Comparison of crystal structures of human androgen
receptor ligand-binding domain complexed with
various agonists reveals molecular determinants
responsible for binding affinity
KARINE PEREIRA DE JE´SUS-TRAN,1PIERRE-LUC COˆTE´,1LINE CANTIN,
JONATHAN BLANCHET,2FERNAND LABRIE, AND ROCK BRETON
Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUL) and Laval
University, Que ´bec, QC G1V 4G2, Canada
(RECEIVED October 12, 2005; FINAL REVISION February 1, 2006; ACCEPTED February 16, 2006)
Androgens exert their effects by binding to the highly specific androgen receptor (AR). In addition to
natural potent androgens, AR binds a variety of synthetic agonist or antagonist molecules with different
affinities. To identify molecular determinants responsible for this selectivity, we have determined the
crystal structure of the human androgen receptor ligand-binding domain (hARLBD) in complex with two
natural androgens, testosterone (Testo) and dihydrotestosterone (DHT), and with an androgenic steroid used
in sport doping, tetrahydrogestrinone (THG), at 1.64, 1.90, and 1.75 A˚resolution, respectively. Comparison
of these structures first highlights the flexibility of several residues buried in the ligand-binding pocket that
can accommodate a variety of ligand structures. As expected, the ligand structure itself (dimension,
presence, and position of unsaturated bonds that influence the geometry of the steroidal nucleus or the
electronic properties of the neighboring atoms, etc.) determines the number of interactions it can make with
the hARLBD. Indeed, THG—which possesses the highest affinity—establishes more van der Waals
contacts with the receptor than the other steroids, whereas the geometry of the atoms forming electrostatic
interactions at both extremities of the steroid nucleus seems mainly responsible for the higher affinity
measured experimentally for DHT over Testo. Moreover, estimation of the ligand–receptor interaction
energy through modeling confirms that even minor modifications in ligand structure have a great impact on
the strength of these interactions. Our crystallographic data combined with those obtained by modeling will
be helpful in the design of novel molecules with stronger affinity for the AR.
Keywords: human androgen receptor; crystal structure; ligand binding pocket; agonists; tetrahydrogestrinone
The androgen receptor (AR) is a member of the nuclear
receptor (NR) superfamily (Mangelsdorf et al. 1995). Like
the other NRs, it is constituted by three main functional
domains: a variable N-terminal domain (NTD), a highly
conserved DNA-binding domain (DBD), and a conserved
ligand-binding domain (LBD) (Jenster et al. 1991). After
binding of an androgen to its LBD, AR rapidly translocates
to the nucleus, where it directly interacts with DNA as a
homodimer, at androgen response elements (ARE) found
in the regulatory regions of target genes. This complex
can thenceforth recruit coactivators (Jenster 1998) through
1These authors contributed equally to this work.
2Present address: Centre de Recherche de l’Ho ˆpital Laval (CRHL),
Ste-Foy, QC G1V 4G5, Canada.
Reprint requests to: Rock Breton, Centre de Recherche en Endo-
crinologie Mole ´culaire et Oncologique, Centre Hospitalier de l’Uni-
versite ´ Laval (CHUL), 2705, boul. Laurier, Ste-Foy, QC G1V 4G2,
Canada; e-mail: email@example.com; fax: (418) 654-2761.
Abbreviations: hAR, human androgen receptor; NR, nuclear receptor;
NTD, N-terminal domain; DBD, DNA-binding domain; LBD, ligand-
binding domain; ARE, androgen response element; LBP, ligand-binding
pocket; DHT, 5a-androstan-3-one, 17b-ol; Testo, testosterone; THG,
Article and publication are at http://www.proteinscience.org/cgi/doi/
Protein Science (2006), 15:987–999. Published by Cold Spring Harbor Laboratory Press. Copyright ? 2006 The Protein Society
the ligand-dependent transactivation function (AF-2) lo-
cated in the LBD and hence control transcription of specific
genes. Through this mechanism, androgens such as testos-
terone (Testo) and 5a-dihydrotestosterone (DHT) regulate
a wide range of physiological responses, most notably male
sexual differentiation and maturation including the de-
velopment, growth, and maintenance of the normal pros-
tate (Mooradian et al. 1987; Keller et al. 1996; Roy et al.
