Tel.: +33 3 90 24 26 79, Fax: +33 3 90 24 27 81, E-mail: npotier@chimie.u-strasbg.fr Abbreviations: ESI-MS, electrospray ionization mass spectrometry; RAR, human retinoic acid receptor a
Trang 1Monitoring ligand-mediated nuclear receptor–coregulator interactions
by noncovalent mass spectrometry
Sarah Sanglier1, William Bourguet2,*, Pierre Germain2, Virginie Chavant2, Dino Moras2,
Hinrich Gronemeyer2, Noelle Potier1and Alain Van Dorsselaer1
1
Laboratoire de Spectrome´trie de Masse Bio-Organique, CNRS UMR 7509, ECPM, Strasbourg, France;2Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS INSERM ULP Colle`ge de France, Illkirch, France
Retinoid receptors are ligand-dependent transcription
factors belonging to the nuclear receptor superfamily
Retinoic acid (RARa, b, c) and retinoid X (RXRa, b, c)
receptors mediate the retinoid/rexinoid signal to the
transcriptional machineries by interacting at the first level
with coactivators or corepressors, which leads to the
recruitment of enzymatically active noncovalent
com-plexes at target gene promoters It has been shown that
the interaction of corepressors with nuclear receptors
in-volves conserved LXXI/HIXXXI/L consensus sequences
termed corepressor nuclear receptor (CoRNR) boxes
Here we describe the use of nondenaturing electrospray
ionization mass spectrometry (ESI-MS) to determine the
characteristics of CoRNR box peptide binding to the
ligand binding domains of the RARa–RXRa
hetero-dimer The stability of the RARa–RXRa–CoRNR
tern-ary complexes was monitored in the presence of different types of agonists or antagonists for the two receptors, including inverse agonists These results show unambig-uously the differential impact of distinct retinoids on corepressor binding We show that ESI-MS is a powerful technique that complements classical methods and allows one to: (a) obtain direct evidence for the formation of noncovalent NR complexes; (b) determine ligand binding stoichiometries and (c) monitor ligand effects on these complexes
Keywords: ESI-MS; noncovalent mass spectrometry; nuc-lear receptors; protein–protein interactions; protein–ligand interactions
Retinoid receptors are ligand-dependent transcription
fac-tors belonging to the nuclear receptor (NR) superfamily [1]
They are involved in vertebrate development, cell
differen-tiation and proliferation or apoptosis [2], and display
significant promise for cancer therapy and prevention [3,4]
Retinoid X (RXRa, b, c) and retinoic acid (RARa, b, c)
receptors form heterodimers, but RXRs can also
hetero-dimerize with a great number of other nuclear receptors [5]
NRs display a modular structure with distinct functional
and structural domains [1], such as the DNA-binding
domain [6] or the ligand binding domain (LBD) [7,8], which corresponds also to the region that interacts with coactiva-tors (CoA) or corepressors (CoR)
Although the molecular mechanisms by which NRs control target gene expression are in principle understood, many aspects remain to be solved This concerns both the details of the ligand-induced early events [9], and the assembly and epigenetic action of multiprotein machineries
on target gene promoters [10] As NRs respond to small ligand molecules and correspond to potent regulators of cell function [11,12], life and death [13,14], they are particularly attractive targets for the design of novel therapeutic agents
A critical step in the drug design process is the elucidation
of the ligand function, as it can be deduced from the characterization of the molecular mechanism(s) that are modulated by ligand binding, such as the interaction with coactivator and corepressor complexes that mediate the transcriptional activity of NRs In the case of retinoid receptors, the unliganded RAR–RXR heterodimers (HD) have been shown to act as repressors of transcription through recruitment of complexes containing corepressors, such as nuclear receptor corepressor [15] or silencing mediator for RARs and thyroid hormone receptor (TRs)
2[16] Upon ligand binding, NRs undergo conformational changes inducing the dissociation of repressor complexes and the subsequent association with coactivator molecules thereby promoting transcriptional activation CoAs are like CoRs; large multidomain proteins, which bind through specific motifs (referred to as NR box or LXXLL motif) to
Correspondence to N Potier, Laboratoire de Spectrome´trie de Masse
Bio-Organique, UMR CNRS 7509, ECPM, Universite´ Louis Pasteur
de Strasbourg, 25 Rue Becquerel, 67082 Strasbourg Cedex 2, France.
