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Research Article
Received: 5 December 2019 Revised: 29 April 2020 Accepted article published: 17 May 2020 Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/ps.5915
Structure dynamics reveal key residues
essential for the sense of 1-dodecanol by Cydia
pomonella pheromone binding protein
2 (CpomPBP2)
Zhen Tian,a,b Yue Li,aTong Zhou,bXuan Ye,aRuichi Liaand Jiyuan Liua
*
Abstract
BACKGROUND: Cydia pomonella, a worldwide quarantine fruit pest, causes great damage to fruit production every year. Sex
pheromone-mediated control of C. pomonella has been widely used. As an indispensable ingredient of commercial sex attrac-
tants, 1-dodecanol (Dod) works to synergize the effect of codlemone in attracting male moths of C. pomonella. The interactions
between Dod and its transporter protein, C. pomonella pheromone-binding protein 2 (CpomPBP2), provide inspiration for
chemical optimizations to improve the synergistic effects of Dod.
RESULTS: In this research, molecular simulations and biological verifications were used in combination to uncover key residues
in CpomPBP2 essential for sensing Dod. After performing 150 ns molecular dynamics (MD) simulations, the C1–C12 chain of
Dod was found to be locked by the van der Waals energy contributed by the hydrophobic residues Phe12, Leu68, and
Ile113, whereas the -OH part of Dod was anchored by the H-bond derived from Glu98 and the salt-bridge derived from
Arg109. Because of the importance of these two electrostatic interactions, Glu98 and Arg109 were further verified as key res-
idues in determining the binding affinity between Dod and CpomPBP2. In addition, interactions unfavorable to the binding
of Dod were described.
CONCLUSION: The research detailed the discovery of key residues involved in CpomPBP2–Dod interactions. Our results provide
guidance and caution for the prospective discovery, optimization, and design of novel chemicals with a similar or stronger syn-
ergistic effect to codlemone in controlling C. pomonella.
Supporting information may be found in the online version of this article.
Keywords: Cydia pomonella; odorant-binding proteins; pheromone synergist; molecular dynamics; pairwise free-energy decomposition;
site-directed mutagenesis
1 INTRODUCTION
Codling moth, Cydia pomonella, is a major cosmopolitan pest of
pome and stone fruits (apple, pear, peach, etc.), causing severe
damage to global fruit production.
1,2
Like other Lepidopteran
insects, C. pomonella relies heavily on chemical signals to regulate
its behavior including host finding, mating, and oviposition.
2,3
To
date, a large amount of research has been undertaken on the con-
trol of pest insects by regulating their behaviors.
4–6
Currently, the most widely used insect behavior regulators are
sex pheromones packaged in slow-release generators.
7–9
For
C. pomonella, pheromone-mediated management strategies
(mating disruption, mass trapping, etc.) have been successfully
applied and have shown promising results in commercial
orchards.
2,10,11
The sex pheromone of C. pomonella is reported
to be a multicomponent mixture, with 1-dodecanol (Dod, CAS:
112-53-8) the second-most abundant compound.
12,13
Even
though Dod alone has no effects on attracting C. pomonella, it aids
in orientation and acts as a synergist of codlemone (E,E-
8,10-dodecadienol, CAS: 33956-49-9), the main component of
the codling moth sex pheromone.
14,15
As reported, Dod acts by
widening the dose range over which male moths of
C. pomonella are optimally attracted to codlemone.
2,13
Dod and
codlemone are used in a blend in current commercial sexual
attractants and mating disruptors for C. pomonella.
2,15,16
Sensilla distributed on insect antennae are dominant olfactory
organs sensing semiochemicals bearing specific chemical sig-
nals.
17,18
In most cases, odorant-binding proteins (OBPs) are
thought to be the main proteins responsible for the recognition
**Correspondence to: J Liu, Key Laboratory of Plant Protection Resources & Pest
Management of the Ministry of Education, College of Plant Protection, Northwest
A&F University, Yangling 712100, Shaanxi, China, E-mail: kingljy0818@hotmail.com
aKey Laboratory of Plant Protection Resources & Pest Management of the Min-
istry of Education, College of Plant Protection, Northwest A&F University, Yan-
gling, China
bCollege of Horticulture and Plant Protection, Yangzhou University, Yangzhou,
China
Pest Manag Sci 2020 www.soci.org © 2020 Society of Chemical Industry
1
and transportation of semiochemicals within the antennal sen-
silla.
19,20
As an OBP, C. pomonella pheromone binding protein
2 (CpomPBP2) is found in the antennae of both sexes.
21
Among
the compounds isolated from female glands and apple volatiles,
Dod exhibited the highest affinity for CpomPBP2. In addition,
CpomPBP2 is superior to other known OBPs from C. pomonella
in binding with Dod.
21,22
The high affinity between CpomPBP2
and Dod indicates that CpomPBP2 may function to transport
Dod across the sensillum lymph to odorant receptors (ORs)
located on the membranes of olfactory neurons.
As a known synergist to codlemone in attracting C. pomonella,
Dod enhances male attraction at low codlemone doses. But how
Dod binds to its transporter CpomPBP2 and what residues are
involved in the binding process remain unknown. To clarify these
questions, a complex model formed by CpomPBP2 and Dod was
initially subjected to 150 ns molecular dynamics (MD) simulations.
