Recognition and Accommodation
at the Androgen Receptor
Coactivator Binding Interface
Eugene Hur1, Samuel J. Pfaff1, E. Sturgis Payne2, Hanne Grøn2, Benjamin M. Buehrer2, Robert J. Fletterick3*
1 Graduate Group in Biophysics, University of California, San Francisco, California, United States of America, 2 Karo Bio, Durham, North Carolina, United States of America,
3 Department of Biochemistry and Biophysics, University of California, San Francisco, California, United States of America
Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of
the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it
preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to
coactivators bearing the leucine-rich ‘‘NR box’’ motifs favored by other nuclear receptors. Under normal conditions,
interactions with these AR-specific coactivators through aromatic-rich motifs underlie targeted gene transcription.
However, during prostate cancer, abnormal association with such coactivators, as well as with coactivators containing
canonical leucine-rich motifs, promotes disease progression. To understand the paradox of this unusual selectivity, we
have derived a complete set of peptide motifs that interact with AR using phage display. Binding affinities were
measured for a selected set of these peptides and their interactions with AR determined by X-ray crystallography.
Structures of AR in complex with FxxLF, LxxLL, FxxLW, WxxLF, WxxVW, FxxFF, and FxxYF motifs reveal a changing
surface of the AR coactivator binding interface that permits accommodation of both AR-specific aromatic-rich motifs
and canonical leucine-rich motifs. Induced fit provides perfect mating of the motifs representing the known family of
AR coactivators and suggests a framework for the design of AR coactivator antagonists.
The androgen receptor (AR) is the cellular mediator of the
actions of the hormone 5-a dihydrotestosterone (DHT).
Androgen binding to AR leads to activation of genes involved
in the development and maintenance of the male reproduc-
tive system and other tissues such as bone and muscle.
However, it is the pivotal role of AR in the development and
progression of prostate cancer that has led to increasing
interest in this nuclear receptor. Presently, hormone-depen-
dent prostate cancer is treated with a combination of
strategies that reduce circulating levels of androgens, such
as the administration of antiandrogens that compete for the
androgen-binding pocket in the core of the C-terminal
ligand-binding domain (LBD). The benefits of these treat-
ments are typically transient, with later tumor growth
associated with increases in expression levels of AR or its
cofactors, or mutations that render AR resistant to antian-
drogens (Gregory et al. 2001; Culig et al. 2002; Lee and Chang
2003). Alternative approaches to inhibiting AR transcrip-
tional activity may therefore lie in disrupting critical protein
associations the receptor needs for full function.
The precise details of how AR binds the dozens of
coregulator proteins reported to associate with different
regions of AR in vivo remain poorly understood (Lee and
Chang 2003). Many nuclear receptors activate transcription
by binding short leucine-rich sequences conforming to the
sequence LxxLL (where ‘‘x’’ is any amino acid), termed
nuclear receptor (NR) boxes, which are found within a variety
of NR coactivators including the p160 family. Hormone
binding to the LBD stabilizes the C-terminal helix of the
receptor, helix 12, in a conformation that completes a
binding surface for these LxxLL motifs (Darimont et al.
1998; Nolte et al. 1998; Shiau et al. 1998; Bledsoe et al. 2002).
The structural elements composing this binding interface,
consisting of helices 3, 4, 5, and 12 of the receptor, are
synonymous with a previously defined hormone-dependent
activation function that lies within the LBD termed activation
function (AF)–2. Association of p160 coactivators allows the
recruitment and assembly of a number of other cofactors that
together modulate the state of chromatin and interactions
with components of the basal transcription machinery to
initiate transcription (Glass and Rosenfeld 2000).
AR, however, utilizes multiple mechanisms to activate gene
transcription. Generally, AR activity is dependent on con-
tributions from multiple transactivation functions that lie
Received March 26, 2004; Accepted June 16, 2004; Published August 24, 2004
Copyright: ? 2004 Hur et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
Abbreviations: AF, activation function; AR, androgen receptor; ARA, androgen
receptor-associated protein; DHT, 5-a dihydrotestosterone; ER, estrogen receptor;
FxxFF, phenylalanine-x-x-phenylalanine-phenylalanine; FxxLF, phenylalanine-x-x-
leucine-phenylalanine; FxxLW, phenylalanine-x-x-leucine-tryptophan; FxxYF, phe-
nylalanine-x-x-tyrosine-phenylalanine; GR, glucocorticoid receptor; GRIP1, gluco-
corticoid receptor-interacting protein 1; GST, glutathione S-transferase; Kd,
equilibrium dissociation constant; LBD, ligand-binding domain; LxxLL, leucine-x-
x-leucine-leucine; N/C interaction, interaction between the N-terminal domain and
the ligand-binding domain; NR, nuclear receptor; NTD, N-terminal domain; SMRT2,
silencing mediator for RXR and TR 2; TIF2, transcriptional intermediary factor 2; TR,
thyroid hormone receptor; WxxLF, tryptophan-x-x-leucine-phenylalanine; WxxVW,
Academic Editor: Ueli Schibler, University of Geneva
*To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
PLoS Biology | www.plosbiology.orgSeptember 2004 | Volume 2 | Issue 9 | e2741303
Open access, freely available online P PL Lo oS S BIOLOGY
within the N-terminal domain (NTD) collectively called AF-1.
