ARTICLES
A unique secondary-structure switch controls constitutive gene repression by retinoic acid receptor
© 2010 Nature America, Inc. All rights reserved.
Albane le Maire1,2,9, Catherine Teyssier1,2,9, Cathie Erb3, Marina Grimaldi4–7, Susana Alvarez8, Angel R de Lera8, Patrick Balaguer4–7, Hinrich Gronemeyer3, Catherine A Royer1,2, Pierre Germain1,2 & William Bourguet1,2 In the absence of ligand, some nuclear receptors, including retinoic acid receptor (RAR), act as transcriptional repressors by recruiting corepressor complexes to target genes. This constitutive repression is crucial in metazoan reproduction, development and homeostasis. However, its specific molecular determinants had remained obscure. Using structural, biochemical and cell-based assays, we show that the basal repressive activity of RAR is conferred by an extended b-strand that forms an antiparallel b-sheet with specific corepressor residues. Agonist binding induces a b-strand–to–a-helix transition that allows for helix H11 formation, which in turn provokes corepressor release, repositioning of helix H12 and coactivator recruitment. Several lines of evidence suggest that this structural switch could be implicated in the intrinsic repressor function of other nuclear receptors. Finally, we report on the molecular mechanism by which inverse agonists strengthen corepressor interaction and enhance gene silencing by RAR. The gene-regulatory capacity of several nuclear receptors (NRs) involves both activation and repression of target-gene transcription. These abilities allow retinoic acid receptor (RAR) to control gene networks critical for metazoan homeostasis, development and reproduction1,2. Gene silencing proceeds via recruitment of nuclear receptor corepressor (N-CoR) or silencing mediator for retinoid and thyroid hormone receptors (SMRT) corepressor-containing complexes to unliganded (apo) DNA-bound receptors, whereas gene activation involves agonist-induced corepressor release and the recruitment of coactivator complexes3,4. The C-terminal NR-interaction region of SMRT5 and N-CoR6 is composed of two or three interaction domains (ID1–ID3)7–9, respectively. Each of them contains corepressor-NR boxes (CoRNR1–CoRNR3) characterized by the core sequence (I/L)XX(V/I)I (where X denotes any residue), which presents a central extended helical motif mediating the interaction with NRs10–12. In addition to this core sequence, flanking residues have been shown to strengthen the interaction and determine NR specificity13. On the NR side, biochemical and structural studies have identified a receptorrecognition surface for the extended (I/L)XX(V/I)I motifs of corepressors formed by residues from helices H3 and H4 (refs. 10–12,14). The generation of a smaller H3-, H4- and H12-containing surface through the agonist-induced repositioning of helix H12 is thought to account for the dissociation of corepressors and the recruitment of coactivators via their shorter LXXLL-interaction motifs. However, in spite of a large body of structural and functional data on the modulation
of NR-coregulator interactions, the reason why some apo receptors recruit corepressors and act as constitutive transcriptional silencers, whereas others do not, has remained elusive despite its major physiological relevance15–19. In contrast to agonists, a subclass of antagonists referred to as inverse agonists has the potential to promote the interaction with corepressors, thus decreasing the basal transcriptional activity of apo receptors20. We recently reported on the different mechanistic bases for the antagonism exerted by two RAR ligands that we classified as neutral antagonist (BMS614) and inverse agonist (BMS493) due to their ability to induce distinct coregulator interaction patterns21. We provided evidence that BMS614 acts as a potent RAR antagonist by efficiently preventing the recruitment of coactivators, whereas it has little effect on the association with corepressors. In contrast, we showed that BMS493 is less efficient than BMS614 in preventing the recruitment of coactivators but strongly enhances corepressor interaction. Compounds with such properties represent unique pharmacological tools to probe the implication of NR-mediated repression in cellbiological phenomena. Recently, some of them have also been used to facilitate the crystallization of several NRs, including the peroxisome proliferator activated receptor A14 (PPARA, PDB 1KKQ), progesterone receptor22 (PDB 2OVH and 2OVM) and estrogen-related receptor A23 (ERRA, PDB 2GPV) in complexes with peptides derived from SMRT or N-CoR CoRNR2 (termed hereafter S- and N-CoRNR2). Whereas these results were obtained on NRs with weak basal repressive activity, we
1Institut
National de la Santé et de la Recherche Médicale, U554, Montpellier, France. 2Centre National de la Recherche Scientifique, UMR5048, Centre de Biochimie Structurale, Universités Montpellier 1 & 2, Montpellier, France. 3Department of Cancer Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Communauté Urbaine de Strasbourg, Illkirch, France. 4Institut de Recherche en Cancérologie de Montpellier, Montpellier, France. 5Institut National de la Santé et de la Recherche Médicale, U896, Montpellier, France. 6Université Montpellier 1, Montpellier, France. 7Centre Régional de Lutte contre le Cancer, Val d’Aurelle Paul Lamarque, Montpellier, France. 8Departamento de Química Orgánica, Facultad de Química, Universidad de Vigo, Vigo, Spain. 9These authors contributed equally to this work. Correspondence should be addressed to W.B. (
[email protected]). Received 14 December 2009; accepted 25 March 2010; published online 13 June 2010; doi:10.1038/nsmb.1855
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1
ARTICLES Table 1 Data collection and refinement statistics RAR–Am580–SRC-1 NR2
RAR–BMS493–N-CoRNR1
C2
C2
a, b, c (Å)
88.31, 61.22, 49.44
104.83, 105.62, 53.38
A, B, G (°)
90.00, 105.38, 90.00
90.00, 89.92, 90.00
Resolution (Å)
35.00–1.80 (1.90–1.80)a
43.40–2.10 (2.21–2.10)
Rsym
0.043 (0.319)
0.064 (0.341)
I / SI
16.2 (3.6)
13.2 (3.0)
93.8 (77.9)
96.6 (98.6)
2.6 (2.5)
2.8 (2.7)
Resolution (Å)
32.02–1.80
43.40–2.10
No. reflections
22,172
32,759
Rwork / Rfree
19.7% / 23.7%
17.2% / 22.9%
No. atoms
2,087
4,262
1,940
3,834
26
130
Data collection Space group Cell dimensions
Completeness (%) Redundancy Refinement
© 2010 Nature America, Inc. All rights reserved.