Tel.: +33 3 90 24 26 79, Fax: +33 3 90 24 27 81,
E-mail: npotier@chimie.u-strasbg.fr
Abbreviations: ESI-MS, electrospray ionization mass spectrometry;
RAR, human retinoic acid receptor a; RXR, human retinoid X
receptor a; CoR, corepressor; CoA, coactivator; NR, nuclear receptor;
NcoR, nuclear corepressor protein; CoRNR, corepressor nuclear
receptor; LBD, ligand binding domain; LBP, ligand binding pocket;
Vc, accelerating voltage; HD, RAR–RXR heterodimer; TR, thyroid
hormone receptor.
*Present address: Centre de Biochimie Structurale, CNRS UMR 5048,
UM1 INSERM UMR 554, Faculte´ de Pharmacie, 15 Avenue
Flahault, 34060 Montpellier, France.
(Received 17 August 2004, revised 12 October 2004,
accepted 27 October 2004)
Trang 2agonist-bound NRs CoRs interact with NRs through
nuclear receptor interacting domains that contain two
helical motifs referred to as corepressor nuclear receptor
boxes (CoRNR1 and CoRNR2, comprising LXXI/
HIXXXI/L sequences) [17–19]
Since 1991 [13], electrospray ionization mass
spectro-metry (ESI-MS) has been regularly used to study
noncova-lent complexes, and an increasing number of studies of
protein–protein, protein–ligand and protein–DNA
com-plexes are now reported (reviewed in [20–22]) However,
such studies are not yet straightforward because some
technical difficulties, such as the presence of nonvolatile
buffers or the fragility of the complexes, are encountered
Nevertheless, ESI-MS appears to be an attractive approach
and offers new possibilities for the study of such complexes,
providing direct evidence for their formation and an
accurate determination of their binding stoichiometry For
instance, in the field of NRs, ESI-MS was previously used
by Witkowska et al [23,24] to detect the estrogen receptor
ligand binding domain in its dimeric noncovalently bound
form Craig et al [25] analyzed by microESI-MS the
binding of the human vitamin D receptor and the retinoid
X receptor-a to the osteopontin vitamin D response
element, and assessed the influence of their endogenous
ligands 1a,25-dihydroxyvitamin D3 and 9-cis retinoic acid
The importance of structural zinc ions for the interaction
between the DNA binding domain of the human vitamin D
receptor with a double-stranded DNA containing the
vitamin D response element from the mouse osteopontin
gene was determined using ESI-MS [25,26] Bourguet et al
[27] used ESI-MS to reveal the presence of an unexpected
ligand in the mRXRaF318A complex that was apparent at
the early stages of the structure refinement and Germain
et al [28] demonstrated by ESI-MS that the RXR subunit
of the HD heterodimer
when RAR is ligand-free Potier et al [29] and other groups
[30–32] described the strength of nondenaturing mass
spectrometry to identify unexpected ligands coming from
the expression host that bind to orphan receptors More
recently, the use of ESI-MS has also been reported for the
characterization of protein–ligand complexes involving
other nuclear receptors [
In the study reported here, we used ESI-MS to
charac-terize the binding of CoRNRs to the LBDs of the HD
heterodimer In particular, the influence of ligand binding to
RAR or RXR on the stability of HD–CoRNR complexes
was investigated by nondenaturing ESI-MS We show that
supramolecular mass spectrometry is a powerful tool to
rapidly and unambiguously determine if a particular peptide
sequence can interact with LBDs of the HD heterodimer
and to directly visualize the influence of various ligands on
the stability of the corresponding ternary complexes These
results could only be obtained after precisely adjusting of the
mass spectrometer interface parameters, which are
des-cribed here in detail
Materials and methods
Materials and chemicals
Expression and purification of the HD heterodimer was
performed as described by Bourguet et al [39] Peptides
containing CoRNR1 and CoRNR2 sequences were syn-thesized and used as model peptides for the corepressor proteins CoRNR1 is a 24 amino acid peptide (THRLIT LADHICQIITQDFARNQV; molecular mass 2806.2 Da) containing the consensus sequence that specifically interacts with the RAR subunit CoRNR2 contains 14 amino acids (NLGLEDIIRKALMG; molecular mass 1542.8 Da) and interacts specifically with the RXR subunit
Ligands were chosen to interact specifically with either RAR (AM80, BMS493 and BMS614) or RXR (HX531) [28,40,41]
