Modeling of the binding of octopamine and dopamine in insect

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Article

Modeling of the binding of octopamine and dopamine in insect monoamine transporters reveals structural and electrostatic differences. Sandra Arancibia, Matias Marambio, Jorge M. Campusano, and Angélica Fierro ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00631 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 3, 2019

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ACS Chemical Neuroscience

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Modeling of the binding of octopamine and dopamine in insect monoamine transporters

2

reveals structural and electrostatic differences.

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Sandra Arancibia1, Matías Marambio1, Jorge M Campusano2, Angélica Fierro1*

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1Bioorganic

and Molecular Modeling Lab, Organic Department, Facultad de Química y de

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Farmarcia, Pontificia Universidad Católica de Chile, Santiago, Chile.

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2Department

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Universidad Católica de Chile, Santiago, Chile.

of Cell and Molecular Biology, Biological Sciences Faculty, Pontificia

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Running title: Structural insights into monoaminergic transporters in insects.

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ACS Paragon Plus Environment

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ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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Octopamine, a trace amine in mammals, is a major neurotransmitter linked to important

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biological processes in insects. Interestingly, one of the molecular entities responsible for

18

octopamine availability, the octopamine transporter (OAT), has not been identified in

19

certain insect species. For instance, no OAT has been reported in the fly Drosophila

20

melanogaster (Dm), and the molecule involved in octopamine reuptake in Drosophila is not

21

known. Here, we used molecular modeling methodologies to obtain three-dimensional

22

insights for the dopamine transporter (DAT) and OAT in a common agricultural pest insect,

23

Trichoplusia ni (Tni). Our results show several similarities but also significant differences in

24

the general structures of the proteins of Dm and Tni. One important difference is observed

25

in the ligand binding cavity, where a negatively charged amino acid present in both

26

dopamine transporters is replaced by a polar neutral residue in the Trichoplusia OAT. This

27

modification could influence both the binding mode and the driving force involved in the

28

transport mechanism of these amines into neurons of these species. We also obtained data

29

that support the idea that octopamine could bind and possibly be transported by DmDAT.

30

The structural characterization of macromolecules from different insect species is

31

fundamental in the agricultural field to gain insights into the design of new compounds for

32

controlling pests.

33 34

Keywords:

Monoaminergic

system,

octopamine,

35

melanogaster, Trichoplusia ni, molecular dynamics.


neurochemistry,

Drosophila

2

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INTRODUCTION

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Based on United Nations projections, the world population is expected to exceed 11 billion

38

by 2100 and life expectancy to increase from 75 to 85 years1. Although efforts have been

39

continuously carried out to increase both quality and quantity of food, it is a fact that the

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land surface that can be used for agricultural purposes is limited2. In addition, pest plagues

41

generate damage to crop yields and, due to the application of chemicals, to soil and water

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quality as well. Consequently, a reduced productivity in crops or animals may generate a

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considerable economic and environmental impact. Thus, studies aimed at improving the

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productivity of current agricultural resources are essential. Likewise, research directed to

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address common problems in agriculture, including resistance to agrochemicals by pest

46

insects, or the generation of new chemicals is needed.

47

In recent decades, the study of specific chemical interactions between biological targets

48

and ligands has been a popular focus in a variety of research areas. In this regard,

49

computational approaches to model three-dimensional structures of macromolecules have

50

proven useful to predict interactions, design new compounds, and validate and/or

51

understand experimental results3-6. In spite of this, molecular simulation of biological

52

systems has been less explored in the agricultural field as a tool to gain a better

53

understanding of pesticide resistance, to design new compounds with potential as pest

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control agents, or to increase selectivity of those chemicals already used in agrochemistry7.

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In silico tools could also help predict whether new agrochemicals discriminate between

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mammals and insects, as well as between beneficial and non-beneficial species.


