Key Residues Involved in the Interaction between Cydia pomonella

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Article

Key residues involved in the interaction between Cydia pomonella pheromone binding protein 1 (CpomPBP1) and Codlemone Zhen Tian, Jiyuan Liu, and Ya-Lin Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02843 • Publication Date (Web): 06 Oct 2016 Downloaded from http://pubs.acs.org on October 7, 2016

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Journal of Agricultural and Food Chemistry

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Key residues involved in the interaction between Cydia pomonella pheromone

2

binding protein 1 (CpomPBP1) and Codlemone

3

Zhen Tian 1a

Jiyuan Liu 1a

Yalin Zhang1*

4 5

1

6

of Education, College of Plant Protection, Northwest A&F University, Yangling

7

712100, Shaanxi, China

8

a

9

*Corresponding author: Ya-Lin Zhang

Key Laboratory of Plant Protection Resources & Pest Management of the Ministry

These authors contributed equally to this work.

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Key Laboratory of Plant Protection Resources & Pest Management of the Ministry of

11

Education, Northwest A&F University, Yangling 712100, Shaanxi, China. Tel./Fax:

12

+86-29-8709-2190. E-mail: [email protected].

13

ACS Paragon Plus Environment


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Abstract: Codlemone exhibited high affinity to CpomPBP1, studying their binding

15

mode can provide insights into the rational design of active semiochemicals. Our

16

findings suggested that residues including Phe12, Phe36, Trp37, Ile52, Ile 94, Ala115

17

and Phe118 were favorable to the binding of Codlemone to CpomPBP1, whereas

18

residues providing unfavorable contributions like Ser56 were negative to the binding.

19

Van der Waals energy and electrostatic energy, mainly derived from the sidechains of

20

favorable residues, contributed most in the formation and stability keeping of

21

CpomPBP1-Codlemone complex. Of the residues involved in the interaction between

22

CpomPBP1 and Codlemone, Phe12 and Trp37, whose mutation into Ala caused

23

significant decrease of CpomPBP1 binding ability, were two key residues in

24

determining the binding affinity of Codlemone to CpomPBP1. This study shed lights

25

on discovering novel active semiochemicals as well as facilitating chemical

26

modification of lead semiochemicals.

27 28

Key words: CpomPBP1, Codlemone, binding free energy decomposition,

29

computational alanine scanning, site-directed mutagenesis

30


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Introduction

32

As a quarantine pest, Cydia pomonella causes great harm to fruit production

33

throughout the world. At present, the control of Cydia pomonella mainly relies on

34

integrated management (IPM) instead of individual method.1-4 Of the utilized IPM

35

method, chemical control plays a leading role.5,6 However, frequent application of

36

pesticides is not advisable due to their potential risks to environment and food safety.

37

Moreover, the biological characteristics of Cydia pomonella including fruit boring,

38

generation overlapping and pesticide resistance severely discount the effects of

39

pesticides.7-10 Controlling pests through behavior disruption, a method derived from

40

the dependence of insects on chemical signals, is promising to impair pesticides

41

application.11,12 Even though this method is provided with evident advantages, its

42

development is greatly limited by the labor-consuming process of searching for

43

physiologically active semiochemical.13-15 The progress-attention tradeoff doesn’t

44

scale linearly, up till now, the commonly applied semiochemical in Cydia pomonella

45

control remains to be Codlemone (E, E-8, 10-Dodecadienol).16, 17

46

As a major sex pheromone component which has been widely used in the

47

monitoring and control of Cydia pomonella,16, 18, 19 Codlemone exhibited selectivity to

48

Cydia pomonella pheromone binding proteins (CpomPBP). In our former studies,

49

Codlemone showed high affinity to CpomPBP1 but poor affinity to other PBPs like

50

CpomPBP2.20, 21 Additionally, no ligand exhibited higher affinity than Codlemone to

51

CpomPBP1 in our tests.20 With respect to these points, we suggest that the protein

52

CpomPBP1 may be the potential transporter of Codlemone in the olfaction system of



53

Cydia pomonella. Focusing on the interaction between Codlemone and CpomPBP1

54

could provide insights into the development and optimization of semiochemicals

55

discovering methodology.

56

In the present study, to reveal the binding mode between CpomPBP1 and

57

Codlemone, the constructed model of CpomPBP1-Codlemone complex is analyzed by

58

molecular dynamic simulations, binding free energy calculation and per residue free

59

energy decomposition in sequence. As a common method in studying protein-ligand

60

interaction, computational alanine scanning (CAS) could splendidly predict key

61

residues involved in the interaction.22-24 In the course of CAS, residue for which

62

alanine mutation increased the binding free energy more than 2kcal/mol is considered

63

as warm- and hot-spot. The prediction of warm-spot and hot-spot could achieve an

64

overall success rate of no less than 80%.25 So to further unveil the key residues

65

involved in the interaction, CAS and site-directed mutagenesis were used in

66

combination.

67

Materials and methods

68

3D structure construction of CpomPBP1

69

The 3D molecular model of CpomPBP1 was built by using Modeller9.10.26

70

Based on crystallographic R-factor (21.8%), sequence identity (50%) and pH state

71

(pH 7.0), BmorPBP-Bombykol complex from Bombyx mori (PDB ID: 1DQE, Chain

72

A, resolution 1.8 Å) was finally selected as the template for homology modelling.27

73

Thereafter, the modelling processes were performed in accordance with our former

74

reports21, 24. The quality of optimized model was assessed by MolProbity and Profile


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3D24, 28.

