Zotti_et_al-2012-Insect_Molecular_Biology

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Structural changes under low evolutionary constraint
may decrease the affinity of dibenzoylhydrazine
insecticides for the ecdysone receptor in
non-lepidopteran insects
M. J. Zotti*†, O. Christiaens*, P. Rougé‡,
A. D. Grutzmacher†, P. D. Zimmer§ and G. Smagghe*
*Department of Crop Protection, Ghent University,
Ghent, Belgium; †Department of Phytosanitary, UFPel,
Pelotas, Rio Grande do Sul, Brazil; ‡UMRUPS-CNRS,
Université deToulouse, Toulouse, France; and
§Department of Agriculture, UFPel, Pelotas, Rio Grande
do Sul, Brazil
Abstractimb_1154 488..501
Understanding how variations in genetic sequences
are conveyed into structural and biochemical proper-
ties is of increasing interest in the field of molecular
evolution. In order to gain insight into this process,
we studied the ecdysone receptor (EcR), a transcrip-
tion factor that controls moulting and metamorphosis
in arthropods. Using an in silico homology model, we
identified a region in the lepidopteran EcR that has
no direct interaction with the natural hormone but
is under strong evolutionary constraint. This region
causes a small indentation in the three-dimensional
structure of the protein which facilitates the binding of
tebufenozide. Non-Mecopterida are considered much
older, evolutionarily, than Lepidoptera and they do not
have this extended cavity. This location shows differ-
ences in evolutionary constraint between Lepidoptera
and other insects, where a much lower constraint is
observed compared with the Lepidoptera. It is pos-
sible that the higher flexibility seen in the EcR of
Lepidoptera is an entirely new trait and the higher
constraint could then be an indication that this region
does have another important function. Finally, we
suggest that Try123, which is evolutionarily con-
strained and is up to now exclusively present in
Lepidoptera EcRs, could play a critical role in
discriminating between steroidal and non-steroidal
ligands.
Keywords: ecdysone receptor, molecular evolution,
dibenzoylhydrazine, evolutionary constraint, differen-
tial binding.
Introduction
Evolution in macromolecule binding site structure has
wide-ranging implications for ligand-binding affinities. Pro-
teins or functionally important regions of proteins (ligand-
binding pockets [LBPs]) may be constrained by strict
structural and/or functional requirements (Tourasse &
Li, 2000; Simon et al., 2002). Similarly, protein-encoding
genes are under selective pressure, which limits the
number of amino acid substitutions that can occur. Sub-
stitution rates are likely to vary by different regions of the
protein owing to differences in selective constraints across
these regions (Capra & Singh, 2007). Indeed, it has been
widely accepted that functionally important regions such
as catalytic sites, dimerization interfaces and binding
domains are strictly conserved, with few accumulated
changes, whereas less important regions evolve more
rapidly owing to more relaxed constraints (Kimura, 1991;
Li, 1997; Knudsen & Miyamoto, 2001; Iwema et al., 2009).
One of the most pressing issues in the field of molecular
evolution is to increase our understanding of how varia-
tions in protein sequences are conveyed into biochemical
properties, potentially leading to adaptation in the existing
function or the appearance of new traits. To address this
issue, three types of information are essential: structural
data for the proteins under study, protein sequences for
use in phylogenetic analysis and, for the calculations of
constraints, ligand-binding assays. In this context, arthro-
pod nuclear receptors (NRs) are appropriate for this type
of investigation.
The members of the superfamily of NR transcription
factors are regulated by diverse mechanisms, including by
ligands, post-translation modifications and associations
with other proteins or DNA (Laudet & Gronemeyer, 2002;
First published online 19 July 2012.
Correspondence: Moises Joao Zotti, Department of Crop Protection,
Ghent University, Ghent, B-9000, Belgium. Tel: +32-9-2646150; fax: +32-
9-2646239; e-mail: mzotti.faem@ufpel.tche.br; zottimoises@yahoo.co.uk
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Insect
Molecular
Biology
Insect Molecular Biology (2012) 21(5), 488–501 doi: 10.1111/j.1365-2583.2012.01154.x
© 2012 Royal Entomological Society488
Gronemeyer et al., 2004; Bridgham et al., 2010; Markov &
Laudet, 2011). The moulting process in arthropods is
regulated by steroid hormone molecules acting via NR
proteins (Nakagawa & Henrich, 2009; Smagghe, 2009;
Fahrbach et al., 2012). The most common moulting
hormone is the ecdysteroid 20-hydroxyecdysone (20E;
Riddiford et al., 2001; Ishimoto & Kitamoto, 2010; Harada
et al., 2011). 20E binds to the ecdysone receptor (EcR),
which forms a functional heterodimer complex with the
ultraspiracle protein (USP [Yao et al., 1993]). NRs, includ-
ing EcRs, contain at least one of the two following modular
domain structures: a highly conserved DNA-binding
domain (DBD) and a moderately conserved ligand-binding
domain (LBD), which in most receptors has a ligand-
regulated transcriptional activation function (Bridgham
et al., 2010; Fahrbach et al., 2012). Several synthetic
insecticides, including dibenzoylhydrazine (DBH), are
non-steroidal ecdysone agonists that exert their effects
by binding to a site overlapping with the 20E-binding site
and activating EcRs (Wing, 1988; Dhadialla et al., 1998;
Nakagawa, 2005).
Recent experiments (Carmichael et al., 2005; Graham
et al., 2007) have revealed that the binding affinities of
tebufenozide (a DBH compound) vary greatly across the
taxonomic orders of arthropods and reflect lepidopteran-
selective toxicity. Additionally, in vitro assays of the binding
affinities of tebufenozide have shown a strong negative
correlation with the phylogenetic distances of the EcR-
LBDs from a lepidopteran reference sequence (Graham
et al., 2009). This has attracted attention because the
moulting process of most insects is regulated by the ste-
roidal hormone 20E.
Carmichael et al. (2005) calculated the LBP surface for
the Bemisia tabaci EcR crystal structure and that for
the Heliothis virescens structure (Billas et al., 2003) and
found an extra region of the pocket in the lepidopteran
receptor that was not present in the hemipteran EcR.
Carmichael et al. (2005) also hypothesized that this varia-
tion in the structure and shape of this region of the LBD
may contribute to the preferential binding of DBHs to the
lepidopteran receptor.
Ecdysone receptor sequences and structures have
been extensively studied, but whether evolutionary con-
straints lead to rearrangements in the size and shape of
the EcR-binding pocket has yet to be explored. To further
investigate whether the molecular evolution of EcR might
explain the difference in binding affinities of tebufenozide
between lepidopteran and non-lepidopteran insects, we
performed a series of analyses using a large set of
sequence and structural data. Molecular modelling and
docking studies were used to compare different binding
modes. We performed a molecular analysis of the EcR-
LBD of Forficula auricularia (FaEcR-LBD). Dermapterans
(earwigs) are considered to be an ancient order of insects
(Buxton, 1974), and their EcRs are phylogenetically
distant from lepidopteran EcRs and, therefore, less likely
to bind DBH compounds (Graham et al., 2009; Shaw &
Wallis, 2010).