PSG5-based Gal-RARa and VP16-RARa expression vectors, and the (17m)5x-bG-Luc reporter gene have been described [42] Gal-NCoR corresponds to a fusion between residues 1–147 of Gal4 and residues 1629–2453 of NCoR
Cell culture and transient transfections COS cells, cultured in Dulbecco’s modified Eagle’s medium/ 5% (v/v) fetal bovine serum, were transiently transfected using the standard calcium phosphate method Results were normalized to coexpressed b-galactosidase
Electrospray ionization mass spectrometry analysis Prior to any ESI-MS analysis, samples were desalted on Centricon PM30 microconcentrators (Amicon, Millipore, Molsheim, France)
Ammonium acetate presents the advantage not only of preserving the ternary and quaternary structures of proteins
in solution but also of being compatible with ESI-MS experiments
ESI-MS measurements were performed on an electro-spray time-of-flight mass spectrometer (LCT, Waters, Manchester, UK)
receptors were verified by mass spectrometry analysis in denaturing conditions: proteins were diluted to 5 pmolÆlL)1
in a water/acetonitrile mixture (1 : 1, v/v) acidified with 1% (v/v) formic acid Mass spectra were recorded in the positive ion mode on the mass range 500–2500 m/z, after calibration with horse heart myoglobin diluted to 2 pmolÆlL)1in a water/acetonitrile mixture (1 : 1, v/v) acidified with 1% (v/v) formic acid The following molecular masses were measured: 29 930 ± 1.8 Da for RARa; 26 735 ± 2.3 Da for RXRa with deletion of the N-terminal methionine and
26 867 ± 2.1 Da corresponding to RXRa These results were in agreement with the molecular masses calculated from the known amino acid sequences
The mass measurements of the noncovalent complexes were performed in ammonium acetate (50 mM, pH 6.5) Samples were diluted to 10 pmolÆlL)1in the previous buffer and continuously infused into the ESI ion source at a flow rate of 6 lLÆmin)1through a Harvard syringe pump Great care was exercised so that noncovalent interactions survive the ionization/desorption process Particularly the acceler-ating voltage (Vc), which controls the kinetic energy communicated to the ions in the interface region of the mass spectrometer, was optimized to 50 V in order to prevent ligand dissociation in the gas phase ESI-MS data were acquired in the positive ion mode on the mass range 1000–5000 m/z Calibration of the instrument was per-formed using the multiply charged ions produced by a
Trang 3separate injection of horse heart myoglobin diluted to
2 pmolÆlL)1 in a water/acetonitrile mixture (1 : 1, v/v)
acidified with 1% (v/v) formic acid The relative abundance
of the different species present on ESI mass spectra were
measured from their respective peak intensities, assuming
that relative intensities displayed by the different species on
the ESI mass spectrum reflect the actual distribution of these
species in solution The reproducibility of the determination
of the relative proportions of the different species was
estimated to be ± 2–3%
Results
Optimization of the ESI-MS operating conditions to
detect heterodimer–ligand–corepressor complexes
To obtain optimal sensitivity without affecting the stability
of noncovalent NR complexes, we first optimized the
operating conditions Indeed, it is critical to prevent
dissociation of weak interactions in the mass spectrometer
when transferring ions from the condensed phase to the gas
phase It has been shown by several groups that the gas
phase stability of noncovalent complexes is greatly
depend-ant on the nature of the interactions involved in the
formation of the complexes [37,43–47] It seems that the gas
phase stability strongly increases with an increased
contri-bution of electrostatic interactions within a noncovalent
complex Therefore, it is important that the Vc, which
controls the kinetic energy communicated to the ions in the interface region of the mass spectrometer, is carefully set [37,47] Operating conditions were optimized on the HD– ligand–corepressor complex with CoRNR1 and BMS493,
an inverse pan-RAR agonist [28,48] that does not interact with RXRs
Mass spectra for the ligand-bound HD–BMS493– CoRNR1 complex at different Vc voltages were generated from samples in which a threefold molar excess of CoRNR1 and a twofold molar excess of BMS493 were added to the
