A structural view of nuclear hormone receptor: Endocrine disruptor interactions

Abstract

Endocrine-disrupting chemicals (EDCs) represent a broad class of exogenous substances that cause adverse effects in the endocrine system by interfering with hormone biosynthesis, metabolism, or action. The molecular mechanisms of EDCs involve different pathways including interactions with nuclear hormone receptors (NHRs) which are primary targets of a large variety of environmental contaminants. Here, based on the crystal structures currently available in the Protein Data Bank, we review recent studies showing the many ways in which EDCs interact with NHRs and impact their signaling pathways. Like the estrogenic chemical diethylstilbestrol, some EDCs mimic the natural hormones through conserved protein-ligand contacts, while others, such as organotins, employ radically different binding mechanisms. Such structure-based knowledge, in addition to providing a better understanding of EDC activities, can be used to predict the endocrine-disrupting potential of environmental pollutants and may have applications in drug discovery.

Full text

REVIEW
A structural view of nuclear hormone receptor: endocrine
disruptor interactions
Albane le Maire •William Bourguet •
Patrick Balaguer
Received: 14 October 2009 / Revised: 3 December 2009 / Accepted: 22 December 2009
ÓBirkha
¨user Verlag, Basel/Switzerland 2010
Abstract Endocrine-disrupting chemicals (EDCs) repre-
sent a broad class of exogenous substances that cause
adverse effects in the endocrine system by interfering with
hormone biosynthesis, metabolism, or action. The molec-
ular mechanisms of EDCs involve different pathways
including interactions with nuclear hormone receptors
(NHRs) which are primary targets of a large variety of
environmental contaminants. Here, based on the crystal
structures currently available in the Protein Data Bank, we
review recent studies showing the many ways in which
EDCs interact with NHRs and impact their signaling
pathways. Like the estrogenic chemical diethylstilbestrol,
some EDCs mimic the natural hormones through conserved
protein–ligand contacts, while others, such as organotins,
employ radically different binding mechanisms. Such
structure-based knowledge, in addition to providing a
better understanding of EDC activities, can be used to
predict the endocrine-disrupting potential of environmental
pollutants and may have applications in drug discovery.
Keywords Nuclear hormone receptors 
Endocrine-disrupting chemicals Environmental
pollutants Crystal structures Hormone
Introduction
Endocrine-disrupting chemicals (EDCs) are exogenous
substances that interfere with the function of hormonal
systems and produce a range of developmental, reproduc-
tive, neurological, immune, or metabolic diseases in
humans and wildlife [1,2]. Many EDCs are man-made
chemicals produced by industry and released into the
environment, for example phthalate or bisphenol A (BPA)
plasticizers, organotins, pesticides, dioxins, polychlorinated
biphenyls, flame retardants, or alkylphenols. Some naturally
occurring EDCs can also be found in plants or fungi, such as
the so-called phytoestrogens: genistein, daidzein, or the
mycoestrogen zearalenone. The sources of exposure to
EDCs are diverse and vary widely around the world. There
are several historical examples of toxic pills or contami-
nation that show a direct causal relationship between a
unique chemical and the manifestation of an endocrine or
reproductive dysfunction (see below). However, these types
of single exposure are not representative of more common
persistent exposures to a broad mix of chemicals and con-
taminants. Industrialized and agricultural areas are typically
characterized by contamination from a wide range of
chemicals that may seep into soil and groundwater. These
complex mixtures enter the food chain and accumulate in
animals and humans. Exposure occurs through drinking
A. le Maire W. Bourguet (&)
INSERM, U554, Centre de Biochimie Structurale,
34090 Montpellier, France
e-mail: bourguet@cbs.cnrs.fr
A. le Maire W. Bourguet
CNRS, UMR5048, Universite
´s Montpellier 1 & 2,
34090 Montpellier, France
P. Balaguer (&)
Institut de Recherche en Cance
´rologie de Montpellier (IRCM),
34298 Montpellier, France
e-mail: patrick.balaguer@valdorel.fnclcc.fr
P. Balaguer
INSERM, U896, 34298 Montpellier, France
P. Balaguer
Universite
´Montpellier 1, 34298 Montpellier, France
P. Balaguer
CRLC Val d’Aurelle Paul Lamarque, 34298 Montpellier, France
Cell. Mol. Life Sci.
DOI 10.1007/s00018-009-0249-2 Cellular and Molecular Life Sciences

water, breathing contaminated air, ingesting food, or con-
tacting contaminated soil. People working with pesticides,
fungicides, and industrial chemicals are particularly
exposed to these toxic substances and thus have a high risk
for developing reproductive or endocrine abnormalities.
EDCs can affect the endocrine systems of an organism in a
wide variety of ways. These include mimicking natural
hormones, antagonizing their action or modifying their
synthesis, metabolism, and transport. Moreover, these
substances can act via multiple pathways including mem-
brane receptors, the aryl hydrocarbon receptor, or the
enzymatic machineries involved in hormone biosynthesis/
metabolism. However, most of the reported harmful effects
of EDCs are attributed to their interference with hormonal
signaling mediated by nuclear hormone receptors (NHRs)
[3–6]. The first example of endocrine disruption was pro-
vided by the pharmaceutical diethylstilbestrol (DES, Fig. 1)
which was used to prevent miscarriage in women with high
risk pregnancies. In the 1970s, prenatal exposure to DES
was linked with the development of vaginal cancer in
so-called DES-daughters and DES-toxic effects were sub-
sequently attributed to the interaction of this compound
with estrogen receptors (ERs) which are members of the
NHR family [7]. Thus, most of the subsequent studies have
focused on NHRs involved in reproductive processes, in
particular ERs and the androgen receptor (AR). More
recently, studies have shown that the activity of the preg-
nane X receptor (PXR), the constitutive androstane receptor
(CAR), the estrogen related receptors (ERRs), the thyroid
hormone receptors (TRs), the retinoid X receptors (RXRs),
or the peroxisome proliferator-activated receptors (PPARs)
can also be affected by EDCs. Based on the conservation of
structural and functional NHR features and on the large
structural and chemical diversity of compounds found in the
environment, one can predict that all members of the NHR
family are potential targets of EDCs.
OH
OH
OH
OH
O
OH
OH OOH N
N
N
NH2
N
N
N
NH2
OH
Estrogen receptors (ERs)
E2 DES GEN PhIP 4-0H-PhIP
Estrogen related receptors (ERRs)
OH
OH
OH
OH
BPA
Retinoid X receptors (RXRs)
OH O
9-cis RA TBT
Sn
Cl
Sn
Cl
O
O
OH
Pregnane X receptor (PXR)
MA
O
OOH
O
OH
O
O
OH
N
N
OH OH
OH
NH
N
OH
OH
O
O
O
O
O
O
O
O
O
OH
OH
O
ZEN
O
O
OH
OH
OH
α-Zearalenol
Cl
Cl
Cl
Cl Cl
Cl
O
Cl
Cl
Cl
Cl
Chlordecone
DES TPT
Hyperforin Colupulone Rifampicin
Fig. 1 Chemical structures of representative EDCs discussed in
this review. E2 17b-estradiol, DES diethylstilbestrol, GEN geni-
stein, PhIP 2-amino-1-methyl-6-phenylimidazo [4-5-b] pyridine,
4-OH-PhIP 4-hydroxy-2-amino-1-methyl-6-phenylimidazo [4-5-b]
pyridine, ZEN zearalenone, BPA bisphenol A, 9-cis RA 9-cis retinoic
acid, TBT tributyltin, TPT triphenyltin, MA methoprene acid
A. le Maire et al.

