still

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block efforts to the developments of OBPs’ structural biology. The prediction of OBP 3D

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structures combined with site-directed mutagenesis provides an alternative strategy. Homology

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modelling can construct a 3D model of designated protein, and molecular docking analysis

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predict the presence of cavities in ligand binding proteins and the amino acid residues in the

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cavities involved in interacting with odorants. For examples, residues such as Tyr111 in OBP1

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of Holotrichia oblita,30 Lys123 in the OBP7 and Phe12 and Trp37 in PBP1of Helicoverpa

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armigera as well as Lys74 and Pro121 in OBP5 of Adelphocoris lineolatus have been

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demonstrated to play crucial roles in ligand-binding and releasing.31-33

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Previously, almost 38 putative OBPs transcripts of mirid bug A. lucorum were identified in

84

our laboratory. The tissue profile suggested that AlucOBP22 was mainly expressed in adult

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antennae of both sexes,34, 35 indicating its possible chemosensory roles in the foraging behavior of

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A. lucorum. In the current work, we focused on the potential ligands of AlucOBP22 and

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characterized the key binding sites

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competitive binding assays, homology modelling, molecular docking and site-directed

responded to host plant terpenoids by using fluorescence



89

mutagenesis technologies. We proposed AlucOBP22 has a specific binding profile and its primary

90

ligand-binding forces are derived from hydrophobic interactions.

91 92

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Odor Chemicals and Insects.

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Odor chemicals used in this study were purchased from Sigma-Aldrich (St Louis, MO, USA;

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purity > 98%) and were dissolved in high-performance liquid chromatography (HPLC) purity

96

grade methanol.

MATERIALS AND METHODS

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The A. lucorum adults were collected from cotton fields at the Langfang Experimental

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Station of Chinese Academy of Agricultural Sciences (CAAS), Hebei Province (39.53°N,

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116.70°E), China. The bug colony was fed with fresh corn and maintained at 29 ± 1°C, 60 ± 5%

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relative humidity (RH), and 14:10 h light: dark cycle.

101 102

RNA Isolation and cDNA Synthesis.

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The total RNAs were isolated from 500 antennae of both male and female adult bugs using Trizol

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reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The

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spectrophotometer (NanoDropTM1000, Thermo Fisher Scientific, and Waltham, MA, USA) and

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1.2 % agarose electrophoresis were employed to assess the quality of the RNA sample. The

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first-strand cDNA was synthesized using the SuperScriptTM III reverse transcriptase system

108

(Invitrogen).

109 110

Recombinant Expression of AlucOBP22.


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Primers with the restriction enzyme sites NcoI and XhoI were designed using Beacon Designer 7.0

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(Table S1). Full-length cDNA encoding AlucOBP22 was amplified with ExTaq DNA polymerase

113

(TaKaRa, Dalian, China). PCR cycling parameters are 94 ℃ for 4 min, followed by 35 cycles of

114

95℃ for 30 s, 60℃ for 30 s, and 72℃ for 1 min, and a final 10 min elongation step at 72℃.

115

PCR products were checked on 1.5 % agarose gels, and then were cloned into pGEM-T easy

116

vector (Promega, Madison, WI, USA). Positive plasmids were checked by sequencing, and then

117

were digested with NcoI and XhoI. The fragment encoding correct AlucOBP22 was sub-cloned

118

into the bacterial expression vector pET32a(+) (Novagen, Madison, WI, USA), and subsequently

119

transformed into E. coli BL21(DE3) cells. Single colony was incubated overnight in 5mL LB

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broth with 100 mg/mL ampicillin. Then culture was diluted 1:100 with fresh LB medium, and

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incubated at 37℃ for 2–3 h until OD600 achieved 0.6. The β-D-1-thiogalactopyranoside (IPTG)

122

was added to a final concentration of 1 mM. After incubation at 37℃ for 6 h, the bacterial cells

123

were harvested by centrifugation at 7,000 g for 30 min, re-suspended in PBS buffer, and sonicated

124

in ice. After centrifugation at 20,000 g for 30 min, the supernatant was collected and purified

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twice by Ni ion affinity chromatography (GE-Healthcare). The quality of recombinant protein was

126

checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

127

Finally, His tag of target protein was removed by recombinant enterokinase (Novagen).