Protein Ligand/ion Water
121
298
B-factors
29.81
29.59
Protein
29.65
28.75
Ligand/ion
17.95
35.86
Water
34.97
37.64
Bond lengths (Å)
0.010
0.010
Bond angles (°)
2.700
1.218
R.m.s. deviations
Data were collected from one crystal for each dataset. aValues
in parentheses are for highest-resolution shell.
report here novel insights into the structural basis of ligand-dependent and ligand-independent corepressor recruitment by human RAR, a receptor that shows high constitutive repression. RESULTS Structures of RARa with coactivator and corepressor fragments We determined the crystal structure (Table 1) of a ternary complex containing the RARA ligand binding domain (RAR LBD) bound to a 19-mer peptide derived from CoRNR1 of N-CoR (N-CoRNR1) and the inverse agonist BMS493, which is known to further stabilize this complex21. In addition, we solved the crystal structure of RAR LBD in complex with the agonist Am580 and a 13-residue fragment containing the NR-interaction motif 2 of the coactivator SRC-1 (SRC-1 NR2). This latter structure shows the canonical active conformation, with the C-terminal activation helix H12 capping the ligand binding pocket and the SRC-1 peptide bound to the surface formed by residues from helices H3, H4 and H12 (Fig. 1a). In the complex with N-CoRNR1 and BMS493 (Fig. 1b), the helices H1–H10 of RAR LBD fold into the archetypal NR helical sandwich resembling the agonist-bound (Fig. 1a) and the previously reported neutral antagonist (BMS614)bound RAR24 structures (Fig. 1c). In contrast, the structure of the region comprising the C-terminal helices H11 and H12 diverges dramatically in the three complexes. A most obvious difference between the various structures is the position of helix H12. Whereas it folds back in a conformation allowing coactivator binding in the presence of the agonist (Fig. 1a) or occupies the coactivator-binding groove in the neutral antagonist–bound structure (Fig. 1c), helix H12 has no defined position and could not be modeled in the RAR–N-CoRNR1 complex with the inverse agonist BMS493 (Fig. 1b). Consistently, monitoring of helix H12 dynamics by fluorescence anisotropy revealed a much higher mobility in the 2
RAR–BMS493–N-CoRNR1 complex (see below for the conformational dynamics study). However, the most unexpected difference resides in the region C-terminal of helix H10. Unlike the agonist (Fig. 1a,d) and neutral antagonist (Fig. 1c) complexes, in which the residues Arg394–Lys399 adopt a helical structure (H11), these residues assume a well-defined extended B-strand (S3) conformation in the corepressor complex, allowing for the formation of an antiparallel B-sheet with N-terminal residues (B1) of N-CoRNR1 (Fig. 1b,e,f). Notably, this unexpected conformational change of helix H11 residues was not observed in the previously reported corepressor-bound NR structures14,22,23. Besides the N-terminal B1, N-CoRNR1 contains a C-terminal fourturn helix A1 (Fig. 2a,b) that docks into the coregulator binding cleft of RAR LBD through conserved residues Leu(+1), Ile(+5) and Ile(+9) as well as Phe(+13), which lie along one face of the helix (CoRNR residues are numbered according to their position in helix A1). Comparison of the helical part of CoRNR motifs in the RAR–N-CoRNR1 and PPAR–S-CoRNR2 structures reveals two notable differences. First, a shift toward RAR H11/S3 of N-CoRNR1 A1 brings residues Ile(+5), Ile(+9) and Phe(+13) into the same spatial environment as Leu(+1), Ile(+5) and Leu(+9) of S-CoRNR2, respectively (Fig. 2b). Second, S-CoRNR2 contains a glycine residue (Gly(−1)), which shortens the helix by one turn compared to that of N-CoRNR1 (Fig. 2a,b). The high degree of sequence conservation suggests that the structural features of N-CoRNR1 are preserved in S-CoRNR1 and N-CoRNR3 (Fig. 2a). In particular, the conserved threonine (Thr(−1)) has a key role, acting as a helix N-capping residue stabilizing the junction between B1 and A1 (Fig. 2c). Overall, comparison of the structures reveals that CoRNR1 and CoRNR2 use different binding mechanisms such that their consensus motifs should be redefined separately as LXXXIXX(V/I) IXXX(Y/F) and LXXIIXXXL, respectively (Fig. 2a). CoRNR1 b1 is indispensable for corepressor recruitment by RAR Our structure reveals an additional and unforeseen binding interface between RAR S3 and N-CoRNR1 B1 that may account for the transcription-repressor function of apo RAR. To test this hypothesis, we first validated the corepressor and CoRNR binding preferences of apo RAR. Using fluorescence anisotropy, we determined the affinities of the four CoRNR motifs for apo RAR, which confirmed previous studies showing that RAR binds more avidly to SMRT than to N-CoR and that this preference is determined by CoRNR1 (ref. 13) (Fig. 3a). In this respect, several residue substitutions may account for the higher binding affinity of S-CoRNR1 relative to that of N-CoRNR1 (Fig. 2a,c). First, the replacement of N-CoRNR1 B1 Ile(−2) and Leu(−3) by two valine residues which are even more potent B-strand formers than Ile and Leu is very likely to strengthen S-CoRNR1 B1. Second, the replacement of the N-CoRNR1 A1 residue Gln(+7) by a charged glutamate capable of forming a salt bridge with Arg(−4) (dotted line in Fig. 2c) is also likely to play a role in the stabilization of S-CoRNR1 B1. These observations suggest that the S3-B1 antiparallel B-sheet is more stable in the RAR–S-CoRNR1 complex and explain the stronger effect of BMS493 on the association between RAR and N-CoRNR1 as compared with that of S-CoRNR1. Whereas the agonist Am580 decreases the affinity of all corepressor motifs for RAR, the inverse agonist BMS493 efficiently enhances interaction with N-CoRNR1 and preserves the high affinity of S-CoRNR1 but has no effect on S- or N-CoRNR2 binding (Fig. 3a). We drew similar conclusions from mammalian two-hybrid experiments in which the wild-type (WT) interaction regions of SMRT and N-CoR or mutants containing only one functional interaction domain (see Online Methods) were assayed for their interaction with
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Figure 1 Structures of RAR LBD in complex with agonist, inverse agonist or neutral antagonist ligands. (a) Overall structure of the RAR LBD–SRC-1 NR2–Am580 complex. (b) Overall structure of the RAR LBD– N-CoRNR1–BMS493 complex. (c) Overall structure of the RAR LBD–BMS614 complex24 (PDB 1DKF). (d) Detail of helix H11 (violet) in the RAR LBD–SRC-1 NR2–Am580 complex. (e) Interface between RAR S3 (violet) and N-CoRNR1 B1 (green). (f) The electron density, 2Fo – Fc (mesh) contoured at 1.2S, is shown for the B-strand S3 of RAR (black) and for the B-strand B1 of N-CoRNR1 (gray). The RAR protein and the peptide are drawn as yellow and gray sticks, respectively.
b
H9
H8
H8
H5 S1
H3 S2
1
d
e
Glu393
H10
© 2010 Nature America, Inc. All rights reserved.