Human NHRs (Table 1) are a family of 48 transcription
factors, many of which have been shown to be activated by
ligands. NHRs regulate cognate gene networks involved in
key physiological functions such as cell growth and dif-
ferentiation, development, homeostasis, or metabolism
[8,9]. As a consequence, dysfunctional NHR signaling
(i.e., receptor mutation or inappropriate exposure to envi-
ronmental pollutants) often leads to proliferative,
reproductive, and metabolic diseases, including hormonal
cancers, infertility, obesity, or diabetes. NHRs are modular
proteins composed of several domains (Fig. 2a), most
notably an N-terminal domain which harbors a ligand-
independent activation function (AF-1), a central DNA
binding domain (DBD), and a C-terminal ligand binding
domain (LBD) hosting a ligand-dependent transcriptional
activation function (AF-2) [8]. In the absence of the cog-
nate ligand, some NHRs are located in the nucleus, bind to
the DNA response elements of their target genes, and
recruit corepressors, while others are located in the cyto-
plasm in an inactive complex with chaperones. Ligand
binding induces major structural alterations of the receptor
LBDs leading to (1) destabilization of corepressor or
Table 1 The 48 human nuclear receptors
NHR Subtypes M, D, H Ligands
a
NR1A1, A2 TR a,bH Thyroid hormones
NR1B1, B2, B3 RAR a,b,cH Retinoic acid
NR1C1, C2, C3 PPAR a,b,cH Fatty acids, leukotriene B4 (a), fibrates (a),
prostaglandin J2 (c), thiazolidinediones (c)
NR1D1, D2 Rev-erb a,bM, D Heme
NR1F1, F2, F3 ROR a,b,cM Cholesterol (a), cholesteryl sulfate (a), retinoic
acid (b), Orphan (c)
NR1H3, H2 LXR a,bH Oxysterols, T0901317, GW3965
NR1H4, NR1H5 FXR a,bH Bile acids (a), fexaramine (a), lanosterol (b)
NR1I1 VDR H Vitamin D, 1, 25-dihydroxyvitamin D3
NR1I2 PXR H Xenobiotics, 16a-cyanopregnenolone
NR1I3 CAR H Xenobiotics
NR2A1, A2 HNF4 a,cD Orphans
NR2B1, B2, B3 RXR a,b,cD Retinoic acids
NR2C1, C2 TR 2, 4 D, H Orphans
NR2E2 TLL M, D Orphan
NR2E3 PNR M, D Orphan
NR2, F1, F2 COUP-TF I, II D, H Orphans
NR2F6 EAR-2 M Orphan
NR3A1, A2 ER a,bD Estradiol-17b, tamoxifen (a), raloxifene (a),
various synthetic compounds (b)
NR3B1, B2, B3 ERR a,b,cM, D Orphan (a), DES (b,c), 4-OH tamoxifen (b,c)
NR3C1 GR D Cortisol, dexamethasone, RU486
NR3C2 MR D Aldosterone, spirolactone
NR3C3 PR D Progesterone, medroxyprogesterone acetate,
RU486
NR3C2 AR D Testosterone, flutamide
NR4A1 NGFI-B M, D, H Orphan
NR4A2 NURR1 M, H Orphan
NR4A3 NOR1 M Orphan
NR5A1 SF1 M Orphan
NR5A2 LRH-1 Orphan
NR6A1 GCNF D Orphan
NR0B1 DAX-1 Orphan
NR0B2 SHP H Orphan
Adapted from [9]
MMonomer, Ddimer, HRXR-heterodimer
a
For ligand specific of a subtype, the concerned subtype is indicated in parentheses
Structural basis of endocrine disruption

chaperone interfaces, (2) exposure of nuclear localization
signals to allow nuclear translocation and DNA binding of
cytoplasmic receptors, and (3) recruitment of coactivators
triggering gene transcription through chromatin remodeling
and activation of the general transcription machinery.
The crystal structures of many NHR LBDs have been
determined, revealing a conserved core of 12 a-helices
(H1–H12) and a short two-stranded antiparallel b-sheet
(s1 and s2) arranged into a three-layered sandwich fold
(Fig. 2b). This arrangement generates a mostly hydropho-
bic cavity in the lower half of the domain which can
accommodate the cognate ligand. In all hormone-bound
LBD structures, the ligand binding pocket (LBP) is sealed
by helix H12. This conformation is specifically induced by
the binding of hormones or synthetic agonists and is
referred to as the ‘‘active conformation’’ because it allows
the dissociation of corepressors and favors the recruitment
of transcriptional coactivators [10–12]. It is noteworthy
that this conformational state can also be achieved by some
constitutively active orphan receptors for which no natural
ligand has been identified. In this active-form, helices H3,
H4, and H12 define a hydrophobic binding groove for short
LxxLL helical motifs (L stands for leucine and x for any
amino acid) found within coactivators (Fig. 2b). In contrast
to agonist binding, interaction with antagonists prevents the
correct positioning of helix H12, thus avoiding association
with the LxxLL motifs of coactivators [10–12].
Several receptors including ERs, RXRa, ERRa, ERRc,
and PXR have been crystallized in complex with various
EDCs. This review provides an overview of the biological
function of these receptors, of their associated environ-
mental disruptors, and of the mechanisms by which some
of these substances bind to NHRs and alter their signaling
pathways at concentrations within the micro- to nanomolar
ranges.
Estrogen receptors
Estrogen receptors and their environmental
ligands (xenoestrogens)
Estrogen receptors (ERaand ERb) are receptors for the sex
hormone, 17b-estradiol (E2, Fig. 1), which play important
roles in the growth and maintenance of a diverse range of
tissues such as the mammary gland, uterus, bone, or the
cardiovascular system. Both ERs are widely distributed
throughout the body, displaying distinct but overlapping
expression patterns in a variety of tissues [13]. ERais
expressed primarily in the uterus, liver, kidney, and heart,
whereas ERbis expressed primarily in the ovary, prostate,
lung, gastrointestinal tract, bladder, and hematopoietic and
central nervous systems [14]. However, ERaand ERbare
coexpressed in a number of tissues including the mammary
gland, thyroid, adrenal, bone, and some regions of the
brain. Although ERaand ERbshare similar mechanisms of
action, several differences in the transcriptional abilities of
each receptor as well as distinct phenotypes between gene-
null animals have been identified, suggesting that these
receptors may regulate distinct cellular pathways [13,15].
Interestingly, when ERs are coexpressed, ERbexhibits an
inhibitory action on ERa-mediated gene expression [16,
17], such that ERbhas been shown to antagonize several
ERa-mediated effects including fat reduction and cellular
proliferation in breast, uterus, or prostate [18–20]. Breast
cancer is one of the leading forms of cancer in the western
world, and it is well documented that the mitogenic actions
of E2 are critical in the etiology and progression of this
pathology. Accordingly, current therapies of breast cancers
are primarily based on estrogen antagonists that interact
with ERaand completely or partially shut down the cor-
responding hormone-responsive pathway. Because both
ERs are expressed in the normal breast, it is pertinent to ask
whether EDCs with different selectivity for ERs present
Fig. 2 Schematic illustration of the structural and functional orga-
nization of NHRs. aNHRs contain a well-conserved DNA binding
domain (DBD), a moderately conserved ligand binding domain
(LBD), and a highly divergent N-terminal A/B region. Two transcrip-
tional activation functions have been described in these receptors: a
‘‘constitutively active’’ AF-1 in region A/B and an AF-2 which
corresponds to a coactivator binding surface formed by helices H3,
H4, and H12 of the LBD whose completion requires the presence of
the hormone. bOverall structure of NHR LBDs exemplified by the
ERaLBD homodimer (PDB code 1GWR) in complex with estradiol
(stick representation) and a coactivator fragment (CoA). H1–H12 and
s1, s2 denote a-helices and b-strands, respectively
A. le Maire et al.