After determining the MD representative conformation of the
CpomPBP2–Dod complex, binding free-energy calculations, pair-
wise per-residue free-energy decomposition, site-directed muta-
genesis, and a competitive binding assay were used in
combination to determine the residues essential for sensing of
Dod by CpomPBP2. During analysis, interactions unfavorable to
the binding of Dod to CpomPBP2 were also highlighted, giving
opportunities for active enhancement of Dod. Our results have
great significance for the prospective discovery of novel chemicals
with functions similar to Dod in enhancing the male-attraction
effect of codlemone, and in boosting the development and wide-
spread use of pheromone-mediated management to control
C. pomonella.
2 MATERIALS AND METHODS
2.1 MD simulations
A 3D model of the CpomPBP2–Dod complex (Fig. S1) constructed
in our previous research was subjected to the Amber12 package
for 150 ns MD simulations.
21,23,24
To prepare for MD simulations,
the CpomPBP2–Dod model was immersed in a rectangular box
of explicit transferable intermolecular potential with 3 points
(TIP3P) water with a minimum solute wall distance of 8 Å.
25
Na
+
ions were added to keep the entire system neutral.
26
The param-
eters and charge of the Dod ligand were optimized using Gen-
eral Amber Force Field (GAFF) and AM1-BCC (semi-
empirical (AM1) with bond charge correction (BCC)) methods,
and counterparts for the protein CpomPBP2 were set using
ff9SB.
27–29
To remove possible unfavorable contacts, the free-
energy of the CpomPBP2–Dod complex was minimized through
successive application of the steepest descent method (5000
steps) and conjugate gradient method (5000 steps). MD simula-
tions of the CpomPBP2–Dod complex were performed in
Amber12 without restraint. During the equilibration phase of
MD simulations, the prepared complex was heated to 300 K in
500 ps in the NVT ensemble (canonical ensemble) and kept at
300 K for 5 ns under constant pressure for unstrained equilibra-
tion. To prevent an abrupt jump in potential energy, the same
conditions as in the equilibration phase were adopted in the pro-
duction phase. Throughout MD simulations, coordinates were
recorded every 10 ps; this helps to keep the conformations uncor-
related. The isothermal isobaric ensemble (NPT) used in the equil-
ibration and production phases was realized by a Berendsen
barostat.
30
MD trajectories produced in the 150 ns were analyzed
using the AmberTools13 package.
23
2.2 Binding free-energy calculation
According to our previous research, the molecular mechanics
Poisson–Boltzmann surface area (MM-PBSA) method is rational
enough to calculate the theoretical binding free-energy (ΔG
bind-
cal
).
24,31,32,33
Thus the MM-PBSA method in Amber12 software
was adopted to estimate ΔG
bind-cal
of the CpomPBP2–Dod com-
plex.
31,34,35
The MM-PBSA method calculates trajectory averages
based mainly on force field energies combined with Poisson–
Boltzmann electrostatics and surface area terms.
36,37
In the MM-
PBSA method, the ΔG
bind-cal
of CpomPBP2–Dod binding was
obtained from the difference between the free-energy of the
CpomPBP2–Dod complex (ΔG
complex
) and unbound CpomPBP2
(ΔG
protein
) and Dod (ΔG
ligand
), as shown in Eqn (1).
31
ΔGbind−cal =ΔGcomplex−ΔGprotein +ΔGligand
ð1Þ
For the CpomPBP2–Dod complex, we calculated ΔG
bind-cal
values for the 15 000 snapshots of the MD trajectories (one snap-
shot for each 10 ps during the 150 ns MD simulations). The final
ΔG
bind-cal
was the average of calculated ΔG
bind-cal
values for these
snapshots.
2.3 Pairwise per-residue free-energy decomposition
Definition of the CpomPBP2–Dod interaction energy spectra is
needed to detail interactions between CpomPBP2 and
Dod. Based on the MD representative conformation of
the CpomPBP2–Dod complex, energy spectra of the
CpomPBP2-12:OH interface were depicted by pairwise per-
residue free-energy decomposition.
38-40
In the current
research, pairwise per-residue free-energy decomposition
was performed using the MM-PBSA method from the
mmpbsa.py module of AmberTools13.
41,42
The whole process
was modified from our former research.
43
2.4 Site-directed mutagenesis
Residue sites for site-directed mutagenesis were selected based
on the comprehensive results of pairwise assays performed
above and computational alanine scanning in our previous
research.
21
Site-directed mutation of CpomPBP2 was implemen-
ted following the instruction manual (Vazyme, China). Briefly,
the wild-type CpomPBP2 gene was subcloned into PMD-19T
vector to generate the PMD19T–CpomPBP2 plasmid. Primers for
site-directed mutagenesis were prepared accordingly
(Vazyme). These primers (Table S1) were used to perform poly-
merase chain reactions (PCR) with PMD19T–CpomPBP2 plasmids
as a template. After removing the templates by DpnI digestion
(37 °C, 90 min), PCR products were treated with Exnase II (37 °
C, 30 min) to promote cyclization. Reaction products were trans-
formed into DH5⊍-competent cells, coated plates, and a select
monoclonal colony. Mutant CpomPBP2 genes were verified by
gene sequencing (Tsingke, China).