HD heterodimer preparation (Fig 1) At low accelerating voltages (i.e for Vc£ 30 V), the HD–BMS493–CoRNR1 noncovalent complex is detected as a major component (Fig 1A) However, the observed peak shapes are broad, which probably results from incomplete desolvation A direct consequence of this is a significant loss in mass accuracy Nevertheless, significant amounts of the HD– BMS493 complex can be detected Increasing the Vc to 50 V significantly improves the signal to noise ratio and still allows the detection of the HD–BMS493–CoRNR1 com-plex as a main component with a molecular mass of
59 884.1 ± 1.4 Da, while the HD–BMS493 complex with a molecular mass of 57 080.2 ± 0.9 Da is only a minor component (Fig 1B) Under these conditions an additional ion series displaying a molecular mass of 59 474.2 ± 0.1 Da emerges and can be attributed to the unliganded HD– CoRNR1 complex Thus, increasing Vc from 30 V to 50 V improves considerably the desolvation and the accuracy of
17+ V
17+ V
16+
18+
16+
15+
17+ V
15+
18+ 17+
16+
15+
Partial peptide dissociation
Ligand dissociation
DM = 408 Da (100%)
Slight ligand dissociation (less than 5%)
A
B
C
Fig 1 Optimization of the operating conditions: ESI-MS mass spectra of the HD–BMS493–CoRNR1 complex at different accelerating voltages (Vc) Positive ESI mass spectra of the HD–BMS493–CoRNR1 diluted to 10 pmolÆlL)1 in AcONH 4 (50 m M , pH 6.5) at (A) Vc ¼ 30 V: the HD–BMS493–CoRNR1 noncovalent complex is detected without any dissociation; (B) Vc ¼ 50 V: slight dissocitation of the ligand (less than 5%) from the HD–BMS493–CoRNR1 complex; (C) Vc ¼ 80 V: 100% dissociation of the ligand from the HD–CoRNR1 and partial dissociation of the peptide from the HD–CoRNR1 complex Peaks labeled with * correspond to the different species with the additional N-terminal methionine.
Trang 4the mass measurement but induces concomitantly some
dissociation of the ligand-bound HD–BMS493–CoRNR1
complex into the unliganded HD–CoRNR1 complex A
further increase of the accelerating voltage to 80 V (Fig 1C)
provokes dissociation of the quaternary complex to HD–
CoRNR1 (major component) and HD (minor component)
complexes No ions corresponding to a ligand-bound species
are detected under these conditions These data indicate that
the interactions involving the ligand are less stable in the gas
phase than those involving the corepressor peptide Whether
HD originates from the gas phase dissociation of the HD–
BMS493 complex or from the HD–CoRNR1 complex
cannot be distinguished
to 120–150 V (data not shown) does not lead to any major
change in the dissociation pattern regarding the complexes,
which suggests that the HD–CoRNR1 complex is quite
stable in the gas phase
The above data reveal that the chosen operating
condi-tions (buffer and control of the Vc voltage) allow the
transfer of intact noncovalent receptor–ligand–corepressor
peptide complexes from solution to the gas phase It should
be noted that such experiments were performed for each
HD–ligand–CoRNR complex described in this paper In all
cases, at low accelerating voltages (30–50 V), HD–ligand–
CoRNR complexes remained intact after passage into the
gas phase Increasing the Vc voltage (up to 80–150 V)
resulted in a stepwise removal of the ligand first, and
subsequent dissociation of the CoR peptide Consequently,
as a compromise between sufficient desolvation, optimal
transmission of the ions and nondestructive gas-phase
collisions was found at 50 V and we retained such
experimental conditions in our further experiments
Interestingly, while various biochemical studies including
circular dichroism, proteolytic digestions or gel mobility
suggested that NRs undergo conformational changes upon
ligand binding, no charge state distribution changes were
detected upon ligand binding or CoR peptide binding
(+15 to +17)
Witkowska et al [23], that ESI-MS might also be useful for
revealing structural changes in protein conformations from
subtle changes in the charge state distribution observed on
ESI mass spectra [20,49–52] So, if conformational changes
occur, these do not sufficiently affect the surface of the
protein exposed to solvent to be detected by mass
spectro-metry This latter assumption is supported by X-ray
crystallographic studies showing no obvious evidence of
new exposure of charged amino acids The mainly local
conformational changes appear not to affect the protein
surface in a way sufficient to be revealed by ESI-MS
RAR–RXR heterodimer forms a 1 : 1 noncovalent
complex with corepressor peptides
The mass spectrum obtained at a threefold molar excess of
CoRNR1 over HD displays two main ion series at mass to
charge ratios between m/z 3000 and m/z 4000 (Fig 2A) The