76

Construct the model of CpomPBP1-Codlemone complex

77

CpomPBP1-Codlemone complex was constructed by molecular docking

78

simulations using program GOLD5.3.29 The 3D model of CpomPBP1, a receptor for

79

docking simulations, was performed 5000 steps minimization with ff99SB force field

80

in Amber12.30 The 3D structure of Codlemone was sketched using Maestro

81

(Schrodinger Inc.) and optimized 2000 steps in Amber12 with GAFF force field prior

82

to the docking simulation.31 The ChemPLP score in GOLD was employed to finely

83

produce the best binding model of these complexes due to its superiority for binding

84

pose prediction.32 All the details of the molecular docking were performed according

85

to our previous reports.21, 24

86

Molecular dynamic analysis of CpomPBP1-Codlemone complex

87

All molecular dynamic (MD) simulations for CpomPBP1-Codlemone complex

88

were performed with Amber12 package.33 Parameters and charges of the ligand

89

Codlemone were optimized by the GAFF and the AMI-BCC method.31,

90

AMBER for bioorganic systems force field (ff99SB) was applied to depict

91

CpomPBP1 protein parameters.35 An appropriate number of counterions were added

92

to ensure the entire system at pH 7.0. For CpomPBP1-Codlemone system, we

93

performed 50 ns MD simulations for production phase without any restraint. Based on

94

the 50ns MD trajectories, MD results were analyzed by Ambertools13 package.

95

Binding free energy calculation

96

34

The

To calculate binding free energy of the constructed CpomPBP1-Codlemone



97

complex, Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) in

98

AMBER 12 was employed.36-38 The detail process was in accordance with our former

99

studies.24, 39

100

Per residue free energy decomposition

101

To unveil the detail interactions of Codlemone and each residue in CpomPBP1,

102

per-residue based free energy contribution was decomposed by MM-PBSA method in

103

Amber12.40 In the process of energy decomposition, energy contribution of each

104

residue was divided into three items including total/sidechain/backbone energy

105

contributions, each item was further broken down into van der Waals energy,

106

electrostatic energy and polar solvation free energy. The details of free energy

107

decomposition were performed according to former reports by our team.41

108

Computational Alanine Scanning

109

Computational alanine scanning (CAS), an effective and reliable protocol in

110

predicting hot-spot,25 was employed to predict key residues involved in the binding of

111

Codlemone to CpomPBP1. The CAS was performed according to the procedures

112

described in the former reports of our team.24, 41

113

Site-directed mutagenesis and protein expression

114

Overlapping extension PCR was employed to get mutated types of CpomPBP1

115

gene.42 Take the residue site A for example, 20-30 nucleotides long forward and

116

reverse primers (AFm and ARm) were designed. Notably, the two primers had better

117

overlap more than 10bp with the target residue being changed into Ala in the

118

overlapping region. Three rounds PCR were required to change site A into Ala. The


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119

first round PCR was conducted by taking P1F and ARm, P1R and AFm each as

120

primer pairs. In the second round, 10 cycles of PCR were performed with the former

121

two PCR products as primers and templates each other. Thereafter, the mutated

122

CpomPBP1 genes were obtained by taking PCR product of the second round as

123

template and P1F/P1R as primer pairs, and confirmed by gene sequencing. According

124

to the results of CAS, 6 residues including Phe12, Phe36, Trp37, Ile52, Ile94 and

125

Phe118 were subjected to site-directed mutagenesis in the present study. With the

126

primers shown as Supporting Information in Table S1, the 6 individually mutated

127

CpomPBP1 ORFs (without signal sequence) corresponding to the selected residue

128

sites were obtained by reference to the procedures described above.

129

To

express

native

and

mutant

CpomPBP1F36A,

CpomPBP1

proteins

CpomPBP1W37A,

(CpomPBP1,

130

CpomPBP1F12A,

CpomPBP1I52A,

131

CpomPBP1I94A and CpomPBP1F118A), CpomPBP1 genes (native and mutant genes)

132

without signal sequences were incorporated into pET-28a (+), the generated constructs

133

were then transformed into competent Rosetta-gami 2 (DE3) strains. Proteins

134

expression and purification were performed as our former reports.20, 21

135

Competitive binding assay and dissociation constants calculation

136

To measure the binding ability changes caused by site-directed mutation,

137

CpomPBP1 proteins (wild- and mutant-type CpomPBP1) gained by prokaryotic

138

expression were subjected to competitive binding assay. Briefly, 50 mM Tris-HCl (pH

139

7.4) solutions containing 2 µM proteins and 2 µM 1-NPN (as probe) were titrated with

140

1 mM Codlemone (final concentrations range from 0 to 64 µM). For each point, two



141

replicates were performed. Response spectra were recorded by Hitachi F-4600

142

spectrofluorimeter with excitation and emission wavelength being 337 nm and

143

350-450 nm, respectively.

144

Data analysis

145

In competitive binding assay, all data were analyzed by Graphpad prism 5.0

146

(Graphpad software, Inc.). Dissociation constants (Kd) were calculated from IC50 by

147

equation (1). Kd= [IC50]/(1+ [1-NPN]/K1-NPN)

148

(1)

149

In this equation, [IC50] stands for the ligand concentration where the ligand

150

quenching the fluorescence intensity of 1-NPN to 50%, [1-NPN] and K1-NPN mean the

151

free concentration of 1-NPN and the Kd of the CpomPBP1/1-NPN complex,

152

respectively.

153 154

Kd values were transformed into experimental free energy (∆∆Gbind-exp) according to the following equation (2): ∆∆Gbind =RTln(Kd-MT/ Kd-WT)

155

(2)

156

In the equation, Kd-MT and Kd-WT stand for the Kd values between Codlemone and

157

mutant-type/wild-type CpomPBP1. R and T mean the ideal gas constant and

158

temperature in Kelvin.

159

Results and discussion

160

3D structure construction of CpomPBP1

161

With the crystal structure of BmorPBP-Bombykol complex as template, 3D

162

model of CpomPBP1 was constructed and optimized by Modeller software.


