Subtype-Specific Modulation of Estrogen ReceptorâCoactivator

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Subtype-specific modulation of Estrogen Receptor– Coactivator in-teraction by phosphorylation Inga M. Tharun, Lidia Nieto, Christian Haase, Marcel Scheepstra, Mark Balk, Sabine Moecklinghoff, Wencke Adriaens, Sonja A. Dames, and Luc Brunsveld ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 03 Nov 2014 Downloaded from http://pubs.acs.org on November 4, 2014

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ACS Chemical Biology

64x36mm (300 x 300 DPI)

ACS Paragon Plus Environment


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Inga M. Tharun,1,‡ Lidia Nieto,1,‡ Christian Haase, 1 Marcel Scheepstra,1 Mark Balk,1 Sabine

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Möcklinghoff,1 Wencke Adriaens,1 Sonja A. Dames,2* Luc Brunsveld1*

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1

Laboratory of Chemical Biology and Institute of Complex Molecular Systems, Department of Biomedical Engineering,

Eindhoven University of Technology, Den Dolech 2, 5612AZ Eindhoven, The Netherlands; 2

Chair of Biomolecular NMR Spectroscopy, Department of Chemistry, Technische Universität München, Lichtenbergstr. 4,

85747 Garching, Germany & Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany; ‡These authors contributed equally; *[email protected]; [email protected]

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ABSTRACT The Estrogen Receptor (ER) is the number one target for the treatment of endocrine responsive breast cancer, and remains a highly attractive target for new drug development. Despite considerable efforts to understand the role of ER post-translational modifications (PTMs), the complexity of these modifications and their impact, at the molecular level, are poorly understood. Using a chemical biology approach, fundamentally rooted in an efficient protein semi-synthesis of tyrosine phosphorylated ER constructs, the complex role of the ER tyrosine phosphorylation is addressed here for the first time on a molecular level. The semi-synthetic approach allows for the site-specific introduction of PTMs as well as biophysical probes. A combination of biophysical techniques, including NMR, with molecular dynamics studies reveal the role of the phosphorylation of the clinically relevant tyrosine 537 (Y537) in ER and the analogous tyrosine (Y488) in ER. Phosphorylation has important effects on the dynamics of the ER Helix 12, which is centrally involved in receptor activity regulation, and on its interplay with ligand and cofactor binding, but with differential regulatory effects of the analogous PTMs on the two ER subtypes. Combined, the results bring forward a novel molecular model of a phosphorylation-induced subtype specific ER modulatory mechanism, alternative to the widely accepted ligand-induced activation mechanism.

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ACS Chemical Biology The ligand-inducible transcription factors Estrogen Receptor (ER)  and  belong to the protein superfamily of Nuclear Recep-

tors (NR) and serve as versatile drug targets, such as in hormone therapy, as cancer treatments, or contraception. NRs share a common architecture of structurally and functionally conserved domains, including a ligand binding domain (LBD), harboring a ligand binding pocket.1 Crystal structures have revealed a conserved -helical rich fold of the LBD and the structural impacts of agonistic and antagonistic ligands.2,3 Together with in vivo and in vitro studies,4 a widely accepted NR activation mechanism has been established.5 A central role in the translation of ligand-binding into the activation of the receptor, is assigned to the structural rearrangement of the most C-terminal -helix, helix 12 (H12), of the ER LBD (hereafter abbreviated as ER). Upon binding of agonists, such as 17-estradiol (E2), the orientation of H12 changes from a state of enhanced free motion and distal orientation (‘open’ conformation) to a stabilized and compact orientation via interactions with helices 3 and 4 (‘closed’ conformation), 2,6 resulting in the formation of the coregulator binding surface (Figure 1, top). Coregulator proteins interact with the LBD via a conserved helical LXXLL sequence motif (“NR box”).7 The agonistic closed conformation of H12 induces the formation of a ‘charge clamp’ and a hydrophobic groove, 5 to which the coregulator binds via its helix’ electrostatic dipole and by the accommodation of the hydrophobic leucine-residues. Other types of ligands induce different H12 orientations, such as the repressive closed conformation, which has been revealed for the antagonist 4-hydroxy-tamoxifen (OHT).8

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Figure 1. Schematic overview of the molecular mechanism of ER LBD-coactivator (LXXLL) interaction induced by an agonistic ligand

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The complex network of ligand-dependent protein-protein interactions (PPIs), involved in ER regulation is additionally regulat-