1999). Defects in AR function are involved in health dis-
orders including prostate cancer’s resistance to androgen
ablation therapy (Quigley et al. 1995; Heinlein and Chang
Because of their anabolic characteristics, androgens
have been used by athletes for a long time (Evans 2004).
It is thus not surprising that chemically modified andro-
gens, often synthesized for pharmacological purposes,
have rapidly given rise to interest in elite sports. Indeed,
athletes have been using modified steroids with a higher
anabolic:androgenic ratio to enhance their performances.
Recently, a novel chemically modified steroid, tetrahy-
drogestrinone (THG), has appeared as a doping agent. A
potent androgen and progestin (Death et al. 2004), THG
is produced by the hydrogenation of gestrinone, a pro-
gestin used to treat endometriosis (Dawood et al. 1997),
and has been identified as the first true ‘‘designer androgen,’’
being custom produced to evade detection (Catlin et al.
2004). Indeed, it was undetectable in urine by standard
antidoping tests until Catlin et al. (2004) developed a specific
test. Using a pangenomic assay, THG has been shown to
modulate hundreds of genes in a time-dependent fashion
almost superimposable to DHT (Labrie et al. 2005).
All androgens, natural or chemically designed, exert
their action via the AR by binding its unique LBD. How-
ever, these various ligands bind AR with very different
affinities, their Ki values ranging from low nanomolar
concentrations for the most potent androgens to micro-
molar concentrations for the weaker ones. Interestingly, it
is almost impossible to predict the strength of the inter-
action between a ligand and a receptor only on the basis
of its structure, since steroids with very similar structures
can possess markedly different affinities for a given
receptor while structurally different ligands could have
similar high affinities. The ligand-binding pocket (LBP)
of the NRs is composed of a large number of residues
making up the binding interface and involved in ligand–
receptor complex formation. It is not clear, however, if all
these residues or only a small subset contribute to the
binding energy. A complete characterization of the factors
contributing to the ligand–AR interaction would thus
greatly help us to understand the basis of the ligand specificity
To date, several crystal structures of the human AR
ligand-binding domain (hARLBD) have been solved in
complex with the natural androgen DHT (Sack et al.
2001) and with chemically modified steroids such as the
agonist metribolone (R1881) (Matias et al. 2000) or the
corticosteroid agonist 9a-fluorocortisol (Matias et al.
2002). In addition, structures of the liganded hARLDB
have been determined in complex with a peptide derived
from physiological coactivators (He et al. 2004; Hur et al.
2004; Estebanez-Perpina et al. 2005). In all these com-
plexes, the LBD adopts the same fold, mainly composed
of a-helices arranged as a three-layered antiparallel
a-helical sandwich, a fold common to all the NRs (Wurtz
et al. 1996). These structures show that the LBP is mainly
composed of hydrophobic residues, the side chains of which
can easily adopt variable positions in order to better fit the
hydrophobic core of the steroid and stabilize it.
LBP is also composed of polar amino acids able to
establish hydrogen bonds at both extremities of the steroid
nucleus of all potent androgens. If several studies have well
demonstrated the importance of these polar residues in
ligand binding and shown the effect of their substitution,
which is associated with disorders that impair androgen-
dependent male sexual differentiation or affect the devel-
opment and/or progression of cancers such as prostate
cancer in the human (De Bellis et al. 1992; Pinsky et al.
1992; Gaddipati et al. 1994; Taplin et al. 1995; Poujol
et al. 2000; Sakai et al. 2000; Chavez et al. 2001; Labrie
et al. 2002, 2005), little is known about the role and the
importance of the hydrophobic residues that form the major
part of the LBP. Because almost all potent androgen
steroids known to bind hAR with high affinity possess the
same polar groups at their nucleus extremities, it is more
than likely that the hydrophobic residues are of paramount
importance, not only in the stabilization of the steroid in its
pocket but also in the high selectivity and specificity
observed for all members of the NR superfamily.