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Ramachandran plot (Figure S1A) and 3D profile analysis (Figure S1B) both

164

suggested stereo chemical rationality of the selected model. As shown in Figure 1, 3D

165

structure of CpomPBP1 was composed of 6 α-helices and several free loops.

166

Thereinto, α1, α4, α5 and α6 formed the binding pocket whose wider open was

167

occupied by the α3, leaving the narrower side being open for the entry of ligands. By

168

reference to our former study on CpomPBP2,21 the obtained model of CpomPBP1

169

exhibited a longer C-terminus which may contribute to different behaviors of these

170

two proteins corresponding to pH variation.

171

In

the

process

of

CpomPBP1

model

construction,

the

template

172

(BmorPBP-Bombykol complex, PDB ID: 1DQE) we used is a dimer. However, a

173

water molecule bridging the hydroxyl oxygen of Bombykol and the oxygen (OG1) of

174

residue Thr111 was only detected in 1DQE Chain A. Through superimposition

175

(models of CpomPBP1 and 1DQE Chain A), a water molecule was detected on

176

residue Val111 in CpomPBP1. Due to the inexistence of polar atoms in Val111

177

sidechain, a water bridge was regarded not to be formed on the residue. So the water

178

on Val111 was not taken into consideration in the following molecular docking and

179

molecular dynamic simulations.

180

Molecular dynamics based stability analysis of CpomPBP1-Codlemone complex

181

CpomPBP1-Codlemone complex obtained by molecular docking was subjected

182

to MD simulations analysis. According to the averaged conformation of

183

CpomPBP1-Codlemone complex during the whole process of MD simulations shown

184

in Figure 2C, a hydrogen bond between the hydroxyl (-OH) oxygen atom of



185

Codlemone (hydrogen bond acceptor) and the NE1 atom (hydrogen bond donor)

186

derived from the sidechain of residue Trp37 was formed with a distance of 2.8 Å.

187

Other residues involved in the interactions between CpomPBP1 and Codlemone were

188

present in the 2D interaction schematic diagram as well (Figure 2D). As shown, the

189

interaction modes were dominantly composed of hydrophobic interaction,

190

electrostatic interaction (including hydrogen bond) and polar interaction etc.

191

In the course of 50ns MD simulations, CpomPBP1-Codlemone complex

192

achieved equilibrium at about 16ns with an averaged RMSD value of 4.80±0.46 Å

193

(Figure 3A). For Codlemone, it reached equilibrium ahead of the complex with the

194

RMSD fluctuating around 2.15 Å (Stdev=0.23 Å) after the time point of 12.5 ns

195

(Figure 3B). These data suggested stability of the complex formed by CpomPBP1 and

196

Codlemone.

197

As shown in Figure 1A, the 3D structure of CpomPBP1 consisted of α-helices

198

and free loops. To study the flexibility and local motion characters on the binding of

199

Codlemone, root-mean-square fluctuation (RMSF) was further analyzed on the basis

200

of MD simulations (Figure 3C). It was evident that helix regions were less flexible

201

than loop regions, especially the last loop composed of residues 132-145

202

(EIAVGEVLAEIAMV) at the C-terminus. During the whole MD simulations, the

203

averaged RMSF were as high as 10.44 Å for residues from 142 to 145 (IAMV).

204

Moreover, the part composed of residue 136-141 (EVLAE) could even spontaneously

205

form a new α-helix. The seventh α-helix formation verified in several other

206

lepidopteran PBPs has been suggested to be in relation to the release of ligands.43, 44


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207

However, considering the distance between the binding sites and the flexible

208

C-terminal tail, the apparent fluctuation of C-terminus shed negligible effects on the

209

binding interface of the complex. Actually, during the sampling time of MD

210

simulations, residues involved in the interaction, like Ser9, Phe12, Trp37, Ile52, Ile94,

211

Trp114 and Phe118, all present little RMSF fluctuation. This further indicated

212

stability of the complex formed by CpomPBP1 and Codlemone.

213

After superimposing the MD representative structure of CpomPBP1-Codlemone

214

complex on the crystal structure of 1DQE Chain A (Figure S2), we found that, in the

215

binding sites of Codlemone and Bombykol, some key residues including Ser9, Phe12,

216

Phe36, Trp37, Ile52, Ser56 and Phe118 were highly conserved. Meanwhile, these

217

residues were conserved ones as well across lepidopteran OBPs, suggesting that they

218

were generally involved in the binding of hydrophobic ligands.27 Besides, 4 pairs of

219

non-conservative

220

CpomPBP1-Codlemone complex and Leu8, Leu62, Val94 and Val111 from 1DQE

221

Chain A were also presented. These variable residues were potentially to be

222

specificity binding determinants. Evidently, residue Thr111 bridging a water molecule

223

in 1DQE Chain A was mutated into Val111 in CpomPBP1-Codlemone complex. In

224

comparison with 1DQE Chain A, we speculated that no water bridge was formed in

225

the corresponding site of CpomPBP1-Codlemone complex due to the inexistence of

226

polar atoms in the sidechain of Val111.

227

Binding free energy calculation

228

residues,

Met8,

Val62,

Ile94

and

Thr111

from

To further check reliability of molecular docking and to analyze binding mode



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229

between CpomPBP1 and Codlemone, MM-PBSA approach was employed to

230

calculate the binding free energy of CpomPBP1-Codlemone complex. According to

231

the

232

CpomPBP1-Codlemone complex formation, hydrophobic interaction (∆EVDW=-33.09

233

kcal/mol) and electrostatic interaction (∆EELE=-7.48 kcal/mol) were two main driving

234

forces and determined the affinity of Codlemone to the binding pocket of CpomPBP1,

235

whereas other items like solvation free energy (including nonpolar part ∆ECAVITY and

236

polar part ∆EEPB) provided limited or even negative contributions. It should be noted

237

that the SEM (standard errors of mean) of each energy item was no more than 0.16

238

kcal/mol, indicating the reliability of calculated binding free energy (∆Gbind-cal). As

239

shown (Table 1), the difference between theoretical binding free energy

240

(∆Gbind-cal=-6.60 kcal/mol) and experimental binding free energy (∆Gbind-exp=-5.15

241

kcal/mol) was not beyond the acceptable limits, suggesting stability of the constructed

242

CpomPBP1-Codlemone complex and dependability of MD simulations.