128 129

Homology modelling and Molecular Docking.

130

The modeling structure of AlucOBP22 (accession number: AMQ76475) was obtained with a

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template of MvicOBP3 (4Z39) using On-line Swiss-model software. The identity between

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AlucOBP22 and MvicOBP3 is 23.17%. Binding cavity was predicted by a ligand mode of



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SYBYL 7.30 software. The molecular conformations of two ligands β-caryophyllene and β-ionone

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were constructed by Sketch mode and optimized using the Tripos force field and

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Gasteiger-Hückel charge. The Surflex-Dock of SYBYL 7.3 was employed to perform the

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molecular docking modeling. The binding cavity was set as "Auto" and the Total Score was used

137

to evaluate the binding affinity between ligand and protein. All molecular modeling between

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AlucOBP22 protein and ligands were conducted on the Silicon Graphics® (SGI) Fuel Workstation

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(Silicon Graphics International Corp., CA, USA).

140 141

The Site-Directed Mutant.

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Six mutants, Leu5Ala (Leucine to alanine at position 5), Ile40Ala (Isoleucine to alanine at

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position40), Met41Ala (Methionine to alanine at position41), Val44Ala (Valine to alanine at

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position44), Met45Ala (Methionine to alanine at position45), Lys70Ala (Lysine to alanine at

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position70) were constructed based on the AlucOBP22 coding sequence, respectively. The first

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five mutants were mutated at target sites that might be involved in AlucOBP22 ligand-binding

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activities, and the Lys70Ala was set up as a negative control. Using pET32a/AlucOBP22 plasmid

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as a template, mutations were generated by employing the Fast Mutagenesis System kit (Beijing

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TransGen Biotech Co., Ltd, Beijing, China). Primers were designed according to the manual of

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the Fast Mutagenesis System kit (Table S1). The correct mutations were checked using

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sequencing verification. All the recombinant mutant proteins were purified similarly as above.

152 153

Fluorescence Competitive Binding Assays.

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All the tested chemicals were selected according to previous reports which suggested they are


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released from host plants of A. lucorum

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performed on a fluorescence spectrometer (F-380, Tianjin, China) in a 1 cm light path quartz

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cuvette with 10 nm slits for both excitation and emission. N-phenyl-1-naphthylamine (1-NPN) as

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the fluorescent probe was excited at 337 nm and emission spectra were recorded between 390 and

159

460 nm. 1-NPN and all compounds were dissolved in HPLC purity-grade methanol with a

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concentration of 1 mM. 2 mM solution of the protein in 50 mM Tris-HCl (pH 7.4) was titrated

161

with aliquots of 1-NPN prepared in advance to final concentrations of 2–20 µM to measure the

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affinity of 1-NPN to the protein. The binding affinities of other chemicals to wild-type protein and

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mutants were measured in competitive binding assays with the protein and 1-NPN at 2 µM in the

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presence of each competitor at 230μM.

36-38

(Table 1). The fluorescence binding assays were

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The fluorescence intensities at the maximum ﬂuorescence emission between 390 and 460 nm

166

were plotted against the free ligand concentration to determine the binding constants. The bound

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chemical was evaluated based on its fluorescence intensity with the assumption that the protein

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was 100 % active with a stoichiometry of 1 : 1 (protein : ligand) saturation. The binding curves

169

were linearized using a Scatchard plot, and the dissociation constants of the competitors were

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calculated from the corresponding IC50 values based on the following equation: Ki = [IC50] / (1 +

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[1-NPN]/K1-NPN), where [1-NPN] is the free concentration of 1-NPN and K1-NPN is the dissociation

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constant of the complex protein / 1-NPN.