2.5 Protein expression and purification
To obtain recombinant wild-type CpomPBP2 (CpomPBP2WT) pro-
tein, pCold III (TaKaRa, Japan) was selected as the protein expres-
sion vector and TransB (DE3) strain (Transgen, China) was taken as
the host cell. After transformation, the host containing pColdIII–
CpomPBP2 plasmids was cultured at 37 °C, 200 rpm. When the
optical density at 600 nm (OD
600
) reached 0.6, the cultured strain
was precooled to 15 °C before addition of isopropyl ⊎-M-
1-thiogalactopyranoside (IPTG; 0.6 mM). Thereafter, the strain
was cultured for 24 h at 15 °C and 160 rpm to induce expression
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2
of CpomPBP2WT. The cultured strain was collected (5000 rpm,
10 min), sonicated (10 s work/10 s stop for 10 min), and centri-
fuged (12 000 g, 30 min). The resultant supernatants were loaded
onto a Ni
2+
-NTA resin column (Qiagen, Germany) for protein puri-
fication. Recombinant CpomPBP2 protein equipped with a His tag
was eluted using 300 mMimidazole. After 15% sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), fractions
containing CpomPBP2 protein were pooled. His-tag cleavage was
achieved by incubating with PreScission protease (GE Healthcare)
at 4 °C for 12 h, followed by a re-purification step using a Ni
2+
-NTA
resin column (Qiagen) to remove the His tag and PreScission pro-
tease. Finally, the purified sample was checked by 15% SDS–
PAGE, dialyzed against 10 mMphosphate-buffered saline (PBS;
pH 7.4), and quantified using a Bradford kit (Solarbio, China). Puri-
fied CpomPBP2 protein was kept at −80 °C for further tests.
Expression and purification of mutant CpomPBP2
(CpomPBP2MT) proteins were performed similarly.
26
2.6 Competitive binding assay
With N-phenyl-1-naphthylamine (1-NPN) as the fluorescent probe, a
competitive binding assay was used to measure changes in affinity
between Dod and recombinant CpomPBP2 proteins
(CpomPBP2WT and CpomPBP2MT).
24,32,44
Briefly, the dissociation
constants between 1-NPN and recombinant CpomPBP2 proteins
were determined by titrating 2 μMprotein solutions with 1 mM
1-NPN (final concentration: 0.5–20 μM). To test the binding affinity
between Dod and CpomPBP2WT/CpomPBP2MT proteins, 10 mM
PBS (pH 7.4) solutions containing 2 μMrecombinant CpomPBP2 pro-
teins and 2 μM1-NPN were titrated against 1 mMDod (final concen-
tration: 0–64 μM) in three replications. Fluorescence responses were
detected using a Hitatchi F-2700 spectrofluorometer (Hitatchi,
Japan),withanexcitationwavelengthof337nmandemissionspec-
tra were recorded between 350 and 500 nm.
2.7 Statistics
GraphPad Prism 6.0 (GraphPad Software, Inc., California, USA) was
used to analyze the competitive binding assay data. Dissociation
constants (K
d
) between recombinant CpomPBP2 proteins and
Dod were calculated using Eqn (2).
22,24
Kd=IC50=1+ 1−NPN½=K1−NPN
ðÞð2Þ
In Eqn (2), IC
50
is the concentration of Dod quenching the fluo-
rescence intensity by half; [1-NPN] is the free concentration of
1-NPN; K
1-NPN
is the K
d
between CpomPBP2 protein and 1-NPN.
The experimental binding free-energy (ΔG
bind-exp
) for the com-
plex formed by Dod and wild-type CpomPBP2 was calculated
using Eqn (3).
24,32
ΔGbind−exp =RT1nKd−WT ð3Þ
In Eqn (2), Rand Tare the ideal gas constant and temperature in
Kelvin, respectively. K
d-WT
is the K
d
value between wild-type
CpomPBP2 protein and Dod.
Changes in the experimental binding free-energy (ΔΔG
bind-exp
)
caused by mutation of target residues were calculated based on
corresponding K
d
values using Eqn (4).
24,43
ΔΔGbind−exp =RT1nK
d−MT =Kd−WT
ðÞ
ð4Þ
In Eqn (4), Ris the ideal gas constant, Tis the temperature in Kel-
vin, K
d-MT
is the K
d
value between mutant CpomPBP2 proteins and
Dod, K
d-WT
is the K
d
value between wild-type CpomPBP2 proteins
and Dod.
3 RESULTS
3.1 The CpomPBP2–Dod complex is stable during MD
simulations
The CpomPBP2–Dod complex (Fig. S1A) constructed in our for-
mer research was subjected to 150 ns MD simulations.