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A vast proportion of the insecticides described in the literature are targeted at proteins and

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molecules present in the insect central nervous system (CNS). Casida et al8, reported that

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most insecticides act on components of insect acetylcholine, glutamate, and -

60

aminobutyric acid neural systems, while less explored targets include octopamine and its

61

receptors (OARs)9-12.

62

Octopamine acts as neurotransmitter in the CNS and has been associated with learning and

63

memory13 as well as with the feeding behavior of insects14. Octopamine has also been

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observed at high concentrations in non-neuronal insect tissues, and several reports

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describe this amine as a peripheral neuromodulator. Octopamine has been additionally

66

involved in the regulation of the activity of the light-emitting organ of the firefly and of the

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endocrine gland in other insects15. It plays a role in the definition of social functions and

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hierarchy in social insects such as the honeybee16. In addition, low levels of octopamine

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have a direct influence on arthropod oviposition17-18.

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The action of monoamines in the insect CNS depends on receptors, metabolic enzymes, and

71

transporters that are involved in the reuptake of neurotransmitters to end their actions.

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Specific octopamine transporters (OATs) have been described in different insects, including

73

Trichoplusia ni19. This is a harmful pest insect that feeds on a wide range of plants of

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economic importance including crucifers and a variety of other vegetables in both field and

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greenhouse settings20. Interestingly, no specific OA transporter has been described in

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Diptera such as the fly Drosophila melanogaster (Dm) or in Hymenoptera such as the

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beneficial insect honeybee21. Thus, the main protein responsible for octopamine reuptake

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and the molecular mechanisms involved in this event are yet to be defined in some insect


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orders. It has been postulated that given the similarities in the chemical structure of

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dopamine and octopamine, in species where no OAT is found, the molecular entity

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responsible for octopamine uptake could be DAT22.

82

The SLC6 family of proteins, which includes transporters for monoamines like dopamine

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and serotonin (DAT and SERT, respectively), have been highly associated with

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neurodegenerative and psychiatric diseases in humans23-24. Important efforts have been

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directed to understanding specific requirements for high affinity ligand binding and their

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mechanisms. These studies have led to the elucidation of the three-dimensional structure

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of these proteins, which is valuable information that could be used to advance our

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knowledge of the structure and functioning of other systems. In particular, no OAT X-ray

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structure or structural information is available in the literature. Thus, the atomic-resolution

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X-ray crystal structure of DAT reported by the Goaux research group25-26, provides valuable

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information that could be used to advance our knowledge on the structure and operation

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of OAT in those insect species where this protein has been described.

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Here, by using molecular simulation methodologies we report for the first time a model for

94

an OAT protein and provide new insights into the macromolecules responsible for the

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reuptake of dopamine and octopamine in T. ni. We also evaluate structural and electrostatic

96

differences of these proteins as compared to DmDAT and further assess the idea that

97

DmDAT is able to bind octopamine. These studies provide new information that contributes

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to our understanding of amine availability in insects and could encouragement the

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development of future generations of molecules for selective control of pests.

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RESULTS AND DISCUSSION

102 103

Similarities and differences in 3D structure of monoamine transporters

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The three-dimensional structures of T. ni DAT and OAT (TniDAT and TniOAT, respectively),

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were obtained by means of homology modeling using the crystal structure of DmDAT as a

106

template, given the amino acid identity of these transporters (DmDAT/TniDAT: 76,47%;

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TniDAT/TniOAT: 48,71% and DmDAT/TniOAT 47,77%). The stereochemical and energetic

108

analysis of chosen models validate our structure as viable. Changes in residues were

109

detected in different segments of the transporters but the most significant differences were

110

observed in segments of the proteins not associated with the lipid membrane, including the

111

extracellular loops 2, 3 and 4 (EL2, EL3 and EL4, respectively) which are represented in

112

different colors in Figure 1A. These differences can be better observed in Figure 1B where

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an alignment of the amino acid sequence of these segments is shown (a full alignment is

114

provided in Figure S1).