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Expression and Purification of AlucOBP22.

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The recombinant AlucOBP22 protein was successfully expressed in vitro. The induced target

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protein was appeared at supernatant. After two rounds of Ni ion affinity chromatography, the well

178

purified recombinant protein was harvested. SDS-PAGE analysis showed that there was a single

RESULTS



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band corresponding to target protein of 15 kDa, which was consistent with the predicted molecular

180

weight of AlucOBP22 (Figure. 1). Subsequently, six mutated proteins were successfully obtained

181

and purified (Figure 1).

182 183

The specific binding of AlucOBP22 to plant terpenoids.

184

The relative affinities of the recombinant AlucOBP22 with 44 candidate odor chemicals were

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evaluated by fluorescence binding assays (Table 1). The dissociation constant of the

186

AlucOBP22/1-NPN complexes was 4.38 ± 0.25 µM. It was found that recombinant AlucOBP22

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could specifically bind to two plant terpenoids, β-ionone and β-caryophyllene, with the Ki of 4.7±

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1.34 µM and 2.43± 0.49 µM, respectively (Table 1).

189 190

AlucOBP22 3D model.

191

Compared to other crystal structures of hemipteran OBPs, OBP3 of Megoura viciae shared higher

192

amino acid identity with AlucOBP22. The root mean square deviation (RMSD) indicated that

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nearly all the residues were located at the rational region (Figure S1). Additionally, the predicted

194

3D structures of AlucOBP22 and MvicOBP3 had higher cover degree and aligned well each other

195

(Figure 2 A, C). Therefore, MvicOBP3 was selected as template protein for homology modeling.

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The predicted 3D model of AlucOBP22 possessed six α-helices, which are respectively located

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between residues Gln2-Gln13 (α1), Thr19-Pro19 (α2), Glu36-Asn49 (α3), His55-Lys70 (α4),

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Lys75-Asp95 (α5) , Gln106-Asn118 (α6), respectively (Figure 2B).

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Molecular docking.


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The molecular docking of AlucOBP22/ligand complex showed that two plant terpenoids, β-ionone

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and β-caryophyllene, were located in the same pocket of AlucOBP22 and there were some strong

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hydrophobic residues surrounding the ligands. Residue such as Leu5, Ile40, Met41, Val44, and

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Met45 in the helixes of 1, 3, and 4 had a short distance and formed solid hydrophobic interactions

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with the ligands. However, some hydrophilic residues such as Lys70 had a far distance and hardly

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formed hydrogen-bone interaction between functional groups of ligands (Figure 3 A and B, Table

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S2).

208 209

Binding affinities of mutants.

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To verify the results of molecular docking and clarified the key amino acids associated to binding

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activities of AlucOBP22 to ligands, we performed site-directed mutant assay and prepared the

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purified AlucOBP22 mutant proteins. The binding constants (Kd) of mutant proteins with 1-NPN

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was 9.76± 0.93µM for Leu5Ala, 1.71± 0.05µM for Ile40Ala, 2.62± 0.61µM for Met41Ala, 6.98±

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0.21µM for Val44Ala, 1.53± 0.48µM for Met45Ala, and 2.85± 0.21µM for Lys70Ala,

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respectively. Compared to the wild-type proteins, four mutants Ile40Ala, Met41Ala, Val44Ala and

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Met45Ala showed significant decreased binding affinities to β-ionone, while two other mutants

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Leu5Ala and Lys70Ala had no remarkable change in binding to β-ionone (Table 2 and Figure 5).

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Four mutants Leu5Ala, Val44Ala, Met41Ala and Ile40Ala showed disabled or decreased binding

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abilities to β-caryophyllene, however, two Met45Ala and Lys70Ala had similar binding affinities

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to the wild-type AlucOBP22 protein (Table 2 and Figure 5).

