Báo cáo khoa học: Monitoring ligand-mediated nuclear receptor–coregulator interactions by noncovalent mass spectrometry pptx

Monitoring ligand-mediated nuclear receptor–coregulator interactions
by noncovalent mass spectrometry
Sarah Sanglier
1
, William Bourguet
2,
*, Pierre Germain
2
, Virginie Chavant
2
, Dino Moras
2
,
Hinrich Gronemeyer
2
, Noelle Potier
1
and Alain Van Dorsselaer
1
1
Laboratoire de Spectrome
´
trie de Masse Bio-Organique, CNRS UMR 7509, ECPM, Strasbourg, France;
2
Institut 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 s uperfamily.
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 c orepressors, which leads to the
recruitment of enzymatically active noncovalent c om-
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 o f CoRNR box peptide binding to the
ligand binding domains of the R ARa–RXRa hetero-
dimer. The stability of the RARa–RXRa–CoRNR
1
tern-
ary complexes was monitored in the presence of different
types of a gonists or a ntagonists for the two receptors,
including inverse agonists. These r esults show unambig-
uously the differential impact of distinct retinoids on
corepressor binding. W e show that ESI-MS is a p owerful
technique that complements classical methods and allows
one to: (a) obtain d irect evidence for the formation o f
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; p rotein–ligand
interactions.
Retinoid receptors are ligand-dependent transcription f ac-
tors belonging to the nuclear receptor (NR) superfamily [1].
They are involved in vertebrate development, cell differen-
tiation and proliferation o r apoptosis [2], and display
significant promise for cancer therapy and prevention [3,4].
Retinoid X (R XRa, 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 r espond 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 c ritical step in the drug design process is t he elucidation
of the ligand function, as i t c an 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 o f N Rs. In the case of retinoid
receptors, th e 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 a nd th yroid hormone receptor (TRs)
2
[16]. Upon ligand binding, NRs undergo conformational
changes inducing the dissociation of repressor complexes
and the sub sequent association w ith coac tivator molecules
thereby promoting transcriptional activation. CoAs are like
CoRs; large multidomain proteins, which bind t hrough
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, F rance.
Tel.: +33 3 90 24 26 79, Fax: +33 3 90 24 27 81,
E-mail:
Abbreviations: ESI-MS, electrospray ionization mass spectrometry;
RAR, human retinoic acid receptor a; RXR, human retinoid X
receptor a; CoR, corepressor; Co A , coactivator; NR, nuclear receptor;
NcoR, nuclear corepressor p rotein; CoRNR, c orepressor 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 , CN RS UM R 5 048,
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)
Eur. J. Biochem. 271, 4958–4967 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04466.x
agonist-bound NRs. CoRs interact with NRs th rough
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 1 991 [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 e vidence for their f ormation 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 rec eptor
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 r eceptor-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 s tructural z inc 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 m ouse osteopontin
gene was determined using ESI-MS [25,26]. Bourguet et al.
[27] used ESI-MS to reveal the presence of an unexpected
ligandinthemRXRaF318A 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
3
can bind its cognate ligand even
when RAR is ligand-free. Potier et al . [29] and other groups
[30–32] described the strength o f 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 [
4
33–38].
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 o r 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 pep tide
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 p arameters, which a re 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 s equence 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, c ultured 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)
5
in 100 m
M
ammonium acetate (pH 6.5).
Ammonium acetate p resents the advantage not only o f
preserving the ternary and quaternary structures of p roteins
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)
6
. Purity and homogeneity of the retinoid
receptors were verified by mass spectrometry analysis in
denaturing conditions: p roteins 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
)1
in a
water/acetonitrile mixture ( 1 : 1, v/v) acidified with 1%
(v/v) formic acid. The f ollowing molecular masses were
measured: 29 930 ± 1.8 Da for RARa;26735±2.3Da

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 m
M
,pH6.5).
Samples were diluted to 10 p molÆlL
)1
in the previous buffer
and continuously infused into the ESI ion source at a flow
rate of 6 lLÆmin
)1
through a Harvard syringe pump. Great
care was exercised so that non covalent interactions survive
the ionization/desorption process. Particularly the a cceler-
ating v oltage (Vc), which controls the k inetic energy
communicated to the ions in the interface region of the
mass spectrometer, was optimized to 50 V i n o rder 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 th e multiply charged ions produced by a
Ó FEBS 2004 ESI-MS to investigate noncovalent complexes (Eur. J. Biochem. 271) 4959
separate injection of horse heart myoglobin diluted to
2pmolÆlL
)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 o n ESI mass spectra were
measured from their r espective peak i ntensities, 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 p roportions of the different species was
estimatedtobe±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 N R complexes, we first optimized the
operating co nditions. I ndeed, i t i s c ritical t o p revent
dissociation of w eak 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 im portant 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 co nditions were op timized on the HD–
ligand–corepressor c omplex 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 BM S493 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 r esults from incomplete desolvation. A
direct consequence o f this i s 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 5 9 474.2 ± 0.1 Da
emerges a nd 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
18 +
16
+
17 +
V
16 +
18 +
16

+
15 +
3
m/z
200 3300 3400 3500 3600 3700 3800 3900
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