first series (r) corresponds to the +15 to +17 charge states
of the HD, with a molecular mass of 56 669.4 ± 2.5 Da
The second series (d) gives rise to a molecular mass of
59 478.3 ± 0.5 Da and can be attributed to the formation
of a 1 : 1 complex between HD and CoRNR1 corepressor
peptide, with a charge state distribution varying from +16
to +18 Using identical experimental conditions for HD–CoRNR2 complexes, two series of multiply charged ions were detected corresponding to the HD (56 664.8 ± 0.3 Da) and the HD–CoRNR2 complex (58 210.2 ± 1.8 Da) (Fig 3A) All data were recorded at a cone voltage
of 50 V (see above) As ESI-MS analyses faithfully reflect solution equilibrium in the previously mentioned experi-mental conditions, we reproducibly observed that only
50% of the detected species correspond to both HD–CoRNR complexes in the absence of ligand Titration experiments, in which increasing amounts of CoRNR peptide (up to fourfold molar excess, data not shown) were added to the HD, reveal no further saturation of HD in the absence of ligand This suggests that a twofold molar excess
of CoRNR peptide is sufficient to reach the equilibrium state under our experimental conditions This ratio (50%) was taken as a reference point in the present study to monitor the effect of various classes of ligands on this equilibrium
Ligand modulation of HD–CoRNR ternary complexes
In order to monitor the effect of different types of ligands on the recruitment of two CoRNR peptides, various ligands were added to each HD–CoRNR complex and the stability
of each ternary complex was recorded by ESI-MS The antagonist activity of the inverse agonist BMS493, the RARa-selective agonist BMS614 and the RXR antagonist HX531 were confirmed by competition of AM80-induced transactivation using the chimeric Gal-RARa as ligand-dependent activator of a cognate 17mer-luciferase reporter gene (Fig 4) These data reveal the antagonistic potential of BMS493 and BMS614 and demonstrate also that HX531 has a weak RARa antagonist activity
Effects of RAR ligand binding on the stability of the HD–CoRNR1 complex Mass spectra obtained for the
HD in presence of CoRNR1 with a twofold molar excess of either the inverse pan-RAR antagonist BMS493 (Fig 2B)
or the RARa-selective agonist AM80 (Fig 2C), revealed that all ligands bind to the HD and to the HD–CoRNR complexes More importantly, they have a dramatic effect
on the initial HD to HD–CoRNR1 ratio (Fig 2A–C) In the presence of a twofold molar excess of BMS493, two ion series are observed The most abundant series (d) has a molecular mass of 59 884.1 ± 1.4 Da corresponding to the binding of one BMS493 molecule [mass difference (DM)
408 Da] to the HD–CoRNR1 complex (Fig 2B) The minor one (r) can be attributed to the fixation of one BMS493 molecule to the free HD No significant signal corresponding to unliganded species is detected Even in the presence of a twofold molar excess of BMS493, no protein/ ligand stoichiometry higher than 1 : 1 is observed, indicating that the specific binding of BMS493 to its cognate receptor
is faithfully revealed by MS and that no nonsite-specific ligand addition resulting from any artefactual aggregation during the electrospray ionization process occurs Similar results were obtained with BMS614, a RARa-selective antagonist, which induced a mass increase of 448 Da for the binding of one BMS614 molecule to the HD and HD– CoRNR complexes (data not shown) As interface condi-tions are optimized so that neither peptides nor ligands dissociate during transfer to the gas phase, we conclude that
Trang 5the HD–BMS493 complex is present in solution at
equilibrium and does not result from the dissociation of
the HD–BMS493–CoRNR1 complex in the interface of the
mass spectrometer
For comparison of the relative proportions of the
different species present in solution, we made the
assump-tion that ionizaassump-tion efficiencies and response factors of all
species are similar, and thus that relative intensities of the
main charge states can serve to estimate the relative
abundances in solution This assumption is supported by
several points: (a) strictly identical experimental conditions
are used for the addition of each ligand (molar excess,
buffer, pH, Vc, etc.); (b) the mass contribution of the ligand
is very small compared to the mass of the HD–CoRNR
complex, thus similar response factors are expected; (c) all