221 222



DISCUSSION



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In the present study, we characterized the binding activities of AlucOBP22 to host plant volatiles

224

of A. lucorum and identified key amino acid residues that contributed to their binding interactions.

225

Our results suggested that AlucOBP22 specifically bound to two plant terpenoids β-ionone and

226

β-caryophyllene, and the hydrophobic residues such as Leu5, Ile40, Met41, Val44, and Met45

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rather than the hydrophilic residues were strongly involved in the AlucOBP22 binding activities.

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These findings will provide new insights to investigate the selective bindings of hemipteran OBPs

229

and define the potential binding sites of mirid bug OBPs to host plant odors.

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OBPs were thought to be highly expressed in insect chemosensory system and were capable of

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binding to host plant volatiles.39-41 AlucOBP22 was previously demonstrated to be expressed in A.

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lucorum adult antennae of both sexes,34 implying it’s fulfill potential roles in perceiving host plant

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odors of A. lucorum. In this study, we assessed the binding abilities of recombinant AlucOBP22 to

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selected host plant volatiles of A. lucorum. The fluorescence binding assays showed that

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AlucOBP22 specifically bound to β-ionone and β-caryophyllene with high binding affinities (Ki

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below 5 µM). The specific binding behaviors indicated that AlucOBP22 could recognize β-ionone

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and β-caryophyllene from various host volatiles. Our previous study suggested that AlucOBP22

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was one of the most abundant OBPs expressed in female and male antennae.34 The high binding

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affinities of β-ionone and β-caryophyllene to the antennae-biased expressed OBPs of adult A.

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lucorum suggested an essential role of these odor molecules in chemoreception of A. lucorum.

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β-ionone and β-caryophyllene were two important constitutive plant terpenoids. The β-ionone was

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a constitutive floral fragrance of host plants5, especially in the tea which was the early spring host

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plant of A. lucorum2, 36, 42 , while β-caryophyllene was released from cottons, summer host plants

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of A. lucorum.37 Both of the two compounds could elicit significant electrophysiological responses


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of adult A. lucorum,43 indicated that A. lucorum adults could detect these two terpenoids to

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locating host plants. AlucOBP22 would be involved in host plant alternation behavior of A.

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lucorum. Furthermore, AlucOBP22 could be a potential target to design behavioral strategy to

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manage the infestation of bugs.

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Understanding the interaction between olfactory proteins and their ligands is crucial for

250

development of new biological active chemicals to control insect pests. The potential binding sites

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of OBPs are considered to be located at hydrophobic pockets, and the van der Waals interactions,

252

hydrophobic interactions and/or formation of hydrogen bonds between functional groups and

253

hydrophilic residues were be important for ligand-binding.30,

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OBPs, AlucOBP22 showed selective and sensitive binding affinities to two plant terpenoids. Thus,

255

homology modelling and molecular docking were employed to explore this specific

256

ligand-binding feature. We found that strong hydrophobic residues were available to interact with

257

the ligands, whereas the hydrophilic residues were far away from the ligands (Figure 2 and 3).

258

Therefore, we proposed that the hydrophobic interactions rather than the hydrogen-bone

259

interaction were responsible for AlucOBP22 specific binding to host plant terpenoids. First, the

260

key binding sites of AlucOBP22 were comprised of hydrophobic residues that could not form

261

hydrogen-bone interaction with oxygenic functional groups. Next, the wild-type AlucOBP22

262

failed to bind the other plant volatiles of alcohol hydroxyl and carbonyl groups (Table 1). Finally,

263

the replacement of hydrophobic residues Leu5, Ile40, Met41, Val44 and Met45 with Ala disabled

264

the hydrophobic interactions and caused the significant decreased binding affinities or completely

265

abolished binding affinities to β-ionone or β-caryophyllene, however, hydrophilic residues

266

replaced by Ala did not affect the AlucOBP22 bindings to the β-ionone or β-caryophyllene

33, 44


Compared to other insect


267

(Figure 5 and Table 2).