(E2) (top). An alternative, ligand-independent ER-coactivator interaction, is triggered by tyrosine phosphorylation (P) (bottom). Both pathways start from a ligand-free flexible ‘open’ conformation and induce a ‘closed’, active conformation, instating a coactivator binding surface (light blue) on the LBD.

ed by post-translational modifications (PTMs).9 Of specific interest in this respect are tyrosine 537 (Y537) in ER and, though less described, the analogous tyrosine 488 (Y488) in ER, which undergo context-specific10 ligand-independent phosphorylation by Src-kinase11–13 and are located at the loop-helix transition – or “capping motif”14 – immediately N-terminal of H12.2,15 Phosphorylation of Y537 and Y488 may therefore exert an important contribution to the dynamics of this C-terminal region and thereby have regulatory influence on ER activity14 (Figure 1, bottom). Mutations of this tyrosine residue or of the amino acid residues in the immediate vicinity have been shown to have dramatic effects on H12 dynamics and ER function.14,16–19 Furthermore, the phosphorylation status of this tyrosine has been implied to be of functional importance mainly for the ER with respect to ligand binding affinity and kinetics, DNA binding, transactivation and PPIs.11–13,20–23 Mutagenesis studies of the tyrosine to aspartic or glutamic acid have shown a significantly increased activity of these mutants in the absence of ligand and can even render the receptor consecutively active.24,25 Clinical studies have recently revealed ER Y537 mutations to be activating in hormone-resistant metastatic breast cancer.26–30 The responsible molecular mechanism has however remained unresolved. H12 dynamics and structural integrity

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underlie a fine-balanced regulation between active and inactive states.2,5,31 Molecular elucidation of the effect of tyrosine phosphor-

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Here, we report the generation of well-defined ER and ER constructs featuring either a phosphorylated or non-

ylation is therefore of great interest, regarding the clinically reported resistance inducing mutations,26–30 cancer cell overexpression of tyrosine kinases responsible for ER phosphorylation,32 and new ligand development. A major limitation for performing this molecular analysis has been the inaccessibility of well-defined phosphorylated ER constructs for biochemical and structural studies.

phosphorylated Y537/Y488 (hereafter summed up as (p)ER) in combination with a site-specifically introduced fluorescent probe or 15

N-labeled amino acids, via an efficient protein semi-synthesis approach (Figure 2).15 Biophysical studies on these semi-synthetic

proteins in combination with molecular dynamics (MD) studies allowed to directly probe the effect of ER-Y537 and ER-Y488 phosphorylation on the molecular properties of ER H12 and its role in modulating the ER–coactivator binding. The results reveal the molecular basis for a phosphorylation dependent pathway for subtype selective ER modulation.

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Protein semi-synthesis and labeling. Previous work showed that enzymatic phosphorylation of the tyrosine leads to incomplete mixtures of phosphorylated proteins.15 An expressed protein ligation strategy was therefore devised to obtain ER constructs featuring a phosphorylated tyrosine and additional molecular probes (Figure 2).15,33 ER protein constructs, constituting helices 1-11 of the ER and  (K302-K529 and L260-K480, respectively) were recombinantly expressed with a C-terminal thioester (Figure 2A). The peptide fragments comprising the loop between helix 11 and 12 and H12 were synthesized by solid phase peptide synthesis (Figure 2B), with and without tyrosine phosphorylation, as well as featuring a site-directed insertion of a C-terminal fluorophore (fluorescein, FAM) or 15N-labeled amino acids. The ER and ER thioesters and the C-terminal peptides were ligated at pH 8.0 with a three-fold excess of peptide under reducing conditions at 4°C. After an incubation time of 16 hrs, the reaction typically went to > 95% completion. In total a library of 10 different ER constructs (Figure 2C, Table S3) was prepared that was used for the molecular studies described below.

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A ERα LBD

B

ERβ LBD

H12 (p)Y α: H-CKNVVPL(p)YDLLLEMLDAHRLHAPT-CONH2 β: H-CKNVVPV(p)YDLLLEMLNAHVLRG-CONH2

H12 (p)Y-FAM α: H-CKNVVPL(p)YDLLLEMLDAHRLK(FAM)-CONH2 β: H-CKNVVPV(p)YDLLLEMLNAHVLK(FAM)-CONH2

H12 (p)Y-15N-labeled β: H-CKNVVPV(p)YDLLLEMLNAHVLRG-COOH

C ERα LBD

ERβ LBD

F

F ERα LBD

ERβ LBD

ERβ LBD

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Figure 2. Protein semi-synthesis of site-specifically labeled phosphorylated ER constructs. A) H12 truncated ER and ER expressed and