Although the AR AF-2 can bind to a restricted set of LxxLL
motifs (Ding et al. 1998; He et al. 1999; Needham et al. 2000)
and is relatively potent (Wang et al. 2001), it usually displays
weak independent activity at typical androgen-regulated
genes, with significant activity observed only in the presence
of high levels of p160 coactivators, as detected in some
prostate cancers (He et al. 1999; Gregory et al. 2001). Instead,
the AR AF-2 exhibits a distinct preference among NRs for
phenylalanine-rich motifs conforming to the sequence FxxLF
(He et al. 2000; He and Wilson 2003). Such motifs have been
identified in the AR NTD and in an AR cognate family of
coactivators that includes AR-associated protein (ARA) 54,
ARA55, and ARA70 (He et al. 2000, 2002b; Lee and Chang
2003). The NTD FxxLF motif (residues 23–27) mediates a
direct, interdomain, ligand-dependent interaction between
the NTD and LBD (N/C interaction) that is thought to
facilitate dimerization, stabilize androgen binding, and
possibly regulate AF-1 and AF-2 activity (Langley et al.
1998; He et al. 2000). In addition, the NTD also contains a
related hydrophobic motif, WxxLF (residues 433–437), that
nucleates formation of an alternative N/C interaction that
may serve to inhibit AR activity (He et al. 2000, 2002a; Hsu et
Presently, how the AR AF-2 surface can accommodate
residues with bulky aromatic side chains and distinguish
FxxLF motifs from LxxLL motifs is not known. To under-
stand the structural basis of this unusual coactivator
recognition preference, we characterized the full repertoire
of interacting sequences using phage display to define amino
acids preferred at the AR coactivator binding interface.
Crystal structures of the AR LBD in complex with several
phage display–derived peptides reveal the structural basis of
FxxLF motif specificity and an induced fit of the receptor
that allows accommodation of other related hydrophobic
motifs. Comparisons of the structures suggest strategies for
the design of AR coactivator antagonists.
AR Preference for Aromatic Groups in Coregulator
Phage display has been used to study coactivator recog-
nition specificity and to identify coactivator motif sequence
variants preferred by the estrogen receptor (ER), thyroid
hormone receptor (TR) b, and most recently AR (Chang et al.
1999; Norris et al. 1999; Paige et al. 1999; Northrop et al. 2000;
Hsu et al. 2003). Using phage display, we screened more than
2 3 1010randomized peptides against DHT-bound AR LBD.
Selections identified sequences containing hydrophobic
motifs that were primarily aromatic in character, consistent
with another recent study (Hsu et al. 2003) (Figure 1). Of these
aromatic motifs, FxxLF and related motifs with substitutions
of phenylalanine or tryptophan for leucine at positions þ1,
þ5, or both, dominated the selections. (Peptide residues are
numbered in reference to the first hydrophobic residue of the
core motif, which is numbered þ1. Residues preceding the
first hydrophobic residue are numbered negatively in
descending order starting with ?1.) Substitutions of tyrosine
at the þ5 position were also observed, but to a much lesser
extent (unpublished data). At the þ4 position, valines,
methionines, and even the aromatic residues phenylalanine
and tyrosine were observed (Figure 1; unpublished data). In
general, LxxLL motifs were not selected. The LxxLL motif
shown in Figure 1 was derived from prior phage selections
with ER and subsequently demonstrated to bind AR in FRET-
based screens in vitro (unpublished data).
Preliminary characterization of the subset of AR-interact-
ing peptides shown in Figure 1 confirmed that each competed
for binding of in vitro translated AR cofactors to bacterially
expressed AR LBD in pulldown assays, and generally did so
with modestly improved efficiency relative to the native
FxxLF motif from the AR NTD and significantly greater
efficiency than a native LxxLL motif from glucocorticoid
receptor-interacting protein 1 (GRIP1) NR box 3 (P. Webb,
personal communication). The equilibrium dissociation con-
stants (Kd) were directly determined for the interaction
between the AR LBD and FxxLF and LxxLL peptides and one
variant tryptophan-containing peptide, FxxLW, using surface
plasmon resonance (Table 1). The Kdfor FxxLF was 1.1 lM,
similar to the affinities of physiologically derived FxxLF
motifs determined previously by isothermal titration calo-
rimetry (He and Wilson 2003). The affinity of LxxLL was less
than 2-fold weaker, with a Kdof 1.8 lM, but more than three
times stronger than the tightest binding p160-derived LxxLL
motif, NR box 3 of transcriptional intermediary factor 2
(TIF2) (He and Wilson 2003). Surprisingly, the affinity of
FxxLW, with a Kdof 920 nM, was slightly better than FxxLF, in
spite of the presence of the tryptophan residue at the þ5
position. Together, our results are consistent with the notion
that the phage display peptides interact with the same AR
surface that binds FxxLF and LxxLL motifs in native
cofactors, and that they do so with similar or improved
affinities relative to their natural counterparts.
One Site Fits All
To understand the binding mode of different AR coac-
tivators, we determined the crystal structures of DHT-bound
AR LBD without peptide and in complex with each of the
seven peptides listed in Figure 1. All complexes crystallized in
the space group P212121with one molecule per asymmetric
Figure 1. AR LBD–Interacting Peptides Selected by Phage Display
Hydrophobic residues of the core motif are highlighted in yellow.
Residues in bold were ordered in electron density maps.