differential effects on breast cancer proliferation. The
LBDs of ERaand ERbshare a high degree of homology in
their primary amino acid sequence and are very similar in
their tertiary architecture [11,21]. It is therefore not sur-
prising that most compounds tested so far bind to ERaand
ERbwith similar affinities or have similar potencies in
activation of estrogen response element (ERE)-mediated
reporter gene expression [22]. However, some ERa-or
ERb-selective ligands have been identified [23].
High affinity xenoestrogens
For historical reasons, most studies on endocrine disruption
have focused on sex hormone receptors, thus leading to the
discovery of a large panel of substances interfering with
ER signaling. Indeed, the group of molecules identified as
ER endocrine disruptors is highly heterogenous and
includes high affinity ligands (Kd values between 10 pM
and 1 nM), which are pharmaceutical agents contained in
contraceptive pills, human estrogens (E2, estriol, and
estrone), and some natural compounds such as zearalenone
(ZEN) and its metabolites; when available, affinities and
activities of the compounds are reported in Table 2. The
most notorious pharmaceutical EDC is DES (Fig. 1), an
orally active synthetic nonsteroidal estrogen that, due to its
teratogenic effects, was banned in 1971. In utero exposure
to DES provoked abnormal cervical, uterine, and oviduct
anatomy [24], vaginal adenocarcinoma [25], infertility, and
ectopic pregnancy [26]. Ethynylestradiol which is present
in contraceptive pills is commonly found in environmental
samples such as the water of sewage treatment plants or
rivers [27–29]. In spite of its structural differences with E2,
ZEN (Fig. 1) is one of the most active endocrine disruptors
[30]. ZEN is a mycoestrogen with non-estrogenic chemical
structure which is produced by several species of Fusarium
fungi, and it widely contaminates agricultural products [31]
such that ZEN exists in almost all agricultural crops [32].
After consumption, ZEN is metabolized to provide two
major stereo isomers, a- and b-zearalenol [33,34], the
a-metabolite displaying the highest estrogenic activity [30].
Medium/low affinity xenoestrogens
Many other environmental compounds interact with ERs
with medium to low affinity (Kd values between 1 nM and
10 lM). Phytoestrogens are plant-derived substances that
have estrogenic activity [22]. They are classified according
to their chemical structure: isoflavones, flavones, flava-
nones, coumestans, stilbenes, and lignans. The most widely
studied are the isoflavones which are present at high con-
centrations in soy products and red clover [35]. In recent
years, efforts to implement healthier eating habits have
resulted in an increased consumption of soy products and,
hence, increased exposure to phytoestrogens. Genistein
(GEN, Fig. 1) is the principal phytoestrogen in soy and has
a wide range of biological actions. It has a structural simi-
larity with E2 and is an agonist for both ERs, albeit with a
marked preference for ERb[22,36]. The traditionally low
breast cancer incidence rates in Asia are commonly asso-
ciated with the high dietary intake of phytoestrogens, but
the role of phytoestrogens as anticancer or chemopreventive
agents remains under investigation. Other estrogenic
Table 2 EC
50
s of ligands for human ERa,ERb, RXRa, and PXR as reported
ERaERbRXRaPXR
E2 0.02–0.05 nM [24,47] 0.07–0.2 nM [24,47]ND 10lM[98]
DES 0.2 nM [47] 0.4 nM [47]ND10lM[98]
GEN 38 nM [24,47] 6–9 nM [24,47]ND NA[98]
PhIP 30 nM [37]NA[37]NDND
BPA 4 lM[40]5lM[40]2lM
a
[124][10 lM[98]
ZEN 1 nM
b
1nM
b
ND 10 lM[98]
a-Zearalenol 0.1 nM
b
0.1 nM
b
ND 1 lM[98]
Chlordecone 12 lM[39] Antagonist [39]ND ND
TBT ND ND 3 nM [120]ND
Hyperforin ND ND ND 0.11 lM[90]
Rifampicin ND ND ND 0.7 lM[90]
All values are reported as EC
50
s in stably or transiently transfected human cell lines
ND No relevant EC
50
for the ligand/receptor interaction has been reported to our knowledge, NA the ligand is inactive, Antagonist the ligand acts
as an antagonist
a
BPA EC
50
determined by a RXR yeast two-hybrid assay
b
Balaguer P., unpublished results
Structural basis of endocrine disruption