115

With respect to the binding cavities, dopamine transporters in both species show a highly

116

conserved site formed mainly by aromatic and polar amino acids. The aromatic

117

environment is conserved in the binding cavity of TniDAT but at least two important

118

differences were observed with respect to the dopamine transporters: the D121 residue in

119

DmDAT (or D97 in TniDAT), is occupied by a serine residue (S96) in TniOAT. In addition, close

120

to the F271 in TniOAT a histidine residue (H273) is located in the position where a conserved

121

leucine is observed in the dopamine transporters. The role of these changes became

122

evident in the molecular dynamic simulation studies (see below).


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123 A)

B)

EL2 EL4

EL3 EL2

DmDAT TniDAT TniOAT

EL3

DmDAT TniDAT TniOAT

EL4

DmDAT TniDAT TniOAT

S1

C)

DmDAT

TniDAT

N125

Y100

Y124

D46

DA

S421 DA

D97 F43

S426 G425

D22

N101

S422 D121

A93 S380

Y99 Y98 OA

S379 F19

F283

124

D22 F271

D424

S266

F325 S384

DA OA

TniOAT

Y265

125

Figure 1: Neurotransmitter transporter description. A) General three-dimensional structure

126

of monoamine transporter showing the extracellular segment (EL2 green; EL4: pink; EL3:

127

cyan) where the biggest differences were observed. B) Alignment of EL2, EL3 and EL4 amino

128

acid sequences of DmDAT, TniDAT and TniOAT. C) Main interactions defined from docking

129

studies of transporters and their substrates. In red: oxygen atoms; blue: nitrogen atoms;

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white: carbon atoms from the protein; dopamine and octopamine are displayed in green

131

and cyan respectively. *Numbers of amino acids in TniDAT and TniOAT correspond to the

132

model number.

133


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134

Differences in the binding cavity of transporters influence the binding mode of

135

neurotransmitters.

136

Once transporter models were obtained, docking studies for each substrate into the binding

137

cavity were carried out (Figure 1C). Our results show that dopamine is located inside an

138

aromatic cavity formed by F34, Y124, F325 (among others) in DmDAT. In addition,

139

substrates interact with negatively charged amino acids D46 and D121 at less than 2 Å,

140

generating a hydrogen bond with the hydroxyl group and at approximately 3 Å via a

141

coulombic interaction with the ammonium group. It is important to mention that our results

142

are in agreement with the interactions and binding mode reported by Wang et. al,28

143

validating our methodology to obtain transporter-ligand complexes.

144

As in DmDAT, dopamine was positioned in a similar conformation into the cavity in TniDAT

145

and amino acids forming the cavity and the interactions responsible to accommodate the

146

ligand were mainly conserved.

147

The OA/TniOAT complex showed differences in comparison with the DA/DAT complex,

148

although the transporter cavity and the amino acids interacting with the substrate were

149

essentially conserved. One of the most obvious differences is that octopamine initial

150

binding to the pocket was in an inverted position with respect to dopamine. The ammonium

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group of octopamine was located close to D424, generating a coulombic interaction at 2.01

152

Å, and was positioned far from D22 (D46 in DmDAT). In addition, the hydroxyl group located

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in para-position of the aromatic center generates a strong hydrogen bond with Y265 at 1.68

154

Å (distance for a low barrier hydrogen bond, LBHB: dO--H—O 2.58 Å).


8

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155

In all cases, tyrosine and phenylalanine residues are widely distributed into the cavities

156

encouraging interaction with the aromatic center of the neurotransmitters.

157 158

Evolution of transporter-neurotransmitter complexes in an isothermic-isobaric system.

159

Molecular dynamics simulations of DA/DmDAT, DA/TniDAT and OA/TniOAT complexes

160

inserted in a membrane were carried out for 100 ns simulations (RMSD representations in

161

Figure S2 for all systems simulated). In Figure 2, the final conformation of dopamine inside

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the binding cavity in D. melanogaster and T. ni DATs, and of octopamine in TniOAT are

163

displayed. In DAT of both species (Figure 2A and 2B), dopamine showed similar molecular

164

interactions with amino acid residues located in the main cavity. Both systems showed the

165

closer interactions (less than 2 Å) with negatively charged residues (D46 and D121 in DmDAT

166

and D22 and D97 in TniDAT) stabilizing the ammonium group and the catechol moiety.