SRC-1 NR2
ID1 ID2
89
13
1
45
9
S-CoRNR1 N-CoRNR1 N-CoRNR3 Sec. str.
N-CoRNR2 Sec. str.
c CoRNR2 H10
+9 +5 +1
+5
Phe(Tyr)
+13
+9
1
+13
H4
Gln(Glu) +5
+1
S3 H11
+9
CoRNR1
PPAR H12
S2
H12
H11
H10
f
Glu393 N-CoRNR1 1
BMS493
Ile2049 1
Arg394 Ile396 S3
Arg2047
Thr397
Met400 Lys399
Lys399 Leu398 Thr397 Ile396 Val395
Arg2047 Leu2048 Ile2049 Thr2050
Arg394
mutations affect the RAR-corepressor interaction through different mechanisms. Among the three mutations tested, V2141P is the most drastic, as it most likely abrogates the B-strand conformation of B1 residues as indicated by the incapacity of BMS493 to reinforce the weak RAR-SMRT interaction. Through the interactions described above (Fig. 2c), R2139 and T2142 stabilize the topological organization of CoRNR1, and their mutations probably do not markedly affect the B-strand conformation of B1 but instead affect its position relative to helix A1. This interpretation is consistent with the clear inverse-agonist effect of BMS493 on these mutants. Taken together, these structural and functional analyses reveal that the integrity of the CoRNR1 B1 motif must be preserved for optimal association between CoRNR1 and RAR. Although N-CoRNR3 contains most of the structural features of N- and S-CoRNR1, N-CoRNR3 lacks the salt bridge or hydrogen bond observed between residues Arg(–4) (B1) and Glu/Gln(+7) (A1) (Fig. 2c). In N-CoRNR3, these residues are replaced by Thr(−4) and Val(+7) (Fig. 2a). Given our mutational data revealing the importance of this salt bridge for the RAR–SMRT interaction (Fig. 3e), it is very likely that this missing interaction accounts for the low affinity of N-CoRNR3 for RAR13. A single mutation in S3 disrupts interaction with corepressors Having shown the critical role of CoRNR1 B1 residues for interaction with RAR, we evaluated the implication of RAR S3 residues in the
S-CoRNR2
H4
S1
N-CoRNR1
H4 H3
H11
5
b
1
1 S3
RD3
SMRT/N-CoR
S2
H12
ID3 RD2
S1
SRC-1 NR2
H5
H4 H3
H1
H8
H10 H5
H4
RAR in different ligation states (Fig. 3b,c). Am580 Having shown that RAR interacts preferenH11 H12 tially with CoRNR1, we tested whether the Arg394 motif CoRNR1 B1 could have a role in this preference. We first evaluated the affinity Glu401 of interaction with RAR LBD of fluorescent N-CoRNR1 and S-CoRNR1 peptides lacking the N-terminal B1 residues (CoRNR1 short) and found that both peptides had very low affinities for unliganded RAR (Fig. 3d). Subsequently, residues that we previously identified as being potentially important for the stability of S-CoRNR1 B1, namely Arg2139 (−4), Val2141 (−2) and Thr2142 (−1) (Fig. 2a,c), were mutated into alanine, proline and glycine, respectively. Point mutants were analyzed using two-hybrid assays and in the context of a SMRT protein containing a disabled S-CoRNR2 (ID1 + ID2m) to avoid any functional interference with this second interaction motif (see Online Methods). All three mutations (R2139A, V2141P and T2142G) abolished or reduced SMRT interaction with apo RAR LBD (Fig. 3e). Notably, the addition of the inverse agonist BMS493 restored a substantial interaction with mutants R2139A and T2142G but had no effect in the case of V2141P, suggesting that the various RD1
H9
H1
H10
H10
Ile396
a
c
H9
H1
–4 Ile (Val)
+1 H10
RAR
NATURE STRUCTURAL & MOLECULAR BIOLOGY
1
–1 S3
Leu(Val)
Figure 2 Comparison of CoRNR1 and CoRNR2 motifs. (a) Sequence alignment of SMRT and N-CoR interaction domains. Residues of the consensus motifs are numbered according to their position in helix A1. Secondary-structure (Sec. str.) elements of CoRNR1 and CoRNR2 as observed in the crystal structures are indicated. RD, repression domain; ID, interaction domain. (b) Overlay of the RAR LBD–N-CoRNR1–BMS493 (blue) and PPAR LBD–S-CoRNR2–GW6471 (orange) (PDB 1KKQ 14) structures. (c) Close-up view of the RAR LBD–N-CoRNR1 interface. Residues conserved in N- and S-CoRNR1 are colored in blue, whereas differing residues are colored in yellow (residues in S-CoRNR1 are indicated in parenthesis). The hydrogen bond between the conserved helix-capping Thr(−1) and the main chain of helix A1 is shown (dotted line) as well as the hydrogen bond linking Arg(−4) and Gln(+7).