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Recognition of AR Coactivator Motifs
unit and unit cell dimensions similar to those observed in
previous AR LBD crystal structures (Matias et al. 2000; Sack et
al. 2001). Overall structural features of the complexes are
shown in Figure 2. Peptides assumed short a helical
conformations centered on the core hydrophobic motif and
bound in a solvent channel relatively free of crystal contacts
on a groove formed by helices 3, 4, 5, and 12 of the receptor
(Figure 2A). Detailed data collection and refinement statistics,
as well as buried surface areas for each complex, are listed in
Table 2. The structures confirm previous suggestions that AR
utilizes a single binding interface for LxxLL and non-
canonical aromatic-rich motifs (He et al. 2000, 2002a). Only
side chains move to accommodate the array of peptides,
sometimes considerably, with the unbranched side chains of
Lys720, Met734, and Met894 making the largest conforma-
tional changes upon binding of peptide (Figure 2B).
The mechanisms that permit AR to accommodate motifs
with bulky phenylalanine residues were assessed in a crystal
structure of the AR LBD in complex with the FxxLF peptide.
The FxxLF peptide recapitulates the binding mode of p160-
derived LxxLL motifs to other nuclear receptors (Darimont et
al. 1998; Nolte et al. 1998; Shiau et al. 1998; Bledsoe et al. 2002).
The peptide forms a short a helix whose hydrophobic face,
composed of Pheþ1, Leuþ4, and Pheþ5, binds an L-shaped
groove formed by helices 3, 4, 5, and 12 of the LBD that is
composed of three subsites that accommodate each hydro-
phobic residue (Figures 2A and 3A). The conserved charged
residues at either end of the cleft, Lys720 and Glu897, the so-
called charge clamp residues, make electrostatic interactions
with the main chain atoms at the ends of the peptide helix:
Lys720 with the carbonyl group of Pheþ5, and Glu897 with the
amide nitrogens of Pheþ1 and Arg?1 (Figure 3C). Glu897 also
interacts with the side chain of Arg?1. The two interior
Table 1. Rate and Dissociation Constants for the Interaction
between the AR LBD and Selected Peptides
Surface plasmon resonance data were best fit using the two-state conformational
change model (Warnmark et al. 2001, 2002). Dissociation constants were calculated
from rate constants as described previously (Warnmark et al. 2001).
Figure 2. A Structural Profile of the AR
Coactivator Binding Interface
AR–peptide complexes are colored as
follows: FxxLF, yellow; FxxLW, orange;
WxxLF, wheat; WxxVW, purple; FxxYF,
green; FxxFF, blue; LxxLL, pink; un-
(A) Ca trace of the peptides super-
imposed onto the AF-2. For clarity only
the LBD of AR–FxxLF is shown.
(B) Superposition of the LBD of the AR–
peptide complexes in the region of the
coactivator interface. Backbone atoms
are shown as a Ca trace. Side chains of
residues composing the interface are
shown as sticks.
(C) Hydrophobic side chains of the core
motif superimposed as in (B).
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Recognition of AR Coactivator Motifs
residues of the motif, Gluþ2 and Serþ3, are solvent exposed
and do not interact with the receptor.
Comparison of AR alone and AR in complex with FxxLF
(and other aromatic-rich peptides described below) reveals
that the AF-2 cleft reorganizes to accommodate the bulky
peptide side chains (see Figures 2B and 4). The unbranched
side chains of Lys720 and Met734 move from an extended
conformation over theþ5 pocket to one almost perpendicular
to the surface of the protein. The pockets for Pheþ1 and
Pheþ5 are arranged in a line, forming a deep, extended cleft
on the LBD spanning the length of the two side chains on the
face of the peptide helix (see Figures 3A and 4B). Pheþ1,
almost entirely solvent inaccessible, binds face down at the
base of this groove, making hydrophobic contacts with
Leu712, Val716, Met734, Gln738, Met894, and Ile898, which
define the þ1 pocket. The top of the groove, composed of
Val716, Lys720, Phe725, Ile737, Val730, Gln733, and Met734,
narrows to form the þ5 pocket. Met734 and the aliphatic
portion of Lys720 constrict thissubsite, forming van der Waals
interactions with opposite faces of the Pheþ5 benzyl ring.
Together, the þ1 and þ5 residues are almost entirely solvent
inaccessible. In contrast, Leuþ4 binds in a shallow hydro-
phobic patch consisting of Leu712 and Val716 lined at the
ridges by Val713 and Met894 and is largely solvent exposed.
The preference of AR for motifs with aromatic groups over
leucine-rich motifs was assessed with a crystal structure of the
AR LBD in complex with the LxxLL peptide. The structure
reveals similarities between the binding modes of the LxxLL
and FxxLF motifs to AR, and other LxxLL motifs to other
nuclear receptors. The LxxLL motif adopts a helical
conformation, and interactions of the motif with the AF-2
cleft are predominantly hydrophobic, with the three leucine
residues of the motif contributing most of the interactions.
However, significant differences can be seen between the
binding mode of the LxxLL motif to AR and that of p160-
derived LxxLL motifs to other nuclear receptors. First,
flanking residues were largely disordered, with only two N-
terminal flanking residues and one C-terminal residue visible
in electron density maps (see Figures 1 and 3B). This contrasts
with extended structures seen in the p160-derived LxxLL
motifs in complex with their cognate receptors (Darimont et
al. 1998; Nolte et al. 1998; Shiau et al. 1998; Bledsoe et al.