243

Free energy decomposition

binding

free

energy

items

listed

in

Table

1,

in

the

course

of

244

The spectrum of per residue free energy decomposition was obtained by the

245

method of MM-PBSA (Figure 2A, B). It is worth noting that, for each residue, the

246

total energy contribution mainly derived from their sidechains. As shown in Figure 2B,

247

the sidechains of 7 residues including Phe12, Phe36, Trp37, Ile52, Ile94, Ala115 and

248

Phe118 provided more than 0.50 kcal/mol to the total interaction free energy.

249

Additionally, we also detected that residue Ser56, providing unfavorable energy as

250

high as 0.76 kcal/mol, was negative to the binding of Codlemone.


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251

Thereafter, these 7 residues contributing above 0.50 kcal/mol to the total

252

interaction energy were further depicted in Table 2. As shown, energy contributions of

253

the 7 residues were depicted from 3 aspects, namely total energy contribution (T),

254

backbone energy contribution (B) and sidechain energy contribution (S). Each aspect

255

was subdivided into three energy items including van der Waals energy (VDW),

256

electrostatic energy (ELE) and polar solvation free energy (EPB). Due to the

257

hydrophobic interaction between aliphatic chain (especially the two double bonds) of

258

Codlemone and these selected residues, significant van der Waals energy (TVDW>0.50

259

kcal/mol) was detected. For residues Phe12, Phe36, Ile52 and Phe118, the TVDW was

260

even beyond 1.00 kcal/mol. Noticeably, out of the 7 residues, the TVDW contributions

261

of 6 residues were overwhelmingly provided by their sidechains with Ala115 being an

262

exception. Moreover, the sidechain of Trp37 provided -2.09 kcal/mol in the aspect of

263

electrostatic energy (SELE), this agreed well with the stable hydrogen bond interaction

264

between Trp37 sidechain and Codlemone. Polar interactions were also detected

265

between Codlemone and CpomPBP1. As shown in Figure 2C, D, the hydroxyl group

266

and olefins group derived from Codlemone formed polar interactions with residue

267

Ser9 and sidechain of residue Thr73, respectively. Due to the large polar solvent free

268

energy (TEPB) which was negative to ligand binding, Ser9 provided fairly small

269

contribution (Table 2, -0.13 kcal/mol) to the total interaction free energy (TTOT),

270

despite its remarkable contributions of TVDW (-0.50 kcal/mol) and TELE(-0.89

271

kcal/mol). Noticeably, for residue Thr73, the favorable TVDW, weak TELE and tiny loss

272

in TEPB made it a potential site in enhancing the binding of Codlemone to CpomPBP1.



Page 14 of 35

273

Increasing the electrostatic interaction between Thr73 and Codlemone would be an

274

advisable way to optimize Codlemone-derived semiochemicals.

275

formation and stability keeping of CpomPBP1-Codlemone complex were mainly

276

attributed to the hydrophobic interaction and electrostatic interaction.

277

CAS based site-directed mutagenesis

In conclusion, the

278

The 2D schematic diagram (Figure 2D) presented residues involved in the

279

interaction between Codlemone and CpomPBP1. However, it should be noted that

280

interaction related amino acids don’t mean key residues. So to further unveil residues

281

played a key role in the interaction, CAS was performed. In the process of CAS, the 6

282

binding-favorable residues (Phe12, Phe36, Trp37, Ile52, Ile94 and Phe118) were

283

individually replaced by Ala. The calculated free energy changes (∆∆Gbind-cal) caused

284

by the individual site mutation were listed in Table 3. For the tested residues, the more

285

negative ∆∆Gbind-cal resulted from mutation, the more favorable for the binding. The

286

mutation of Phe12 (∆∆Gbind-cal=-4.54 kcal/mol) and Trp37 (∆∆Gbind-cal=-4.25 kcal/mol)

287

both resulted in ∆∆Gbind-cal more than 4.00 kcal/mol and can be regarded as hot-spot,

288

Phe36 (∆∆Gbind-cal=-3.04 kcal/mol) and Phe118 (∆∆Gbind-cal=-3.56 kcal/mol) belonged

289

to

290

(∆∆Gbind-cal=-2.79 kcal/mol) were not beyond the upper limit of null-spot.

291

Surprisingly enough, residue Ile94 contributing the most to the total interaction

292

energy (Table 3) only caused a ∆∆Gbind-cal of -2.79 kcal/mol on its mutation and was

293

categorized into the null-spot. This can be attributed to the speculation that

294

hydrophobic interaction derived from Ile94 was possibly approximate to the

the

warm-spot,

whereas

Ile52

(∆∆Gbind-cal=-1.77


kcal/mol)

and

Ile94

Page 15 of 35


295

counterpart of Ala, mutating Ile94 into Ala didn’t shed significant effects on the

296

interaction between the site 94 and Codlemone. To conclude, Phe12, Phe36, Trp37

297

and Phe118 were predicted to be candidate key residues in the interaction.