The present analysis enabled us to verify previous
predictions (Carmichael et al., 2005) that suggested a
role for the binding pocket extension in explaining the
different binding affinities of DBHs for lepidopteran and
non-lepidopteran EcRs. Indeed, we show that for lepi-
dopteran EcRs, this extended cavity, which does not
directly interact with the natural hormone, is located in a
region under strong evolutionary constraint. In contrast,
in non-Mecopterida EcRs the corresponding region is
under low evolutionary constraint. This low constraint
could mean that this region of the receptor has under-
gone more structural changes but, even though as the
non-Mecopterida are consideredmuch older evolution-
arily than Lepidoptera, they do not have this extra
binding cavity. The higher constraint seen in Lepidoptera
could then be an indication that this region has arisen
during EcR evolution and became constrained to another
important function which is as yet unknown. These
results have general implications for the evolution of
EcRs and provide the first evidence derived by molecu-
lar evolutionary analysis in support of an explanation for
the differential binding affinity of insecticide ligands to
macromolecules.
Results
Molecular analysis of the Forficula auricularia and
Tribolium casteneum ecdysone receptor ligand-binding
domains and phylogenetics
We did not obtain the full-length FaEcR-LBD because we
probably overlooked ~30 bp at the end of the coding
sequence. We performed 3′rapid amplification of cDNA
ends (RACE) using several optimizations, but results were
inconclusive. The chosen amino acid sequences of the
EcR-LBD members were used in an alignment (Fig. 1).
Another alignment with additional species was used to
determine the conservation profile (data not shown). The
EcR-LBD sequences exhibited a high overall conservation
(64% and 76.8% identity and similarity, respectively) that
was even higher when we compared the FaEcR-LBD and
Locusta migratoria EcR-LBD (LmEcR-LBD; 85.2% and
92.8% identity and similarity, respectively [Table S1]). The
FaEcR-LBD sequence showed the highest identity to
those of more basal insects, such as LmEcR-LBD, which
was followed by the Blatella germanica EcR-LBD (BgEcR-
LBD; 83.9% and 91.9% identity and similarity, respec-
tively) and that of Tribolium castaneum (TcEcR-LBD;
83.5% and 90.2% identity and similarity, respectively). The
amino acid identity was lower when we compared the
Structural evolution and binding of ligands 489
© 2012 Royal Entomological Society, 21, 488–501
FaEcR-LBD with those from lepidopteran and dipteran
insects; specifically, the sequences displayed 59.6% and
60.6% identity with the EcR-LBDs of Manduca sexta
(MsEcR-LBD) and H. virescens (HvEcR-LBD), respec-
tively, and 64.4% with that of Drosophila melanogaster
(DmEcR-LBD).
The phylogenetic analysis is in agreement with previous
findings (Bonneton et al., 2003, 2006; Zotti et al., 2012).
We observed a distinct separation of the more divergent
Mecopterida superorder (which includes Diptera and Lepi-
doptera) from those of other insects and arthropods
(Fig. 2). These results demonstrated that during molecular
evolution, the FaEcR-LBD branched off earlier than
LmEcR- and BgEcR-LBDs, which could have led to dis-
similar sequences, although, these three sequences dem-
onstrated the highest identity and similarity with each other.
By analysing the phylogenetic tree in Fig. 2, we observed
that FaEcR-LBD is much more similar to the LmEcR-LBD,
the TcEcR-LBDs and to other non-Mecopterida ortho-
logues than to the Mecopterida EcR-LBDs, even though
the EcR has undergone for at least six speciation events
between these non-Mecopterida orders and diverged
before Crustacea and Chelicerata. This observation con-
tradicts the known phylogeny, whose topology permits
the assignment of a sister-group relationship between
Hymenoptera and Mecopterida. In fact, this deviation from
the normal topology, which is shown in Fig. 2, is attributable
to a long-branch attraction that is caused by an accelera-
tion of the evolutionary rate that occurred in the stem
lineage of Mecopterida. This differential tree topology was
previously described by Christiaens et al. (2010) for EcR
and E78 (ecdysone-induced protein) from Acyrthosiphon
pisum. These results agree with other findings (Bonneton
et al., 2008), who have demonstrated that some NRs in
the Mecopterida group, particularly EcR, underwent an
increase in evolutionary rate.
Figure 1. Sequence alignment of the ecdysone
receptor (EcR) ligand-binding domains (LBDs). The
alignment includes EcR-LBDs from Diptera (D),
Hymenoptera (H), Hemiptera (he), Cladocera (cl),
Lepidoptera (L), Coleoptera (C), Orthoptera (O),
Neuroptera (N) and Dermaptera (de). The helices
positions were based on the EcR-LBD crystal
structure of Tribolium castaneum (RCSB Protein Data
Bank code 2NXX). The organism abbreviations are:
DmEcR: Drosophila melanogaster (D), AmEcR:
Apis mellifera (H), LmEcR: Locusta migratoria (O),
NvEcR: Nezara viridula (he), DamEcR:
Daphnia magna (cl) (GenBank accession no.
BAF49033), BtEcR: Bemisia tabaci (he), HvEcR:
Heliothis virescens (L), CcEcR: Chrysoperla carnea
(N), FaEcR: Forficula auricularia, TcEcR:
Tribolium castaneum (C). The strictly conserved
amino acid residues forming the ecdysone-binding
site for PonA are indicated by (�) (residues involved
in hydrogen bonds), (#) (residue involved in a
water-mediated hydrogen bond and (�) (residues
involved in stacking interactions).
490 M. J. Zotti et al.
© 2012 Royal Entomological Society, 21, 488–501
Model and docking of the ecdysone receptor
ligand-binding domainof Tribolium casteneum
Nine amino acid residues of TcEcR-LBD participate in the
binding of PonA through a network of eight hydrogen
bonds (Glu330, Thr362, Thr365, Arg402, Val417 and
Tyr427) and stacking interactions (Phe416 and Trp543)
(Fig. 3E), and Asn521 interacts with ecdysone via a water-
mediated hydrogen bond. We identified a network of seven
hydrogen bonds (Glu20, Thr52, Thr55, Arg92, Ala107 and
Tyr117) supporting the binding of PonA in Bemisia tabaci
(Bt)EcR-LBD (Fig. 3G). Because of the conserved charac-
ter of these residues, the docking experiments that were
performed with ecdysone yielded similar binding schemes
for the modelled FaEcR-LBDs (Fig. 3F, H). In the FaEcR-
LBD models, the aliphatic chain of PonA is anchored in a
large lobe that is located at the bottom of the pocket via
hydrophobic interactions that involve the residues Ile47,
Met88, Met89 and Met121. Those residues in equivalent
positions in Bemisia-based FaEcR-LBD also hydrophobi-
cally interact with PonA. A network of hydrogen bonds with
hydrophilic residues (Asp20, Arg91, Ala106 and Tyr116)
and a stacking interaction with an aromatic residue
(Phe105) participate in the binding of PonA (Fig. 3F, H).