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Phosphorylation modulates coactivator binding subtype specific. Fluorescence Polarization (FP) has seen application for

purified to yield a C-terminal thioester. B) Library of 10 H12 peptides, carrying a tyrosine (5x) or a phosphotyrosine (5x) and additionally a C-terminal fluorescein-label (FAM, ‘F’) or 15N-labeled amino acid residues (bold and underlined). C) Final two sets of 5 phosphorylated (pY) and 5 non-phosphorylated (Y) semi-synthetic ER and ER constructs featuring diverse biophysical probes.

NRs on PPARγ-LBD16 as well as full length ER.31 In the FP assay devised here, binding events of ligand or coactivator that influence H12 positioning, orientation, and dynamics, result in changes in the fluorophore polarization. A low polarization indicates a more flexible structure and an increased polarization relates to a rigidification of the H12 region, in line with a closed conformation

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of the LBD (Figure 1). Five different coactivator peptides, based on native NR coregulators involved in cellular ER pathways, were

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Comparison of the peptide binding affinities of ER in its phosphorylated and non-phosphorylated state (ER vs. pER) re-

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used to probe for potential sequence-dependent effects (Table 1). In the serial coactivator peptide titrations (Figure 3, S4 and S5), the presence of E2 generally lowered the KD values of the ER variants for the interaction with the tested coactivator peptides (Table 1). This observation is in line with the general consensus that ligand binding stabilizes the agonistic conformation of the protein and enhances the affinity for LXXLL motifs (Figure 1, top).

veals small modulatory effects of the tyrosine phosphorylation. Phosphorylation for example slightly decreased the affinities of ER without E2 for peptide (2) and (4) while having no effect on the interaction with peptide (1). In contrast to ER, phosphorylation of ER in the absence of E2 strongly increased the affinity for almost all tested coactivator peptides to the same order of magnitude as E2 binding. (ER -E2 vs. pER -E2). Subsequent E2 binding to the phosphorylated ER constructs resulted only in a small further enhancement of the pER-LXLLL binding affinities (pER -E2 vs. pER +E2). The ER tyrosine phosphorylation thus introduces agonist-like affinities towards the coactivator-peptides, implying that it induces conformational or dynamic alterations that result in a shift of the conformer-equilibrium towards a “closed” agonist-like state even in the absence of ligand. Insights in the underlying structural and dynamic alterations of the protein can be obtained from the polarization values at low coactivator peptide concentrations (Figures 3, S4, and S5). High levels of polarization (190 ± 10 mP) presumably confer to a more closed conformation of H12 and low levels of polarization (160 ± 10 mP) to a more flexible open H12 conformation. High starting levels of polarization were observed in the presence of E2 for both phosphorylated and non-phosphorylated ER. This indicates that the ER-H12 adopts a closed conformation in the presence of E2. Analysis of the ER variants in the ligand free state revealed low polarization levels at low cofactor concentrations. Interestingly, the phosphorylation induces the transition of the ligand-free pERH12 towards the closed conformation at lower coactivator peptide concentrations.

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Figure 3. Representative fluorescence polarization (FP) data obtained from titrations of FAM-labeled ER and ER with coactivator pep-

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A different pattern was observed for the polarization levels of ER. At low coactivator peptide concentrations the starting levels

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tides. A) Peptide (1) titrated to FAM-ER. B) peptide (3) titrated to FAM-ER, both in their phosphorylated and non-phosphorylated state (circles and squares, respectively) as well as in the presence (black and blue) and absence (green and red) of agonist 17-estradiol (E2).

of three of the four ER proteins reside around 160 mP, only the ligand-bound and phosphorylated ER (pER) shows high starting polarization levels. In contrast to ER, the E2-bound ER-H12 is thus in a dynamic open state and requires phosphorylation to induce the closed conformation. This observation adds another level of complexity to the activation model of ER, as saturation of the ligand binding site with E2 does not imperatively induce a closed conformation to ER. The modulatory effect of the phospho-group on ER and ER dynamics is also reflected in levels of the polarization values at high cofactor concentrations. Interestingly, these levels differ depending on the ER phosphorylation status. The cofactor-bound conformation is thus also fine-tuned by the phosphorylation status. This observation is in line with the differential cofactorpreferences which previously evolved for the phosphorylated and non-phosphorylated ER in a cofactor in vitro evolution approach.33

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Table 1. KD values (M) for the binding of coregulator peptides to fluorescently labeled phosphorylated or non-phosphorylated ER and ER constructs in the absence or presence of estradiol (E2); Colours are shown as in Figure 3; n.d., not determined

i

(Peptide)i

(1)

(2)

(3)

(4)

(5)

ER -E2

6.1 ± 0.9

2.0 ± 0.5

n.d.