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ARTICLES I396E mutation abrogates completely any interaction with SMRT or N-CoR, with the 24 inverse agonist BMS493 exerting a weak but 12 12 substantial stabilization of N-CoR binding 10 20 8 8 (Fig. 4c). Fluorescence anisotropy data reveal 6 that the RAR I396E mutant shows a severely 10 4 4 decreased affinity for all CoRNR peptides 2 and that BMS493 increases its affinity for 0 0 0 Gal–N-CoR ID1+ ID1 + ID1m Gal–SMRT ID1+ ID1 + ID1m N-CoRNR1 and S-CoRNR2 (Fig. 4d). ID2 ID2m + ID2 ID2 ID2m + ID2 We then examined the ability of RARI396E VP16–WT RAR VP16–WT RAR to interact with coactivators and the effect d e WT of the neutral antagonist BMS614 on this R2139A S-CoRNR1 V2141P interaction. We found that, in contrast to the N-CoRNR1 T2142G deletion mutants, the I396E mutation does No ligand CoRNR1 300 12 Am580 CoRNR1 short not impair the ligand-dependent recruitBMS493 250 ment of TIF-2 (Fig. 4e), in agreement with 8 200 fluorescence anisotropy data indicating that 4 150 the SRC-1 LXXLL-containing peptide (SRC-1 2 NR2) is recruited to agonist-bound WT 0 Gal–SMRT WT ID1 R2139A V2141P T2142G 0 and mutant receptors with similar affinities ID1+ ID2m SMRT N-CoR VP16–WT RAR (Fig. 4f). As expected, the neutral antagonist BMS614 does not promote appreciable interFigure 3 CoRNR1 B1 residues are indispensable for corepressor recruitment by RAR. (a) Affinities action with the coactivator (Fig. 4e,f). Finally, of N- and S-CoRNR motifs for wild-type (WT) RAR measured by fluorescence anisotropy. transient-transactivation assays showed that (b,c) Mammalian two-hybrid assays in HeLa cells were used to evaluate interactions of the wild-type (ID1 + ID2) and mutants (ID1 + ID2m or ID1m + ID2) NR interaction regions of SMRT (b) and the expression of full-length RAR I396E N-CoR (c) with WT RAR in various ligation states. (d) Affinities of B1-deleted CoRNR motifs (CoRNR results in a substantial basal stimulation of short) for RAR LBD measured by fluorescence anisotropy. (e) Mammalian two-hybrid assays in HeLa transcription as compared with that obtained cells were used to evaluate interactions of WT RAR in various ligation states with NR-interaction in nontransfected or WT RAR–transfected regions of intact SMRT ID1 + ID2m (ID1 is the only functional interaction motif) or containing cells (Fig. 4g,h). Furthermore, this activity single point mutations in ID1. Error bars, s.e.m. is either increased in an agonist (Fig. 4g) or decreased in a neutral antagonist (Fig. 4h) B1–S3 interface by studying RAR deletion mutants. In the context of concentration-dependent manner, confirming that RAR I396E canSMRT and N-CoR containing both interaction domains, we examined not bind corepressors but retains its coactivator recruitment function. the recruitment to an H12-deleted RAR (RAR $H12) and a RAR lack- Hence, this RAR variant provides a unique tool to probe the role of ing both S3 and H12 (RAR $S3 $H12). In agreement with previous RAR-mediated gene silencing in embryonic development15 by genereports5,10,25,26, RAR $H12 recruited corepressors more efficiently rating genetically modified animal models. than WT RAR, whereas the inverse agonist BMS493 further stabilized this interaction (Fig. 4a,b). In contrast, the agonist Am580 induced Conformational dynamics of H12 in RAR–corepressor complexes concentration-dependent corepressor dissociation in both cases, To further characterize the role of H12 in the mechanisms of even though higher concentrations (1–10 MM) are required in the corepressor association and release, we attached a fluorescein moiety case of RAR $H12, possibly due to reduced affinity of Am580 for to the C terminus of RAR LBD through intein chemistry27,28 and analythe truncated receptor. We interpreted this ligand-dependent dis- zed the dynamic properties of helix H12 in the presence of various sociation from RAR $H12 as an indication of the presence of the ligands and coregulator fragments (Supplementary Table 1). We RAR S3–CoRNR1 B1 interface, which is destabilized upon agonist measured the time-resolved anisotropy decay using multifrequency binding through the formation of helix H11. The weak recruitment phase and modulation fluorometry. The fluorescence lifetime of the of SMRT and N-CoR by RAR $S3 $H12 supports this interpreta- dye (4 ns) was insensitive to conditions of ligation or peptide binding. tion (Fig. 4a,b). Furthermore, in contrast to the RAR $H12 situa- The anisotropy decays, regardless of ligation conditions, could all be tion, BMS493 failed to increase the interaction of RAR $S3 $H12 well fitted to a model with two correlation times. The long correlation with corepressors, suggesting that the structural basis of inverse ago- time (Tc1), corresponding to the global rotation of the complexes, was nism relies on S3 stabilization (see below for a comparison of neutral found to be 17 o 2 ns for all the datasets, and the short correlation antagonist– and inverse agonist–bound RAR structures). time (Tc2) was found to be 0.65 o 0.07 ns, corresponding to the fast To address the functional role of RAR S3 in the context of the full- rotation of helix H12 and the dye. When RAR is unliganded, ~56% length receptor, we looked for a specific point mutation that would of the depolarization of fluorescence is due to the longest correlation destabilize S3 without affecting H11, which has critical functions time Tc1. Upon saturation with N-CoRNR1 (and even more so with in ligand binding and coactivator recruitment. Examination of the S-CoRNR1), the fractional contribution of the slow global rotations RAR–N-CoRNR1 structure revealed that Ile396 points toward the decreases, whereas that of the fast rotational motion Tc2 increases. solvent in the helical H11 conformation but toward the hydrophobic This indicates that the interactions between the core of the protein ligand binding pocket in the B-strand S3 conformation (Fig. 1d,e). and the C terminus are destabilized by the corepressor peptides. In the We reasoned that the replacement of this aliphatic residue, which presence of the corepressor peptides, the addition of the inverse agoacts as strong B-strand former by the charged B-strand breaker nist BMS493 further increases the fast dynamics. In contrast, BMS493 glutamate, would favor the S3-to-H11 transition. Notably, the single alone does not modify the dynamics of helix H12 as measured in Relative reporter activity
No ligand Am580 BMS493
N
N
-C
oR
N
c 16
R 2
R 1
2
R
oR
N
-C
N
C
S-
oR
N
R
1
Kd (µM)
C
S-
oR
© 2010 Nature America, Inc. All rights reserved.