Two additional hydrogen bonds (Gln21 and Arg91-NH2)
were identified in the FaEcR-LBD model based on the
Bemisia EcR crystal structure (Fig. 3H).
There is only one crystal structure of the EcR-LBD of
T. castaneum and another of the EcR-LBD of B. tabaci,
which are both described with PonA as the ligand; there-
fore, these crystal structures were used to dock
tebufenozide and PonA. We used the T. castaneum struc-
ture as a template by accounting for its phylogenetic rela-
tionship with that suggested in the phylogenetic tree;
however, we also modelled the F. auricularia EcR-LBD
based on B. tabaci structure as a control. As previously
reported (Soin et al., 2009; Amor et al., 2012), different
solutions resulted from the docking of tebufenozide to
the ecdysone-binding site of TcEcR-LBD. A solution that
seems to be particularly relevant includes the binding of
the ethyl-phenyl ring (B ring of the DBH) of the agonist in
a cavity, which extends the binding site of TcEcR-LBD to a
location that is opposite to the cavity that harbours the
alkyl chain of the ecdysteroid (Fig. 3I). This is in agree-
ment with the binding scheme that was previously pro-
posed for tebufenozide in complex with the HvEcR-LBD
(Billas et al., 2003). In contrast to the model that was
reported before (Soin et al., 2009) for the EcR-LBD of the
coleopteran weevil (Anthonomus grandis), in the present
work, tebufenozide failed to bind to the FaEcR-LBD. The
ethyl-phenyl ring of tebufenozide attempts to accommo-
date in a second, less-extended lobe, which is present at
the bottom of the ecdysone-binding pocket of FaEcR-LBD
and at the opposite side of the lobe that harbours the
aliphatic chain of PonA (Fig. 3J, M). However, both
FaEcR-LBD models, which lack this less-extended lobe at
the bottom of the ecdysone-binding pocket, clearly differ
from the AgEcR-LBD (Soin et al., 2009). Tebufenozide
failedto bind to the EcR-LBD of B. tabaci, which also lacks
this extend lobe responsible for properly anchoring the
ethyl-phenyl ring (Fig. 3L).
Calculations of evolutionary rates
We performed an analysis of a comparison of evolutionary
constraint profiles that are based on the alignment of the
EcR-LBDs of 53 arthropod taxa in different sets of calcu-
lations (Fig. S1). The predicted evolutionary constraints
were identified and plotted (Fig. 4A). In the x-axis is pre-
sented the protein position derived from the amino acid
alignment. In the y-axis is presented the relative con-
straints and evolutionary rates. In the Fig. 4B and C the
Figure 2. A phylogenetic tree was constructed based
on amino acid sequences of the ecdysone receptor
ligand-binding domains (EcR-LBDs) from
lepidopterans, dipterans, mecopterans,
siphonapterans and trichopterans (grouped in
Mecopterida 32 taxa), hemipterans, hymenopterans,
orthopterans, neuropterans, strepsipterans,
dermapterans, crustaceans and arachinidans. The
GenBank accession numbers are described in
Table S1. The tree was made using the
neighbour-joining method using CLUSTAL-X multiple
alignment program. The indicated numbers are
bootstrap values >50 as percentage of 1000
replicates and the scale bar indicates 0.05 change
per residue. The evolutionary distances were
computed using the Jones-Taylor-Thornton (JTT)
matrix-based method. All positions containing gaps
and missing data were eliminated from the dataset
resulting in 225 complete sites.
Structural evolution and binding of ligands 491
© 2012 Royal Entomological Society, 21, 488–501
homologous evolutionary constraint regions were delin-
eated by vertical grey lines and a single-letter code (A-L)
encompassing a 20-position-wide window. Specifically,
regions B, E, and I are among those that contained the
lowest constraints (Fig. 4A). This result agrees with the
poor conservation profile that was observed in the EcR
alignment of these regions. Interestingly, when the calcu-
lations for region F were performed individually, a high
evolutionary rate, and by extension, a low constraint
were observed in the non-Mecopterida group (Fig. 4C).
However, the same effect was not observed in Lepi-
doptera or in all of the EcRs (Fig. 4A, B, respectively).
When we examined these graphs in detail, we noticed
that the evolutionary constraint from Lepidoptera was
much steeper, with sharp slopes up and down, than in
their counterpart regions in non-Mecopterida EcR-LBDs
(Fig. 4C). This may be a direct consequence of the accel-
eration in the evolutionary rate of this protein, and it may
be specific for this group of insects, which was described
above (see also Experimental procedures).
Comparison between ligand-binding pockets
The LBP comparison (Fig. 5) aimed to identify those re-
gions (Fig. 4) not involved in the binding of natural hormone
analogue. Consistent with the analysis of Carmichael et al.
(2005) based on structures available at the time, those
residue/regions of the LBP that do not interact directly with
the natural hormone analogue are more susceptible to
bearing structural changes. This has been proved experi-
mentally and is assumed to underline the selectivity of DBH
insecticides (Carmichael et al., 2005). However, up to now
this has only been observed for the Lepidoptera EcR-LBD.
In the present study, we used the information extracted
from the LBD structures that may shed light on the mecha-
nism of action of tebufenozide and its differential binding
affinities across insect orders, especially for the earwig
F. auricularia. In general, we noticed that the regions in the
lining of the ligand pockets tend to maintain their physico-
chemical features, especially in those that interact with
PonA through hydrogen bonding (Fig. 5). Forty-one amino
Figure 3. Ribbon diagram of ecdysone receptor
ligand-binding domains (EcR-LBDs) of: Tribolium
castaneum (TcEcR-LBD) (a), Bemisia tabaci
(Bt)EcR-LBD (c) and Forficula auricularia
(FaEcR-LBD) models (b, d). The 12 a-helices
building the three-dimensional fold of the receptors
are differently coloured and numbered H1-H12; the
two short strands of b-sheet are coloured purple and
numbered b1 and b2. N and C correspond to the N-
and C-terminus of the polypeptide chain, respectively.
The ecdysone complex to the EcR-LBD is shown in
pink. Clips showing the binding of ecdysone to the
ligand-binding groove of TcEcR-LBD (e), BtEcR-LBD
(g) and FaEcR-LBDs (f, h). Residues interacting with
ecdysone (pink-coloured) by hydrogen bonds
(purple-coloured), water-mediated hydrogen bonds
(green-coloured) and stacking interactions
(yellow-coloured sticks) are labelled, unless otherwise
stated. Some of the labelled residues are not
apparent on the clipping plane. Clips showing the
binding of tebufenozide (red stick) to the
ligand-binding groove of TcEcR-LBD (i), BtEcR-LBD
(l) and FaEcR-LBDs (j, m). The absence of steric
clash upon binding of tebufenozide to the binding
pocket of TcEcR-LBD is indicated by the green star.