To better understand the role of the hydrophobic resi-
dues found in the LBP, we have determined and compared
the crystal structures of the hARLBD in complex with
three agonist ligands of similar structure but possessing
different levels of affinity for the receptor. Here we report
the crystallographic structure of the human ARLBD in
complex with natural androgens Testo and DHT and with
the designer steroid THG (Fig. 1). Close inspection and
comparison of these high-resolution complex structures
have allowed identifying in both molecules, ligand and
receptor, molecular determinants that appear to be impor-
tant for high-affinity binding of androgens. To confirm
the relative importance of each of these determinants
identified by crystallography, ligand–receptor interaction
energy has been estimated by modeling and energy mini-
mization. Our results, which led us to propose possible
roles for these determinants, will be used in drug design
strategies, especially for the conception of new AR
antagonists with higher affinity for the human androgen
Pereira de Je ´sus-Tran et al.
Protein Science, vol. 15
in CNS (Bru ¨nger et al. 1998). Five percent of the data was
randomly selected and excluded from the refinement procedure
(Bru ¨nger 1992). After each refinement cycle, the model was
manually corrected using O (Jones et al. 1991) with the 2Fo? Fc
and Fo? Fccalculated maps. Ligand and water molecules were
progressively added to the model. Since Testo– and THG–
hARLBD complexes crystallized in the same P212121 space
group and had similar unit cell parameters as the DHT–
hARLBD complex, the initial model for Testo– and THG–
hARLBD was generated with a rigid body protocol from
REFMAC (Murshudov et al. 1997) using the DHT-complex
structure. The refinement procedure for these ligands was the
same used for DHT. Before the addition of the ligand in the
model, the density of Testo, DHT, and THG was clearly visible
in all maps. Sulfate ions were added to the three complex
models; DTT and glycerol molecules were observed in the
Testo– and THG–hARLBD structures and a HEPES molecule
was only added to the THG complex model. The quality of all
models was monitored with PROCHECK (Laskowski et al.
1993), and the final refinement results and statistics are shown
in Table 1. The volume of the cavity occupied by the ligands
was calculated with SPDBV (Kaplan and Littlejohn 2001), and
all figures were generated with Molscript (Kraulis 1991) and
SPDBV. Protein Data Bank accession codes are 2AM9 for
Testo–hARLBD, 2AMA for DHT–hARLBD, and 2AMB for
In order to find structural elements explaining the variation in
androgen receptor affinity of the three ligands, we calculated the
interaction energies of the complexes using ZMM (http://
www.zmmsoft.com; Zhorov 1983; Zhorov and Bregestovski
2000). The receptor was represented by a double-shell model,
as previously described in Zhorov and Lin (2000), based on our
three crystallographic structures. The inner flexible shell was
composed of hARLDB amino acids having at least one atom
within 8 A˚of the ligand, thus selecting 56 residues. Residue
internal coordinates of the flexible shell were allowed to move
during minimization steps. The other amino acids of the model
were included in the outer rigid shell, in which they were not
allowed to vary during energy minimization. Water molecules
were removed from the models, but the hydration effects were
simulated by an implicit method (Lazaridis and Karplus 1999).
Partial charges of the ligands were determined with the AM1
method of the MOPAC software (Dewar et al. 1985). The models
were then energetically minimized using the Monte Carlo mini-
mization (MCM) protocol (Li and Scheraga 1987) with the
AMBER force field (Weiner et al. 1984).
The goal of this modeling was not to find the global energy
minimum of the system but to estimate the ligand–receptor
interaction energy with minimal changes of the crystal struc-
tures. For this purpose, we used a multistep relaxation method,
which only affects the flexible shell, with very short MCM
trajectories (which were terminated after 10 consecutive energy
minimizations). In the first relaxation step, only hydrogen atoms
were allowed to move. In the second and third steps, side chains
and backbone torsion angles were respectively allowed to vary.
Finally, in the fourth step, the constraints were removed for all
variables (torsion angles, bond angles, and free particle move-
ment). In all steps however, non-hydrogen atoms were not
allowed to move by >1 A˚from their crystallographic coordinates,
a constraint achieved through a flat-bottom energy function.
This work was supported by Endorecherche Inc. P.-L.C. is the
recipient of a Master’s scholarship provided by the Natural
Sciences and Engineering Research Council of Canada. We
thank Dr. Sylvain Gauthier and his research group for THG
synthesis; ZMMSoft for providing us with the ZMM molecular
modeling program; and Ms. Sylvie Me ´thot for careful reading of
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Structure of hARLBD in complex with various agonists