species exhibit nearly the same charge states and are
detected with the same m/z ratios, suggesting that no strong
discrimination effects of the ion species upon focalization in
the interface of the mass spectrometer should occur Table 1 summarizes the relative abundances of all species in the absence or presence of the corepressor peptide The fact that the relative abundance of the HD–CoRNR1 complex significantly increases in the presence of RAR-antagonist
or inverse agonist ligands (75% and 56% of HD–CoRNR1
in the presence of, respectively, BMS493 or BMS614 instead
of 43% in the absence of ligand) indicates that these RAR-ligands stabilize the HD–CoRNR1 ternary complex Above
60 V (data not shown), the ligand is dissociated from all species in the gas phase while the ratio between HD and HD–CoRNR1 stays unchanged
To compare these data with an analysis of the ligand-induced complex formation in living cells in vivo, we performed mammalian cell two-hybrid experiments in which we monitored the interaction of a VP16 activation domain-linked RARa with a Gal-NCoR hybrid that was capable of activating a 17mer-based luciferase reporter
17+
16+
•
No ligand
15+
16+
351 Da
15+
17+
3200 3250 3300 3350 3400 3450 3500 3550 3600 3650 3700 3750 3800 3850 3900
m/z
•
•
•
• •
•
•
•
+1 +2 +1
17+
+2 +1 +1
Reference
17+
DM =
DM =
408 Da •
•
16+
Stabilizing Effect
RAR-antagonist BMS493
RAR-agonist AM80
RXR-antagonist HX531
Destabilizing Effect
No apparent Effect
*
*
*
*
*
*
*
*
*
A
B
C
D
Fig 2 Effect of ligand additions on the HD–CoRNR1 ternary complex Positive ESI mass spectra of HD–CoRNR1 The mass spectra were acquired
at Vc ¼ 50 V and at a pressure in the interface of 2.5 mbar (A) Before any ligand addition After addition of twofold molar excess
the HD–CoRNR1 complex is stabilized upon BMS493 binding; (C) AM80: the HD–CoRNR1 complex is completely destabilized upon AM80 binding; (D) HX531: no apparent effect on the stability of the HD–CoRNR1 complex.
Trang 6(Fig 5) Transcription activation indicated NCoR–RARa
interaction strongly increased upon exposing the cells to
BMS493, while only a weak effect was seen in presence of
BMS614, and little, if any, in the presence of HX531
(Fig 5) We conclude that the MS analysis faithfully
monitors the modulation of receptor–corepressor
inter-action, as it occurs in living cells
After the addition of a twofold molar excess of the
RARa-specific agonist AM80 in the presence of CoRNR1
(Fig 2C), one single ion series (r) displaying a mass
increase of about 351 Da above the molecular mass of the
HD is then observed No ions corresponding to the
ligand-bound HD–AM80–CoRNR1 or to nonliganded HD are
detected Therefore, MS analysis faithfully reveals the
previously observed effects of RAR agonists, antagonists
and inverse agonists on corepressor–HD interaction
Effects of RAR ligand binding on the stability of the HD– CoRNR2 complex ESI mass spectra obtained after the addition of a twofold molar excess of different ligands and CoRNR2 are presented in Fig 3 for the inverse pan-RAR agonist BMS493 (Fig 3B) and the RARa agonist AM80 (Fig 3C) In the absence of ligand a mixture of HD (47%) and HD–CoRNR2 (53%) was observed (Fig 3A) As for CoRNR1, the addition of RAR-specific ligands induces a dramatic change in the relative amount of the various complexes While the agonist AM80 and the antagonist BMS614 induce a complete destabilization of the ternary HD–CoRNR2 complex, no similar effect is exerted by the inverse agonist BMS493 Indeed, 22% of all observed species correspond to the HD–BMS493–CoRNR2 com-plex In all cases, a single ligand molecule is quantitatively bound to the HD complex
3200 3250 3300 3350 3400 3450 3500 3550 3600 3650 3700 3750 3800 3850 3900m/z
16+
17+
+
DM =
DM =
408 Da
•
•
•
17+
15+
17+
16+
351 Da
15+
17+
•
•
+2
+2
+2
+2 +1 +1
+2
+1
*
*
*
*
*
*
*
A
B
C
D
Reference
Destabilizing Effect
Destabilizing Effect
No apparent Effect
No ligand
RAR-antagonist BMS493
RAR-agonist AM80
RXR-antagonist HX531
Fig 3 Effect of ligand additions on the HD–CoRNR2 ternary complex Positive ESI mass spectra of HD–CoRNR2 The mass spectra were acquired
at Vc ¼ 50 V and at a pressure in the interface of 2.5 mbar (A) Before any ligand addition After addition of twofold molar excess
the HD–CoRNR2 complex is destabilized upon BMS493 binding; (C) AM80: the HD–CoRNR2 complex is completely destabilized upon AM80 binding (D) HX531: no apparent effect on the stability of the HD–CoRNR2 complex.