Regions where steric clash occurs upon binding of
tebufenozide to FaEcR-LBD and BtEcR-LBD are
indicated by the red star.
492 M. J. Zotti et al.
© 2012 Royal Entomological Society, 21, 488–501
acids are involved in the FaEcR-LBD pocket formation
(Tribolium based); however, despite the less-extended lobe
at the bottom of the ecdysone-binding pocket, a larger
binding site (1.373 Å2) is formed. The FaEcR-LBD pocket
(Bemisia based) is similar regarding its surface area
(1358 Å2), interactions with the ligand PonA and packed
character. Some small differences in the surface area were
observed depending on what structure was used as tem-
plate; however it is difficult to assess how the differences
between the initial templates that were used to generate
the models are conveyed into Forficula modelled
structures.
Thirty-eight amino acids participate in the BtEcR-LBD
pocket formation, producing a 1041 Å2 cavity. A smaller
binding pocket is formed in the TcEcR-LBD (1.178 Å2),
and it is even smaller if we consider the HvEcR-LBD
(875 Å2) (Fig. 5). Specifically, the pocket architecture that
was extracted through amino acids in non-Mecopterida
insects (FaEcR, BtEcR and TcEcR) displays a slightly less
packed character around the ligand-binding groove. This
is clearer if we consider that in HvEcR-PonA, a shorter
surface area and, by extension, fewer residues are
involved in the pocket formation, even though all of the
structures contain PonA as a ligand (Fig. 5). In addition,
during the surface calculations for non-lepidopteran
LBDs, we observed a few ‘useless’ surfaces, which were
involved in small channels, and other shapes that do not
contribute to the ecdysone ligand-binding groove itself
(data not shown). It seems that in the HvEcR-LBD, these
‘useless’ amino acids are excluded from the pocket
arrangement, and the HvEcR-LBD maintained only those
residues that had a high efficiency regarding their contri-
bution to the surface area, shape and position. These
observations may be a consequence of the ligand-
dependent pocket that is adopted by this structure upon
binding either steroidal or non-steroidal ligands (Billas
et al., 2003). This induced-fit binding scheme becomes
clear when comparing the amino acids that are involved in
the pocket rearrangement in both situations of Fig. 5 (grey
box), where the volume of the whole cavity upon non-
steroidal binding remains unchanged (875 Å2 to 877 Å2,
~2 Å2) even though fewer residues (34 to 25 residues)
participate in its formation. In fact, until now, the structures
of other NR LBDs have indicated that only small, local
adaptations of the receptor LBD to its ligand can occur.
Moreover, a region that lacks an apparent interaction
with PonA was detected, and it is identified by the grey box
(Fig. 5). This is noteworthy because the interactions with
PonA are nearly uniform over the pocket surface and
across the EcR-LBD structures apart from in this particular
region. However, some interactions (Val124, Asp127, and
Leu128) appear only in theHvEcR-LBD, which is com-
plexed with a non-steroidal molecule. These interactions
are followed by a greater surface in the pocket formation
3
2.5
1.5
0.5
Protein Position
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−1
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1
0
31
0
32
0
33
0
34
0
35
0
36
0
37
0
38
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39
0
40
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41
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43
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44
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Figure 4. Comparison of the evolutionary analysis of the ecdysone
receptor ligand-binding domains (EcR-LBDs) reveals regions predicted
to be important for binding affinity of dibenzoylhydrazine compounds.
Smoothed evolutionary rates and their constrained regions across
EcR-LBDs are shown in the graphs. The homologous evolutionary
constraint regions and constraints are delimited by vertical grey lines
and indicated as a single-letter code (A-L). The values from manipulated
average of amino acids substitution in each aligned position along the
protein are plotted on the y axis. The protein positions are plotted on the
x axis. The evolutionary rates and constraints for all available EcR-LBDs
are plotted separately (4a). For ease of comparison, the evolutionary
rates and constraints are plotted on the same graph (4b and 4c,
respectively) wherein the groups are displayed as follows: Lepidoptera
EcR-LBD (red line), other Mecopterida (green line), non-Mecopterida
(blue line) and all available EcRs (black line).
Structural evolution and binding of ligands 493
© 2012 Royal Entomological Society, 21, 488–501
Figure 5. Residues forming the ligand-binding pocket of Forficula auricularia (Fa) ecdysone receptor ligand-binding domains (EcR-LBDs), Tribolium
casteneum (Tc)EcR-LBD, BtEcR-LBD and Heliothis virescens (Hv)EcR-LBDs and their surfaces contribution for the pocket formation. The residues
involved in the pocket formation (number of residues) as well as their surface areas (Å2) are presented in the end of each column. The interactions with
ligand are indicated as follows: Polar interaction (Pi), van der Waals (vdW), charge interaction (ch) and hydrogen bond ( ). Amino acids in grey boxes
are involved in the second lobe formation at bottom of the ecdysone-binding pocket. For easy comparison between surfaces, a conditional grey scale
was used to emphasize the contribution of each residue. The corresponding regions of Fig. 4 (A-L) were added for ease of comparison. MC and SC
represent the surface contributions to the pocket wall by residue main chain atoms plus residues side chain atoms, respectively. DBH,
dibenzoylhydrazine.
494 M. J. Zotti et al.
© 2012 Royal Entomological Society, 21, 488–501
(Fig. 5), which contribute for the indentation observed in
this structure (Fig. 6E); (Carmichael et al., 2005). Further,
we investigated those residues involved in the steric hin-
drance upon docking of tebufenozide. In contrast to the
TcEcR-LBD and HvEcR-LBD, the FaEcR-LBD completely
lacks the second lobe at bottom of ecdysone binding
pocket. This lobe is indicated by blue and yellow stars
where it occurs (Fig. 6). This extent cavity is supposed to
anchor B-ring of tebufenozide in TcEcR-LBD; however
there is no place in FaEcR for anchoring this ring in the
ecdysone-binding pocket so preventing correct binding. If
we look at the residues surrounding the binding pocket of
the FaEcR-LBD we find two important residues preventing
the opening of a lobe. Residue Gln208 is oriented with its
CG portion (purple arrowhead) towards protein. Second,
the side chain of Met128 (orange arrowhead; Leucine in
other EcRs) is located where the lobe extension could
occur.
Finally, we predicted the impact of amino acid substitu-
tion at a chosen position of the EcR-LBDs by means of a
multivariate analysis of protein polymorphism (Fig. S2).