Kd (µM)
4
No ligand Am580 BMS493
30
Relative reporter activity
b
No ligand Am580 BMS493
Relative reporter activity
a 26
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ARTICLES b
120 80 40
c
60
Relative reporter activity
160
Relative reporter activity
Relative reporter activity
a
40
20
0
0 – – – Ligands Am580 BMS493 Am580 BMS493 Am580 BMS493 RAR H12 RAR S3 H12 VP16– WT RAR
– – – Am580 BMS493 Am580 BMS493 Am580 BMS493 RAR S3 H12 RAR H12 WT RAR
Ligands VP16–
Gal–SMRT
RARI396E
Kd (M)
20
e
No ligand No ligand Am580 BMS493
15 10
S-CoRNR1
S-CoRNR2
10
0 VP16–
WT RAR
RAR I396E
WT RAR
RAR I396E
Gal–N-CoR
Gal–SMRT
N-CoRNR1
f
No ligand Am580 BMS614
6
20
4
15 10 5
0 VP16– WT RAR RAR S3 H12 RAR H12
N-CoRNR2
No ligand Am580 BMS614
2
0
RAR I396E
Gal–TIF-2
g
WT RAR
RAR I396E
SRC-1 NR2
Figure 4 RAR S3 residues are indispensable for corepressor 16 recruitment and release. (a,b) Mammalian two-hybrid assays in 160 HeLa cells were used to evaluate interactions of the RAR deletion 12 120 mutants (RAR $H12 and RAR $S3 $H12) with SMRT (a) and N-CoR (b). (c) Mammalian two-hybrid assays in HeLa cells were 8 80 used to evaluate interactions of Gal–SMRT or Gal–N-CoR with 4 40 VP16–WT RAR or VP16–RAR I396E. (d) Affinities of N- and S-CoRNR motifs for WT RAR and RAR I396E measured by 0 0 fluorescence anisotropy. (e) Mammalian two-hybrid assays in COS Am580 – – – BMS614 – – – cells were used to detect interactions of the coactivator TIF-2 with Control WT RAR RAR I396E Control WT RAR RAR I396E WT RAR, RAR $S3 $H12, RAR $H12 and RAR I396E. (f) Affinities of the SRC-1 NR2 motif for WT RAR and RAR I396E as measured by fluorescence anisotropy. (g) Transactivation assays in HeLa cells transiently transfected with retinoic acid response element reporter ((RARE) 3×–tk–Luc) alone and with WT RAR or RAR I396E. Am580 concentration range, 10−9–10−7 M. (h) Transactivation assays in HeLa cells transiently transfected with (RARE) 3×–tk–Luc alone and with WT RAR or RAR I396E. BMS614 concentration range, 10−9–10−7 M. All error bars are expressed as s.e.m. Fold activation
h
Fold activation
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5 0
20
Kd (M)
WT RAR 25
30
Gal–N-CoR
Relative reporter activity
d
No ligand Am580 BMS493
40
apo RAR. Notably, the dynamics of the C terminus for the neutral antagonist (RAR–BMS614; Fig. 1c) and agonist (RAR–Am580–SRC-1 NR2; Fig. 1a)-bound complexes are identical to those measured for the apo RAR, suggesting that, in the unliganded receptor, helix H12 is somehow stabilized, most likely through nonspecific hydrophobic interactions with the coregulator-binding groove of the receptor. We note that the dynamic properties of RAR differ from those measured for PPAR28 and for RXR27. In these proteins, a slow, ligand-dependent intermediate–time scale motion putatively corresponding to helix H12
a
b BMS614
L6-7 Leu305
BMS493 H11 Leu398 Val395
Figure 5 Structural basis of inverse agonism in RAR. (a) Chemical structures of the neutral antagonist BMS614 (left) and the inverse agonist BMS493 (right). (b) Superposition of the ligand binding sites of the RAR–BMS614 (yellow, ligand; orange, protein; PDB 1DKF24) and RAR–BMS493 (cyan, ligand; blue, protein) complexes.
NATURE STRUCTURAL & MOLECULAR BIOLOGY
Val395 Ile396
Thr397 Leu398 S3
dynamics was observed in addition to the slow global Brownian tumbling and the very fast rotation of the dye. This intermediate-scale motion is much faster for RAR and hence is indistinguishable from the local rotational dynamics of the dye. Structural basis for the inverse agonist activity of BMS493 To gain additional insight into the molecular basis of the inverseagonist activity of BMS493, we compared the structure reported in the present article with that of RAR LBD bound by the neutral antagonist BMS614 (ref. 24). Although BMS164 and BMS493 are very similar in their chemical H5 structures (Fig. 5a), they show slightly different binding modes in RAR LBD, which most IIe273 Arg316 likely accounts for their differing actions on coregulator recruitment21. Whereas the A rings of both ligands superpose well, a steric clash between the carbonyl group of BMS614 Ser232 and Ile273 forces rings B, C, D and E to adopt Ser287 a different position in BMS614 compared to H3 BMS493 (Fig. 5b). In the neutral-antagonist complex, interactions between BMS614 and Val395 and Leu398 stabilize the helical conformation of H11. Of note, these stabilizing interactions are also observed in the agonistbound (RAR–Am580–SRC-1 NR2) complex.
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© 2010 Nature America, Inc. All rights reserved.
DISCUSSION Why do some unliganded receptors repress transcription, whereas others do not? The answer primarily involves the inhibitory role of helix H12, as receptors lacking the C-terminal region, like the heme receptor RevErb, act as strong repressors7,29,30, and deletion of helix H12 converts nonrepressive receptors to potent repressors31–33 or further strengthens the interaction between corepressors and RAR5,25,33 (Fig. 4a,b). This raises the question of why helix H12 fully inhibits corepressor binding to most NRs, whereas it only reduces interaction with others. Here we have shown that a short region C-terminal of RAR helix H10 adopts a B-strand conformation (S3) that specifically interacts with CoRNR1 B1 residues. Our data suggest that the formation of this B-sheet interface is important for (i) increasing the availability of the coregulator groove of apo RAR by removing helix H12, which might be stabilized through establishment of hydrophobic contacts with this surface (Fig. 1c and Supplementary Table 1), (ii) strengthening the interaction by increasing the contact surface with corepressors (Fig. 1e) and (iii) CoRNR1 helix A1 docking, as such packing is prevented by steric hindrance provided by helix H11 (Figs. 1b and 3d). In this respect, the preservation of the helical H11 conformation in antagonist-bound PPAR, ERR and progesterone receptor structures14,22,23 explains why antagonist-driven corepressor interaction is generally mediated by the short helical motif of CoRNR2. In the current structural model of the ligand-induced coregulator swap, the repositioning of helix H12 of the NR has a central role in both corepressor release and coactivator recruitment. Here, we show that a novel secondary-structure transition from B-strand S3 to A-helix H11 is the master regulator of corepressor dissociation from RAR, whereas H12 is primarily involved in the interaction with coactivators. Together, our data shed light on a previously unknown regulatory paradigm for RAR action that helps to explain how this
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In the inverse-agonist complex, these interactions are lost, but novel van der Waals contacts of Ile396 and Leu398 with the phenyl D ring of BMS493 stabilize the B-strand S3 conformation, thus favoring corepressor binding via CoRNR1. Because of their antagonistic extensions (rings D and E in BMS614, ring D in BMS493), both BMS614 and BMS493 prevent the positioning of helix H12 in the active orientation, thus antagonizing coactivator binding. In this respect, it is noteworthy that the bulky extension of BMS614 protrudes markedly more from the ligand binding pocket toward helix H12 (when in the agonistbound conformation; Fig. 1a) than that of BMS493. This difference very likely contributes to the higher efficiency of BMS614 in preventing coactivator binding through a higher efficacy in the displacement of helix H12 from its active position.