21
As
shown in Fig. 1(A), the CpomPBP2–Dod complex reached equilib-
rium at ~ 50 ns, and its averaged root-mean-square deviation
(RMSD) fluctuated around 4.30 Å (SD =0.22 Å). Specifically for
the ligand Dod in the CpomPBP2–Dod system, equilibrium was
achieved immediately after initiation of the 150 ns MD simula-
tions with an RMSD of 1.93 ±0.15 Å (Fig. 1B). Equilibrium in the
CpomPBP2–Dod system indicated the stability and rationality of
the complex formed by CpomPBP2 and Dod.
To obtain further information on the structure of the
CpomPBP2–Dod complex, root-mean-square fluctuation (RMSF)
analysis was carried out to reveal its flexibility and local motion
characteristics (Fig. 1C). As previously reported,
21
the 3D model
of CpomPBP2 contained two secondary structure elements,
namely ⊍-helices and free loops (Fig. S1A). Considering the results
in Figs 1(C) and S1, it is easy to show that ⊍-helices were more rigid
Figure 1 Molecular dynamics (MD) simulations of the CpomPBP2–Dod
complex. (A) Root mean square deviation (RMSD) values for the
CpomPBP2–Dod complex in the process of 150 ns MD simulations.
(B) RMSD values for the ligand Dod in the process of 150 ns MD simula-
tions. (C) Root mean square fluctuations for each residue derived from
the CpomPBP2–Dod complex in the process of 150 ns MD simulations.
Dod, 1-dodecanol; CpomPBP2–Dod, CpomPBP2–Dod complex.
Key residues in CpomPBP2–Dod interaction www.soci.org
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3
than loops, which was particularly evident in the N-terminus and
C-terminal tail. It should be noted that, due to their large distance
from the binding pocket of CpomPBP2, the dramatic flexibility of
the N- and C-termini has no apparent effect on the interaction
contacts of the CpomPBP2–Dod complex, further suggesting sta-
bility of the complex formed by CpomPBP2 and Dod.
3.2 Binding mode analysis of the CpomPBP2–Dod
complex
An average-linkage algorithm and pairwise RMS were applied in
conjunction to cluster the MD trajectories produced during the
150 ns MD simulations. As shown in Table S2 and Fig. S2, five clus-
ters were obtained, with Cluster I and Cluster II having the highest
rates of 44% and 30.4%, respectively. It is interesting that Dod pos-
sessed similar binding modes in the representative conformations
of the two dominant clusters. As shown in Fig. S3, the alkyl group
of Dod was located in the hydrophobic pocket composed of resi-
dues including by Phe12, Leu68 and Ile113. By contrast, the
hydroxyl group (-OH) was found to interact with hydrophilic resi-
dues including Glu98 and Arg109. Specifically, the O1 atom from
-OH of Dod formed an H-bond interaction with the OE atom of
the Glu98 side-chain. A salt-bridge interaction was detected
between -OH of Dod and the side-chain of Arg109. Tiny differ-
ences between the binding modes of Dod in the representative
conformations of Cluster I and Cluster II also exist. Taking the H-
bond as an example, the distance of O─O atoms forming the H-
bond changed from 1.9 Å in Cluster II to 1.7 Å in Cluster I (Fig. S3).
Considering the higher occupation rate and stronger H-bond
interaction, the conformation derived from Cluster I was closer
to the representative conformation extracted from the 150 ns
MD trajectories (Fig. 2).
3.3 Theoretical binding free-energy calculation
Based on the MD representative conformation (Fig. 2), theoretical
binding free-energy (ΔG
bind-cal
) of the CpomPBP2–Dod complex
was calculated using the MM-PBSA method.
31,36,37,45,46
As
revealed by the free-energy items listed in Table 1, van der Waals
interactions (ΔE
vdW
=−34.83 kcal mol
–1
) were the most domi-
nant forces driving formation and maintenance of the
CpomPBP2–Dod complex. Considering the strong salt bridge
(between Arg109 and -OH of Dod) and the H-bond (between
Glu98 and -OH of Dod) in the CpomPBP2–Dod system, the free-
energy contribution derived from electrostatic interactions
(ΔE
ELE
) was fairly evident, with a value of −7.78 kcal mol
–1
.In
the representative conformation of the CpomPBP2–Dod complex,
the nonpolar contribution to the solvation free-energy (ΔE
SURF
)
was also favorable for binding of Dod to CpomPBP2. According
to the results of an empirical model, ΔE
SURF
of the CpomPBP2–
Dod complex was estimated to be as high as −28.53 kcal mol
–1
.
However, free-energies unfavorable for the CpomPBP2–Dod
interaction were also detected. For example, unfavorable energy
from the polar part of solvation free-energy (ΔE
EPB
) reached up
to 13.84 kcal mol
–1
. The SEM of each free-energy item was limited
to 0.40 kcal mol
–1
(Table 1), indicating the high accuracy of the
theoretical binding free-energy calculated using the MM-PBSA
method.
3.4 Pairwise per-residue free-energy decomposition
Based on the MD representative conformation of the CpomPBP2–
Dod complex, the pairwise energy contribution (ΔE
pair
) of each
residue was estimated using pairwise per-residue free-energy
decomposition. It is clear in Fig. 3(A) that five residues (Phe12,
Leu68, Glu98, Arg109, and Ile113) made relatively significant
ΔE
pair
contributions (≥−2.00 kcal mol
–1
) compared with other
residues, so the pairwise energy contributions between these five
residues and Dod were selectively analyzed (Fig. 3B). As shown in
Fig. 3(B) and Table 2, Glu98 and Arg109 contributed the highest
ΔE
pair
, with values of −4.17 and −3.95 kcal mol
–1
, respectively.