The physicochemical profile for the positions that demon-
strated a direct interaction with PonA is strictly conserved,
and this was also true for the key positions that are
involved in the pocket formation. All of the single-site
replacements across the FaEcR-LBD have only a low
impact on this profile.
Discussion
The DBH class of insecticides has been commercialized
for agricultural purposes owing to its excellent binding
affinity for EcR/USP heterodimers in lepidopteran insects.
However, these insecticides exhibit little or no toxicity in
other taxonomic orders of insects, which include many
beneficial species. In the present report, despite the
canonical conservation of the amino acids in the
ecdysone-binding domain, our docking results suggest
that the restricted size of the cavity at the bottom of the
ecdysone-binding pocket from both F. auricularia and B.
tabaci (FaEcR-LBDs and BtEcR-LBD) caused a steric
clash upon the docking of tebufenozide into ecdysone-
binding site (Fig. 3J, L, M). This prevented the ethyl-
phenyl ring of the tebufenozide from being accommodated
by the receptor. A steric clash also occurred upon the
docking of the closely related methoxyfenozide molecule
Figure 6. The ligand binding pockets of the Forficula auricularia (Fa) ecdysone receptor ligand-binding domain (EcR-LBD) model with PonA bond (a),
Tribolium casteneum (Tc)EcR-LBD with PonA bond (b), Bemisia tabaci (Bt)EcR-LBD with PonA bond (c), Heliothis virescens (Hv)EcR-LBD with PonA
bond (d) and HvEcR-LBD with BYI06830 bond (e). The pockets are shown in equivalent coordinates. The amino acids discussed in the text are
displayed in ‘stick’ representation and coloured by parent colour. The ligands are shown in pink coloured ‘sticks’. Pockets were generated using
DISCOVERY Studio 2.5 (Accelrys) with the default 1.4-Å probe radius. The purple, orange and green arrowheads indicate changes in residue
conformations. The side-chain conformation of the residues Gln208 in FaEcR-LBD and Gln212 in TcEcR-LBD (purple arrowheads) are oriented toward
protein volume, producing smaller surfaces than their counterpart residues of HvEcR-LBDs. In FaEcR-LBD the residue Met128 (orange arrowhead;
Leucine in other structures) is oriented towards the aliphatic chain of PonA, hampering the opening of a small extra lobe. This extra lobe is observed in
TcEcR-LBD (blue star) where Metionine is replaced by Leu131. In HvEcR-LBD a smaller lobe is also observed (yellow star). The green arrowhead of
BtEcR-LBD indicates that the Met120 (Glutamine in other structures) hinders the opening of an indentation in that location. The superimposition of the
PonA- (aqua) and BYI06830-bond (olive green) HvEcR-LBD structures with different views related by a 90° rotation around the vertical axis (f, g, h). The
pocket was generated as described above but only for BYI06830-bond structure. The change of Tyr123 position is presented by blue arrowheads.
Structural evolution and binding of ligands 495
© 2012 Royal Entomological Society, 21, 488–501
into the ecdysone-binding site of the FaEcR-LBDs (data
not shown). Similarly, in a previous study, tebufenozide
failed to efficiently bind the EcR-LBD from dipteran
D. melanogaster owing to the lack of a second lobe at
the bottom of the ecdysone-binding pocket (Soin et al.,
2010). As a result, in vitro assays using dipteran S2
cells transfected with an ecdysone response element
(ERE)-dependent reporter construct for screening active
compounds, tebufenozide and other DBH analogues have
demonstrated response activities correlated with those
observed in lepidopteran Bm5 cells, although the overall
level of activity is 2–3 orders of magnitude higher in Bm5
cells (Soin et al., 2010). The lack of a cavity capable of
harbouring the ethyl-phenyl ring of tebufenozide has also
been observed in the EcR of the crustacean Crangon
crangon (brown shrimp; Verhaegen et al., 2010).
As previously shown by Soin et al. (2009), tebufenozide
reached activities higher than 50% compared with 1 mM of
20E in EcR reporter assays. This demonstrates that
tebufenozide has affinity to Tribolium EcR-LBD as shown
by docking studies. However, this ecdysone agonist
targets mainly lepidopteran insects with a limited number
of coleopteran insects (Dhadialla et al., 1998). Addition-
ally, a recentdocking study (Bengochea et al., 2012) dem-
onstrates that halofenozide is well docked on Tribolium
EcR-LBD. In this context, the question is raised of why
both ligands are properly docked without any steric clash
on Tribolium EcR-LBD, but only halofenozide shows in
vivo activity on coleopteran insects. This observation may
be because of its higher intrinsic potency to activate the
coleopteran EcR, which reached activity twice as high
as that observed for tebufenozide (Soin et al., 2009).
Our models are alone incapable of explaining why
tebufenozide has much lower activation properties in non-
lepidopteran receptors than in lepidopteran receptors. In
addition, a properly docked tebufenozide means it may be
efficient in activating EcRs, but in fact how great its effi-
ciency can reach is a matter of further investigation. Simi-
larly, the occurrence of steric hindrance means that the
molecule does not contain the most appropriately physi-
cochemical and structural requirements for a particular
pocket, but to what extent this affects the affinity is also a
matter of discussion. Furthermore, the EcR is, by itself,
incapable of high-affinity DNA-binding or transcriptional
activation. These activities are dependent on heterodimer
formation with USP (Yao et al., 1993). The high-affinity
DNA binding by the EcR complex is preceded by several
events, such as binding of response elements and coac-
tivators. For these events, subtle differences in the overall
structure of the EcR complex upon binding of ligands may
be translated into rather large differences in activity
regarding the target gene activation. These differences
cannot be accessed only by docking studies and lie
beyond ligand-receptor interactions.