61.7 ± 0.4

n.d.

ER +E2

0.4 ± 0.04

0.2 ± 0.04

n.d.

3.6 ± 0.7

n.d.

pER -E2

4.4 ± 0.05

3.7 ± 0.6

n.d.

101 ± 9

n.d.

pER +E2

0.3 ± 0.04

0.6 ± 0.1

n.d.

3.2 ± 0.4

n.d.

ER -E2

1.8 ± 0.1

14 ± 1

13 ± 1

26 ± 2

6.9 ± 0.4

ER +E2

0.030 ± 0.001

0.18 ± 0.02

0.12 ± 0.02

0.24 ± 0.05

0.05 ± 0.01

pER -E2

0.08 ± 0.01

0.24 ± 0.02

0.32 ± 0.02

0.76 ± 0.07

0.16 ± 0.02

pER +E2

0.025 ± 0.004

0.13 ± 0.03

0.07 ± 0.01

0.09 ± 0.01

0.05 ± 0.01

peptide sequence and protein origin (1) PRQGSILYSMLTSAKQT (NR0B1); (2) LTERHKILHRLLQEGSPSD (NCOA1); (3)

SKGQTKLLQLLTTKSDQ (NCOA2); (4) AEEPSLLKKLLLAPANT (PGC-1); (5) AANNSLLLHLLKSQTIP (NRIP1)

On-chip coactivator peptide binding array34 was used to evaluate ER binding to multiple recognition sequences (Figure 4 and S6-S8, Table S5). The different protein constructs were incubated on a membrane which carries 53 different coactivator and corepressor peptides, immobilized on individual spots. The binding levels of phosphorylated and non-phosphorylated ER and ER were quantified, each in the presence and absence of ligand. The relative changes in binding levels between two different states e.g. phosphorylated vs. non-phosphorylated or ligand-bound vs. ligand-free ER - are plotted in Figure 4. Addition of E2 to ER and to ER induced a strong increase in ER binding levels to the majority of the peptides (Figure 4A-B). The response of pER to E2 (Figure 4C) appears even slightly stronger than for non-phosphorylated ER, in line with the fluorescence polarization study. In contrast, the changes of pER binding levels upon addition of E2 are minor (Figure 4D), also in line with the small effects observed for this event in the FP assay. The effects of phosphorylation on coactivator binding are displayed in Figure 4E-H. In the absence of E2, phosphorylation of ER has minor effects on the binding levels (Figure 4E). In contrast, phosphorylation of ER in the absence of E2 leads to a strong increase in cofactor binding levels (Figure 4F). This again points to a ligand-independent activation of ER. The effect of phosphorylation on ER in the presence of E2 revealed a small overallincrease of cofactor recruitment. Specifically, ER-phosphorylation induced an increase of binding to a set of, generally weakly binding, corepressor sequences, vide infra (highest bars in Figure 4G, Figure S8 Table S5). Phosphorylation of ER in the presence of E2, does not lead to any, additional, changes in the binding levels (Figure 4H). This platform thus reveals the effects of ER and ER phosphorylation on a large group of native cofactor peptides and the results are in line with the FP results and highlight an ER subtype specific regulation mechanism by tyrosine phosphorylation.

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Figure 4. Changes in ER (left) and ER (right) binding levels towards a set of 53 coregulator sequences, screened by an on-chip binding array upon addition of E2 (A-D) and upon phosphorylation (E-H). Changes in binding levels upon E2 stimulation by ER (A) and ER (B) are by the phosphorylated pER (C) and pER (D) Changes in binding levels upon phosphorylation of ER and ER in the absence of E2 (E and F, respectively) and in the presence of E2 (G and H, respectively). Details can be found in Supporting Figure 8 and Supporting Table 5.