2002). Second, the LxxLL peptide backbone forms hydrogen
bonds with only one of the two conserved charge clamp
residues, Lys720. A shift in the position of the LxxLL peptide
helix precludes direct interactions with Glu897 (see Figures
2A and 3D). This shift results from changes in the geometry of
theþ1 andþ5 subsites mediated by Met734, which moves 2.5A˚
toward the þ1 pocket (see Figures 2B and 4C) and enables
binding of a leucine at the þ5 subsite by a simultaneous
widening and shallowing of the pocket. This movement of
Met734 causes displacement of the þ1 residue, resulting in a
rotation of the peptide helix away from helix 12, toward helix
3. A slight translation of the peptide helix also occurs away
Table 2. Summary of Structures and Crystallographic Statistics
Unit cell dimensions (A˚)
98.2 (88.4) 99.3 (98.6) 99.9 (99.6) 98.8 (89.6) 99.8 (85.3) 99.9 (99.9) 97.2 (93.6) 99.3 (99.2)
7.2 (57.1)9.7 (54.2)5.2 (65.0)8.4 (59.6)
21.3 (2.0)20.4 (3.1)54.1 (3.4)19.1 (2.2)
Total1012 9261197 937
r.m.s.d. bond lengths (A˚)
r.m.s.d. bond angles (8)
Average B-factor (A˚2):
Buried surface area (A˚2):
PDB accession code
aNumbers in parenthesis denote values for the highest resolution shell.
bRsym= RjI ? ,I.j / R (I).
cRcryst= R jFo? Fcj / R jFoj, where Foand Fcare observed and calculated structure factors, respectively; Rfreewas calculated similarly with a randomly selected set of
reflections consisting of 5% of total reflections that were excluded from refinement.
dValues for side chain atoms only.
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Recognition of AR Coactivator Motifs
from helix 12 because of the shorter side chain length of
leucine (see Figure 2A).
Side chains of residues flanking the first leucine of the
motif make additional hydrophobic interactions with the AR
surface (see Figure 3B). Trpþ2 reaches over Met734, clamping
the methionine in between itself and Leuþ1. Leu?1 extends
over Met894, abutted against Glu893. These interactions
likely explain the moderate affinity of AR for this particular
LxxLL motif despite suboptimal complimentarity with the
residues of the core motif (as discussed below) and the loss of
main chain interactions with Glu897.
WxxLF, FxxLW, and WxxVW
To understand how the AR AF-2 accommodates tryptophan
residues, structures of AR in complex with peptides contain-
ing tryptophan substitutions at theþ1 orþ5 position, or both,
were determined (Figure 5). Surprisingly, WxxLF, analogous
to the only tryptophan-containing motif known in vivo,
WHTLF in the AR NTD, was relatively disordered, with the
peptide displaying the highest B-factor and least well defined
density, suggesting that it binds with the lowest affinity (Table
2). Nonetheless, each of the tryptophan peptides adopted
similar helical conformations. As described above for the
LxxLL motif, substitutions at theþ1 andþ5 positions for non-
phenylalanine residues result in shifts of the peptide helix (see
Figure 2A). Consequently, backbone interactions with Lys720
are maintained, but interactions with the other charge clamp
residue, Glu897, are lost. Once again, however, flanking
residues within the peptide make additional contacts with
the AR surface, and, unlike the LxxLL peptide, these contacts
include Glu897. In FxxLW and WxxVW, the?2 serine (Figure
6) forms a bidentate hydrogen-bonding interaction, making
hydrogen bonds to both Glu897 and the backbone amide
group of the þ2 residue. Ser?2 of WxxLF similarly interacts
with Glu897, but is too distant for helical-capping interactions
with the þ2 amide group. Instead, Glu893, in a more typical
interaction with theþ1 amide nitrogen, caps the WxxLF helix
(Figure 6B). Thus, tryptophan substitutions are tolerated, but
they induce a shift in the peptide backbone that precludes
interactions with one of the charge clamp residues. This
suboptimal interaction is compensated partially by interac-
tions of flanking residues with the AR surface.
FxxFF and FxxYF
Finally, effects of substitutions at the þ4 position were
assessed in structures of AR in complex with peptides
Figure 3. Interactions of FxxLF and LxxLL with the AR LBD
(A and B) FxxLF (A) and LxxLL (B) bound to the AR AF-2 interface.
FxxLF and LxxLL are shown as yellow and pink Ca coils, respectively.
Helices 3, 4, and 5 of the LBD are shown as blue ribbons; Helix 12 is
shown in green. LBD residues interacting with peptides are depicted
as white sticks. For clarity only peptide side chains making significant
interactions with the LBD are shown.
(C and D) Hydrogen-bonding interactions between backbone atoms
of FxxLF (C) and LxxLL (D) with Glu897 of the LBD. Peptide alpha
carbons are labeled.
Figure 4. Induced Fit of the AR AF-2 Interface
Surface representations of the AR AF-2 interface. The unbound structure is shown in (A), the FxxLF bound in (B), and the LxxLL bound in (C).
Side chains of the hydrophobic residues of the core motifs of FxxLF and LxxLL are shown as spheres.
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Recognition of AR Coactivator Motifs
containing FxxFF and FxxYF motifs (Figure 7). Surprisingly,
the binding mode of FxxFF to AR resembled that of the
tryptophan peptides more closely than the binding mode of
FxxLF (see Figures 2A and 7B). Like the tryptophan peptides,
interactions with Glu897 are mediated by Ser?2 instead of the
peptide backbone (see Figure 6D). Deviations from ideal
helical geometry allow Pheþ4 to bind facedown in the þ4
pocket with the benzyl ring stacked against Val713.