The variation in the tebufenozide-binding affinity
probably reflects sequence-dependent differences in the
topography of the LBP at residues other than those that are
critical for the binding of 20E-like molecules. Even small
changes in the position or the nature of the residues that
interact with a particular ligand can have a large impact on
the binding affinity of that ligand (Billas et al., 2009). In the
present study, a detailed investigation of the pocket lining
has allowed us to identify the region responsible for an
indentation in the Lepidoptera EcR-LBD cavity and its
evolutionary rate was calculated. Pocket surface calcula-
tions using a probe radius of 1.4 Å revealed the residues
responsible for this cavity indentation in Lepidoptera and
those responsible for hampering the opening of this cavity
in other insects (Figs 5 and 6). Five amino acids produced
a combined effect in the opening of a cleft between helixes
H7 and H10 that is required to accommodate the B-ring of
tebufenozide (grey boxes, Fig. 5). In particular, the residue
Gln211 in the HvEcR-LBD contributes 89.6 Å2 to the
pocket surface. In addition, the side-chain rotameric con-
formations adopted by Gln208 and Gln212 in the FaEcR-
and TcEcR-LBDs differ from their counterparts in the
HvEcR-LBD, which make smaller contributions. The side
chain of the FaEcR-LBD Gln208 is oriented parallel to the
pocket surface with its long polar side chain directed
toward the protein (Fig. 6). In the TcEcR-LBD, the cavity
shrinkage was not as scattered as in the FaEcR-LBD
because of a compensatory surface in the pocket’s inden-
tation that was produced by Leu131. The residue Leu128
in HvEcR-LBD with BYI06830 bond clearly contributes to a
significant surface area (73.7 Å2), which supports the
pocket rearrangements upon binding of non-steroidal
ligand (Fig. 5). Residue Leu128, conserved in other
insects but replaced by methionine in FaEcR-LBD, notably
prevents the opening of an extra lobe in this receptor. The
side chain of Met128 in FaEcR-LBD is oriented towards
the aliphatic chain of PonA and downwards to the bottom
of the ecdysone-binding pocket. This extra lobe occurs in
TcEcR-LBD and HvEcR-LBD (blue and yellow stars;
Fig. 6) where the corresponding side chain of Leu131,
which is smaller, is oriented away from the aliphatic chain
of PonA, while Leu128 is oriented toward side chain of
Val92. In BtEcR-LBD the corresponding side chain of
Leu129 is quite similar to that observed in TcEcR-LBD,
even though this extra lobe is not observed because of the
rotameric conformation adopted by Met210 (green arrow-
head), which is occupied by glutamine in other structures.
It is worth mentioning that the residue Val92, conserved in
lepidopteran insects but replaced by methionine in other
insects, is considered to be essential for the specificity of
BYI06830 to Lepidoptera (Fig. 6). As previously shown by
Soin et al. (2009) this residue interacts via hydrophobic
interaction with tebufenozide docked in the EcR-LBD
model of the coleopteran A. grandis. Similarly, this
496 M. J. Zotti et al.
© 2012 Royal Entomological Society, 21, 488–501
methionine potentially interacts hydrophobically with
tebufenozide in our model of FaEcR-LBD (data not
shown). In addition, our inspection of these surfaces
revealed a previously unobserved contribution by Asp127
in the HvEcR (DBH bond) that is not present in the other
structures. The appearance of surfaces only in the struc-
ture containing a DBH bond may be a consequence of the
high flexibility that this structure adopts. This flexibility
allows the residues to be rearranged in such a manner that
the entire surface area can be explored and the proper
LBP can be formed around the ligand. It is possible that
this flexibility in the HvEcR-LBD interferes with the binding
of DBH, and variation in flexibility could explain the range
of DBH affinities for the EcRs in non-lepidopteran insects.
According to this hypothesis, it is expected that dipterans
and non-Mecopterida EcR-LBDs lack flexibility, therefore
only minor local adaptations can occur. If this is the case,
it may be possible that the affinity for non-steroidal ago-
nists lies in the rigid structure.
We also investigated whether evolutionary rates/
constraint could lead to structural rearrangements in
an attempt to explain the previous predictions (Graham
et al., 2009), which correlate phylogenetic distances
and tebufenozide-binding affinities. To address this, the
species with similar ecdysone activity of tebufenozide
were grouped, and then evolutionary rates calculated (see
Experimental procedures). Evolutionary constraint com-
parisons reveal individual variation that depends on the
group in which the calculation is performed. In the present
study, different sets of EcR sequences were compared.
For the HvEcR-LBD, two different crystal structures have
been described; one structure binds PonA, and the other
binds BYI06830, a DBH compound. The LBDs of these
two crystal structures only partially overlap (Billas et al.,
2003). This demonstrates that the LBD can adopt different
binding cavities, illustrating the flexibility and adaptability
of this protein that enables its conformation around a
ligand and results in the proper shape of the LBP. In the
second structure, a small indentation of the pocket wall
occurs into the HvEcR-LBD/DBH protein volume. This
indentation, located between residues Val124, Leu128
and Gln211, is not observed in non-lepidopteran insects
(Carmichael et al., 2005). It appears that structural vari-
ability in the vicinity of this indentation across taxonomic
orders may contribute to variation in the binding affinity of
tebufenozide. Indeed, it is due to the lack of an extended
cavity in this vicinity that tebufenozide fails to efficiently
bind FaEcR-LBD.
We noted that the amino acids involved in this indenta-
tion lie between residues 450 and 460 (region F; Fig. 4A)
and do not interact with PonA but do electrostatically inter-
act with DBH in lepidopteran EcRs (Fig. 6). If we look at
the region of this indentation (region F) in the Fig. 4B,
clearly we can see that the evolutionary rates display a
negative correlation with binding affinities of tebufenozide.
This prediction is virtually identical to that established by
Graham et al. (2009) who alsofound a negative correla-
tion with binding affinities of tebufenozide and phylogenet-
ics distances of the EcR-LBDs from a lepidopteran
reference sequence. Here, the order in evolutionary rates
of the EcR-LBDs at region F is non-Mecopterida > Other
Mecopterida > Lepidoptera which negatively correlates
with binding affinities of tebufenozide wherein the order
is Lepidoptera > Other Mecopterida > non-Mecopterida
(Carmichael et al., 2005). In this graph, there is no other
appropriate region to be used in such correlation. At least
two residues (Val124 and Leu128), mentioned by Car-
michael et al., 2005 as important in remodelling the cavity
shape, are located at this region, but also, as reported in
their study, this region holds the residues involved in the
opening of an extra lobe that could properly anchor
tebufenozide or to cause a steric hindrance.
Our constraint calculations clearly reflect the structural
relative importance of this region F in lepidoptaran EcR-
LBDs (Fig. 4C). It may be that this evolutionarily con-
strained region plays a role in the high flexibility of the
lepidopteran EcRs, and the binding of DBH compounds by
this receptor could be a consequence. It represents a
switch in surface area ofc > 275% between two HvEcR-
LBDs and provides the largest contribution to the pocket
formation, even if we consider other structures (Fig. 5).
We suggest that besides those residues mentioned before
(Val124, Leu128 and Gln211), Tyr123 and Asp127 could
also be important in the flexibility mechanism seen in
lepidoptaran EcRs. Using mutagenesis of the EcR from
Choristoneura fumiferana-LBD, Kumar et al. (2002)
identified a mutant EcR-LBD V128Y (V124Y – Heliothis
sequence in our alignment), which responds well to tet-
rahydroquinoline compounds but weekly to DBH ligands.
This valine is also found in Nezara viridula thus cannot by
itself account for the tebufenozide affinities/ligand recog-
nition in Lepidoptera. A second mutation A110P (A106P –
Heliothis sequence in our alignment) was assumed by
Billas et al. (2003) as important in explaining the loss of
activity for the steroidal ligands, but not for non-steroidal
ligands. Tyr123 has up to now been found exclusively in
lepidopteran EcRs. Yet, as shown in the present study, this
residue it is under strong evolutionary constraint, making a
significant contribution to the pocket surface. In addition, a
switch in the backbone makes a significantly change on its
position and provides an entry point for the DBH B-ring,
which is not possible with its adopted orientation in the
EcR-LBD PonA bond (Fig. 6G). Even though a number of
other changes to the pocket architecture of the HvEcR-
LBD upon binding of BYI06830 occur, we tentatively
suggest that because of the Tyr123 location, which is at
the tip of the H8 and at the edge of the protein surface,
may be involved in tebufenozide recognition.