Local structural effect of tyrosine phosphorylation. CD spectroscopy was employed to monitor the effect of phosphorylation on the secondary structure of the isolated C-terminal protein sequences, for both ER and ER (H12 and H12, respectively). Based on crystal structures of ER and ER, this C-terminal region consists of a loop and a short H12 in the agonist-bound state.2,15 The CD spectra of the non-phosphorylated peptides (Figure 5A, green) feature a signature typical for α-helical peptides with θ222/θ208 ratios of 0.6 and 0.9 (H12 and H12, respectively). Tyrosine phosphorylation leads to a CD signature with a lowered αhelical character (Figure 5A, red) for both peptides. The θ222/θ208 ratios decreased to 0.5 for both phosphorylated sequences (pH12 and pH12), suggesting a disruption, at least to some extent, of the initial length of helix 12, by the phosphorylation.



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Figure 5. A) Far-UV CD spectra of the C-terminal peptides of ER and ER in non-phosphorylated (green lines, H12 and H12, respectively) and phosphorylated state (red lines, pH12 and pH12, respectively). B) Sequence alignment of amino acids of the loop region preceding H12 for several human steroid NRs and hRXRa. * Position of a Proline conserved among steroid NRs.

Phosphorylation of amino acid side chains within α-helices can have stabilizing or destabilizing effects on the secondary structure, depending on the location within the helix as well as the sequence context. The tyrosine of interest is located at the N-terminus of the H12 core motif of ER and ER and is part of the H12 capping motif.14 Additionally, this tyrosine is part of the intramolecular H12 corepressor motif P(L/V)YDLLLEML (amino acid sequence of the ER/ core motif with the corepressor motif in bold), and its molecular status will thus impact the role and conformation of H12. The other steroid NRs feature a conserved proline at the ER tyrosine position (Figure 5B, * symbol).1,35 This proline contributes to the orientation of H12 and acts as H12 cap. In ER, the tyrosine thus takes over the role of the proline cap and the effectiveness thereof is potentially mediated via its phosphorylation. ER and ER actually feature a proline residue 2 positions more N-terminal of the tyrosine (Figure 5B). The different location of this proline with respect to the H12 core motif of the other steroid NRs implies differences in the (nonphosphorylated) H12 length and consequently differences in helicity of the intramolecular cofactor binding motifs. The general importance thereof has been associated with different cofactor interaction tendencies.35,36 We have observed, for instance, an enhanced binding of NCOR1 and NCOR2 corepressor sequences in the case of ligand-bound phosphorylated ER (Figure 4G, Figure S8G, Table S5). These results support the hypothesis that the competition of the corepressor binding motif, located in H12, with the corepressor sequences is reduced by disruption of the helicity of the internal corepressor motif in H12. Crystal structures of ligand-free ER are not available. However, in the ligand-free structure of the NR RXR,37 H11 extends into H12 without being interceded by a flexible loop. Remarkably, RXR and ER both feature the aforementioned common proline at the same upstream position (Figure 5B). This implies that in the unliganded state the H12 of ER extends until this proline and that


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tyrosine phosphorylation shortens the length of H12, to that typically observed in the agonistic state in the crystal structure15 and in

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line with the proline in the other steroid NRs.1,35 Ligand binding may thus induce a partial unfolding of the ER H12 helical segment, to enable the shortened H12 to fold back on to the LBD. Tyrosine phosphorylation facilitates this process and consequently directs the equilibrium of ER conformers to the agonistic state. Local structural and dynamic changes upon phosphorylation. Solution NMR provides a powerful tool to monitor conformational and dynamic changes of a protein fold on a per-residue basis. NMR spectroscopy on NR-LBDs is challenging due to the size of the protein domain and their instability at high concentrations and has only seen limited application to NRs.38,39 To monitor the effect of Y488 phosphorylation on the conformation of ER on a local level, we used ER constructs containing three 15N-labeled amino acids in the C-terminal region. We chose to focus on ER since it showed more pronounced dynamic and interaction differences upon phosphorylation and is more stable and better soluble in solution than the ER. Figure 6A-B shows the crystal structure of human ER phosphorylated at Y488 in the presence of E2 and peptide (2) (PDB-id 3OLL) and a close up of the region around H12. (For the non-phosphorylated form and a superposition of both structures see Figure S9.) 15N-K482 is located in the loop region directly following H11. 15N-V487 is directly preceding Y488, and 15N-A497 resides at the end of the short -helix H12 that is flanking the binding sites for the coactivator peptide (2) and E2. Eight different protein samples were prepared for NMR studies; non-phosphorylated and phosphorylated ER in the free state, in the presence of only E2 or peptide (2), and in the presence of both E2 and peptide (2). Figure 6C-D shows superpositions of the 1

H-15N-HSQC spectra of the non-phosphorylated and phosphorylated ER in the four states as well as the spectra for each state

separately. The 1H-15N-HSQC spectra show strong signals for V487 and A497, which could be clearly assigned based on the 15NNOESY spectra of the free and complexed states (Figure S10-12). The signal(s) for K482 showed almost no NOE correlations and were mainly assigned based on the exclusion principle.