298

Based on site-directed mutagenesis, mutant CpomPBP1 proteins including

299

CpomPBP1F12A,

CpomPBP1F36A,

CpomPBP1W37A,

CpomPBP1I52A,

300

CpomPBP1I94A and CpomPBP1F118A were obtained to verify the results of CAS

301

(Figure S3). By reference to in vitro competitive binding assay, the Kd between

302

Codlemone and CpomPBP1 (including wild- and mutant-type CpomPBP1) was

303

calculated (Figure 4). As shown, wildtype CpomPBP1 exhibited high binding affinity

304

to Codlemone (Figure 4G) with Kd being 3.22 µM. However, this affinity was

305

weakened to varying degree on the individual mutation of selected residue (Phe12,

306

Phe36, Trp37, Ile52, Ile94 and Phe118). Out of the 6 mutant proteins,

307

CpomPBP1W37A, CpomPBP1F12A and CpomPBP1F118A were most unfavorable

308

to the binding of Codlemone with Kd being 23.87 µM, 16.71 µM and 15.09 µM,

309

respectively. As for other three residues including Phe36, Ile52 and Ile94, their

310

mutation didn’t cause remarkable negative effects to the binding ability of CpomPBP1,

311

with Kd being 7.80 µM for CpomPBP1F36A, 5.72 µM for CpomPBP1I52A and 5.35

312

µM for CpomPBP1I94A.

313

According to formula (2), the dissociation constants between Codlemone and

314

CpomPBP1 proteins (wild- and mutant-type CpomPBP1) were transformed into

315

experimental free energy (∆∆Gbind-exp). As a whole, the values of ∆∆Gbind-exp and

316

∆∆Gbind-cal were qualitatively agreed, and the differences between them were



317

acceptable as well (Table 3), suggesting the reliability of theoretical binding free

318

energy calculation. According to the criterions of hot-spot (∆∆Gbind ≥4.00 kcal/mol),

319

warm-spot (2.00 kcal≤ ∆∆Gbind ≤4.00 kcal/mol) and null-spot (∆∆Gbind ≤2.00

320

kcal/mol), it could be concluded that Phe12 (4.11 kcal/mol) and Trp37 (4.92 kcal/mol)

321

were hot-spots, Phe36 (2.38 kcal/mol) and Phe118 (3.88 kcal/mol) were warm-spots,

322

and the two Ile residues (Ile52 and Ile94) fell into the range of null-spot with their

323

corresponding ∆∆Gbind-exp and ∆∆Gbind-cal no more than 2.00 kcal/mol. This further

324

verified the statement that changing Ile94 into Ala didn’t cause significant decrease to

325

the binding ability of CpomPBP1, even though the residue contributed the most to the

326

total interaction energy (Table2). The negative effects of changing Phe36 into Ala

327

were not as significant as expected. In our binding assays, Phe36 belonged to

328

warm-spot rather than hot-spot as predicted in CAS (Table 3). However, the

329

difference between ∆∆Gbind-exp and ∆∆Gbind-cal of CpomPBP1F36A (no more than 1.00

330

kcal/mol) was within the range of error. As for residue Phe118, both ∆∆Gbind-exp and

331

∆∆Gbind-cal caused by its mutation were close to 4.00 kcal/mol, so it can be treated as a

332

potential key residue.

333

To combine the results of CAS and site-directed mutagenesis, it could be

334

concluded that Phe12 and Trp37 were two key residues involved in the interaction

335

between Codlemone and CpomPBP1. However, for BmorPBP-Bombykol complex

336

which was the template we used in CpomPBP1 model construction, changing Trp37

337

into Ala didn’t cause significant effects on the binding of Bombykol to BmorPBP.45

338

According to our CAS assay, the His gate composed of His70 and His95, which was


Page 16 of 35

Page 17 of 35


339

suggested to be important to the ligands binding of ApolPBP from Antheraea

340

polyphemus,46 did not shed significant effects on the interaction between Codlemone

341

and CpomPBP1. Such a difference indicated that, for lepidopteran PBPs, different

342

mechanisms are involved in their interactions with corresponding ligands.

343

Significance

344

The present research conducted by the combination of molecular docking, MD

345

simulations, per-residue free energy decomposition, CAS and site-directed

346

mutagenesis shed light on the detailed interaction between Codlemone and

347

CpomPBP1. Binding free energy analyses unveiled that van der Waals energy

348

(hydrophobic interaction) and electrostatic energy (hydrogen bond interaction) were

349

two key factors in determining the binding affinity of Codlemone to CpomPBP1.

350

Binding free energy decomposition determined the favorable and unfavorable

351

residues in the binding of Codlemone. These discoveries are of significance to

352

developing novel methods for the control of Cydia pomonella.

353

Studies on the interaction modes between CpomPBP1 and Codlemone are

354

beneficial to further active semiochemicals searching. As we all know, active

355

semiochemicals are traditionally screened by insect behavior bioassays, this process is

356

time- and labor-consuming. However, unveiling CpomPBP1-Codlemone interactions

357

allows for the development of more efficient searching methods. Take the commonly

358

used pharmacophore modeling in pharmaceutical industry for example, the

359

pharmacophore model could be constructed on the basis of the interactions between

360

CpomPBP1 and Codlemone. This model could be used to search for potentially active



361

semiochemicals from a large database (ZINC for example). This could significantly

362

expand the source of semiochemicals and improve the chance of active

363

semiochemicals discovery. What is more, uncovering key interactions of

364

CpomPBP1-Codlemone complex could provide new insights into the rational design

365

and modification of novel active semiochemicals for Cydia pomonella. In the present

366

study, a residue (Ser56) negative to Codlemone binding was revealed as well.

367

Avoiding the interactions between ligands and the unfavorable residue (Ser56) could

368

be regarded as a potential avenue to improve the binding affinity of candidate

369

semiochemicals.