Table 2 shows that the ΔE
pair
of Glu98 and Arg109 was predomi-
nantly derived from their remarkable electrostatic energy contri-
butions (−6.87 kcal mol
–1
for Glu98, −3.28 kcal mol
–1
for
Arg109). Such high electrostatic energy contributions are in line
with H-bond and salt-bridge interactions formed between -OH
of Dod and Glu98/Arg109. For Glu98, it should not be ignored that
a high polar solvation energy (2.27 kcal mol
–1
) unfavorable to the
binding of Dod was detected, even though its contribution to the
total ΔE
pair
was remarkable (Table 2). Unfavorable van deer Waals
interactions derived from Glu98 and Arg109 were detected also.
For Glu98 in particular, the unfavorable van der Waals energy
was close to 1.00 kcal mol
–1
(Table 2).
In addition to Glu98 and Arg109, three other hydrophobic resi-
dues (Phe12, Leu68, and Ile113) showed a superior pairwise
energy contribution, with ΔE
pair
ranging from −2.00 to
−3.00 kcal mol
–1
. For Phe12 in particular, ΔE
pair
was as high as
−2.90 kcal mol
–1
. For the three residues, the van der Waals and
nonpolar solvation energy contributions were all significant (>
−1.00 kcal mol
–1
) and favorable for binding of Dod to CpomPBP2.
Specifically, the favorable van der Waals energy (−1.65 kcal mol
–
1
) provided by Phe12 was due to hydrophobic contact between
Phe12 and the C1–C6 part of the Dod alkyl group (Figs 3B and
S4). The large contributions of Ile113 (−1.28 kcal mol
–1
) and
Leu68 (−1.22 kcal mol
–1
) to van der Waals forces can be attrib-
uted to their hydrophobic interactions with the C7–C9 and C10–
C12 regions of Dod, respectively (Table 2, Figs 3B and S4). Notably,
the electrostatic energy (−0.049 kcal mol
–1
) and polar solvation
energy (−0.21 kcal mol
–1
) derived from Ile113 were both weak
but favorable for binding of Dod to CpomPBP2. The introduction
of polar and charged groups like hydroxyl (-OH) to the C9 site of
Dod would be feasible to boost the binding affinity between
CpomPBP2 and Dod (Fig. S4).
3.5 Site-directed mutagenesis and protein expression
After comprehensive consideration of the results produced by
pairwise free-energy decomposition and computational ala-
nine scanning (reported in our previous research),
21
four res-
idues, Phe12, Glu98, Arg109 and Ile113, were selected for
Figure 2 Representative conformation of the CpomPBP2–Dod complex
produced based on the molecular dynamics trajectories. Representative
residues Phe12, Leu68, Glu98, Arg109, and Ile113, are marked on the bind-
ing interface. The ligand Dod is shown as a sphere–stick model.
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4
site-directed mutagenesis. Wild-type and mutant CpomPBP2
genes were cloned into pCold III vectors and expressed in
TransB (DE3) strains. It should be noted that pCold III replaced
pET-28a(+) used in our previous report. As a cold shock plas-
mid, pCold III could promote the soluble expression of target
proteins at low temperatures. Meanwhile, TransB (DE3) strains
aid formation of disulfide bonds which are important for the
correct folding of CpomPBP2. Because of the double effects
of vector and host strain, soluble expression of wild-type
and mutant CpomPBP2 proteins was successfully realized
(Figs S5 and S6). For convenience, wild-type CpomPBP2 pro-
tein was abbreviated as CpomPBP2WT, CpomPBP2 proteins
mutated at the sites of Phe12, Glu98, Arg109, and Ile113 were
named CpomPBP2F12A, CpomPBP2E98A, CpomPBP2R109A,
and CpomPBP2I113A.
3.6 Calculating experimental binding free-energy
The experimental binding free-energy (ΔG
bind-exp
) of the
CpomPBP2–Dod complex was calculated using the K
d
value
between wild-type CpomPBP2 protein and Dod (K
d-WT
). Using a
competitive binding assay, K
d-WT
was determined as 0.18
±0.09 μM(Fig. 4A), giving a ΔG
bind-exp
for the CpomPBP2–Dod
complex of −9.57 kcal mol
–1
(Table 1). The value of ΔG
bind-exp
was in qualitative agreement with ΔG
bind-cal
(−11.27 kcal mol
–1
)
calculated using the MM-PBSA method, and the difference was
within acceptable limits (2.00 kcal mol
–1
). The consistency of
ΔG
bind-exp
and ΔG
bind-cal
further indicated the stability and ratio-
nality of the complex formed by CpomPBP2 and Dod.