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Figure 6. Structure of the human phosphorylated ER in complex with E2 (orange) and peptide (2) (blue ribbon) (PDB-id 3OLL). A) Ribbon representation of the full ligand-binding domain in grey. B) Close up of the ligand-binding region. The three 15N-labeled residues (K482, V487, A497) are depicted in green, and pY488 in red. Helices 11 and 12 are indicated by the labels H11 and H12. The stretch around pY488 is presumably stabilized by polar interactions with the positively charged side chain of K300 (dark blue), whereas K482 (green) may make polar interactions with E291 and E371 (both in magenta). C and D) Comparison of the 1H-15N-HSQC spectra of ER (C) and pER (D) in the free state (black), in the presence of peptide (2) (red), in the presence of estradiol (E2, green), and in the presence of E2 and peptide (2) (blue). The upper plot in C) and D) shows a superposition of the four spectra. Lines label peaks that represent additional conformational states of the respective residue. Dotted lines indicate that the respective peaks could not be fully unambiguously assigned.


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In previous NMR studies of the PPAR LBD the resonances for residues of H12 were not visible.38,40 For the ER, all three 15Nlabeled residues could be observed in the apo form if the spectra were recorded at low temperatures (10°C), expected to lower the backbone dynamics. In the 1H-15N HSQC spectra of the non-phosphorylated ER (Figure 6C) A497 shows generally two neighboring peaks, indicating the existence of two distinct conformers in slow chemical exchange. Binding of E2 resulted in overall broader and lower peak intensities (green spectrum), suggesting H12 resonances to be in intermediate chemical exchange. In contrast, binding of peptide alone or in combination with E2 resulted in a clear increase of V487 and A497 signal intensities (red and blue spectra), which may be explained by a stabilization of the conformation of this segment compared to the apo ER. This is consistent with the observed changes in the FP studies upon titration with peptide (Figure 3B) and the increase of the NOE intensities in the 15

N-NOESY-HSQC data in the presence of peptide. Ligand or peptide binding thus lead to differences in the populations of the

sampled conformations and the time scales on which they are populated.41 Figure 6D shows the 1H-15N-HSQC spectra of the phosphorylated ER in the four different states. In the spectrum of the apo form, a single peak is visible for A497. Whereas K482 and V487 appeared to show multiple peaks, which indicate the presence of local structural heterogeneity in this segment. This is in line with the observation that tyrosine phosphorylation reduces the helical content of this sequence (Figure 5A). Addition of only peptide (2) resulted in even more peaks for K482 and a shoulder in the peak assigned to A497 (red spectrum), while binding of E2 alone or with peptide (2) reduced the number of peaks (green and blue spectra). Importantly, the set of peaks observed for K482 in the free and peptide (2)-bound state merged into a single resonance when E2 was added, around the same position as the one observed for the non-phosphorylated protein. In the ligand bound state K482 potentially makes polar interactions with E291 at the N-terminal end of -helix 2 and with E371 in the loop between helices 6 and 7, and pY488 with the positively charged side chain of K300 (Figure 6B). However, in the free form or in the presence of only the peptide, increased flexibility could enable polar interactions of K482 with the phosphate group of Y488. The reduction of the number of peaks to a single resonance may thus be explained by a reduced conformational heterogeneity and a stronger population of an agonistic-like conformational ensemble. 15

N-relaxation data were recorded for all eight samples to monitor the dynamic structural changes 42 that occur upon lig-