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Figure 6 Proposed model for ligand-dependent and ligand-independent corepressor recruitment by NRs based on the crystal structures of RAR and progesterone receptor (PR). (a) In apo NRs, the C-terminal helix H12 masks the corepressor (CoR) interaction surface. (b) Formation of a B-sheet interface between RAR S3 and CoRNR1 B1 removes H12 and unmasks the hydrophobic groove to which the four-turn helix A1 binds. (c) An inverse agonist further strengthens the interaction by stabilizing the S3-B1 interface. (d) Agonist binding induces the S3-to-H11 secondary-structure switch, release of the CoR and folding back of helix H12, which allows coactivator (CoA) binding (e). (f) PR does not form the B-sheet interface with CoRNR1, and the CoR interaction surface remains masked. (g) Antagonist binding induces H12 displacement and CoRNR2 binding. The presence of helix H11 prevents CoRNR1 from binding. (h,i) As in RAR, agonist binding induces formation of the active conformation of PR (h) and CoA recruitment (i).
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receptor fine-tunes its target-gene network. Notably, several lines of evidence suggest that the mechanistic model (Fig. 6) implicating a versatile S3/H11 secondary-structure, allowing for constitutive interaction with corepressors, could apply to other NRs acting as transcriptional repressors, such as RevErb7,13, the vitamin D receptor (VDR)25,26,34 or the orphan receptor Tailless (TLL/TLX)35. It has been shown previously that, like RAR, both RevErb13 and VDR34 interact with corepressors via CoRNR1. Such specificity most likely reveals the presence of a B-sheet interaction between the putative S3 residues of VDR or RevErb and residues B1 of CoRNR1. Accordingly, mutations in the putative S3 region of RevErb (L606A and R610A; Supplementary Fig. 1) have been reported to reduce the in vitro and in vivo interaction of this receptor with corepressors36. In the same vein, deletion of the 13 residues at the C terminus of RevErb (from His602 to Gln614; Supplementary Fig. 1), which includes the putative S3 region, has been shown to abolish N-CoR interaction7. Along these lines, two TLL derivatives carrying mutations in the putative S3 region, designated as the C1 motif in TLL (from Phe423 to Gly435; Supplementary Fig. 1) have been recently reported to be largely inactive when expressed in vivo, whereas deletion of the sole H12 motif (from Leu442 to Ile452; Supplementary Fig. 1) did not modify TLL activity, suggesting that the C1/S3 motif is essential for the specific repression function of TLL/TLX35. Finally, mutations in S-CoRNR1 B1 (R4W and V2A; Fig. 2a) have been shown to impair VDR-mediated repression34. All together, these previously reported mutagenesis data, which we could readily reinterpret in light of our structural data, support the notion that the mechanism defined for RAR also could be operative with these receptors. METHODS Methods and any associated references are available in the online version of the paper at http://www.nature.com/nsmb/. Accession codes. Protein Data Bank: Atomic coordinates and structure factors for RAR–Am580–SRC-1 NR2 and RAR–BMS493– N-CoRNR1 have been deposited under accession codes 3KMR and 3KMZ.
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ARTICLES Note: Supplementary information is available on the Nature Structural Molecular Biology website.
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ACKNOWLEDGMENTS We thank G. Labesse for helpful discussions, C. Clerte for help with time-resolved fluorescence anisotropy experiments and Mitchell Lazar (Univ. of Pennsylvania) for providing us with Gal–N-CoR constructs. We acknowledge the experimental assistance from the staff of European Synchrotron Radiation Facility (ESRF) (ID14-2 beamline) during data collection. This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, Université Montpellier 1 & 2, the French National Research Agency (ANR-07-PCVI-0001-01) and the Association pour la Recherche sur le Cancer (ARC 1056). C.T. and H.G. (laboratoire labélisé) are supported by the Ligue contre le cancer. Work in the laboratories of H.G. and A.R.d.L. is supported by EPITRON, an Integrated Project funded by the European Union under the 6th Framework Programme (LSHC-CT-2005-518417). AUTHOR CONTRIBUTIONS A.l.M., C.T. and W.B. purified proteins and grew crystals; A.l.M. and W.B. solved the structures; A.l.M., C.T. and C.A.R performed fluorescence anisotropy experiments; C.T., C.E., M.G., P.B. and H.G. made the DNA constructs; S.A. and A.R.d.L. synthesized BMS493; C.T. performed cell-based assays; P.G. analyzed data; W.B. planned the project, analyzed the data and wrote the manuscript; H.G. and C.A.R. edited the manuscript; all authors commented on the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at http://www.nature.com/nsmb/. Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/. 1. Mark, M., Ghyselinck, N.B. & Chambon, P. Function of retinoid nuclear receptors: lessons from genetic and pharmacological dissections of the retinoic acid signaling pathway during mouse embryogenesis. Annu. Rev. Pharmacol. Toxicol. 46, 451–480 (2006). 2. Germain, P. et al. International Union of Pharmacology. LX. Retinoic acid receptors. Pharmacol. Rev. 58, 712–725 (2006). 3. Gronemeyer, H., Gustafsson, J.A. & Laudet, V. Principles for modulation of the nuclear receptor superfamily. Nat. Rev. Drug Discov. 3, 950–964 (2004). 4. Perissi, V. & Rosenfeld, M.G. Controlling nuclear receptors: the circular logic of cofactor cycles. Nat. Rev. Mol. Cell Biol. 6, 542–554 (2005). 5. Chen, J.D. & Evans, R.M. A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377, 454–457 (1995). 6. Horlein, A.J. et al. Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377, 397–404 (1995). 7. Zamir, I. et al. A nuclear hormone receptor corepressor mediates transcriptional silencing by receptors with distinct repression domains. Mol. Cell. Biol. 16, 5458–5465 (1996). 8. Seol, W., Mahon, M.J., Lee, Y.K. & Moore, D.D. Two receptor interacting domains in the nuclear hormone receptor corepressor RIP13/N-CoR. Mol. Endocrinol. 10, 1646–1655 (1996). 9. Cohen, R.N. et al. The specificity of interactions between nuclear hormone receptors and corepressors is mediated by distinct amino acid sequences within the interacting domains. Mol. Endocrinol. 15, 1049–1061 (2001). 10. Hu, X. & Lazar, M.A. The CoRNR motif controls the recruitment of corepressors by nuclear hormone receptors. Nature 402, 93–96 (1999). 11. Perissi, V. et al. Molecular determinants of nuclear receptor-corepressor interaction. Genes Dev. 13, 3198–3208 (1999).