268

In addition, we found that different hydrophobic residues in the binding sites contributed

269

differently to the interactions between the OBP and ligands (Table 2). This distinct importance

270

might be explained by the strength of the hydrophobic interactions and the distance between

271

residues and the ligands: the stronger hydrophobic interactions of the residues and the closer

272

distance with the residues to ligands, the higher binding forces. Notably, for different ligands, the

273

hydrophobic residues involved in binding interactions might be different. Leu5Ala significantly

274

affected the binding activities of AlucOBP22 to β-caryophyllene rather than to β-ionone. By

275

contrast, Met45Ala significantly decreased binding abilities of AlucOBP22 to the β-ionone but not

276

to the β-caryophyllene. Therefore, the molecular mechanisms of the specific binding might depend

277

on the specific interaction between OBPs and distinct ligands.

278

Overall, our results revealed the binding sites of AlucOBP22 in recognition of host plant

279

terpenoids and suggested that hydrophobic interactions are crucial for hemipteran OBP

280

ligand-binding. These findings will lay a foundation to investigate the molecular mechanisms of

281

ligand-OBP interactions in hemipteran mirid species, which further help to develop new biological

282

active analogs for pest management.

283 284

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Supporting Information

286

Table S1 Primers used in this study.

287

Table S2 Distance of residues to ligands

288

Figure S1 The root mean square deviation analysis

ASSOCIATED CONTENT


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

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Hangwei Liu and Hongxia Duan contributed equally to this work.

291

Corresponding Authors

292

Yongjun Zhang, E-mail: [email protected]

293

Liang Sun, E-mail: [email protected]

294

ORCID

295

Yongjun Zhang: 0000-0003-4276-9072

296

Notes

297

The authors declare no competing financial interest.

AUTHOR INFORMATION

298 299

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300

This work was supported by the National Natural Science Foundation of China (31772176,

301

31772207, 31672038 and 31621064), the Central public-interest Scientiﬁc Institution Basal

302

Research Fund (1610212018010), the Young Elite Scientist Sponsorship Program by CAST

303

(2018QNRC001),Research Foundation of State Key Laboratory for Biology of Plant Diseases and

304

Insect Pests (SKLOF201719).

ACKNOWLEDGEMENTS

305 306 307 308 309 310



311

FIGURE AND TABLE LEGENDS

312 313

Figure 1 SDS-PAGE analysis of recombinant target proteins. M, Molecular weight marker; 1,

314

Non-induced PET32a / AlucOBP22; 2, Induced Crude PET32a / AlucOBP22; 3, Supernatant of

315

PET32a / AlucOBP22; 4, Inclusion bodies of PET32a / AlucOBP22 ; 5, Purified wild-type

316

AlucOBP22 protein; 6-12, Purified mutants of Leu5Ala, Ile40Ala, Met41Ala, Val44Ala,

317

Met45Ala, and Lys70Ala, respectively.


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Figure 2 Homology modeling of AlucOBP22. A, Sequence alignment between AlucOBP22 and

320

MvicOBP3. The a-helices are displayed as squiggles, and strictly identical residues are highlighted

321

in white letters with a red background, and residues with similar physico-chemical properties are

322

shown in red letters. B, 3D structure of AlucOBP22, the six a-helices are labeled with yellow. C,

323

The superimposed structures of AlucOBP22 and MvicOBP3, the crystal structures of MvicOBP3

324

is shown in yellow.



325 326

Figure 3 Molecular docking of AlucOBP22 with β-ionone (A) or β-caryophyllene (B).

327

β-ionone or β-caryophyllene are shown with brown and green stick model, respectively.

328

Hydrophobic and hydrophilic residues are labeled.

329 330

Figure 4 Binding curve and Kd of wild-type AlucOBP22and six mutants with 1-NPN.