and/peptide binding. The respective 15N-T1, -T2 and 1H-15N-NOE values of the 15N-labeled residues K482, V487, and A497 are listed in Table S7 together with an additional explanation. In line with literature,40 the 15N-relaxation data for ER indicates that the C-terminal region is overall rather flexible. Clearly negative 1H-15N NOE values for K482 in all states suggest that the loop region harboring this residue is very flexible in both the phosphorylated and non-phosphorylated proteins and that ligand binding does not significantly restrict this flexibility. In comparison, V487 and A497 showed overall higher 1H-15N NOE values around 0 that slightly increased upon binding of both E2 and/or peptide (2). The observed difference in the 15N-T1 and -T2 times suggest that phosphorylation modulates the populated conformational equilibrium of the H12 region, which is the basis for the observed higher affinity for coactivator peptides in the absence of E2 (Table 1). The observed reduction in the number of peaks as well as the further observed changes in 15N-relaxation times upon E2 and/or peptide (2) binding may correspond to a stabilization of an ensemble of multiple conformational states to one with reduced populations in some of the conformational states. 40 Molecular Dynamics highlight stabilizing effect phosphorylation. MD has proven to be a useful tool in the analysis of NR dynamics.43,44 To further elucidate the atomistic mechanism of the phosphorylation mediated modulation of the ER, MD simulations were performed in different molecular contexts: the ER/coactivator peptide complex in absence (1) and presence of E2 (2), the phosphorylated ER/coactivator peptide complex in absence (3) and presence of E2 (4), in all the aforementioned cases with the starting coordinates of H12 in a ‘closed’ conformation; and the ER/coactivator peptide complex in absence of E2 for the non-



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phosphorylated (5) and phosphorylated (6) forms with H12 initial coordinates in an ‘open’ conformation. 10-20 ns MD simulations

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were run using the ER X-ray structures15 as initial models. The conformational stability of non-phosphorylated and phosphorylated ER in the different complexes was analyzed by inspecting the main chain root mean square deviations (RMSDs), as a function of the simulation time (Figure 7A). The simulation trajectories of the E2-bound proteins with H12 in the ‘closed’ conformation showed very similar trends, with no major difference caused by Y488 phosphorylation. In contrast, an increase of the RMSD was observed for the complexes in the absence of E2. A RMSD value of 2.57 Å was obtained for the non-phosphorylated ER and of 2.11 Å for the phosphorylated ER. For the model with H12 in an open conformation, RMSD values of 2.93 Å and 2.65 Å were obtained for the non-phosphorylated and phosphorylated models, respectively. Tyrosine phosphorylation thus stabilizes the agonistic fold of the ER-coactivator peptide complex in the absence of E2. The root mean square fluctuation (RMSF) of C atoms relative to the average structure over the 20 ns simulations was also calculated as an indicator of structural flexibility (Figure 7B-C). B-factors obtained from the MD simulations for the ER-E2-coactivator peptide complexes were well in agreement with crystallographic B-factors (Figure 7B-C and S13). Whereas the majority of the protein helices showed overall rather small conformational fluctuations, all the loop regions showed higher RMSF values. Loop H8-H9 was the most flexible during the simulations, which agrees with the crystallographic data, where part of this loop is unresolved. Importantly, the loop H11-H12 and H12 behaved differently in the analyzed trajectories. The nonphosphorylated ER-coactivator peptide complex in the absence of E2 showed overall significantly higher RMSF values than the phosphorylated one. Particularly, residues of the regions H2, H3, loop H6-H7, H6, H7, and H12 showed a higher mobility for the non-phosphorylated ER. In contrast the simulation with the phosphorylated ER in the absence of E2 showed very similar RMSF values to the E2 bound proteins. The RMSF differences between the simulations can further be illustrated if mapped on the respective three-dimensional structures (Figure 7D). Residues in green highlight regions showing a decreased conformational flexibility, and those in red an increase when comparing the RMSF values among the different simulations. Phosphorylation of ER reduces the conformational flexibility to a similar extent as E2-binding. Subsequent binding of E2 to the phosphorylated ER protein does not further restrict the conformation of loop H11-H12 and H12. This observation explains the little changes in cofactor binding levels previously observed for pER in response to E2 (Figure 4D). Atomic analysis of RMSD values and phi/psi dihedral angles distributions in the peptide-bound proteins showed that Y488 phosphorylation exhibits also local effects in the vicinity of H12. In the case of the non-phosphorylated protein, the residues of the C-terminal H12 alpha-helix showed a wider range of phi-, psi- values, with rapid 3-10/helix transitions (Figure S14), suggesting an increased mobility for H12, compared to the phosphorylated counterpart. Phosphorylation of Y488 is able to stabilize the Cterminal H12 alpha-helix, as seen in the RMSD trajectories presumably via a destabilization of the loop upstream of H12 (Figure S14 and S15). Moreover, an increased mobility of the loop preceding H12 was observed upon phosphorylation, in line with the CD data (Figure 5A). In fact, in the peptide-bound state of the protein, in absence of E2, phosphorylation of Y488 results in two different populations of phi/psi angles for the residues in the segment K482-V485. Phosphorylation of Y488 also modulates to some extent the salt bridge network within the vicinity of Y488 in ER (Figure 6B). For instance, K482 of the non-phosphorylated ER makes mainly polar interactions with E291 at the N-terminal end of H2. In contrast, K482 of the phosphorylated ER also makes contacts with E371, in the loop between H6 and H7, giving rise to an increased flexibility around K482. The negative local charge of the phosphorylated Y488 is felt by the nearby K300, only in the presence of E2, giving conformations with shorter distances between these two residues (Figure S16). In agreement with the experimental results, the MD simulations thus corroborate the observations that phosphorylation induces local and global ordering effects, resulting in an increase in the overall stability.