By contrast, the conformation of FxxYF was the closest to
FxxLF (see Figure 2A). Other than FxxLF, only FxxYF makes
direct backbone interactions with Glu897. Unlike the face-
down orientation of Pheþ4 observed in the FxxFF peptide,
Tyrþ4 is bound edgewise into the shallow þ4 pocket, making
interactions with Val713, Val716, and the aliphatic portion of
Lys717. FxxYF was the most ordered of all the peptides, with
12 out of 15 residues observed in the electron density (see
Figures 1 and 7A). Significant interactions were observed
involving residues other than hydrophobic residues of the
motif. Lysþ2 and Metþ6 are predominantly solvent exposed,
extending out over the protein surface. Metþ6 is bound on
top of Pheþ5, while Lysþ2 makes a water-mediated hydrogen
bond with Asp731. Thr?3 of the peptide defines a new
subsite, with the hydroxyl group forming a hydrogen bond to
Gln738 and the methyl group making hydrophobic contacts
in a pocket formed by Glu897, Ile898, and Val901. Similar
interactions were observed in the glucocorticoid receptor
(GR)–TIF2 complex involving the ?3 glutamine of the TIF2
NR box 3 motif (Bledsoe et al. 2002). However a valine to
asparagine substitution at the residue corresponding to 901
in AR creates a pocket with a more polar character in GR
Restrictions of the Three Subsites
Together, the structures described above permit an assess-
ment of the way that individual subsites of the AR AF-2 cleft
Figure 6. Interactions of Ser?2 with Glu897
Interactions between Ser?2 of the peptides (A) FxxLW, (B) WxxLF,
(C) WxxVW, and (D) FxxFF and Glu897 of the LBD. Peptide alpha
carbons are labeled.
Figure 7. Interactions of FxxYF and FxxFF with the AR LBD
FxxYF (A) and FxxFF (B) bound to the AR AF-2 interface. FxxYF and
FxxFF are shown as yellow and orange Ca coils, respectively. The LBD
is depicted as in Figure 3.
Figure 5. Interactions of the Tryptophan Motifs with the AR LBD
FxxLW (A), WxxLF (B), and WxxVW (C) bound to the AR AF-2
interface. FxxLW, WxxLF, and WxxVW are shown as orange, beige,
and purple Ca coils, respectively. The LBD is depicted as in Figure 3.
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Recognition of AR Coactivator Motifs
accommodate hydrophobic groups. The indole rings of
tryptophan and the phenyl rings of phenylalanine fit into
their pockets analogously with the þ1 and þ5 residues bound
facedown and edgewise, respectively, into the AF-2 cleft. On
the other hand, the position of theþ4 residue is variable, with
binding in this shallow pocket largely dictated by the position
of the peptide backbone caused by the bound conformations
of the þ1 and þ5 residues (see Figure 2C). Small shifts in the
position of the N-terminal of helix 12 can be seen, which
reposition Met894 for more optimal contacts withþ4 residues
bound at that subsite (see Figure 2B).
The binding mode detected in the þ1 pocket is the most
conserved of the three hydrophobic subsites (see Figure 2C).
The benzyl moiety of the indole side chains superimpose with
the corresponding benzyl side chains of the phenylalanine-
rich motifs, effectively mimicking interactions of a phenyl-
alanine residue. However, the presence of a hydrogen-
bonding partner on the indole side chain enables an
additional polar interaction not seen in the phenylalanine-
rich motifs between the indole nitrogen and Gln738 (see
Figure 5B). Unexpectedly, this additional interaction in the
þ1 pocket does not occur with Trpþ1 of WxxVW (see Figure
5C). While similarly distanced to make the same interaction,
the plane of the indole ring is rotated about 208 relative to
that of WxxLF, causing it to be at a poor angle for strong
hydrogen bonding to Gln738.
Binding of tryptophans in the þ5 pocket is slightly more
variable (see Figure 2C). Trpþ5 of WxxVW is bound similarly
to phenylalanine residues at the same position. Only the six-
membered ring of the indole group is fully buried in the
pocket. The five-membered ring of the indole side chain
sticks out, solvent exposed. In contrast, theþ5 indole group of
FxxLW is rotated almost 908, resulting in burial of both rings
of the indole group, as well as the formation of a strong
hydrogen bond between the indole nitrogen and Gln730 (see
Figure 5A). Binding in this orientation appears to be highly
favorable, as the FxxLW peptide deviates from helical
geometry at the þ5 position to do so.
The crystal structures reported here reveal how AR binds
coactivator motifs with bulky aromatic hydrophobic groups
and permit construction of a profile of the AR coregulator
interface (see Figure 2). In some ways, this interface resembles
those of other nuclear receptors: it is an L-shaped hydro-
phobic cleft comprised of three distinct subsites that bind
hydrophobic groups at theþ1,þ4, andþ5 positions in cognate
peptides. Moreover, the so-called charge clamp residues
(Lys720 and Glu897) bracket the cleft. Nonetheless, the AR
coregulator recognition site is unique in that it rearranges
upon motif binding to form a long, deep, and narrow groove
that accommodates aromatic residues at the þ1 and þ5
positions (Figure 9). Sequence alignments of AR with other
NRs suggest that a unique combination of substitutions at
Val730, Met734, and Ile737 combine to permit the formation
of a smoother, flatter interaction surface that displays a
higher complimentarily to aromatic substituents than to
branched aliphatic (see Figure 8). Of these, methionine, the
only unbranched hydrophobic amino acid and the most
accommodating, at a key position between the þ1 and þ5
sites, allows the AR AF-2 interface to vary the size and shape
of its pockets to associate with a more diverse set of
coregulators. GR also contains a methionine residue at this
position, raising the possibility that it may also employ
induced fit to broaden motif recognition. While naturally
occurring mutations in AR have yet to be observed at Met734,
it is interesting to note that mutations at Val730 and Ile737
have been reported in patients with prostate cancer and
androgen insensitivity, respectively (Newmark et al. 1992;
Quigley et al. 1995; Gottlieb et al. 1998).