3.7 Hot-spot determination
Before evaluating changes in the binding affinity caused by site-
directed mutation, K
d
values between the fluorescent probe
1-NPN and wild-type/mutant CpomPBP2 proteins, namely
K
1-NPN
, were determined and compared. As expected, individual
replacement of Phe12, Glu98, Arg109, and Ile113 by Ala had no
significant effect on the binding of 1-NPN to CpomPBP2 proteins
(Fig. S6). Consequently, for convenience, the K
1-NPN
value of wild-
type CpomPBP2 protein was taken as the sole K
1-NPN
in subse-
quent competitive binding assays. As shown in Fig. 4, individual
mutation of Phe12, Glu98, Arg109, and Ile113 into Ala was unfa-
vorable for the binding of Dod. Of the four mutant CpomPBP2
proteins, CpomPBP2E98A and CpomPBP2R109A showed mini-
mum affinity to Dod, with K
d
values of 135.97 ±9.56 and
147.15 ±6.91 μM, respectively. The K
d
values for CpomPBP2E98A
and CpomPBP2R109A were further transformed into changes in
the experimental binding free-energy (ΔΔG
bind-exp
) using Eqn
(3) (Table 2). According to the thresholds for hot-spots
(ΔΔG
bind
≥4.00 kcal mol
–1
), warm spots (2.00 kcal mol
–1
≤
ΔΔG
bind
<4.00 kcal mol
–1
), and null spots (ΔΔG
bind
<2.00 k-
cal mol
–1
) provided by Moreira et al.,
47
Glu98 (ΔΔG
bind-
exp
=4.08 kcal mol
–1
) and Arg109 (ΔΔG
bind-exp
=4.13 kcal mol
–1
)
were classified as hot-spots (Table 3), suggesting they have key
roles in the binding of Dod to CpomPBP2 proteins. The key roles
Table 1 Theoretical and experimental binding free-energy
a
for the binding of Dod to wild-type CpomPBP2 protein
Contribution ΔE
ele
ΔE
vdW
ΔE
EPB
ΔE
ESURF
ΔG
gas
ΔG
sol
ΔG
bind-calb
ΔG
bind-expc
CpomPBP2WT
d
−7.78 (0.19) −34.83 (0.21) 13.84 (0.27) −28.53 (0.06) −42.61 (0.21) 31.33 (0.34) −11.27 (0.40) −9.57 (0.28)
a
All values are shown in kcal mol
–1
with corresponding SEM in parentheses.
b
ΔG
bind-cal
is the theoretical binding free-energy of the CpomPBP2–Dod complex.
c
ΔG
bind-exp
is the experimental binding free-energy of the CpomPBP2–Dod complex.
d
CpomPBP2WT is the wild-type CpomPBP2 protein.
Figure 3 Pairwise per-residue free-energy decomposition of the
CpomPBP2–Dod complex. (A) Energy spectra presenting the energy con-
tribution of each residue in the CpomPBP2–Dod complex. Five residues
(Phe12, Leu68, Glu98, Arg109, and Ile113) contribute beyond −2.00
kcal mol
–1
free-energy. (B) Energy spectra presenting the interactions
formed between representative residues and groups of Dod. C1–C12
chain of Dod mainly interacts with hydrophobic residues represented by
Phe12, Leu68, and Ile113. The hydroxyl group of Dod mainly forms electro-
static interactions with Glu98 and Arg109.
Key residues in CpomPBP2–Dod interaction www.soci.org
Pest Manag Sci 2020 © 2020 Society of Chemical Industry wileyonlinelibrary.com/journal/ps
5
of Glu98 and Arg109 in determining the affinity between Dod and
CpomPBP2 protein can be attributed to their strong electrostatic
interactions with -OH of Dod (Figs 2 and S4, Table 2). On mutation
of Glu98 and Arg109 to Ala, the salt-bridge derived from Arg109
and the H-bond derived from Glu98, which work to anchor the
-OH of Dod, were disrupted, resulting in the low binding ability
of CpomPBP2E98A and CpomPBP2R109A to Dod (Fig. 4). Muta-
tion of Phe12 and Ile113 to Ala had a much weaker negative effect
on the binding ability of CpomPBP2. From Table 2, it is evident
that the ΔΔG
bind-exp
values corresponding to CpomPBP2F12A and
CpomPBP2I113A fell within the warm spot range (Table 3), sug-
gesting that hydrophobic interactions provided by side-chains
of Phe12 and Ile113 were not strong enough to make them key
residues in determining the binding ability of CpomPBP2 protein.
4 DISCUSSION
Even though pheromone-mediated management is thought of as
an eco-friendly method of controlling C. pomonella, it is more
commonly used as a supplement to chemical control.
48
Applica-
tion of pheromone synergists to improve the activity of phero-
mone lures is feasible and effective. In fact, application of
synergists in commercial pheromone lures is fairly common. In
Grapholita molesta sex attractants containing three active ingredi-
ents (E8-dodecenyl acetate, Z8-dodecenyl acetate and Z8-dode-
cenol), Z8-dodecenol is works as a synergist.
49
However, as an
auxiliary material in insect attractants, the presence of phero-
mone synergists and research into their roles are easily ignored.
Currently, data on pheromone synergists are quite limited. Yang
et al. reported that racemic linalool, E-farnesene, and Z3-hexenol
could synergize a response to codlemone in C. pomonella. Notice-
ably, the synergistic effects of these three compounds were not
inferior to that of Dod.