Page 15 of 18

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Figure 7. A) RMSD trajectories and B-C) RMSF values, obtained along 20 ns simulations for the different models with H12 in the ‘closed’ conformation. D) Illustration of the differences obtained in RMSF values for the different models with H12 in the ‘closed’ conformation



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on the respective three-dimensional structures. Highlighted in green are residues with increased structural stabilization whereas in red are

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residues that show a destabilization.

Conceptual model of tyrosine phosphorylation effects. The accepted model of NR activation is based on a ligand-induced activation that shifts the conformational equilibrium from a population of inactive conformers to the active form.5 The data reported in this manuscript bode for an extension of this model to incorporate the role of post-translation modifications. Specifically for ER, the tyrosine phosphorylation accounts for a similar shift of the conformational equilibrium to the active form (Figure 1). Depending on the ER subtype, this shift is of different strength, with for ER a stabilization of the agonistic state when bound to an agonist and for ER an almost full agonistic state solely upon phosphorylation (in the absence of agonist). The subtype specific effect will in part result from interplay between H12 and the LBD core. The molecular effect of the phosphorylation can be traced back to a destabilization of the loop preceding H12 and the disruption of the H12 capping motif, enabling the orientation of H12 on the ER surface in an agonistic state. The ER tyrosine phosphorylation event takes over the role of a proline residue present in the other steroid NRs, in limiting the H12 length for an optimal agonistic receptor conformation. The tyrosine 537 in ER and potentially 488 in ER are regulatory sites for ER activity via phosphorylation dependent mechanisms. Biochemical and cellular studies on ER have previously indicated a key role for this amino acid in controlling protein activity and conformation 20,23,25 and Y537 has recently been identified as a frequently occurring key mutation site in metastatic breast cancer patients.26,27,29 The molecular basis of the role of the tyrosine located at the beginning of H12 and its phosphorylation has remained elusive. The preparation and structural and functional characterization of a library of ER/ constructs in both nonand phosphorylated state and decorated with diverse molecular probes allowed dissecting the molecular role of this tyrosine in the interplay with its phosphorylation. Tyrosine phosphorylation leads to a remarkable ligand-independent subtype specific increase in coactivator affinity, highlighting an alternative, PTM mediated, activation mechanism for ER, in line with cell biological observations.24,25 This suggests that ER activity can be regulated by the tyrosine phosphorylation alone, for example in cancer cells with upregulated kinase activity. 32 The differences observed for the phosphorylation effects between ER and ER probably indicate an allosteric interplay with the non-homologous surrounding amino acids in the protein fold as the basis for the differential regulatory effects of an analogous PTM on the two ER subtypes. This molecular picture shows how a single tyrosine phosphorylation in the ER can lead to severe structural and functional changes of the protein. Such insights are at the basis to develop novel antagonists which are less prone to lead to resistance or avoid tyrosine phosphorylation, and provide a first molecular relationship between the kinase activity state of a specific cell type and its ER activity.

Supporting Information. Full details of the protein expression, protein semi-synthesis and purification; solid phase peptide synthesis; biochemical characterization of the coactivator sequences, including FP and an on-chip cofactor peptide binding assay; NMR methods, samples and summary of the 15N-relaxation data; MD methods and analysis of the data. This material is available free of charge via the Internet at http://pubs.acs.org.

This work was supported by grants from Netherlands Organisation for Scientific Research via Gravity program 024.001.035 and ECHO grant 711011017, the German Research Foundation to S.A.D. (DA 1195/3-1, SFB1035 project B04) and Marie Curie Actions to L.N. (PIEF-GA-2011-298489). S.A.D acknowledges further financial support from the TUM diversity and talent management office and from the project ‘metabolic dysfunction’ of the Helmholtz Zentrum München.

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