Structural evolution and binding of ligands 497
© 2012 Royal Entomological Society, 21, 488–501
Although, as demonstrated by Billas et al. (2003), the
outer to inner switch of F105 and Y111, which are not
located at region F, are responsible for the flexibility
mechanism of HvEcR-LBD upon binding of BYI06830.
But, both of these residues are so far conserved in EcR
sequences across all orders, thus cannot alone account
for the protein flexibility. The present constraint calcula-
tions data show that differences in constraints between
different groups of EcRs can tentatively be correlated
with the different affinity of receptors for tebufenozide.
We observed that the constraints in non-Mecopteria and
Other Mecopterida become interwoven at region F, while
Lepidoptera remain clearly separated and evolutionarily
constrained. Interestingly, when the constraint was calcu-
lated using all EcR sequences, including lepidopteran
sequences, no constraint was observed in the region F.
This may be attributable to the effect of the homogeniza-
tion wherein the strong constraint found in Lepidoptera
was revoked by the no/low constraint found in other
Mecopterida and non-Mecopterida.
In addition, we documented a region in the non-
Mecopterida EcR that apparently lacks evolutionary con-
straint in the location of some of the amino acids involved
in the binding pocket. The low constraint seen in non-
Mecopterida EcR could mean that this region of the recep-
tor has undergone more structural changes. Those amino
acids in the counterpart region of the Lepidoptera
EcR-LBDs are involved in the indentation responsible
for anchoring the B-ring of DBH; however, the non-
Mecopterida EcRs are considered much older, evolution-
arily, than Lepidoptera and these receptors do not have
this extra binding cavity. The lack of this indentation in the
pocket hinders the binding of tebufenozide.
Finally, the EcR-LBD from lepidopteran H. virescens
has been reported to be very flexible, and yet, as we
reported here, the region that surrounds the B-ring of
DBHs is located within a constrained region. So it is also
possible that the higher flexibility seen in Lepidoptera is an
entirely new trait. The higher constraint could then be an
indication that this region does have another important
function in these Lepidoptera. In this situation, the flexible
structure (Billas et al., 2003), coupled with the constrained
region, plays a critical role for tebufenozide binding. It
appears that the flexibility of the receptor may largely
contribute to shaping the cavity to properly bind the
tebufenozide in lepidopteran insects. In the future, it would
be very interesting to determine whether this receptor
flexibility occurs in insect orders other than Lepidoptera.
Experimental procedures
RNA isolation and cDNA synthesis
The total RNA was isolated from whole insects (F. auricularia
adults and nymphs) at different developmental stages using TRI
reagent® (Sigma, Bornem, Belgium) based on the single-step
liquid phase separation method that was reported before (Chom-
czynski & Sacchi, 1987). This RNA was incubated with DNase
(1 mg/1 ml) and buffer for 15 min at 37 °C, then for 15 min at 65 °C
to inactivate the DNase. Finally, the mixture was placed on ice for
1 min and collected by brief centrifugation. The cDNA synthesis
was performed using 1.5 mg of total RNA with anchored oligo-dT
and the reverse transcriptase SuperscriptTM II RT (Invitrogen,
Carlsbad, CA, USA) in a 20-ml reaction.
Molecular analysis of the Forficula auricularia ecdysone
receptor ligand-binding domain
The degenerate primers, 5′-TGCGGHGAYMGDGCNTCYGG-3′
(F1) and 5′-GAAGTVATGATGYTNMGNATG-3′ (F2), were
designed based on the amino acid sequences CGDRASG and
EVMMLRM that are located in the EcR-DBD and EcR-LBD,
respectively. We also designed a reverse degenerate primer,
5′-ACGTCCCAKATYTCWKCNARVAA-3′ (R1), which was based
on the nucleotide sequence located that is located in the EcR-
LBD. The alignments of the amino acid and nucleotide sequences
were made using the EcRs from 12 insects (Fig. S3).
In the first round of PCR, the primer combination F2_R1 was
used for 30 cycles. The PCR products were purified using the
E.Z.N.A® Cycle pure kit (Omega Bio-tek Inc., Doraville, GA, USA)
and sequenced by LGC genomics (Berlin, Germany). Using this
partial sequence, we designed a reverse specific primer,
5′-CTAAGGATGGTCGTTCTG-3′, which was used in combina-
tion with the degenerate primer F1. PCR product sequencing was
performed as described above. In addition, using this partial
sequence of the EcR-LBD, we designed a gene-specific primer,
5′-GAGTTACGCACACTTGGCAATC-3′, which was used in com-
bination with a universal amplification primer for the RACE, using
the 3′RACE system kit (Invitrogen™ Life Technologies, Ghent,
Belgium) for 35 cycles. The PCR products were purified and
sequenced as described above.
Alignment, phylogenetic tree generation and evolutionary
constraint calculations
The amino acid sequences for the EcR-LBDs of 53 taxa
(Table S1) were collected from the GenBank database and used
in a phylogenetic analysis and in the alignment of Fig. 1.The
chosen sequences were aligned by the CLUSTALx program
version 2.0 (Larkin et al., 2007). The evolutionary distances were
computed under the Jones-Taylor-Thornton (JTT) matrix-based
method (Jones et al., 1992) in units of the amino acid substitu-
tions per site. All of the positions that contained gaps and missing
data were eliminated from the dataset. In total, 225 positions were
presented in the final dataset. The phylogenetic tree was created
by the neighbour-joining method using the MEGA4 program
(Tamura et al., 2007). A bootstrap analysis with 1000 replicates
for each branch position was used to assess support for the
nodes in the tree (Felsenstein, 1985). The method used to cal-
culate the evolutionarily constrained regions has been previously
described (Simon et al., 2002). The final alignment for the
selected group (EcR-LBDs) and tree were used to calculate
single-site evolutionary rate values using the Bayesian-based
program RATE4SITE (Pupko et al., 2002; Mayrose et al., 2004).