370

Not only what mentioned above, key residues associated to the binding of

371

Codlemone to CpomPBP1 can be also used in controlling Cydia pomonella. We can

372

pay some attention to the in vivo refinement of CpomPBP1 to reduce the binding

373

affinity between CpomPBP1 and Codlemone, this production could weaken the

374

behavior response to sex pheromones released by female moths, consequently

375

resulting in mating disruption. This controlling method is similar to releasing infertile

376

pests into the nature and has potential value in the integrated management of Cydia

377

pomonella.

378

Supporting Information Available: Supplements to the determination of template

379

for homology modeling, supplements to competitive binding assay, primers for

380

sit-directed mutagenesis (Table S1), dissociation constants between 1-NPN and

381

wild-/mutant-type CpomPBP1 (Table S2), rationality assessment of CpomPBP1

382

model (Figure S1), superimposition of CpomPBP1 and BmorPBP models (Figure S2),


Page 18 of 35

Page 19 of 35


383

and mutation of CpomPBP1 (Figure S3) are contained in supporting information. This

384

material is available free of charge via the Internet at http://pubs.acs.org.

385

Acknowledgements

386

We are grateful to Dr. John Richard Schrock from Emporia State University

387

(Emporia, KS, USA) for critically reading this manuscript and providing helpful

388

suggestions. We also thank Dr. Zhengwei Wu for his help in insect collection and

389

rearing.

390

Funding sources

391

This research was supported by the Special Fund for Agro-scientific Research in

392

the Public Interest of China (No.200903042).

393

Conflict of Interest

394

The authors declare no competing financial interest.

395



396

References

397

1.

398

Cydia pomonella (L.)(Lepidoptera: Olethreutidae) in British Columbia, Canada. Acta

399

Phytopathol. Entomol. Hung. 1992, 27, 219-222.

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Lepidoptera: Tortricidae) and its role in integrated pest management, with emphasis

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formulations and adjuvants as solar protectants for the granulovirus of the codling

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moth, Cydia pomonella (L). J. Invertebr. Pathol. 2006, 93, 88-95.

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codling moth, Cydia pomonella (Lepidoptera: Tortricidae): implications for pest

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control and the sterile insect release programme. Evol. Appl. 2011, 4, 534-544.

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Agricul. Food. Chem. 2000, 48, 88-92.

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Lacey, L. A.; Unruh, T. R., Biological control of codling moth (Cydia pomonella,

Arthurs, S.; Lacey, L.; Behle, R., Evaluation of spray-dried lignin-based

Chidawanyika, F.; Terblanche, J. S., Costs and benefits of thermal acclimation for

Avilla, J.; Teixidò, A.; Velázquez, C.; Alvarenga, N.; Ferro, E.; Canela, R.,

Stará, J.; Kocourek, F., Insecticidal resistance and cross-resistance in populations

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Sauphanor, B., Diversity of insecticide resistance mechanisms and spectrum in

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Tortricidae), in the Australian Capital Territory. Aust. J. Zool. 1963, 11, 323-367.

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10. Mailloux, M.; LeRoux, E., Further observations on the life-history and habits of

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the codling moth, Carpocapsa pomonella (L.)(Lepidoptera: Tortricidae), in apple

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11. Charmillot, P. J.; Hofer, D.; Pasquier, D., Attract and kill: a new method for

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12. Lösel, P. M.; Penners, G.; Potting, R. P.; Ebbinghaus, D.; Elbert, A.;

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Scherkenbeck, J., Laboratory and field experiments towards the development of an

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Exp. Appl. 2000, 95, 39-46.

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13. Knight, A. L.; Light, D. M., Attractants from Bartlett pear for codling moth,

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14. Ansebo L; Coracini MDA; Bengtsson M; Liblikas I; Ramirez M; Borg-Karlson

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AK; Tasin M; Witzgall P, Antennal and behavioural response of codling moth Cydia

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15. Yan, F.; Bengtsson, M.; Witzgall, P., Behavioral response of female codling

Reyes, M.; Franck, P.; Charmillot, P. J.; Ioriatti, C.; Olivares, J.; Pasqualini, E.;

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moths, Cydia pomonella, to apple volatiles. J. Chem. Ecol. 1999, 25, 1343-1351.

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16. Witzgall, P.; Stelinski, L.; Gut, L.; Thomson, D., Codling moth management and

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chemical ecology. Annu. Rev. Entomol., 2008, 53, 503-522.

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17. Witzgall, P.; Kirsch, P.; Cork, A., Sex pheromones and their impact on pest

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management. J. Chem. Ecol. 2010, 36, 80-100.

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18. Ebbinghaus, D.; Lösel, P.; Lindemann, M.; Scherkenbeck, J.; Zebitz, C.,

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Detection of major and minor sex pheromone components by the male codling moth

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19. Judd, G. J.; Gardiner, M. G.; DeLury, N. C.; Karg, G., Reduced antennal

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sensitivity, behavioural response, and attraction of male codling moths, Cydia

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pomonella, to their pheromone (E, E)-8, 10-dodecadien-1-ol following various

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pre-exposure regimes. Entomol. Exp. Appl. 2005, 114, 65-78.

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20. Tian, Z.; Zhang, Y., Molecular characterization and functional analysis of

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pheromone binding protein 1 from Cydia pomonella (L.). Insect Mol. Biol. 2016, doi:

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21. Tian, Z.; Liu, J.; Zhang, Y., Structural insights into Cydia pomonella pheromone

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binding protein 2 mediated prediction of potentially active semiochemicals. Sci. Rep.

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22. Moreira, I. S.; Fernandes, P. A.; Ramos, M. J., Computational alanine scanning

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mutagenesis—an improved methodological approach. J. Comput. Chem. 2007, 28,

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644-654.

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23. Ramos, R. M.; Moreira, I. S., Computational alanine scanning mutagenesis: an


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improved methodological approach for protein-DNA complexes. J. Chem. Theor.

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Comput. 2013, 9, 4243-4256.