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12. Nagy, L. et al. Mechanism of corepressor binding and release from nuclear hormone receptors. Genes Dev. 13, 3209–3216 (1999). 13. Hu, X., Li, Y. & Lazar, M.A. Determinants of CoRNR-dependent repression complex assembly on nuclear hormone receptors. Mol. Cell. Biol. 21, 1747–1758 (2001). 14. Xu, H.E. et al. Structural basis for antagonist-mediated recruitment of nuclear corepressors by PPARA. Nature 415, 813–817 (2002). 15. Weston, A.D., Blumberg, B. & Underhill, T.M. Active repression by unliganded retinoid receptors in development: less is sometimes more. J. Cell Biol. 161, 223–228 (2003). 16. Rosenfeld, M.G., Lunyak, V.V. & Glass, C.K. Sensors and signals: a coactivator/ corepressor/epigenetic code for integrating signal-dependent programs of transcriptional response. Genes Dev. 20, 1405–1428 (2006). 17. Astapova, I. et al. The nuclear corepressor, NCoR, regulates thyroid hormone action in vivo. Proc. Natl. Acad. Sci. USA 105, 19544–19549 (2008). 18. Nofsinger, R.R. et al. SMRT repression of nuclear receptors controls the adipogenic set point and metabolic homeostasis. Proc. Natl. Acad. Sci. USA 105, 20021–20026 (2008). 19. Jepsen, K. et al. SMRT-mediated repression of an H3K27 demethylase in progression from neural stem cell to neuron. Nature 450, 415–419 (2007). 20. Germain, P., Staels, B., Dacquet, C., Spedding, M. & Laudet, V. Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58, 685–704 (2006). 21. Germain, P. et al. Differential action on coregulator interaction defines inverse retinoid agonists and neutral antagonists. Chem. Biol. 16, 479–489 (2009). 22. Madauss, K.P. et al. A structural and in vitro characterization of asoprisnil: a selective progesterone receptor modulator. Mol. Endocrinol. 21, 1066–1081 (2007). 23. Wang, L. et al. X-ray crystal structures of the estrogen-related receptor-G ligand binding domain in three functional states reveal the molecular basis of small molecule regulation. J. Biol. Chem. 281, 37773–37781 (2006). 24. Bourguet, W. et al. Crystal structure of a heterodimeric complex of RAR and RXR ligand-binding domains. Mol. Cell 5, 289–298 (2000). 25. Wong, C.W. & Privalsky, M.L. Transcriptional silencing is defined by isoform- and heterodimer-specific interactions between nuclear hormone receptors and corepressors. Mol. Cell. Biol. 18, 5724–5733 (1998). 26. Li, H., Leo, C., Schroen, D.J. & Chen, J.D. Characterization of receptor interaction and transcriptional repression by the corepressor SMRT. Mol. Endocrinol. 11, 2025–2037 (1997). 27. Nahoum, V. et al. Modulators of the structural dynamics of the retinoid X receptor to reveal receptor function. Proc. Natl. Acad. Sci. USA 104, 17323–17328 (2007). 28. Kallenberger, B.C., Love, J.D., Chatterjee, V.K. & Schwabe, J.W. A dynamic mechanism of nuclear receptor activation and its perturbation in a human disease. Nat. Struct. Biol. 10, 136–140 (2003). 29. Raghuram, S. et al. Identification of heme as the ligand for the orphan nuclear receptors REV-ERBA and REV-ERBB. Nat. Struct. Mol. Biol. 14, 1207–1213 (2007). 30. Yin, L. et al. Rev-erbA, a heme sensor that coordinates metabolic and circadian pathways. Science 318, 1786–1789 (2007). 31. Gurnell, M. et al. A dominant-negative peroxisome proliferator-activated receptor gamma (PPARG) mutant is a constitutive repressor and inhibits PPARG-mediated adipogenesis. J. Biol. Chem. 275, 5754–5759 (2000). 32. Marimuthu, A. et al. TR surfaces and conformations required to bind nuclear receptor corepressor. Mol. Endocrinol. 16, 271–286 (2002). 33. Zhang, J., Hu, X. & Lazar, M.A. A novel role for helix 12 of retinoid X receptor in regulating repression. Mol. Cell. Biol. 19, 6448–6457 (1999). 34. Kim, J.Y., Son, Y.L. & Lee, Y.C. Involvement of SMRT corepressor in transcriptional repression by the vitamin D receptor. Mol. Endocrinol. 23, 251–264 (2009). 35. Moran, E. & Jimenez, G. The tailless nuclear receptor acts as a dedicated repressor in the early Drosophila embryo. Mol. Cell. Biol. 26, 3446–3454 (2006). 36. Renaud, J.P., Harris, J.M., Downes, M., Burke, L.J. & Muscat, G.E. Structurefunction analysis of the Rev-erbA and RVR ligand-binding domains reveals a large hydrophobic surface that mediates corepressor binding and a ligand cavity occupied by side chains. Mol. Endocrinol. 14, 700–717 (2000).