167

These interactions remain during the entire simulation time. In addition, an aromatic

168

environment and hydrogen bonds network contribute to obtain a final equilibrated system.

169

In contrast to what is observed in dopaminergic systems, octopamine located in the

170

interaction site of the TniOAT shows an important change. When the lowest docked-energy

171

conformation of octopamine was submitted to 100 ns of molecular dynamic simulation, the

172

ligand flipped its orientation into the cavity by 180o. This is clarified in the graphical

173

representation in the middle of Figure 2C where the distances associated with ligand-amino

174

acid interactions became shorter (in some cases distance values exhibit a 6 Å or even 10 Å

175

change). The change in conformation occurred very early in the simulation (see Figure S3),

176

showing that octopamine adopted a conformation into TniOAT comparable to that


9


177

observed for dopamine in DAT. Possibly, the presence of S96 in OAT in the position where

178

a negatively charged amino acid is observed in DAT (D121 or D97 for Dm or Tni respectively)

179

conferred more flexibility to OA inside the transporter. A hydrogen bond histogram for

180

octopamine in TniOAT supports the hypothesis that this amine establishes fewer and/or

181

weaker interactions in the binding site (Figure 2).

182 A) DA/DmDAT

B) DA/TniDAT

D46

S380

Y100

F325

D22

H274

Y99

V273

S320 A117

C) OA/TniOAT Y265

D22

F43

D121 Y124

F19

D97

F319

S367

F283

Y277 F19

10

10

10

8

8

6 4

Distance (Å)

Distance (Å)

Distance (Å)

8

6 4

2

6 4

2 0

50

100

2

0

Time (ns)

50

100

0

50 Time (ns)

Time (ns) F43

183

D46

D121

Y124

S320

F325

D22

D97

Y100

F283

S380

F19

12

10

10

10

8

8

8

6 4

6 4

2

2

0

0

0

50 Time (ns)

100

HBonds

12

HBonds

12 HBonds

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Page 10 of 27

D22

Y265

Y99

100

H274

Y277

6 4 2 0

0

50 Time (ns)

100

0

50

100

Time (ns)

184

Figure 2: Final conformation from molecular dynamic simulations of A) DA/DmDAT, B)

185

DA/TniDAT and C) OA/TniOAT. In the middle panel, a graphical representation of the

186

substrate atoms and closer amino acid distance vs. time. On the bottom panel, histograms

187

of the number of hydrogen bonds during the simulation are displayed. *Numbers of amino

188

acids in TniDAT and TniOAT correspond to the model number


10

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189 190

Octopamine and DmDAT establish similar interactions to those observed for dopamine

191

and its transporter

192

As described in the introduction, octopamine, a phenethylamine whose physicochemical

193

properties are mainly conserved in dopamine, plays a crucial role in several functions in

194

insects. Although it has been proposed that DAT, at least a proportion of octopamine

195

transport can be carried out by DAT, the macromolecule responsible for octopamine

196

reuptake remains undefined in several species27-28.

197

Considering our results above, the chemical characteristics and the versatility observed for

198

octopamine in OAT, and the amino acid composition of the binding site of DmDAT, we

199

carried out docking studies and molecular dynamic simulations of OA inside DmDAT under

200

the same conditions used above.

201

Our results show an energetically favorable complex (binding energy -3.17 kcal/mol) and a

202

similar binding of octopamine in DmDAT as compared to dopamine. Figure 3A shows

203

octopamine and dopamine inside the binding cavity in DmDAT. After a 100 ns molecular

204

dynamic simulation, the lateral chains of some amino acids far from the binding cavity are

205

differently oriented (Figure 3B) but octopamine remains in the cavity and interactions with

206

F43, D46, D121, Y124, F319 and F325 are conserved. Thus, our structural and energetic

207

results show that octopamine can generate similar interactions as dopamine in the binding

208

site of DmDAT (Figure 3A).