The same characteristics that make the AR AF-2 ideal for
binding of longer, aromatic side chains also make it less well
suited for binding of shorter, branched side chains. Although
changes in the position of Met734 widen the groove towards
theþ5 subsite to permit binding of leucine residues, the gross
features of the groove remain largely the same (see Figure 9B).
As a result, theþ1 andþ5 leucines bind in a smooth, elongated
groove and interactions between the þ1 and þ5 residues on
the face of the peptide helix, or with a hydrophobic ‘‘bump’’
present in other receptors caused by a isoleucine to leucine
substitution between theþ1 andþ5 subsites, are absent. Thus,
a smaller proportion of the available surface area is available
for van der Waals interactions.
Unlike the conserved interaction modes of aromatic
residues with the þ1 and þ5 sites, binding interactions at
the þ4 site are variable and characterized by nonspecific
interactions. This finding agrees with the relatively high
conservation of residues at the þ1 and þ5 positions of AR-
interacting motifs and suggests that these residues drive
peptide interaction with the LBD, whereas the þ4 site is less
critical. Indeed, theþ4 pocket is shallow, surface exposed, and
relatively featureless, explaining the assortment of residues
selected at the þ4 position. It is likely that any hydrophobic
residue that does not clash with surrounding residues would
be suitable at this subsite.
While peptide motif recognition is governed by hydro-
phobic interactions, polar interactions from backbone atoms
Figure 8. Sequence Alignment of the AF-2 Region of NRs
Residues composing the coactivator interface of AR are highlighted
in yellow. The absolutely conserved glutamate and lysine composing
the charge clamp are highlighted in pink and blue, respectively.
Residue numbering is that of AR.
PLoS Biology | www.plosbiology.org September 2004 | Volume 2 | Issue 9 | e2741309
Recognition of AR Coactivator Motifs
and residues outside the core motif also contribute. With the
exception of FxxFF, motifs containing phenylalanines at the
þ1 and þ5 positions present canonical main chain inter-
actions with both charge clamp residues, Lys720 and Glu897.
This finding stands in contrast to predictions of previous
studies (Alen et al. 1999; He et al. 1999; Slagsvold et al. 2000;
He and Wilson 2003), which concluded that Lys720 was
dispensable for FxxLF binding and that Glu897 was required
for binding to FxxLF and LxxLL motifs. Lys720 comprises a
significant portion of the þ5 subsite, making important van
der Waals interactions with the Pheþ5 benzyl group in
addition to hydrogen bonds to the motif backbone. These
results suggest that Lys720 is required for binding of FxxLF
motifs. However, it may be that enough binding energy is
provided by the other residues of theþ5 subsite (i.e., Met734),
as well as by the other subsites themselves, such that removal
of Lys720 would have little effect on binding. Observations
that Lys720 plays a greater role in LxxLL motif binding are
likely due to the fact that there is less surface area
contributing to van der Waals contacts in LxxLL motifs.
Disrupting binding contributions from Lys720 would thus
have a more detrimental effect on binding.
On the other hand, Glu897 interacts with the FxxLF
peptide backbone, but is disengaged from the LxxLL peptide
backbone. One possible explanation for the apparent
requirement for Glu897 in LxxLL binding is that it might
interact with residues outside of the core motif. The
corresponding glutamate of GR, Glu 755, forms hydrogen
bonds with the?3 asparagine of TIF2 NR box 3 (Bledsoe et al.
2002), and Glu897 of AR participates in noncanonical
interactions with the hydroxyl group of a Ser?2 residue that
was selected in all of our tryptophan-containing peptides.
This is especially intriguing given that the only WxxLF motif
known in vivo, located in the AR NTD, also possesses a Ser?2
residue. WxxLF also makes backbone interactions with an
alternate charge clamp residue, Glu893, pointing towards
adaptability in AR AF-2 charge clamp formation.
Sequence alignment ofNR coactivator sequencesshows that
positively charged residues are favored N-terminal to the core
hydrophobic motif while negatively charged residues are
favored C-terminal to the motif (He and Wilson 2003). Our
phage-selected peptides are consistent with this trend.
Arginines and lysines were observed at the N-terminal ?1
position in all peptides, except for LxxLL, in which Arg was
present at the ?3 position. Moreover, four out of seven
peptides contained negatively charged aspartate or glutamate
residues C-terminal to the core motif. While previous studies
residues flanking coactivator signature motifs of coactivators
and charged residues surrounding the AF-2 cleft modulated
binding to the receptor (He and Wilson 2003), we find that the
flanking charged residues are typically disordered in the
electron density, with only Arg?1 of FxxLF interacting with
Glu897, and Lysþ2 of FxxYF forming a water-mediated
Figure 9. Surface Complimentarity of
Hydrophobic Motifs in the AR, ERa, and
GR AF-2 Clefts
(A) AR–FxxLF, (B)AR–LxxLL, (C) ERa–
GRIP1 (LxxLL) (Shiau et al. 1998), and
(D) GR-TIF2 (LxxLL) (Bledsoe et al.
2002). The inside surfaces of the AF-2
cleft in AR, ERa, and GR are depicted.
The LBD is additionally shown as a Ca
trace with key side chains shown as white
sticks. Phenylalanines and leucines of the
FxxLF and LxxLL motifs are shown as
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Recognition of AR Coactivator Motifs
hydrogen bond to Asp731. Thus, if charge–charge interactions
between flanking peptide residues and the AR surface occur,
they are too weak to be detected crystallographically.