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24. Liu, J.; Yang, X.; Zhang, Y., Characterization of a lambda-cyhalothrin

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metabolizing glutathione S-transferase CpGSTd1 from Cydia pomonella (L.). Appl.

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Microbiol. Biote. 2014, 98, 8947-8962.

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25. Moreira, I. S.; Fernandes, P. A.; Ramos, M. J., Computational alanine scanning

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644-654.

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26. Šali; Blundell, T. L., Comparative protein modelling by satisfication of spatial

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restraints. J. Mol. Biol. 1993, 234, 779-815.

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27. Sandler, B. H.; Nikonova, L.; Leal, W. S.; Clardy, J., Sexual attraction in the

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silkworm moth: structure of the pheromone-binding-protein-bombykol complex.

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Chem. Biol. 2000, 7, 143-151.

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28. Chen, V. B.; Arendall, W. B., Molprobity: all-atom structure validation for

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macromolecular crystallography. Acta Crystallogr. D. 2010, 66, 12-21.

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29. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R., Development and

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validation of a genetic algorithm for flexible docking. J. Mol. Biol. 1997, 267,

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727-748.

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30. Hummer, G.; Rasaiah, J. C.; Noworyta, J. P., Water conduction through the

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hydrophobic channel of a carbon nanotube. Nature 2001, 414, 188-190.

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31. Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A., Development

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and testing of a general amber force field. J. Comput. Chem. 2004, 25, 1157-1174.



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32. Korb, O.; Stutzle, T.; Exner, T. E., Empirical scoring functions for advanced

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protein- ligand docking with PLANTS. J. Chem. Inf. Model. 2009, 49, 84-96.

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33. Case, D.; Darden, T.; Cheatham III, T.; Simmerling, C.; Wang, J.; Duke, R.; Luo,

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R.; Walker, R.; Zhang, W.; Merz, K., AMBER 12; University of California: San

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Francisco, 2012.

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34. Jakalian, A.; Jack, D. B.; Bayly, C. I., Fast, efficient generation of high-quality

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atomic charges. AM1‐BCC model: II. Parameterization and validation. J. Comput.

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Chem. 2002, 23, 1623-1641.

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35. Berendsen, H. J.; Postma, J. v.; van Gunsteren, W. F.; DiNola, A.; Haak, J.,

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Molecular dynamics with coupling to an external bath. J. Chem. Phys. 1984, 81,

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3684-3690.

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36. Kollman, P. A.; Massova, I.; Reyes, C.; Kuhn, B.; Huo, S.; Chong, L.; Lee, M.;

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Lee, T.; Duan, Y.; Wang, W., Calculating structures and free energies of complex

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molecules: combining molecular mechanics and continuum models. Accounts Chem.

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Res. 2000, 33, 889-897.

499

37. Hou, T.; Wang, J.; Li, Y.; Wang, W., Assessing the performance of the MM/PBSA

500

and MM/GBSA methods. 1. The accuracy of binding free energy calculations based

501

on molecular dynamics simulations. J. Chem. Inf. Model. 2010, 51, 69-82.

502

38. Hou, T.; Wang, J.; Li, Y.; Wang, W., Assessing the performance of the molecular

503

mechanics/Poisson Boltzmann surface area and molecular mechanics/generalized

504

Born surface area methods. II. The accuracy of ranking poses generated from docking.

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J. Comput. Chem. 2011, 32, 866-877.


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506

39. Yang, X. Q.; Liu, J. Y.; Li, X. C.; Chen, M. H.; Zhang, Y. L., Key amino acid

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associated with acephate detoxification by Cydia pomonella carboxylesterate based

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on molecular dynamics with alanine scanning and site-directed mutagenesis. J. Chem.

509

Inf. Model. 2014, 54, 1356-1370.

510

40. Gohlke, H.; Kiel, C.; Case, D. A., Insights into protein–protein binding by

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binding free energy calculation and free energy decomposition for the Ras–Raf and

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Ras–RalGDS complexes. J. Mol. Biol. 2003, 330, 891-913.

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41. Liu, J.; Chen, X.; Zhang, Y., Insights into the key interactions between human

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protein phosphatase 5 and cantharidin using molecular dynamics and site-directed

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mutagenesis bioassays. Sci. Rep. 2015, 5, 12359, doi:10.1038/srep12359.

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42. Ho, S. N.; Hunt, H. D.; Horton, R. M.; Pullen, J. K.; Pease, L. R., Site-directed

517

mutagenesis by overlap extension using the polymerase chain reaction. Gene 1989, 77,

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51-59.

519

43. Damberger, F. F.; Michel, E.; Ishida, Y.; Leal, W. S.; Wuthrich, K., Pheromone

520

discrimination by a pH-tuned polymorphism of the Bombyx mori pheromone-binding

521

protein. P. Natl. Acad. Sci. USA 2013, 110, 18680-18685.

522

44. Di Luccio, E.; Ishida, Y.; Leal, W. S.; Wilson, D. K., Crystallographic observation

523

of pH-induced conformational changes in the Amyelois transitella pheromone-binding

524

protein AtraPBP1. Plos One 2013, 8, e53840.

525

45. Leal, W. S.; Chen, A. M.; Ishida, Y.; Chiang, V. P.; Erickson, M. L.; Morgan, T. I.;

526

Tsuruda, J. M., Kinetics and molecular properties of pheromone binding and release. P.

527

Natl. Acad. Sci. USA 2005, 102, 5386-5391.



528

46. Katre, U. V.; Mazumder, S.; prusti, R. K.; Mohanty, S., Ligand binding turns moth

529

pheromone binding protein into pH sensor: effects on the Antheraea polyphemus

530

PBP1 conformation. J. Biol. Chem. 2009, 284, 32167-32177.