substances are present in foodstuffs, including the
heterocyclic amine 2-amino-1-methyl-6-phenylimidazo
[4-5-b]pyridine (PhIP) contained in cooked meat. PhIP
(Fig. 1) which induces tumors of the breast and prostate in
mice and which has been characterized accordingly as a
compound with significant estrogenic activity inducing
transcription of E2-regulated genes [37,38]. Certain
insecticides and herbicides such as dichloro-diphenyl-
tricholoethane (DDT), methoxychlor, hexachlorocyclo-
hexanes, and related compounds are also suspected to act as
environmental estrogenic chemicals. Chemically stable and
strongly lipophilic, organochlorine compounds tend to
accumulate in lipid-rich tissues and induce endocrine dis-
ruption at exposure levels measured in the environment.
Interestingly, some pesticides such as chlordecone (Fig. 1)
or methoxychlor display ERaagonistic, but ERbantago-
nistic, activities [39], thus increasing the risk of cancer
development. Finally, many industrial compounds display
estrogenic activity. These include BPA (Fig. 1), alkyl-
phenols, and benzophenones. BPA, a monomer of
polycarbonate plastics, is one of the highest-volume
chemicals in commerce. Polycarbonates are used in many
consumer products, including food and water containers,
baby bottles, medical tubing, or epoxy resin, and small
amounts of BPA can migrate from polymers to food or
water, especially when heated. Interestingly, BPA displays
estrogen-like activities at nanomolar doses, but the mech-
anism by which it exerts its biological actions remains
enigmatic. Although BPA binds ERs, its binding affinity is
several orders of magnitude lower than that of E2 [22,40],
and it has been suggested that BPA could also act through
binding to membrane ERs, G-protein coupled receptor 30
[41], or ERRc[42]. Alkylphenols are degradation deriva-
tives of alkylphenols ethoxylates which are widely used
surfactants and detergents in domestic and industrial prod-
ucts. Alkylphenols are present at significant levels in
samples of every environmental compartment examined,
including fish muscle tissue [43], and are also generally
ubiquitous in food. Benzophenones used in topical sun-
screen preparations have also been shown to activate ERs
with a stronger affinity for ERb[44,45].
Structural basis of xenoestrogen action
Natural estrogens
In the ERs, the hormone binding pockets are lined with 20
mostly hydrophobic residues that interact with the steroid
scaffold [46,47]. A few polar residues located at the two
ends of the pockets form hydrogen bonds with the polar
groups at the 3- and 17-positions of estrogens (Fig. 3a).
Selectivity of hormone binding derives from both the shape
of the hydrophobic portion of the pocket and the presence
of receptor-specific hydrogen-bond networks. In contrast to
other steroids which possess a 3-keto group, estrogens have
a 3-hydroxyl group. Thus, part of the specificity of E2 for
ERs is supported by a network of water-mediated hydrogen
bonds involving a hydrogen-bond acceptor in ERs (E353 in
ERa) while a hydrogen-bond donor (glutamine residue) is
present in other steroid receptors. At the other end of the
ligand, the 17-hydroxyl group of E2 is hydrogen-bonded to
H524 (ERa).
Xenoestrogens in food: the example of GEN and PhIP
As noted above, GEN (Fig. 1) is an isoflavonoid phytoes-
trogen which binds to both ER isotypes with a preference
for ERb[22,48]. Although it is a non-steroidal compound,
GEN possesses a diphenolic structure that shares structural
similarity with the steroidal core of estrogens. Conse-
quently, structures of GEN-bound ERaand ERbreveal that
it binds to both ERs nearly identically and in a manner
reminiscent of that observed for the natural hormone [46,
49–51]. The phenol ring of GEN mimics the hydroxyl
group of the A-ring of E2 and interacts with residues E353,
R394, and a highly ordered water molecule, whereas the
isoflavone portion superimposes on the C and D rings of E2
and makes a hydrogen bond with H524 (Fig. 3b). Because
there are only two conservative residue substitutions in the
hormone binding pockets of the two receptor subtypes
(ERaL384/ERbM336 and ERaM421/ERbI373), the
explanation of the 40-fold ERbselectivity of GEN could
not be convincingly explained by visual inspection of
crystal structures. Overall, it appears that ligand selectivity
arises from a combination of subtle differences involving
sequence differences outside the binding pocket that alter
the shape of the cavity [52] and proximal ligand–amino
acid contacts [49]. Using quantum chemical calculations,
Manas et al. [49] demonstrated that, although conservative,
the two substitutions are capable of contributing signifi-
cantly to the observed ERbselectivity of GEN on the basis
of differential electronic interactions mediated by the sul-
fur-containing side chain of methionines relative to purely
aliphatic side chains of leucine and isoleucine residues
[49]. GEN-bound ERs have been crystallized both in the
presence and absence of a coactivator fragment [49,50].
Interestingly, the activation helix H12 was observed in two
different orientations in the two complexes (Fig. 3c). In the
presence of the coactivator fragment, H12 adopts the
canonical active conformation, whereas it is found in the
antagonist-like conformation in the binary ER-GEN com-
plex. This inactive conformation results from the
unwinding of helix H11 and the consecutive lengthening of
the loop connecting H11 and H12. These structural data
account for the observation that GEN acts as a partial AF-2
A. le Maire et al.

agonist, antagonizing E2 but displaying a residual agonist
activity in some cells [48] (Balaguer et al., unpublished
data). As demonstrated for other partial AF-2 agonists [53]
and in contrast to pure agonists, GEN appears unable to
induce the receptor active conformation on its own, and the
presence of a coactivator is required to fully stabilize this
conformational state. Thus, the overall activity of partial
AF-2 agonists like GEN is determined by the concentration
of coregulators which may vary between cell types or
tissues [54].
Similarly, the mutagenic compound PhIP and one of its
metabolites 4-OH-PhIP have been crystallized with ERa
[38]. PhIP is a heterocyclic amine with no hydroxyl groups
(Fig. 1) and thus differs significantly from classical ER
ligands. The phenyl ring occupies a position similar to that
adopted by the A- and B-rings of E2 but does not form the
typical hydrogen bonds with R394 and E353 (Fig. 3d). The
remaining portion of the ligand mimics the C- and D-rings
of E2. Except for the NH
2
moiety which interacts favorably
with H524, the hydrogen-bonding potential of the other
Fig. 3 Structural determinants of ligand recognition by ERs. aClose-
up view of the E2 binding pocket in ERa(PDB code 1GWR). ERa
residues involved in hormone binding as well as the E2 molecule are
colored in yellow.bSuperposition of ERaand ERbLBPs in complex
with E2 (yellow, PDB code 1GWR) and GEN (blue, PDB code
1QKM), respectively. Residues involved in E2 and GEN binding are
colored in yellow and blue, respectively. cClose-up view of the
positions of helix H12 in two GEN-ERbcomplexes in the presence
(blue, PDB code 1X7J) and absence (green, PDB code 1QKM) of a
coactivator fragment. dSuperposition of ERaLBPs in complex with
PhIP (yellow, PDB code 2QXM) and 4-OH-PhIP (pink, PDB code
2QSE). Residues involved in PhIP and 4-OH-PhIP binding are
colored in yellow and pink, respectively. eSuperposition of ERa
LBPs in complex with PhIP (blue, PDB code 2QXM) and E2 (yellow,
PDB code 1GWR), respectively. fSuperposition of ERband ERa
LBPs in complex with GEN (blue, PDB code 1X7J) and DES (green,
PDB code 3ERD), respectively. Residues involved in GEN and DES
binding are colored in blue and green, respectively. WWater
molecule. Helix and residue numbers are indicated
Structural basis of endocrine disruption