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A) A)

Page 12 of 27

B)

K59 R52

Y124

Y542

D46

D312

F43

Y537

D121

F319 F318 F319

F325

DA Dopamine Octopamine OA

F533

209 210

Figure 3: General conformation obtained after molecular dynamic simulations of A)

211

dopamine, DA (green) and octopamine, OA (cyan) in DmDAT (light and dark gray for DA or

212

OA complex respectively). B) Main displacement of DmDAT residues during simulation with

213

DA (pink) and OA (cyan).

214 215

Electronic distribution of transporters is substrate-dependent

216

The reuptake process in one direction (from the extracellular to the intracellular space)

217

could be considered a non-equilibrium process mediated by an energetically expensive

218

conformational change of the whole system. At least two elements are needed for the

219

reuptake process to take place: first, it is necessary that the macromolecule interacts with

220

its substrate, and second, a driving force (DF) is required to specify the direction of the

221

movement of that substrate. Results described above deliver structural information on the

222

favorable interactions between ligands and transporters. An additional layer of complexity

223

to understand these interactions is reveled by the study of the electronic distribution in

224

these proteins when bound to substrates.


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225

Using Adaptive Poisson-Boltzmann Solver (APBS) calculations to estimate the electrostatic

226

potential in a central slide of the protein at 100 ns of simulation shows a positive potential

227

(blue) in the transporter segment interacting with hydrophilic phospholipid heads and a

228

negative distribution (red) inside the macromolecule. These results are reasonable if we

229

take into consideration that neurotransmitters are protonated at physiological pH, and the

230

DF is related to the electrostatic potential (ESP) inside the transporter.

231

Interestingly, a more negative profile was obtained for DA/DmDAT in comparison with the

232

Tni-transporter. In addition, when the electrostatic potential was studied in the main cavity

233

of the transporters, it was observed that a positive peak was followed by a negative one in

234

all cases (0  Z  20, in Figure 4), which could be a necessary condition so that monoamine

235

transporters interact with their corresponding substrates in the binding site.

236 B

A

C

DA/TniDAT

DA/DmDAT

OA/TniOAT

-40

-20

20 -50 -100 -150

237

Z axis

40

Electrostatic Potential (kT/e)

50

100 50 -40

-20

20 -50 -100 -150

40


100

100 Electrostatic Potential (kT/e)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Z axis

50

-40

-20

20

40

-50 -100 -150 Z axis

238

Figure 4: Electrostatic potentials in ligand-transporter complexes. At the top of the figure,

239

a slide of electrostatic potential map is shown. Red to white to blue color represent a


13


240

transition from higher to lower charge concentration. At the bottom, the corresponding

241

graphical representation of electrostatic potential through the Z axis for DA/DmDAT (A),

242

DA/TniDAT (B) and OA/TniOAT (C) is displayed.

243 244

In order to corroborate how the substrate influences the electrostatic potential of the

245

transporter and following the hypothesis that OA generates interactions with DmDAT, we

246

compared the charge distribution of DA/DmDAT and OA/DmDAT complexes. In Figure 5, we

247

can observe a more negative charge profile distributed inside DAT when dopamine is

248

located in the binding cavity (as was described in Figure 4) in comparison to the situation

249

when octopamine is in the same cavity. As observed previously, in both systems it is possible

250

to detect a positive potential followed by a negative electronic distribution, although the

251

magnitude was considerably lower for OA/DmDAT in comparison with DA/DmDAT (Figure

252

5B). A comparison of the final conformations of DmDAT complexed with either dopamine

253

or octopamine shows that the main differences are observed as displacements in charged

254

and aromatic residues, including R52 (4.6 Å), K59 (4.2 Å), D312 (2.6 Å), F318 (5.0 Å), F319

255

(7.6 Å), Y533 (5.7 Å), Y537 (4.0 Å) and Y542 (7.9 Å).