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Figure 5 Competitive binding curves of wild-type AlucOBP22 and the mutants with β-ionone

333

(A) or β-caryophyllene (B). A mixture of recombinant AlucOBP22 protein and 1-NPN in 50 mM

334

Tris-Hcl buffer (pH 7.4) both at the concentration of 2 μM was titrated with 1 mM solutions of

335

each competing ligand to the final concentration range of 2 to 30 µM.

336 337 338 339 340 341 342 343 344 345 346



347

348 349

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Table 1 Affinities of selected odor chemicals with recombinant AlucOBP22 Chemicals

CAS number

cis-3-hexen-1-ol trans-2-hexenal 3-cyclohexene-1-methanol 3-methyl-1-butanol 1-pentanol 2-ethyl hexanol capryl alcohol citronellol linalool 2-hexanol (-)-terpinen-4-ol cineole 1-hexanol valeraldehyde benzaldehyde octanal decane caproaldehyde nonanal dodecyl aldehyde heptanal 3,4-dimethyl-benzaldehyde

928-96-1 6278-26-3 1679-51-2 123-51-3 71-41-0 104-76-7 111-87-5 106-22-9 78-70-6 626-93-7 20126-76-5 470-82-6 111-27-3 110-62-3 100-52-7 124-13-0 124-18-5 66-25-1 124-19-6 112-54-9 111-71-7 5973-71-7

IC50 (µM) ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud.

Ki (µM) ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud. ud.

Chemicals

CAS number

4-ethylbenzaldehyde 2-hexanone 2-heptanone 3-hexanone 2-octanone 6-methyl-5-hepten-2-one (+)-α-pinene limonene myrcene β-ionone β-pinene β-caryophyllene camphene ocimene α-humulene carvacrol naphthalene 2-methylnaphthalene indole acetophenone m-xylene undecane

4748-78-1 591-78-6 110-43-0 589-38-8 111-13-7 110-93-0 7785-70-8 5989-27-5 123-35-3 14901-07-6 127-91-3 87-44-5 79-92-5 13877-91-3 6753-98-6 499-75-2 91-20-3 91-57-6 120-72-9 98-86-2 108-38-3 1120-21-4

IC50 (µM) ud. ud. ud. ud. ud. ud. ud. ud. ud. 6.63±1.9 ud. 3.44±0.7 ud. ud. ud. ud. ud. ud. ud. ud. ud. ud.

Ki (µM) ud. ud. ud. ud. ud. ud. ud. ud. ud. 4.7±1.34 ud. 2.43±0.49 ud. ud. ud. ud. ud. ud. ud. ud. ud. ud.

Affinities (mean ± SD) measured in μM. IC50, Ligand concentration displacing 50% of the fluorescence intensity of the AlucOBP22/1-NPNcomplex; Ki, dissociation constant. ud, undetected.


Page 21 of 27

350 351


Table 2 Binding affinities of host plant volatiles to AlucOBP22 wild-type and six mutants. β-ionone Wild-type Leu5Ala Lys70Ala Ile40Ala Met45Ala Met41Ala Val44Ala

352 353 354 355

β-caryophyllene

IC50 (µM)

Ki (µM)

IC50 (µM)

Ki (µM)

6.63±1.9 6.87±1.02 10.37±2.19 14.76±1.59 15.84±2.54 19.09±0.93 22.84±0.61

4.7±1.34 a 5.8±0.87 ab 6.56±1.37 abc 7.61±0.81 bc 8.11±1.22 c 11.63±0.57 d 18.03±0.48 e

3.44±0.7 ─ 3.53±0.09 6.95±1.73 4.48±1.0 6.67±0.49 ─

2.38±0.47 ab ─ 2.24±0.06 a 3.52±0.83 bc 2.2±0.55 a 4.1±0.32 c ─

Ligand concentration >30µM for half-maximal relative fluorescence intensity was represented as ‘‘–’’. Data within a column followed by the same letter are not significantly different (Tukey’s HSD test, P=0.05).

356 357

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The TOC graphic


The key amino residues determining binding activities of the odorant

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