nitrogen atoms of the heterocyclic ring is not satisfied by
interaction with the receptor LBP. Compared with the ERa-
E2 complex, the structure with PhIP shows a substantial
shift in the last three turns of helix H11 against which helix
H12 docks in the active conformation (Fig. 3e). This loss
of stabilizing contacts between the two secondary struc-
tural elements likely renders helix H12 more dynamic in
solution [38]. Together, these structural data explain the
functional properties (affinity and activity) of PhIP. Inter-
estingly, the PhIP metabolite 4-OH-PhIP differs from its
parent compound by a hydroxyl group which reproduces
the interactions made by the A-ring hydroxyl of E2. This
structural observation leads to the prediction that 4-OH-
PhIP should display a better affinity towards ER and also a
higher estrogenic activity than its parent compound.
Pharmaceuticals: the example of DES
In contrast to the partial agonists described above, the
synthetic nonsteroidal DES (Fig. 1) functions as a full
agonist. The interaction of DES with ERa[47] resembles
that of GEN or E2 with the two conserved hydroxyl groups
involved in hydrogen bonds with E353, R394, and a water
molecule on one side and H524 on the other side (Fig. 3f).
Nevertheless, although the DES B ring occupies the same
position as the GEN C ring, the phenolic A ring and the
ethyl groups of DES do not superimpose the isoflavone ring
of GEN, as a rotation angle of roughly 50°is observed
between the aromatic rings (Fig. 3f). Thus, DES forms
additional non-polar contacts with the LBD. In particular,
contacts with A350 (H3), L384 (H5), F404 (S1), or L428
(H7) may account for the higher affinity of DES for the
receptor [14], whereas specific interactions made with
W383 (H4) or L540 (H12) may help stabilize the active
conformation and explain the full agonistic profile of this
compound.
Estrogen-related receptor c
Estrogen-related receptors and their environmental
ligands
ERRcis a member of the ERR subfamily of orphan
receptors, which are closely related to ERs [55]. The ERR
family includes three members, ERRa, ERRb, and ERRc.
The three receptors are very similar, with 90% sequence
identity in the DBD and more than 60% in the LBD. ERRa
is highly expressed in muscle, heart, bone, and adipose
tissue, as well as in the central nervous system [56]. ERRc
is expressed in a tissue-restricted manner, for example very
strongly in the mammalian brain during development, and
then in the brain, lung, and many others tissues during
adulthood. In terms of structure, ERRs are very close to
ERs. Sequence alignment reveals a 60% identity in the
DBD regions and a moderate similarity (\35%) of the
LBDs, consistent with the incapacity of ERRs to bind E2
[57]. Nevertheless, it has been demonstrated that ERRs can
interfere with estrogen signaling [55]. Indeed, ERs and
ERRs recognize the same DNA binding elements, share
common target genes and are coexpressed in many tissues
[58,59]. Recent publications report that ERRamay rep-
resent a biomarker of poor prognosis in breast and ovarian
cancers suggesting an involvement of the receptor in cell
proliferation. In contrast, exogenous overexpression of
ERRband ERRcin prostate cancer cell line results in
inhibition of proliferation [60]. Furthermore, treatment
with an ERRb/cagonist has been shown to promote this
antiproliferative effect, consistent with ERRcbeing a
favorable prognosis factor [61]. Additionally, several lines
of evidence suggest that ERRs play a central role in reg-
ulating energy metabolism. ERRs are expressed in tissues
associated with lipid metabolism and high energy demands,
their transcriptional activity is highly dependent on the
presence of coregulators implicated in the control of met-
abolic programs, genetic studies in mice reveal that their
presence is essential for the generation of energy and
related tissue-specific functions, and functional genomics/
proteomics studies have associated ERRs with the control
of vast metabolic gene networks, in particular those
involved in mitochondrial biogenesis and function [62].
The rise in the incidence of metabolic syndromes correlates
with the rise in the use and distribution of industrial
chemicals that may play a role in generation of obesity
[63], suggesting that EDCs and ERRs may be linked to this
epidemic crisis.
To date, the ERRs have not been shown to interact with
any physiologically relevant small molecules, suggesting
that these receptors manifest constitutive activity [64,65],
and, indeed, crystallographic analyses of ERRs indicated
that these receptors adopt the transcriptionally active con-
formation in the absence of any ligand [65]. The
compounds screened for activity on the ERRs were known
endocrine disruptors with estrogen-like activity. Two
organochlorine pesticides, toxaphene and chlordane, were
found to act as weak antagonists of ERRa[66]. DES
inhibits the constitutive activity of all three ERRs [67,68].
The phytoestrogen kaempferol is an ERRaand cantagonist
[69]. In contrast to the numerous reported ERR antagonists,
few agonists of ERR have been identified. However, phy-
toestrogens (GEN, daidzein) function as non-selective and
low affinity ERR agonists [70]. Finally BPA, bisphenol E
(BPE), and others phenols were recently reported to bind
with high affinity to ERRc[42,64].
A. le Maire et al.

Structural basis of BPA- and DES-ERR interactions
Structure of the unliganded ERRc
The crystal structure of unliganded ERRcLBD [65,71]
superimposes well with that of the E2-bound ERaLBD,
revealing that the receptor can adopt a transcriptionally
active conformation in the absence of ligand (Fig. 4a). This
conformation allows an interaction with LxxLL motifs of
coactivators via a charge clamp comprised of K284 (H3)
and E452 (H12). The structure reveals that ERRcexhibits a
relatively small putative LBP (Fig. 4a) delimited by 22
amino acids and measuring 280 A
˚
3
in volume compared
with 480 A
˚
3
for ERa[71]. This observation may explain
why most of the exogenous compounds interacting with
ERRs act as antagonists rather than agonists (see the
description of the DES-bound structure bellow). The empty
ERRcpocket is formed by hydrophobic and a few polar
(Y326, N346) or charged (E275, R316) residues. The
substitution of several residues between ERaand ERRc
LBPs [L525/F435 (H11), G521/A431 (H11), and L540/
F450 (H12)] leads to the decrease of the cavity volume for
ERRcthat precludes E2 binding to the orphan receptor.
Nevertheless, it was predicted that ERRccould bind small
phenol-containing ligands because the two charged resi-
dues which form a hydrogen bonding network anchoring
the 3-OH group of the A-ring of estradiol in ERa(E353 in
H3 and R394 in H5) are conserved in ERRc(E275 and
R316).
Structure of BPA-bound ERRc
Although a natural ligand remains to be found for ERRc,
several synthetic ligands have been identified for this
receptor [42,64]. All display a conserved phenol ring such
as BPA, a small symmetric molecule with two phenol rings
and two methyl groups linked to a central sp
3
carbon atom
(Fig. 1). The structure of ERRcin complex with BPA
(Fig. 4b) has been solved, revealing an overall protein
conformation indistinguishable from that of the unliganded
receptor [72–74]. A close look at the LBP shows that, as
previously anticipated, one of the two phenol-hydroxyl
groups of BPA forms hydrogen bonds with residues E275
(H3) and R316 (H5) while the second is hydrogen bonded
to N346 in H7. A hydrogen bond between Y326 and N346
holds N346 in position to interact with the second phenol
group of BPA. Interestingly, this asparagine residue is not
conserved in ERRaand b, thus accounting for the specific
ERRc-BPA interaction. Moreover, comparison of ERRc
and ERRaLBPs reveals a much smaller cavity for the latter
[75]. The replacement of two alanine residues (A272 and
A431) in ERRcby a phenylalanine (F328) and a valine
(V491) in ERRaaccounts for this size reduction and fur-
ther explains why BPA does not bind to the latter. BPA
binding provokes only minimal LBP rearrangements.
E275, which appears disordered in the unliganded struc-
ture, adopts a unique conformation to make a hydrogen
bond with one OH group of BPA, and the side chain of
L345 moves away from the pocket upon BPA binding to
open up the cavity and make room for the second phenol
group of BPA. In summary, ERRcpossesses a LBP to
which BPA can bind with high affinity and specificity
while preserving the constitutively active conformation of
the receptor. The limited impact of BPA binding on
receptor structure is consistent with the fact that this ligand
does not enhance or disrupt coactivator binding and thus
appears as a functionally silent ligand [72,73]. Thus, the
classical mechanism of NHR activation involving the
Fig. 4 Structural determinants of ligand recognition by ERRs.
aOverall structure of unliganded ERRcLBD (PDB code 1KV6) in
cartoon representation. The unoccupied LBP is highlighted in black.
bSuperposition of ERRcLBPs in absence of ligand (yellow, PDB
code 1KV6) and in complex with BPA (green, PDB code 2E2R).
Residues involved in BPA binding are colored in green and
equivalent residues in the unliganded receptor are colored in yellow.
cSuperposition of ERRcLBP in absence of ligand (yellow, PDB code
1KV6) and of the DES-bound form (blue, PDB code 1SP9). Residues
involved in DES binding are colored in blue and equivalent residues
in the unliganded receptor are colored in yellow
Structural basis of endocrine disruption