50
However, no further reports are available.
Taking Dod as a representative molecule of codlemone synergists,
our research aims to provide suggestions for the prospective dis-
covery of novel synergists with function similar to or stronger than
Dod by revealing details of the interaction between CpomPBP2
and Dod.
Using 150 ns MD simulations, a stable conformation close to the
actual structure of the CpomPBP2–Dod complex was obtained.
Acquisition of a reliable conformation is of great significance for
subsequent experiments, including binding mode analysis, bind-
ing free-energy calculation, free-energy decomposition, and hot-
spot determination. As in other research on the structure of insect
OBPs,
26,51,52
van der Waals (ΔE
vdW
) and electrostatic energy
(ΔE
ELE
) were two dominant driving forces in the formation and
maintenance of the CpomPBP2–Dod complex. As shown in
Table 1, ΔE
vdW
and ΔE
ELE
values were as high as −34.83 and
−7.78 kcal mol
–1
, respectively. Through further pairwise free-
energy decomposition (Fig. 3, Table 2), it can be determined that
Phe12, Leu68, Glu98, Arg109, and Ile113 were the top five resi-
dues in terms of pairwise free-energy contribution, with hydro-
phobic residues (Phe12, Leu68, and Ile113) being top donors of
van der Waals energy, and hydrophilic residues (Glu98 and
Arg109) being dominant providers of electrostatic energy. Con-
sidering above results, it could be deduced that the long carbon
chain (C1–C12) of Dod is reinforced by hydrophobic contacts
derived from hydrophobic residues represented by Phe12,
Leu68, and Ile113. As for the -OH in Dod, it is generally anchored
by the H-bond and salt-bridge interactions provided by Glu98
and Arg109, respectively. Similar phenomena were also found in
interactions between other alcohol pheromones and their corre-
sponding pheromone binding proteins (PBPs). Take the phero-
mone bombykol from Bombyx mori for example, the 16-carbon
alcohol is bound in the BmorPBP (PBP from B. mori) cavity through
numerous hydrophobic interactions and a hydrogen bond
formed between -OH and Ser56.
51
Even though the value of ΔE
vdW
was much higher than ΔE
ELE
in
the CpomPBP2–Dod complex (Table 1), two residues (Glu98 and
Arg109) forming electrostatic interactions with -OH of Dod, rather
than hydrophobic residues (Phe12, Leu68, and Ile113), were veri-
fied as key residues in determining the binding ability of
CpomPBP2. Confirmation of Glu98 as a hot-spot supports the
assumption that residues forming H-bond with ligands are usually
crucial to the protein-ligand interactions.
24,32,51
As a matter of
experience, in protein-ligand interactions involved with insect
OBPs, key residues are usually those forming either H-bond or
hydrophobic interactions with ligand groups.
26,32,44,53
However,
Arg109 providing a salt-bridge interaction, a form of electrostatic
interaction rarely described in protein–ligand interactions
involved with insect OBPs, was determined as a second key resi-
due for the CpomPBP2–Dod complex.
Table 2 Decomposition of binding free-energy on a pairwise per-residue level
a
Dod - Residue van der Waals Electrostatic Polar solvation Non-polar solvation Total
Phe12 −1.65 0.014 0.16 −1.42 −2.90 ±0.51
Leu68 −1.22 0.40 −0.34 −1.06 −2.22 ±0.37
Glu98 0.80 −6.87 2.27 −0.36 −4.17 ±0.96
Arg109 0.19 −3.28 −0.32 −0.54 −3.95 ±1.64
Ile113 −1.28 −0.049 −0.21 −1.18 −2.72 ±0.33
a
Energies are shown as contributions from van der Waals energy, electrostatic energy, polar solvation energy, non-polar solvation and their sum for
the CpomPBP2–Dod complex. All values are given in kcal mol
–1
.
Table 3 Experimental binding free-energy changes of the
CpomPBP2–Dod complex caused by site-directed mutagenesis
Protein
a
Cpom
PBP2F12A
Cpom
PBP2E98A
Cpom
PBP2R109A
Cpom
PBP2I113A
ΔΔG
bind-expb
2.91 4.08 4.13 3.26
a
CpomPBP2F12A, CpomPBP2 E98A, CpomPBP2R109A, and CpomPB-
P2I113A are CpomPBP2 proteins mutated into Ala at the residue sites
of Phe12, Glu98, Arg109, and Ile113 respectively.
b
ΔΔG
bind-exp
is the experimental binding free-energy change, all
values are shown in kcal mol
–1
.
www.soci.org Z Tian et al.
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6
In addition to the favorable interactions mentioned above,
interactions unfavorable to the binding of Dod to CpomPBP2
were also discussed. In drug discovery, replacing the unfavorable
structural parts of prototype molecules with favorable groups is
well-established and widely used technique, even though related
techniques remain unusual in the field of agriculture. In a report
on the development of anti-tubercular drugs, Liu et al. found that
introducing H-bond acceptors into the benzothiazole ring of
TCA1 may lead to the generation of new H-bonds and decrease
unfavorable interactions. In fact, the inhibitory effects of TCA1
were improved eightfold when nitrogen was introduced into
the C4 position of the TCA1 benzothiazole ring.