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Ligands and peptides. Am580 was kindly provided by Reinhold Tacke (University of Würzburg). BMS493 was synthesized by A.R.d.L. The fluorescent peptides N-CoRNR1 (fluorescein–RLITLADHICQIITQDFAR), N-CoRNR2 (fluorescein–DPASNLGLEDIIRKALMGSFD), N-CoRNR1 short (LADHICQIITQDFAK–fluorescein), SRC-1 NR2 (fluorescein– RHKILHRLLQEGS) and the N-CoRNR1 peptide (RLITLADHICQIITQDFAR) were purchased from EZbiolab. The fluorescent S-CoRNR1 peptide (tetramethylrhodamine–RVVTLAQHISEVITQDYTR) was from Neosystem, and the fluorescent S-CoRNR2 peptide (fluorescein–TNMGLEAIIRKALMGKYDQWEE) was synthesized by J.F. Guichou (Centre de Biochimie Structurale). Plasmids. Gal–TIF-2.5 (harboring the nuclear interacting domain of human TIF-2 and referred to as Gal–TIF-2), Gal–SMRT (ID1 + ID2, containing human SMRT from 982 to the C terminus), the (17m)5×–G–Luc reporter, and the (RARE)3×– tk–Luc genes have been described previously21. Gal–N-CoR (ID1 + ID2 containing mouse N-CoR from 1629 to the C terminus) as well as Gal–N-CoR derivatives harboring mutated interaction domains (ID1 + ID2m and ID1m + ID2) have been described previously10. We generated Gal–SMRT derivatives harboring mutated interaction domains (ID1 + ID2m and ID1m + ID2) by using QuikChange site-directed mutagenesis with Pfu DNA polymerase (Stratagene), replacing ID1 residues Val2150 and Ile2151 with alanine and ID2 residues Ile2353 and Ile2354 with alanine. We used Gal–SMRT ID1 + ID2m to generate a single point mutation in ID1 by QuikChange site-directed mutagenesis with PfuUltra DNA polymerase (Stratagene). We PCR-amplified residues 176–393 (RAR $S3 $H12), 176–403 (RAR $H12) and 176–421 (WT RAR) of human RARA and ligated into NVP16 using a BglII site or into pET15b using NdeI-BamHI sites. We generated the RAR I396E mutant into pSG5–RARA by PCR-assisted site-directed mutagenesis with Deep Vent DNA polymerase (New England Biolabs) and into NVP16–RARA (176–421) or pET15b–RARA (176–421) by QuikChange sitedirected mutagenesis. We verified all constructs by DNA sequencing. Resolution of the RAR LBD–BMS493–N-CoRNR1 structure. We purified the human RARA LBD (residues 176–421) as previously described27. We mixed the protein with a two-fold molar excess of BMS493 and a three-fold molar excess of the N-CoRNR1 peptide and concentrated it to 3.5 mg ml−1. We mixed the complex in a 1:1 ratio with reservoir solution consisting of 18% (w/v) PEG 3350, 0.15 M NH4Cl and crystallized the complex using the sitting-drop vapor diffusion technique. We mounted a single crystal from mother liquor onto a cryoloop, soaked in the reservoir solution containing an additional 25% (v/v) glycerol and flash-froze it in liquid nitrogen. We collected diffraction data using an ADSC Quantum 4 detector at the ID14-2 beamline of the ESRF at 2.1-Å resolution. We processed diffraction data using MOSFLM37 and scaled them with SCALA from the CCP4 program suite38. We obtained the initial phases by molecular replacement with MOLREP38 using the structure of the RARA–RXRA heterodimer24 (PDB 1DKF). Initial Fo – Fc difference maps had strong signal for the ligand and for the peptide, which we could fit accurately into the electron density. We built the model with Coot39 and refined it with REFMAC38 using restrained refinement and individual B-factor refinements. Resolution of the RAR LBD–Am580–SRC-1 NR2 structure. We mixed the protein with a two-fold molar excess of Am580 and a three-fold molar excess of
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the SRC-1 NR2 coactivator peptide and concentrated it to 7 mg ml−1. We mixed the complex in a 1:1 ratio with reservoir solution consisting of 15% (w/v) PEG 6000, 5% (v/v) glycerol. We mounted a single crystal from mother liquor onto a cryoloop, soaked it in the reservoir solution containing an additional 25% (v/v) glycerol and flash-froze it in liquid nitrogen. We collected diffraction data using an ADSC Quantum 4 detector at the ID14-2 beamline of the ESRF at 1.8-Å resolution. We processed diffraction data using MOSFLM37 and scaled them with SCALA from the CCP4 program suite38. We solved the structure and refined it using the protocol described above. Preparation of RAR LBD for fluorescence anisotropy experiments. We performed protein labeling and purification as previously described27,28. Steady-state fluorescence anisotropy. We performed assays using a Safire microplate reader (TECAN) with the excitation wavelength set at 470 nm and emission measured at 530 nm for fluorescein-tagged peptides and with the excitation wavelength set at 530 nm and emission measured at 580 nm for the tetramethylrhodamine-tagged peptide. The buffer solution for assays was 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 5 mM DTT and 10% (v/v) glycerol. We initiated the measurements at the highest concentration of protein (10 MM) and diluted the protein sample successively two-fold with the buffer solution. For each point of the titration curve, we mixed the protein sample with 4 nM of fluorescent peptide and 30 MM of ligand (final concentrations). We fitted binding data using a sigmoidal dose-response model (GraphPad Prism, GraphPad Software). The reported data are the average of at least three independent experiments. Time-resolved fluorescence anisotropy decay. We carried out time-resolved fluorescence with a KOALA multi-frequency fluorometer (ISS, Inc.) phase locked with a ps mode-locked titanium sapphire Tsunami laser with a 10-W pump (Spectra-Physics) tuned to 900 nm, then picked at 4 MHz and doubled to 450 nm for excitation of fluorescein. We used fluorescein at pH 8.0 with a lifetime of 4 ns as a reference compound. We detected emission at 530 nm through a monochromator using 16-nm slits. We analyzed data using the Globals, Unlimited Software (University of Illinois, Urbana-Champaign) in terms of a double exponential anisotropy decay. Transient transfection assays. We cultured HeLa and COS cells in DMEM with Glutamax and 10% (v/v) FCS and transfected them using JetPei transfectant (Ozyme). After 24 h, we changed the medium to a medium containing the indicated ligands or vehicle. We lysed cells and assayed them for reporter expression 48 h after transfection. We used the luciferase assay system according to the manufacturer’s instruction (Promega). In each case, we normalized results to coexpressed B-galactosidase. We carried out each transfection in duplicate and repeated each three to six times.
37. Leslie, A.G. The integration of macromolecular diffraction data. Acta Crystallogr. D Biol. Crystallogr. 62, 48–57 (2006). 38. CCP4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994). 39. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
doi:10.1038/nsmb.1855