The evolutionary rates were calculated independently for each
selected set, which included all of the available EcRs, Lepi-
doptera, other Mecopterida (Diptera, Mecoptera, Siphonaptera
498 M. J. Zotti et al.
© 2012 Royal Entomological Society, 21, 488–501
and Trichoptera) and non-Mecopterida. We used lepidopteran
sequences instead of Mecopterida as a whole because the simi-
larity in the activity of tebufenozide between dipteran and non-
Mecopterida insects (Hormann et al., 2008; Nakagawa et al.,
2009). The single-position rates were smoothed using a ten-
position-wide window. Thus, the value at the centre was given the
highest value relative to the weight, and decreased linearly for
the values on both sides of the edge of the window. Based on the
assumption that high evolutionary constraint is under low evolu-
tionary rates, the values were converted to relative constraints by
normalizing them until regions became smoothed; they were
inverted by subtracting the values from 1 and were plotted against
the EcR-LBD alignment. To produce the evolutionary constraint
for the individual groups, calculations were performed using the
ConSurf server (http://consurf.tau.ac.il) (Landau et al., 2005).
Among the results, the score variety file was used in the deter-
mination of the constraints. In brief, the data were first normalized
by local weighted scatterplot smoothing using a polynomial
regression and weights that were computed from the Gaussian
density function. Next, the single position values were smoothed
using the same sliding-window average scheme that was
described above. To assess important positions at the single-site
level in the EcR alignment, a multivariate analysis of protein
polymorphisms was conducted as previously reported (Stone &
Sidow, 2005); this analysis estimates the variance at each given
position to predict the impact of amino acid substitutions. In brief,
this analysis converts amino acid information at each alignment
position into a single vector that summarizes the evolutionary
importance of the six following physicochemical properties: hydr-
opathy, polarity, charge, volume, and free energy in both alpha-
helix and beta-strand conformations (Binkley et al., 2010).
Modelling of Forficula auricularia ecdysone receptor
ligand-binding domainEcR-LBD and ligand-docking studies
Multiple amino acid sequence alignments were created with
CLUSTAL-X software (Thompson et al., 1997) using the Risler’s
structural matrix for homologous amino acid residues (Risler
et al., 1998). Molecular modelling of the EcR-LBDs from the der-
mapteran F. auricularia (FaEcR-LBD) were performed on a
Silicon Graphics O2 R10000 workstation using the programs
INSIGHTII, HOMOLOGY and DISCOVER3 (Accelrys, San Diego, CA).
Because our sequence is not a full-length sequence, it was
supplemented with the last 17 amino acids from T. castaneum
(species phylogenetically close related to F. auricularia). The
atomic coordinates of T. castaneum TcEcR-LBD in complex with
PonA (RCSB Protein Data Bank code 2NXX) (Iwema et al., 2007)
and B. tabaci EcR-LBD in complex with PonA (RCSB Protein
Data Bank code 1Z5X) (Carmichael et al., 2005) were used to
build a three-dimensional (3D) model of the receptor cavity. The
high identities (83.5%, 76.3%) and similarities (90.2%, 85.6%) of
FaEcR-LBD to TcEcR-LBD and BtEcR-LBD, respectively, allowed
us to build an accurate 3D model. Steric conflicts were corrected
during the model-building procedure using the rotamer library
(Ponder & Richards, 1987) and the search algorithm that was
implemented in the HOMOLOGY program (Mas et al., 1992) to
maintain a proper side-chain orientation. An energy minimization
of the final model that was performed with 200–250 cycles of
steepest descent using the consistent valence force field (cvff) of
DISCOVER. PROCHECK (Laskowski et al., 1993) was used to
assess the geometric quality of the 3D model. More than 80% of
the residues of the two modelled FaEcR-LBDs were correctly
assigned on the best allowed regions of the Ramachandran plot,
and the remaining residues were located in the generously
allowed regions of the plot with the exception of the residues
Asn219 of FaEcR-LBD Tribolium based model, which occurred
in the non-allowed region (data not shown). Molecular car-
toons were drawn with PyMol (W.L. DeLano, http://pymol.
sourceforge.net). The Protein cavity volume analysis and amino
acid surface area calculations (similar to a Connolly solvent
surface) were conducted with Discovery Studio 2.5 (ACCELRYS)
using a 1.4-Å probe radius unless otherwise stated.
TcEcR-LBD and BtEcR-LBD in complex with PonA were used
as templates for docking both PonA and the agonist tebufenozide
(DBH-analog) to FaEcR-LBDs. The docking simulation was per-
formed with INSIGHTII, which used DISCOVER3 as a force field.
Clipping planes of TcEcR-LBDs, FaEcR-LBD and BtEcR-LBD
which were each complexed to PonA and tebufenozide, were
rendered with PyMol.
Acknowledgements
Appreciation is expressed to the Funding Agency from the
Brazilian Ministry of Education (CAPES) and to the
National Council of Scientific and Technological Develop-
ment (CNPq) for supporting a PhD grant to M.J. Zotti. This
project was also supported by the Special Research Fund
of Ghent University and the Fund for Scientific Research-
Flanders (FWO-Vlaanderen, Belgium) to G. Smagghe.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article under the DOI reference:
10.1111/j.1365-2583.2012.01154.x
Figure S1. Evolutionary rates were calculated for all available ecdysone
receptor ligand-binding domains (EcR-LBDs) (A), Lepidoptera EcR-LBDs
(B), other Mecopterida EcR-LBDs (C) and non-Mecopterida EcR-LBDs (D).
The evolutionary rates were plotted as a function of its relative position on
EcR-LBD alignment. The vertical grey lines indicate each 20-position-wide
window, which is also indicated by a single-letter code (A-L). The values
from manipulated average of amino acids substitution in each aligned
position along the protein are plotted in the y axis. The protein positions are
plotted in the x axis.
Figure S2. Multivariate analysis of protein polymorphism is shown as a
heat map. The predicted impact of amino acid substitution across align-
ment positions of the Fig. 1 are presented in a colour scale as follows from
dark blue (low impact) to red (high impact). Amino acids are present in one
letter code. The positions in bold and underlined are single-alignment
positions involved in hydrogen bond with PonA. The remaining positions
are some involved in the pocket formation.
Figure S3. The alignment of the amino acids used in primer design was
done using the ecdysone receptors from 12 insects. The degenerate
primers were designed based on the amino acid sequences highlighted in
red. The species used were as follows: [Panorpa germanica, AAZ38143.1],
[Hydropsyche incognita, AAZ38144.1], [Xenos vesparum, AAZ38146.1],
[Leptinotarsa decemlineata, BAD99296.1], [Tenebrio molitor,
CAA72296.1], [Tribolium castaneum, NP_001107650.1], [Camponotus
japonicus, BAF79665.1], [Nasonia vitripennis, NP_001152829.1], [Chilo
suppressalis, BAC11714.1], [Aedes aegypti, EAT38529.1], [Drosophila
melanogaster, ACZ94341.1], [Bradysia coprophila, ACY80739.1].
Table S1. Percentages of amino acid identities and similarities relative to
the Dermapteran Forficula auricularia E domain of the ecdysone receptor
ligand-binding domain (highlighted in blue). Species with highest and
lowest identity and similarity are underlined and bold, respectively. The
branching pattern of the phylogenetic relationships among the arthropod
species is presented in the Fig. 2.
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Structural evolution and binding of ligands 501
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