Trang 7Effects of RXR ligands on the stability of HD–CoRNR
complexes When a twofold molar excess of the RXR
antagonist HX531 is added (Figs 2D and 3D), the
inter-pretation of the mass spectra becomes significantly more
difficult because multiple species are detected HD and
ternary HD–CoRNR1 complexes were observed both as
liganded and unliganded species, with the liganded species
involving one or even two HX531 molecules In the presence
of this RXR ligand, no major difference concerning the initial HD to HD–CoRNR1 ratio is observed in the mass spectra The addition of a twofold molar excess of a RXR-specific ligand induces some stabilization of the HD–CoRNR1 complex (51% in the presence of HX531
vs 43% in the absence of any ligand; Table 1) Because of the presence of a second ligand molecule bound to each detected complex, no clear conclusions regarding the influence of a RXR-specific ligand on the stability of the HD–CoRNR1 complex can be drawn
Discussion
ESI-MS allows monitoring of ligand modulations
on HD–CoRNR complexes Due to its ability to detect each species present in solution, ESI-MS appears to be a powerful tool to follow coregulator association and dissociation to NR upon binding of different classes of ligands In addition to previous studies [28,37], we now show that nondenaturaing mass spectro-metry can be used, not only to monitor binding of coregulator peptides but also to assess the effect of ligand binding on the stability of the corresponding NR–coregu-lator complexes
10 The interpretation of the ESI mass spectra provides several facts
(CoRNR1 or CoRNR2) bind to the HD in the absence of any ligand, and (b) ESI-MS data allow one to directly monitor the effect of ligand binding on the association/ dissociation pattern of the heterodimer and the corepressor RAR-specific ligands display a strong influence on the stability of heterodimer–CoRNR complexes, comparing well with previous reports showing that induction of corepressor release is part of the mechanism of action of RAR agonists [1,53] Conversely, the stabilization of the corepressor complex by RAR inverse agonist enhances transcriptional silencing [28,48] These MS data are fully in keeping with the corresponding results obtained by transient transactivation studies Such conclusions are not yet routine and it is of most importance to control that the solution phase image is not distorted during the ESI-MS analysis and that observed peaks on ESI mass spectra in vacuo can
be related to species effectively present in solution
transactivation relative to Am80 10
100
125
75
50
25
0
Ligands [logM]
Gal - RARα + Am80 10–8M
5 x Gal4
Gal4
Luc βG
BMS614 HX531
Fig 4 RARa antagonist potential of synthetic retinoids Transient
transactivation assays were performed to assess the antagonist activity
of BMS493 (j), BMS614 (n), and HX531 (m) COS cells were
co-transfected with reporter (17m)5x-bG-Luc and Gal-RARa The
reporter was activated with 10 n M Am80 (100%) and increasing
con-centrations of the respective retinoid were added, as indicated.
Table 1 Influence of the addition of ligands on the stability of the noncovalent HD–CoRNR complexes The relative abundance of the species are obtained by summing the peak intensities of the three predominant charge states of the complexes Reproducibility of the values is within 2–3%.