re-localization and stabilization of helix H12 in the active
conformation does not explain how ERRccould mediate
the estrogenic effects of BPA. Rather, thermal stability
studies revealed that BPA binding leads to global ther-
modynamic stabilization of ERRcLBD, a phenomenon
which could increase steady state levels of the receptor and
impact both its cellular half-life and biological activity
[71–73].
Structure of DES-bound ERRc
In contrast to BPA, DES deactivates ERRs and acts as a so-
called inverse agonist by disrupting the basal interaction
between ERRs and coactivators [68]. The crystal structure
of the DES-bound ERRc(Fig. 4c) shows that DES-medi-
ated inverse agonism is based on the rearrangement of the
side chain of F435 (H11), which upon ligand binding flips
out and sterically interferes with H12, thus displacing it
from its agonist position [76]. Interestingly, the recently
reported crystal structure of ERRcLBD in complex with
the synthetic agonist GSK4716 [71] revealed an unex-
pected rearrangement of the phenol binding residues E275
and R316, which allows access to an additional pocket of
390 A
˚
3
resulting in the formation of a single combined
pocket of 610 A
˚
3
. This structure reveals that ERRccan
accommodate larger ligands than previously anticipated.
Pregnane X receptor
Pregnane X receptor and its environmental ligands
PXR, also known as steroid and xenobiotic receptor (SXR)
and pregnane activated receptor (PAR), is activated by
xenobiotics and acts as a master regulator of phase I to III
of drug metabolism. PXR is involved in the biosynthesis,
distribution, and metabolism of steroids, bile acids, and
xenobiotics [77]. Activated PXR binds to gene promoters
as a heterodimer with RXR and induces the expression of
target genes such as CYP3A. This receptor plays a prom-
inent role as protector of the endocrine system from
chemical perturbation by sensing increases in the concen-
tration of a multitude of EDCs and inducing detoxification
pathways to prevent other NHRs from interactions with
these chemicals. Literature has described PXR activation as
‘‘Jekyll and Hyde’’ or ‘‘ying and yang’’ [78,79] to illustrate
both its beneficial and prejudicial effects. In fact, activation
of PXR can be positive, as it accelerates the detoxification
process and consequently the elimination of xenobiotics. In
contrast, the premature metabolism of active compounds
such as hormones or drugs means that target responses will
not be activated and can lead to harmful effects or adverse
interactions. The metabolism of inactive compounds can
also lead to the synthesis of active metabolites, such as the
transformation of methoxychlor by CYP2C11, a PXR-
induced enzyme, into phenolic estrogenic compounds [80].
It has also been observed that coregulatory proteins work in
concert with ligands to stabilize PXR LBD such that the
ligand-induced transcriptional response would depend on
which coregulator binds to PXR LBD [81]. This observa-
tion suggests that promoter context (i.e., the combination of
coregulators associated with a target gene promoter) is an
important parameter in determining the transcriptional
activity of PXR. Support for this notion comes from greater
PXR activation by pregnenolone on a CYP3A4 PXR
response element (PXRE) than on a PXRE from the mul-
tidrug resistance protein 1 (MDR1) gene which correlates
with recruitment of the coactivators SRC-1 and not AIB-1
[82]. Overall, it is difficult to conclude whether the xeno-
biotic activation of PXR is predominantly negative or
positive, but clearly PXR plays an essential role in endo-
crine disruption. Unlike most NHRs that tend to be
specialized to bind few ligands with structural homologies,
PXR is able to bind a large number of structurally diverse
ligands with a wide range of affinities [83–87]. PXR binds
a multitude of drugs such as the antibiotic rifampicin
[88–91], the anti-cancer taxol [92,93], the anti-cholesterol
SR12813 [91,94], the barbituric phenobarbital [90], the St
John’s Wort anti-depressor hyperforine (Fig. 1)[95,96],
and many more, recently reviewed in [97]. Furthermore,
numerous studies have focused on its ability to bind
environmental compounds, such as pesticides [91], natural
and synthetic estrogens and alkylphenols [87,98–100],
polychlorinated biphenyls [98,101], brominated flame
retardants [102], or antimicrobial triclosan [98].
Structural basis of PXR-xenobiotics interactions
By virtue of this low ligand selectivity and its dual
response characteristics, PXR differs from the receptors
previously discussed. Contrary to ER and RXR, which
possess high affinity-specific physiological ligands or ERR
which has no known natural ligand, PXR is activated by a
variety of structurally distinct endogenous and exogenous
compounds. Crystallographic studies have revealed several
unique characteristics of PXR that account for its promis-
cuous ligand binding properties. First, PXR possesses a
large LBP which can accommodate compounds with larger
volumes than that of classical NHR ligands (823 Da for the
macrocyclic rifampicin vs 272 for E2). Second, several
loops clustering at the bottom of the LBD confer a high
plasticity allowing the receptor LBP to adopt different
shapes according to the bound ligands. Three of these
flexible elements are found in a PXR-specific sequence of
approximately 60 residues inserted between helices H1 and
H3. This segment folds as a two-stranded antiparallel
A. le Maire et al.

b-sheet (s1 and s1’, residues 210–228), a disordered loop
(177–197), and two flexible loops (198–210 and 229–235)
that appear to be characterized by either rather high ther-
mal B factors (indicating structural mobility) in the
unliganded and hyperforin-bound PXR [85,94] or com-
plete disorder in the structures with rifampicin [103] and
colupulone [104] (Fig. 5a, b). Another region adjacent to
the PXR LBP with high structural dynamics resides
between s4 and H7 (residues 309–321). Thus, it appears
that PXR displays a flexible portion of its LBP that allows
the receptor to modify its shape and volume to bind ligands
via an induced-fit mechanism. For example, the binding
cavity of unbound PXR is 1,294 A
˚
3
in volume but expands
to 1,544 A
˚
3
in the hyperforin-PXR complex. Third, the
unliganded PXR LBP contains 28 residues including eight
polar amino acids with the potential to form hydrogen
bonds with ligands. Among them, three polar (S247, Q285,
and H407) and three hydrophobic (M243, W299, and
F420) residues are consistently involved in ligand binding.
The remaining contacting residues depend on the size and
the chemical nature of the bound ligands. For example,
rifampicin contacts 18 residues while hyperforin interacts
with 13 amino acid side chains. Rifampicin which is 40%
larger than hyperforin and occupies regions not filled by
the latter makes specific interactions with residues V211,
L239, L308, and R410 (Fig. 5c, d). Interestingly, PXR is
also able to bind smaller endogenous molecules such as
steroidal hormones. The recently reported crystal structure
of PXR LBD in complex with estradiol reveals that E2 fills
only a very small part of the LBP, leaving 1,000 A
˚
3
unoccupied [87]. Moreover, the structure shows that the
position of the bound ligand as well as the key stabilizing
interactions differ significantly from that found in the
E2-bound ER complex structure. Notably, E2 in PXR binds
closely adjacent to H12 in an orientation nearly perpen-
dicular to that observed in the ER complex, and key
E2-contacting residues in ER (E353, R394, and H524 in
ERa) are replaced in PXR such that the 3-OH group on the
A-ring forms an hydrogen bond with S247, whereas the 17
OH-group on the D-ring is hydrogen bonded to R410 and
Fig. 5 Structural determinants of ligand recognition by PXR.
aClose-up view of the flexible elements found specifically in PXR.
They are inserted between helices H1 and H3 and comprise two-
stranded antiparallel b-sheet (s1 and s10), a disordered loop and two
flexible loops. These elements are visible in the structure of
unliganded PXR (green, PDB code 1ILG) whereas they are too
disordered to be visible in the structure of PXR bound to rifampicin
(purple, PDB code 1SKX). bThe structure of PXR LBD in complex
with hyperforin (PDB code 1M13) is rendered by thermal displace-
ment parameters (B factor) ranging from blue (low) to red (high).
cClose-up view of PXR LBP in complex with rifampicin (PDB code
1SKX). PXR residues involved in rifampicin binding are colored in
purple and rifampicin is shown in yellow.dClose-up view of PXR
LBP in complex with hyperforin (PDB code 1M13). PXR residues
involved in hyperforin binding are colored in green and hyperforin is
shown in yellow
Structural basis of endocrine disruption