54
Based on accumulated experiences in drug discovery, the exis-
tence of unfavorable interactions provides opportunities for the
discovery and design of novel chemicals with stronger synergistic
effects than codlemone. For example, the unfavorable polar sol-
vation energy derived from Glu98 reached 2.27 kcal mol
–1
(Table 2), so in the prospective chemical optimization of Dod,
groups loaded to Dod should be selected from those unable to
generate polar solvation interactions with Glu98. Introducing a
nonpolar group into the hydroxyl oxygen of the Dod molecule is
recommended. Moreover, the unfavorable van der Waals energy
contributed by Glu98 and Arg109 was also remarkable (Table 2).
As shown in Fig. S4, C12 derived from the backbone of Dod was
spatially close to the side-chains of Glu98 and Arg109. Therefore,
actions to load hydrophilic groups onto the site (C12) of Dod may
enhance the CpomPBP2–Dod interaction. Unfavorable electro-
static and polar solvation energies derived from Phe12 were
small, but did exist. The affinity between CpomPBP2 and Dod
would benefit from the presence of polar groups with the C3 or
Figure 4 Binding curves of 1-dodecanol to wild-type (A) and mutant (B) CpomPBP2 proteins. CpomPBP2WT, wild-type CpomPBP2 protein;
CpomPBP2F12A, CpomPBP2 E98A, CpomPBP2R109A, and CpomPBP2I113A are CpomPBP2 proteins mutated into Ala at the residue sites of Phe12,
Glu98, Arg109, and Ile113 respectively.
Key residues in CpomPBP2–Dod interaction www.soci.org
Pest Manag Sci 2020 © 2020 Society of Chemical Industry wileyonlinelibrary.com/journal/ps
7
C4 of Dod located around the phenyl group of the Phe12 side-
chain. Unfavorable energy was also derived from electrostatic
interactions between Leu68 and the C10–C12 part of Dod
(Table 2). Uncharged and nonpolar groups are advised for the
C10–C12 sites when optimizing Dod as avoidance of electrostatic
interactions with the Leu68 side-chain helps promote the binding
affinity between Dod and CpomPBP2.
Finally, it should be noted that the value of K
d-WT
obtained in
this research was more than ten times lower than reported previ-
ously.
21
The increase in binding ability of recombinant CpomPBP2
protein can be attributed to improvements in the adopted pro-
karyotic expression system. As mentioned above, the host strain
(TransB) and expression vector (pCold III) act to promote the yield
and quality of soluble CpomPBP2 protein. Moreover, after remov-
ing the His tag, the two amino acids (Gly and Pro) attached to the
N-terminus of recombinant CpomPBP2 protein have little effect
on the binding ability of CpomPBP2, especially considering that
the N-terminus of CpomPBP2 is far from the binding cavity.
21
The improved quality of the recombinant CpomPBP2 protein con-
tributed to the higher binding affinity to Dod in this research.
5 CONCLUSIONS
In summary, it is reasonable to state that electrostatic energy and
van der Waals energy were two major forces driving formation of
the CpomPBP2–Dod complex. Attempts to disrupt these interac-
tions, the electrostatic interaction in particular, decreased the bind-
ing affinity between CpomPBP2 and Dod. As dominant donors of
electrostatic interactions, Glu98 and Arg109 were confirmed as
two key residues determining the binding of Dod to CpomPBP2
protein. Not only that, energies unfavorable to the binding of Dod
were also noted, including the unfavorable polar solvation energy
derived from Glu98 and unfavorable electrostatic energy provided
by Leu68. Such negative interactions provide cautions for the pro-
spective chemical optimization of Dod. As an ingredient of
C. pomonella sex pheromone, Dod works to synergize the effect
of codlemone in attracting male moths, our results on the interac-
tion between Dod and CpomPBP2 protein could boost the design
and discovery of novel chemicals with similar functions.
SUPPORTING INFORMATION
Prototype of the CpomPBP2–Dod complex (Fig. S1); Cluster
analysis of the CpomPBP2–Dod complex produced in MD simula-
tions (Fig. S2); Superimposition of representative conformations
derived from dominant clusters (Fig. S3); The distances from
atoms on the backbone of Dod to the closest residue atoms
(Fig. S4); SDS–PAGE analysis of purified wild-type and mutant
CpomPBP2 proteins (Fig. S5); Binding curves of 1-NPN to wild-
type and mutant CpomPBP2 proteins (Fig. S6); Primers used for
protein expression and site-directed mutagenesis (Table S1); Clus-
ter analysis of the CpomPBP2–Dod complex based on the MD
simulations trajectories (Table S2).
ACKNOWLEDGEMENTS
This research was supported by the National Natural Science
Foundation of China (31801797), the Natural Science Foundation
of Jiangsu Province (BK20180902), the Key Research & Develop-
ment Project of Shaanxi Province (2019NY-186), the Fundamental
Research Funds for the Central Universities (2452018008), and the
Sci-Tech Planning Project of Yangling Demonstration Zone
(2018NY-02).
SUPPORTING INFORMATION
Supporting information may be found in the online version of this
article.
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