No ligand
Relative amounts of complexes detected by ESI-MS (%)
Trang 8If the observed complex results from specific interactions
in solution, it must be sensitive to modifications of the
experimental conditions affecting its stability The fact that
different ligands induce (a) a substantial change in the mass
spectrum and (b) distinct effects on the stability of
HD–corepressor complexes, provides a good support for
a structurally-specific interaction To establish that the
interactions detected by ESI-MS in presence of RXR
ligands are really the result of in-solution interactions rather
than artefactual nonspecific associations occurring during
the ESI process [54], rigorous control experiments were
performed In particular, separate titration experiments
performed with the RXR ligand and the monomeric RXRa
subunit revealed that, even in the presence of an excess of
ligand (a threefold molar excess), no binding of a second
HX531 molecule is observed on RXRa (data not shown)
At this level, two hypotheses may be advanced to explain
the binding of a second ligand molecule to the heterodimer:
either the second ligand molecule is anchored directly in the
free RAR-binding site [this option is strongly supported by
the antagonistic action of HX531 on AM80-induced
Gal-RARa transactivation (Fig 4)], or the second ligand
molecule is nonspecifically interacting at the protein surface
Because the single RAR subunit tends to precipitate under
ESI-MS compatible conditions, experiments with
mono-meric RAR could not be achieved
Relationship between the type of interactions involved
in the formation of HD–ligand–CoRNR complexes and their gas phase stabilities
The ESI-MS detection of complexes involving few electro-static contacts between the LBP and the ligands is not straightforward As described in the literature [37,43–46], the stability of noncovalent complexes in the ESI-MS process depends strongly on the type of interaction (elec-trostatic contacts, hydrogen bonds, Van der Waals interac-tions) involved in the formation of the complex During ion transfer from the solution to the gas phase both electrostatic interactions and hydrogen bonds are emphasized, while complexes whose formations in solution are mainly driven
by hydrophobic effects appear to be weakened [55,56] The present study shows that HD–CoRNR and HD–ligand noncovalent complexes have different gas phase stabilities The Vc, which controls the energy communicated
to the ions in the interface of the mass spectrometer, needs
to be set to quite low values (30–50 V) to detect the interaction between NRs and ligands, whereas higher Vc values can be used to observe HD–CoRNR complexes, suggesting that these latter complexes are more stable in the gas phase Such behavior is in complete agreement with crystallographic data showing that interactions between NRs and ligands are mainly hydrophobic, whereas NR– CoRNR
12 interactions involve more polar contacts Until now, it has been
contacts involved in a complex formation and its subse-quent MS detection For the receptors of retinoids, the carboxylic group of the ligand is the only moiety engaged in polar interactions with the LBP Two hypotheses can be made to explain the ESI-MS observations: (a) few electro-static contacts are sufficient to maintain the NR–ligand interactions in the gas phase, and (b) the LBP conformation that totally surrounds the ligand prevents its ejection from the pocket Whether dissociation experiments might be used
to directly describe the type of interactions involved in the formation of a given noncovalent complex constitutes a new challenge
Conclusion
In this study, we demonstrate that noncovalent mass spectrometry is a powerful technique to obtain direct evidence of the formation of NR complexes and to monitor the effect of ligand binding on the stability of the resulting complexes Thanks to its unique advantage of providing direct insights into all species present in solution, even moderate changes induced by the addition of a given substrate (ligand, peptides, metallic ions, small organic molecules, etc.) in the association/dissociation pattern can be directly revealed on the ESI mass spectra Therefore, this nondenaturing ESI-MS approach will efficiently comple-ment other structural methods such as NMR and crystal-lography ESI-MS is a technique that can be extended to a wide range of noncovalent protein–protein or protein–ligand complexes Combining rapidity, sensitivity and accuracy, it will play an increasingly important role not only for the analysis of nuclear receptor action and (orphan) receptor ligand detection, but also for transcription regulation in general, which is triggered by noncovalent interactions
10000
15000
5000
0
etOH Am80
BMS493 BMS614 HX531 Gal-NCoR + VP16 - RARα
5 x Gal4
Gal4
Luc βG
NCoR
RAR VP16
Fig 5 Enhanced interaction between RARa and NCoR induced by
synthetic retinoids Mammalian two-hybrid assay with Gal-NCoR as
bait and VP16-RARa as prey were performed to assess the influence of
indicated synthetic retinoids on interaction between RARa and NCoR
in a cellular context.
Trang 9We are grateful to Pascal Eberling for the synthesis of the CoRNR1
corepression peptide, to Dr Lazar for the kind gift of the CoRNR2
peptide and to Drs Chris Zusi and Koichi Shudo for ligands S.S.
acknowledges the CNRS and Lilly for financial support Work in the
laboratory of H.G is supported by grants from the European
Commission (QLK3-CT2002-02029, HPRN-CT2002-00268), the
Fon-dation de France, the Association for International Cancer Research,
the Association pour la Recherche sur le Cancer, the ULP, the
INSERM, the CNRS, and Bristol-Myers Squibb.
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