S208 (not shown). Globally, the capacity of PXR to
accommodate a variety of chemical scaffolds critically
involves the deformability of its cavity together with the
unique distribution of hydrophobic, polar, and charged
residues over the LBP. CAR is another receptor that
responds to several endo- and xenobiotics [105,106].
However, the smaller and less conformable LBP of CAR is
most probably the reason why this receptor recognizes
fewer EDCs and plays a lesser role than PXR as a xeno-
sensor. Together, these structural observations contribute to
the better understanding of how PXR can detect structur-
ally and chemically different compounds. However, they
also point to the fact that, due to the high plasticity of PXR
LBP, modeling of the interaction between this receptor and
EDCs in view of computational prediction of receptor–
ligand interaction might be difficult.
Retinoid X receptors
Retinoid X receptors and their environmental ligands
RXRa,b, and coccupy a particular position in the NHR
superfamily, as they are the common heterodimerization
partners for one-third of the 48 family members (Table 1).
Consequently, RXRs are involved in the control of multiple
signaling pathways in both ligand-dependent and -inde-
pendent manners [107]. RXRs form three different types of
dimers: RXR homodimer, permissive heterodimers, and
non-permissive heterodimers. RXR permissive heterodi-
mers (e.g., RXR-PPAR, RXR-LXR, or RXR-PXR) are
activated upon ligand binding to RXR even in the absence
of the partner receptor ligand, whereas non-permissive
heterodimers (e.g., RXR-RAR, RXR-VDR, or RXR-TR)
cannot be activated by the RXR ligand alone, and RXR
serves as a silent partner in absence of partner ligand.
Differential corepressor interaction accounts for the diverse
activation profiles of permissive and non-permissive het-
erodimers [108]. However, in both cases, RXR ligands and
ligands of the partner receptors can act synergistically to
activate heterodimers [109,110]. This regulatory control of
nuclear signaling pathways by multiple RXR heterodimers
allows environmental RXR ligands to potentially trigger a
multitude of adverse effects on human health.
RXRs are activated by 9-cis retinoic acid (9-cis RA;
Fig. 1) as well as docosahexaenoic acid [111,112]. In
addition, synthetic RXR ligands, referred to as rexinoids,
are already used or are being developed for cancer therapy
and treatment of metabolic diseases [113]. On the other
hand, RXR can also be activated by environmental exo-
genous chemicals, including some organotin compounds
(Fig. 1), which in so doing act as endocrine disruptors
[114–116]. The RXRa/PPARcheterodimer was reported to
play a major role in mediating the deleterious effects of
organotins which are ubiquitously present throughout the
environment due to their widespread use since the 1960s in
many industrial and agricultural processes [117]. Impor-
tantly, these studies demonstrated that the two biocides,
tributyltin (TBT; Fig. 1) and triphenyltin (TPT; Fig. 1), are
able to activate this heterodimer at nanomolar concentra-
tions thereby inducing various toxicities ranging from
adipogenesis in mammals [118,119] to the development of
male reproductive organs in female gastropods [120,121].
Interestingly, a recent study revealed that in fact TBT
activates all three RXR/PPARa,c,dheterodimers essen-
tially through its interaction with RXR [122]. Another
example of an EDC that modulates RXR function is
methoprene which is an insect growth regulator in
domestic and agricultural use as a pesticide. At least one
metabolite of methoprene, methoprene acid (MA, Fig. 1),
directly binds to and activates mammalian RXRs [123].
Finally, BPA and other phenols (4-tert-octylphenol,
nonylphenol mixture) showed high induction of RXR
directly or after being metabolized [124].
Structural basis of xenorexinoids action
MA binds to and activates the three subtypes of RXR with
a lower affinity than 9-cis RA [123]. In contrast, TPT and
TBT, two of the most active organotin compounds, bind to
and activate RXRs as efficiently as 9-cis RA (Table 1).
Structures of the complexes between RXR and TBT [122],
TPT (described in this review) and MA [125], were
obtained, and their comparison with that of the 9-cis RA-
bound RXR [126] allowed the description of the binding
mode of these EDCs and the explanation of their different
binding affinities. In RXR, 9-cis RA is buried in an
essentially hydrophobic pocket formed by residues located
on helices H3, H5, H7, and H11, and the b-turn (Fig. 6a).
RXR features an L-shaped pocket which requires a sharp
bend or a twist of the polyene side chain in the retinoid
skeleton to allow binding. The pocket is sealed by R316
which forms an ionic interaction with the carboxylate
group of 9-cis RA on one side and by helices H7, H11 and
H12 on the other side. Thus, the restrictive structural,
chemical, and conformational features of the RXR LBP are
perfectly adapted to accommodate the 9-cis isomer and not
the elongated all-trans isomer of retinoic acid. Interest-
ingly, the pesticide MA displays molecular characteristics
similar to 9-cis RA which allow interaction with RXR,
namely a conformable aliphatic chain and a carboxylate
moiety. In the crystal structure [125], it was observed that,
contrary to 9-cis RA, MA does not interact with H12
directly, an observation that could account for the poor
agonist-driven H12 recruitment. MA adopts an L-shaped
conformation to fit the RXR LBP by two consecutive 90°
A. le Maire et al.