Finally, the AR AF-2 surface is an attractive target for
pharmaceutical design. Selective peptide inhibitors that bind
the AF-2 surface of liganded ERa, ERb, and TRb have been
developed (Geistlinger and Guy 2003), and similar a-helix–
mediated protein–protein interfaces have successfully been
targeted with tight binding small molecule inhibitors (Asada
et al. 2003; Vassilev et al. 2004). Drugs that directly interfere
with coactivator binding or formation of the AR N/C
interaction would likely inhibit AR activity, perhaps even in
androgen-resistant prostate cancers in which conventional
therapies have failed. Strategies for designing AR coactivator
antagonists are revealed in spite of the changes to the
structure at the interface. Together theþ1,þ4, andþ5 subsites
contribute the majority of buried surface area of the peptide–
LBD interaction (Table 2). Inhibitors may be designed by
varying hydrophobic constituents at these hotspots. The þ1
andþ5 subsites of AR have a unique preference for aromatic
side chains and provide the most viable starting points for
designing AR-specific inhibitors. Aromatic groups, possibly
with polar constituents to exploit hydrogen bonding inter-
actions with Gln733 and Gln738 in the þ1 and þ5 subsites,
respectively, may provide promising leads. Indeed, initial
screens have yielded compounds that bind to theþ1 subsite in
such a manner (E. Este ´banez-Perpin ˜a ´, personal communica-
tion). Poorly conserved binding and a lack of strong
structural features at the þ4 subsite suggest that this site
may be incorporated for achieving other characteristics
important for inhibitors besides fit. Synthetic strategies that
link together groups that bind with moderate affinity to the
þ1, þ5, and possibly þ4 subsites may yield tight binding
inhibitors of AR coactivator association.
Materials and Methods
Protein purification. Expression and purification of the AR LBD
for crystallization were performed essentially as described (Matias et
al. 2000). The cDNA encoding the chimp AR LBD (residues 663–919—
human numbering), which displays 100% identity to the human form
in protein sequence, was cloned into a modified pGEX-2T vector
(Amersham Biosciences, Piscataway, New Jersey, United States) and
expressed as glutathione S-transferase (GST) fusion protein in the E.
coli strain BL21 (DE3) STAR in the presence of 10 lM DHT. Induction
was carried out with 30 lM IPTG at 17 8C for 16–18 h. E. coli cells were
lysed in buffer (10 mM Tris, [pH 8.0], 150 mM NaCl, 10% glycerol, 1
mM TCEP, 0.2 mM PMSF) supplemented with 0.5 lg/ml lysozyme, 5
U/ml benzonase, 0.5% CHAPS, and 10 lM DHT. All buffers for
further purification steps contained 1 lM DHT. Soluble cell lysate
was adsorbed to Glutathione Sepharose 4 Fast Flow resin (Amersham
Biosciences), washed with buffer containing 0.1% n-octyl b-glucoside,
and eluted with 15 mM glutathione. After cleavage of the GST moiety
with thrombin, final purification of the AR LBD was carried out using
a HiTrap SP cation exchange column (Amersham Biosciences). Eluted
AR LBD was dialyzed overnight at 4 8C against buffer containing 50
mM HEPES (pH 7.2), 10% glycerol, 0.2 mM TCEP, 20 lM DHT, 150
mM Li2SO4, and 0.1% n-octyl b-glucoside, then concentrated to
greater than 4 mg/ml for crystallization.
Purification of AR LBD for use in phage affinity selection was
carried out as above without the final dialysis and concentration steps.
The expression construct contained the AR LBD as an inframe fusion
with GST in a modified pGEX-2T vector containing both a flexible
region and an AviTag sequence (Avidity, Denver, Colorado, United
States) allowing in vivo biotinylation. The GST–AR LBD fusion
expression plasmid was cotransformed with a plasmid-encoding E. coli
biotin ligase (Avidity) into BL21 (DE3) STAR cells. Protein expression
was carried out as above but with induction supplemented with 50 lM
biotin to ensure quantitative biotinylation of AR LBD.
Phage affinity selections and peptide identification. Phage affinity
selections were performed essentially as described (Paige et al. 1999).
Biotinylated AR LBD (10 pmol/well) was incubated in streptavidin-
coated Immulon 4 96-well plates (Dynatech International, Edgewood,
New Jersey, United States) in TBST (10 mM Tris-HCl [pH 8.0], 150
mM NaCl, 0.05% Tween 20) with 1 lM DHT for 1 h at 4 8C. Affinity
selections were performed in TBST containing 1 lM DHT. M13
phage distributed among 24 libraries displaying a total of greater
than 231010different random or biased amino acid sequences were
added to the wells containing immobilized AR LBD and incubated for
3 h at 4 8C. After washing, bound phage were eluted using pH 2
glycine. Enrichment of phage displaying target-specific peptides was
monitored after each round of affinity selection using an anti-M13
antibody conjugated to horseradish peroxidase in an ELISA–type
Synthetic peptides corresponding to the deduced amino acid
sequences from receptor-specific phage were tested for their ability
to interact with purified AR LBD using a FRET–based assay format.
Peptides were synthesized according to the deduced amino acid
sequence displayed on phage with an additional C-terminal amino
acid sequence consisting of SGSGK to allow the attachment of a
biotin tag (Anaspec, San Jose, California, United States). Flourophor
conjugates were prepared by incubating either biotinylated peptides
with streptavidin-cryptate (Cis Bio International, Bagnols Sur Ceze
Cedex, France), or biotinylated AR LBD with streptavidin-XL665 (Cis
Bio). Interaction between peptide and AR LBD was monitored by the
ratio of energy transfer by excitation at 320 nm and emission at 625
nm and 665 nm.