DA/DmDAT

OA/DmDAT

B)

-40


A)


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Page 14 of 27

100

0

5

10

15

20

50

-50 -20

20 -50

40

-100

-100 -150

-150

Z axis DA/DmDAT

OA/DmDAT

DA/DmDAT

OA/DmDAT

256


14

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257

Figure 5: Electrostatic potential analysis in the Z axis of ligand-transporter complexes after

258

100 ns of molecular dynamic simulations. A) and B) show qualitative and quantitative

259

electrostatic potential description of a slide in the middle of transporter for DA and OA

260

inside DmDAT. A close up of Electrostatic Potential vs. Z axis display the profile into the main

261

binding site in complex with the neurotransmitter.

262 263

We observe differences in electrostatic profiles when octopamine is inside TniOAT as

264

compared to the situation when octopamine is in DmDAT, revealing the importance of the

265

electronic distribution around a specific ligand. Despite the fact that more experimental and

266

computational evidence is required, our findings suggest that octopamine could generate

267

favorable interactions with DAT in D. melanogaster. These data are consistent with the idea

268

that this amine could be a ligand for this transporter.

269

In addition, these results strengthen the idea that the amino acid identity of the transporter

270

is not enough to compare the structure of macromolecules bound to ligands, given that in

271

spite of the high amino acid identity between DmDAT and TniDAT (greater than 75%), it is

272

possible to detect important differences in the electrostatic profile of both proteins when

273

interacting with dopamine or octopamine. Because of this ability to detect differences, we

274

believe that these graphical and quantitative studies offer us a strong basis to speculate

275

about the specific differences between the transporters of different species.

276 277

CONCLUSION


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Page 16 of 27

278

Despite the fact that structural information of membrane proteins has increased during the

279

last decades through the availability of techniques such as X-ray diffraction, NMR and more

280

recently cryo-EM, there are numerous macromolecules whose structures have not yet been

281

solved. Thus, computational methodologies play a crucial role in generating structural and

282

functional information.

283

In this article we have focused on obtaining a three-dimensional description and electronic

284

insight into the monoamine transporters of Drosophila melanogaster and the pest

285

Trichoplusia ni when bound to their corresponding substrates. By doing so, we provide the

286

first structural description of an OAT protein in any animal. Our findings indicate that the

287

structural comparison of macromolecules is not sufficient to establish parameters

288

associated with their interaction with potential ligands and/or with their function, as

289

observed in our study when comparing DmDAT vs TniDAT, two macromolecules that share

290

the same function and exhibit a high amino acid sequence identity. Despite these

291

similarities, our electrostatic potential studies support the idea that qualitative and

292

quantitative differences exist in these transporters. Interestingly, when only considering the

293

binding cavity, a similar profile in electrostatic potential was observed in all transporters

294

complexed with neurotransmitters suggesting a similar charge distribution when the

295

substrates are located into the cavity. The use of computational methodologies is applicable

296

to the study of mosquitoes, ticks, and other arthropods to evaluate how inhibitors and/or

297

other substrates affect electrostatic potential. We believe that these methodologies offer

298

promising new insights that could facilitate the design of the next generation of pesticides.

299


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300

METHODS

301

Parameter generation for Dopamine and Octopamine

302

The compounds were constructed using Spartan129 in a protonated state. The partial

303

charges were corrected using ESP methodology. Topology and parameters for the ligands

304

were obtained using Swissparam server30, which use CHARMM27 force field and database

305

for organic compounds.

306

Homology Modeling

307

The crystal structure of dopamine transporter from Drosophila melanogaster at 2.89 Å of

308

resolution25 was used as a template (PDB code 4XP1) to obtain homology models of

309

dopamine and octopamine transporters of Trichoplusia ni. The amino acid sequences of

310

monoamine transporters and crystal structure of the template protein were extracted from

311

NCBI and PDB database31. Target protein and template sequences were aligned through

312

multiple alignments for different species, and an alignment by function was built.