bond rotations, and thereby mimics the sharp cis-bend of
9-cis RA (Fig. 6b). In the same manner as 9-cis RA, the
carboxylate is anchored by an ionic interaction with R387
from helix H5. The methoxy iso-butyl moiety is smaller
than the b-ionone ring of 9-cis RA, and therefore makes
fewer hydrophobic contacts with the receptor. These dif-
ferences in interactions with the receptor most likely
account for the lower affinity observed for MA in com-
parison with 9-cis RA. In contrast to MA, the two
organotin compounds, TBT or TPT, interact strongly with
RXRs and act as full RXR agonists whereas they neither
structurally nor chemically resemble 9-cis RA (Fig. 1).
Organotins form a group of more than 250 tin compounds
containing a variety of mono-, di-, tri-, or tetra-substituted
organic groups and, contrary to classical rexinoids (RXR-
specific ligands), they do not contain an acidic head group
for interaction with R316. The previously reported struc-
ture of RXR in complex with TBT [122], and the crystal
structure of TPT-bound RXR presented here, reveal that, as
compared with 9-cis RA, the two organotins occupy only a
small part of RXR LBP (Fig. 6c). Moreover, the structures
show that the high affinity of the organotins for RXR
derives mainly from the formation of a covalent bond
between the tin atom of organotins and the sulfur atom of a
cysteine residue (C432) in helix H11. The remaining
interactions involve van der Waals contacts between
almost all organotin carbon atoms and RXR residues.
Interestingly, although TBT and TPT interact with only a
subset of binding pocket residues, they are engaged in
enough essential contacts to efficiently stabilize RXRa
in its active conformation. The particular position of C432
in helix H11 likely accounts for the high agonist activity of
TBT and TPT. Indeed, several studies have pointed out the
importance of this secondary structural element which
should adopt a helical conformation for full receptor acti-
vation [38,127,128]. The presence of a cysteine residue at
this particular H11 position is unique to RXRs and may
allow for the stabilization of the helical conformation of
H11 and of the active receptor form by TBT and TPT.
Other NHRs including PPARcand the glucocorticoid
receptor (GR) have been identified as organotin targets
[114,118,119,129]. PPARcand GR contain cysteine
residues at different positions which could help to anchor
the organotins in the LBPs. However, these cysteine resi-
dues could fix the tin compounds in regions of the LBPs
which do not allow efficient stabilization of the active
receptor conformation. Accordingly, organotins were
shown to act as partial agonist or antagonist for PPARc
[122] and GR [129], respectively, indicating that tin-con-
taining compounds could use the specific Sn-S interaction
to modulate the transcriptional activity of a number of
NHRs, the functional outcome being dictated by the
structure of the organotin and the position of the anchoring
cysteine in the LBP.
Concluding comments
Deregulation of NHR-mediated transcription accounts for
the deleterious effects of many EDCs. Thus, characteriza-
tion of the harmful interaction between receptors and
environmental compounds, both at the structural and
functional levels, as well as the development of robust in
vivo, in vitro, and in silico screening methods, are impor-
tant for assessment of the toxic potential of large numbers
of chemicals [5,130–132]. In this context, computer-aided
technologies which allow activity prediction of endocrine
disruptors and environmental risk assessment have been
developed. Computational tools very often include the use
of quantitative structure–activity relationship (QSAR)
methods in which the chemical structure of a compound is
quantitatively correlated with its biological activity through
Fig. 6 Structural determinants of ligand recognition by RXRs.
aClose-up view of the 9-cis-RA binding pocket of RXRa(PDB
code 1FBY). RXR residues involved in 9-cis-RA binding are colored
in yellow and 9-cis-RA is shown in orange.bSuperposition of RXRa
and RXRbLBPs in complex with 9-cis-RA (orange, PDB code
1FBY) and methoprene acid (violet, PDB code 1UHL), respectively.
Residues involved in 9-cis-RA and methoprene acid binding are
colored in yellow and pink, respectively. cSuperposition of RXRa
LBPs in complex with 9-cis-RA (orange, PDB code 1FBY) and TPT
(blue, PDB code 3KWY). Residues involved in 9-cis-RA and TPT
binding are colored in yellow and blue, respectively
Structural basis of endocrine disruption

a number of molecular descriptors, including the molecular
weight and parameters, to account for hydrophobicity,
topology, or electronic properties [133–137]. The growing
number of crystal structures of NHR LBDs in the presence
of various ligands greatly facilitates the understanding of
ligand binding and receptor modulation. This structural
information can also be used with modeling and docking
tools to predict the interaction of EDCs with NHRs
[138–142]. However, several levels of structural com-
plexity limit the current application of these computational
approaches.
The examples reviewed in the present article indicate
that EDCs can be roughly divided into three classes
depending on their structural and chemical proximity with
endogenous hormones. The first class contains all EDCs
mimicking the natural hormones through conserved pro-
tein–ligand contacts. Representative examples for ERs and
RXRs are DES, GEN, and MA (Fig. 1) which display most
of the structural and chemical requirements for interaction
with their respective receptors in a hormone-like fashion.
Other EDCs such as a-zearalenol (Fig. 1) most probably
fall into this category [143]. The second class contains
compounds displaying only a fraction of the anchoring
chemical groups exhibited by the endogenous hormone as
exemplified by PhIP and its metabolite 4-OH-PhIP [144].
Lastly, the third class corresponds to chemicals which
display molecular properties distinct from the endogenous
ligands but that still bind to NHRs by employing radically
different binding mechanisms. The best experimentally
validated example of this concept is the case of TBT and
TPT. Although they do not possess the classical features of
rexinoids, namely a carboxylic head and a long aliphatic
tail, these organotin compounds bind RXRs with high
affinity and are able to efficiently activate these receptors
through the establishment of a covalent link with a cysteine
residue of the LBP. Similarly, due to a chemical structure
unrelated to that of E2, the pesticide chlordecone (Fig. 1)
should exert its estrogenic activity through ER via a non-
estrogen-like binding mechanism.
Computational methods can be effective in recognizing
the putative endocrine activity of compounds of the two-
first classes of EDCs. As an example, ERRccan be bound
by a high variety of phenols [42,69], and modeling and
docking tools can provide valuable help in selecting ERRc
binding compounds from this large family of environ-
mental contaminants [145,146]. In contrast, prediction of
the endocrine disruptive activity of the last category of
compounds will likely be much less straightforward.
Another difficult case is PXR which is activated by a
variety of structurally distinct endogenous and exogenous
compounds. PXR ligands can be categorized into potency
groups, weak, moderate, and strong [83,84,87,91,99],
and modeling and docking tools could be used to predict
the affinity of new chemicals for PXR. However, due to the
structural flexibility of its LBP, predicting the responses of
PXR to environmental chemicals remains a difficult task.
One of the major challenges of computational methods
could be the prediction of all the potential target receptors
for a given chemical compound. Nevertheless, the example
provided by the structures of E2 bound to PXR and ER
revealing that the two receptors bind the same hormone in
remarkably distinct manners suggests that one cannot
easily take advantage of the information provided by the
structure of a given receptor/ligand complex to predict the
binding mode of the same ligand to another receptor.
Finally, compounds that target so-called orphan receptors
might also be difficult to identify owing to the significant
and hardly predictable conformational changes generally
associated with ligand binding.
To date, only a few EDC-bound NHRs have been
crystallized as compared with the 140,000 synthetic
chemicals used in consumer products. Thus, it appears that
efforts in elucidating the mechanisms of NHR/EDC inter-
actions by crystallography and other structural methods
must be pursued in order to deal with difficult cases and to
increase our knowledge of the structural mechanisms and
molecular interactions used by different receptors and a
wide range of structurally and chemically diverse com-
pounds. As exemplified by organotin compounds, such
studies can also reveal unforeseen binding modes and
provide guidelines for the rational design of novel NHR
modulators. Together with the improvement of computa-
tional methods, this mounting structural data should
increase the effectiveness of in silico screening strategies.
As the European Union’s 2006 Registration, Evaluation,
Authorization, and Restriction of Chemicals (REACH)
regulation aims to assess the toxicity of more than 100,000
synthetic chemicals, there is a strong demand for such
computational tools which would allow reduction of the
cost of the evaluation as well as animal lives [147].
Acknowledgments This work was supported by funds from the
INSERM, CNRS, Universite
´Montpellier 1 & 2, the French National
Research Agency (ANR-07-PCVI-0001-01 to W.B.), the Agence
Franc¸aise de Se
´curite
´Sanitaire de l’Environnement et du Travail
(AFSSET, RD-2005-007 to P.B.) and the European Union Commis-
sion (CASCADE FOOD-CT-2004-506319 to P.B.). We thank
Catherine Teyssier, Pierre Germain, and Catherine A Royer for crit-
ical reading of the manuscript.
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Structural basis of endocrine disruption