531 532 533 534 535


Page 26 of 35

Page 27 of 35


536

Figure Captions

537

Figure 1. Structure of CpomPBP1. (A) The constructed 3D model of CpomPBP1. N

538

is the N-terminus, C is the C-terminus, and helices α1-α6 are labeled, of which α1, α4,

539

α5 and α6 converge to form the binding pocket. (B) The amino acid sequence

540

alignments of Cydia pomonella CpomPBP1 and Bombyx mori BmorPBP obtained

541

with Clustal W and refined based on the CpomPBP1 structure. The identical residues

542

are highlighted with star below the letters.

543

Figure 2. Interaction analyses for CpomPBP1-Codlemone complex. (A) The total

544

energy contributions of each residue in CpomPBP1; (B) The sidechain energy

545

contributions of each residue in CpomPBP1; (C) The average conformation of

546

CpomPBP1-Codlemone complex; (D) The 2D interaction diagram of Codlemone with

547

the binding pocket of CpomPBP1.

548

Figure 3. The 50 ns molecular dynamic analyses of CpomPBP1-Codlemone complex.

549

(A) The RMSD changes for Codlemone molecule during the equilibration phase of

550

MD simulation; (B)The

551

CpomPBP1/Codlemone

552

CpomPBP1/Codlemone complex.

553

Figure 4. Binding curves of Codlemone to the 6 mutated CpomPBP1. (A-G) Binding

554

curves of Codlemone to CpomPBP1F12A (F12A), CpomPBP1F36A (F36A),

555

CpomPBP1W37A (W37A), CpomPBP1I52A (I52A), CpomPBP1I94A (I94A),

556

CpomPBP1F118A (F118A) and CpomPBP1WT, respectively. The mutation of each

557

residue makes the binding ability of CpomPBP1 decreased to varying degrees.

RMSD complex;

values for the (C)The

backbone

per-residue


of atoms of

RMSF

for

the


Page 28 of 35

Table 1 The calculated and experimental binding free energy for the Codlemone binding to wildtype CpomPBP1 Contributiona

∆EELE

∆EVDW

∆EEPB

WTb

-7.48

-33.09

17.81

(0.088) (0.089) (0.082) a

∆ECAVITY ∆GGAS ∆GSOL ∆Gbind-cal ∆Gbind-exp -1.64

-40.57

33.97

-6.60

-5.15

(0.023)

(0.12)

(0.11)

(0.15)

(0.02)

All values are given in kcal/mol, values in the parentheses mean the standard errors.

∆EELE, ∆EVDW, ∆EEPB and ∆ECAVITY stand for electrostatic interaction energy, hydrophobic interaction energy, polar solvation energy and nonpolar solvation free energy. ∆Gbind-cal and ∆Gbind-exp are theoretical and experimental free energy changes, respectively. b

WT stands for wildtype CpomPBP1.


Page 29 of 35


Table 2 Decomposition of binding free energy on a per-residue level a Residue SVDW BVDW TVDW SELE

BELE

TELE

SEPB

BEPB

TEPB

STOT

BTOT

TTOT

Phe12 -1.942 -0.112 -2.054 -0.335 -0.055 -0.390 1.295 0.109 1.404 -0.983 -0.058 -1.041 Phe36 -1.443 -0.087 -1.530 -0.120 0.073 -0.047 0.971 -0.127 0.844 -0.593 -0.140 -0.733 Trp37 -0.510 -0.036 -0.546 -2.086 0.035 -2.051 1.510 -0.017 1.493 -1.086 -0.018 -1.104 Ile52 -0.731 -0.129 -0.860 0.075 -0.050 0.025 0.010 0.152 0.162 -0.646 -0.027 -0.673 Ile94 -1.246 -0.139 -1.385 -0.109 -0.006 -0.115 0.054 -0.001 0.053 -1.302 -0.146 -1.448 Ala115 -0.290 -0.337 -0.628 0.010 0.073 0.083 -0.008 -0.006 -0.014 -0.288 -0.270 -0.558 Phe118 -1.653 -0.089 -1.742 -0.100 0.060 -0.040 1.079 0.010 1.089 -0.674 -0.019 -0.693 Ser9

-0.17 -0.34 -0.50 -0.55 -0.34 -0.89

Thr73 -0.33 -0.15 -0.48 a

0.02

-0.05 -0.03

0.72

0.53

1.26 0.009 -0.14 -0.13

0.14

-0.01

0.13

-0.17 -0.22 -0.38

per residue energy contribution was depicted from three aspects including

backbone(B), sidechain (S) and total (T) energy contribution, for each aspect, the energy contribution was further decomposed into van der Waals energy (VDW), electrostatic energy (ELE)and polar solvent free energy (EPB). In the table, TOT means the total energy contribution of each aspect. All values are given in kcal/mol.



Page 30 of 35

Table 3 The theoretical and experimental ∆∆Gbinda for mutant CpomPBP1/Codlemone Proteinb

F12A

F36A

W37A

I52A

I94A

F118A

∆∆Gbind-cal

4.54

3.04

4.25

1.77

2.79

3.56

∆∆Gbind-exp 4.11

2.38

4.92

1.68

1.53

3.88

a

All values are given in kcal/mol, ∆∆Gbind-cal and ∆∆Gbind-exp are theoretical and

experimental free energy change, respectively. b

F12A, F36A, W37A, I52A, I94A and F118A are abbreviations for CpomPBP1F12A,

CpomPBP1F36A,

CpomPBP1W37A,

CpomPBP1I52A,

CpomPBP1F118A, respectively.


CpomPBP1I94A

and

Page 31 of 35


Figure 1



Figure 2


Page 32 of 35

Page 33 of 35


Figure 3



Figure 4


Page 34 of 35

Page 35 of 35


TOC Graphic


Key Residues Involved in the Interaction between Cydia pomonella

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