Surface plasmon resonance. Affinities of peptides to the AR LBD
were determined with a Biacore (Piscataway, New Jersey, United
States) 2000 instrument. A peptide derived from silencing mediator
for RXR and TR 2 (SMRT2) served as a negative control. 1 mM
peptide stock solutions in DMSO were diluted into HBS-P buffer (10
mM HEPES [pH 7.4], 150 mM NaCl, 0.005% Surfactant P20) to
generate 10 lM working solutions. HBS-P buffer was flowed through
the cells to achieve a stable baseline prior to immobilization of the
biotinylated peptides. To achieve the binding of approximately 250
RU of peptides to individual cells, working solutions of peptides were
diluted to 100 nM in HBS-P buffer. Unbound streptavidin sites were
blocked by injection of a 1 mM biotin solution at a rate of 10 ll/min.
Purified AR LBD was diluted into HBS-P buffer to a concentration
of 10 lM and injected into all four Flowcells using the Kinject
protocol at a flow rate of 10 ll/min (contact time 360 s, dissociation
time 360 s). Following the dissociation phase, the surface of the chip
was regenerated to remove residual AR LBD by QuickInject of buffer
containing 10 mM HEPES and 50% ethylene glycol (pH 11).
Following the establishment of a stable baseline, the same procedure
was repeated using a series of AR LBD dilutions (5 lM, 1 lM, and 300
nM) in an iterative manner. Analysis of the data was performed using
BIAevaluation 3.0 software (Biacore). The SMRT2 signals were
subtracted as background from the three remaining peptide signals.
Data were best fit using the two-state conformational change model
(Warnmark et al. 2001, 2002).
Crystallization, data collection, and refinement. Purified, concen-
trated AR LBD was combined with 3x to 6x molar excess of peptide
and incubated 1 h at room temperature before crystallization trials.
Complexes were crystallized using the hanging drop vapor diffusion
method. Protein–peptide solution was combined in a 1:1 ratio with a
well solution consisting of 0.6–0.8 M sodium citrate and 100 mM Tris
or HEPES buffer (pH 7–8). Crystals typically appeared after 1–2 d,
with maximal size attained within 2 wk. For data collection, crystals
were swiped into a cryo-protectant solution consisting of well
solution plus 10% glycerol before flash freezing in liquid nitrogen.
The addition of ethylene glycol to a well concentration of 10%–20%
was later found to both improve crystal quality and enable the
freezing of crystals directly out of the drop.
Datasets were collected at 100K at the Advanced Light Source
(Lawrence Berkeley Laboratory, Berkeley, California, United States),
beamline 8.3.1, with either a ADSC Quantum 315 or Quantum 210
CCD detector. Data were processed using Denzo and Scalepack
(Otwinowski and Minor 1997). Molecular replacement searches were
performed with rotation and translation functions from CNS
(Brunger et al. 1998). Initial searches for AR–FxxLF were performed
using the structure of AR–R1881 (PDB: 1E3G) with R1881 omitted
from the search model. Subsequent searches for all other complexes
were performed using the refined LBD structure from the AR–FxxLF
complex. To minimize the possibility of model bias, FxxLF peptide
and DHT were omitted from all molecular replacement searches.
Protein models were built by iterative rounds of simulated annealing,
conjugate gradient minimization, and individual B-factor refinement
PLoS Biology | www.plosbiology.org September 2004 | Volume 2 | Issue 9 | e2741311
Recognition of AR Coactivator Motifs
in CNS followed by manual rebuilding in Quanta 2000 (Accelrys, San Download full-text
Diego, California, United States) using rA-weighted 2Fo? Fc, Fo? Fc,
and simulated annealing composite omit maps. Superposition of
structures was performed with LSQMAN (Kleywegt 1996). Buried
surface area calculations were performed with CNS. All figures were
generated with PyMOL (DeLano 2002). Coordinates and structure
factors for all complexes have been deposited in the Protein Data
Bank. Accession numbers are listed in Table 2.
The Swiss-Prot (http://www.ebi.ac.uk/swissprot) accession numbers for
the gene products discussed in this paper are AR (P10275), ARA54
(Q9UBS8), ARA55 (Q9Y2V5), ARA70 (Q13772), ER (P03372, Q92731),
glucocorticoid receptor-interacting protein 1 NR box 3 (Q61026 ),
GR (P04150), NR box 3 of TIF2 (Q15596), and TR b (P10828).
The Protein Data Bank (http://www.rcsb.org/pdb) accession num-
bers for the structures used in this paper are FxxFF (1T73), FxxLF
(1T7R), FxxLW (1T79), FxxYF (1T7M), LxxLL (1T7F), unbound
(1T7T), WxxLF (1T74), and WxxVW (1T76).
We would like to thank Erin Anderson-Chisenhall for assistance in
protein purification, James Holton and the staff at ALS beamline
8.3.1 for assistance in data collection, and Paul Webb for critical
review of the manuscript. This work was supported by funds from the
Prostate Cancer Foundation and National Institutes of Health grant
R21 CA95324 to RJF.
Conflicts of interest. The authors have declared that no conflicts of
Author contributions. EH, BB, and RF conceived and designed the
experiments. EH, SP, ESP, HG, and BB performed the experiments.
EH, SP, ESP, HG, BB, and RF analyzed the data. BB and RF
contributed reagents/materials/analysis tools. EH, BB, and RF wrote
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Recognition of AR Coactivator Motifs