313

The MODELLER9v6 program32 was used to construct 3D models of the OAT and DAT

314

transporters of Trichoplusia ni. In this study, 200 Modeller runs were carried out using

315

standard parameters and the outcomes were ranked on the basis of the internal scoring

316

function of the program. The best model was chosen as the target to evaluate the main

317

interaction with the respective substrates.

318

Model evaluation

319

Analyses of geometry, stereochemistry and energy distribution were carried out for each

320

model. The VMD program33 was used to evaluate the 3D structure distribution and general

321

physical chemical characteristics (i.e. hydrophobicity in transmembrane segments,


17


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322

aromatic residues in the membrane-water interface). Then, the stereochemical and energy

323

qualities of the homology models were evaluated using the PROSAII server34, Procheck35

324

and Molprobity36. These programs confirmed the accuracy of the models.

325

Molecular Docking

326

In order to obtain information regarding the main protein-ligand interactions, the molecular

327

docking of octopamine and dopamine with TniDAT and TniOAT (respectively) was done

328

using AutoDock 437 suite. In general, the grid maps were calculated using the autogrid4

329

option and were located in the center of the transporter cavity. The volumes for the grid

330

maps were 40 x 40 x 40 points (a grid-point spacing of 0.375 Å). The autotors option was

331

used to define the rotating bonds in the ligand. An initial population of 1500 random

332

individuals with a population size of 100 individuals, a maximum number of 2.5 x 106 energy

333

evaluations, a maximum number of 27,000 generations, a mutation rate of 0.02 and a cross-

334

over rate of 0.80 were employed in the Lamarckian genetic algorithm (LGA) dockings. The

335

complexes with docked compound were built using the lowest docked-energy binding

336

positions.

337

Molecular dynamics simulations

338

The transporters were analyzed in the H++ server38 to compute pK values of ionizable

339

groups and missing hydrogen atoms according to the specified pH of the environment. Then

340

each

341

phosphoethanolamine (POPE) membrane, solvating with water model TIP3, and ions were

342

added creating an overall neutral system in a 0.15 M NaCl solution. The ions were equally

343

distributed in a water box. The final systems were submitted to molecular dynamics (MD)

macromolecule

was

inserted

into

a

1-palmitoyl-2-oleoyl-sn-glycero-3-


18

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344

simulations for 100 ns using NAMD 2.639. The NPT ensemble was used to perform MD

345

calculations. Periodic boundary conditions were applied to the system in the three

346

coordinate directions. A pressure of 1 atm and a temperature of 298 K were maintained.

347

Electrostatic potential

348

The electrostatic potential studies were determined using Adaptive Poisson Boltzmann

349

Solver40 (APBS v 0.5.1) in AutoDockTools. The model was subjected to conversion of partial

350

charges and atomic radii with PDB2PQR and then a linearized traditional PBE was calculated

351

using solute and solvent dielectric coefficients of 2.0 and 78.5, respectively.

352 353

ASSOCIATED CONTENT

354

Supporting Information Full amino acid sequence alignment for monoamine transporters,

355

RMSD values to protein-ligand complexes simulated during 100 ns, changes during the

356

simulation of OA inside of OAT.

357 358 359

AUTHOR INFORMATION

360

Corresponding Author

361

Phone: +56223541171. Email: [email protected]

362

ORCID

363

https://orcid.org/0000-0002-6507-4188

364

Author contributions

365

SA and MM performed the homology modeling, docking studies and molecular dynamics.

366

JC and AF designed the research project and wrote the manuscript.


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367

Funding

368

This work was supported by Fondecyt 1161375 and VRI-Puente Nº P1805. SA is supported

369

by a CONICYT Doctoral fellowship.

370

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Modeling of the binding of octopamine and dopamine in insect

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