,pH6.5)at(A)Vc¼ 30 V: the
HD–BMS493–CoRNR1 noncovalent co mplex is detected without any dissociation; (B) Vc ¼ 50 V: slight dissocitation of the ligand (less than 5%)
from th e 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.
4960 S. Sanglier et al.(Eur. J. Biochem. 271) Ó FEBS 2004
the mass m easurement 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 t he 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 pep tide. Whether
HD originates from the gas phase dissociation of the HD–
BMS493 complex or from the HD–CoRNR1 complex
cannot be distinguished
7
. Increasing the accelerating voltage
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 Vcvoltage)allowthe
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 v oltage (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 c harge state distribution changes were
detected upon ligand binding or CoR peptide binding
(+15 t o +17)
8
by MS. Indeed, it has been proposed by
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 d istribution 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 c orepressor
peptide, with a charge state distribution varying from +16
to +18. Using identical experimental conditions for
HD–CoRNR2 complexes, two series o f multiply charged
ions were detected corresponding to the HD (56 664.8 ±
0.3 Da) and t he 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 a nalyses 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 H D 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. T his 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 t he effect of different types of ligands on
the recruitment of t wo 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 a gonist BMS614 and the RXR antagonist
HX531 were confirmed by competition o f 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. 2 C), 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–CoRN R1 ratio (Fig. 2A–C). In
the presence of a twofold molar excess of BMS493, two ion
series are observed. The most abundant series (d)hasa
molecular mass of 59 884.1 ± 1.4 Da corresponding to the
binding of one BMS493 molecule [mass difference (DM)
9
¼
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 i n 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 re vealed by MS and that no Ônonsite-specificÕ
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 i nterface condi-
tions are optimized so that neither peptides nor ligands
dissociate during transfer to the gas phase, we con clude that
Ó FEBS 2004 ESI-MS to investigate noncovalent complexes (Eur. J. Biochem. 271) 4961
the 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 c omparison of t he relative proportions of the
different species present in solution, we made the assump-
tion that ionization efficiencies a nd 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 a ddition 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 o f 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), t he ligand is dissociated fro m all
species in the gas phase while the r atio 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 m ammalian cell two-hybrid e xperiments in
which w e monitore d t he interaction of a VP16 activation
domain-linked R ARa with a Gal-NCoR hybrid t hat w as
capable of activating a 17mer-based luciferase reporter
17 +
16
+
•
•
•
No ligand
18 +
16
+
15
+
16 +

351 Da
15 +
17
+
3200 3250 3300 3350 3400 3450 3500 3550 3600 3650 3700 3750 3800 3850 3900
m/z
•
•
•
•
•
•
•
•
+2 +2
+1
+2
+1
+2
16 +
17
+
+2
+1
+1
Reference
17 +
DM=
DM=
408 Da

•
•
•
15 +
16
+
18
+
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 H D–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 t wofold molar excess
16
of (B) BM S493:
the HD–CoRNR1 complex is stabilized upon BMS493 binding; (C) AM80: the HD–CoRNR1 complex is completely destabilized upon AM80
binding; (D) HX531: no ap parent effect on the stability of the HD–CoRNR1 comple x.
4962 S. Sanglier et al.(Eur. J. Biochem. 271) Ó FEBS 2004
(Fig. 5). Transcription activation indicated N CoR–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 e xcess o f t he
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 d ifferent ligands and
CoRNR2 are presented in Fig. 3 for the inverse pan-RAR
agonist BMS493 (Fig. 3B) an d t he RAR a 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 o f 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 b y 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 qu antitatively
bound to the HD complex.
3200 3250 3300 3350 3400 3450 3500 3550 3600 3650 3700 3750 3800 3850 3900
m/z
16 +
17 +

+
DM=
DM=
408 Da
16 +
16 +
•
•
•
17 +
15
+
17 +
17
+ 15 +
•
•
16+
16 +
351 Da
15
+
17
+
•
•
•
•
+2
+2

16 +
16
+
+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 spect ra of HD –CoRNR2. The m ass 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
17
of (B) BMS493:
the HD–CoRNR2 complex is d estabilized upon BMS493 binding; (C) AM80: the H D–CoRNR2 complex i s completely destabilized upon AM80
binding. (D) HX531: no apparent effect on the stability of the HD–CoRNR2 complex.
Ó FEBS 2004 ESI-MS to investigate noncovalent complexes (Eur. J. Biochem. 271) 4963
Effects of RXR ligands on the stability of HD–CoRNR
complexes. WhenatwofoldmolarexcessoftheRXR
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 a nd
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 t he
influence of a RXR-specific ligand on t he stability o f 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 a nd 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 o f the corresponding NR–coregu-
lator complexes
10
. The interpretation of the ESI mass spectra
provides several facts
11
: (a) both corepressor peptides
(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 s trong influence o n 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 o f most i mportance to c ontrol 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
–8
M (100%)
100
125
75
50
25
0
0–5–8 –7 –6
Ligands [logM]
Gal - RARα + Am80 10
–8
M
5 x Gal4
Gal4
LucβG
RARα
BMS493
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 re spective retinoid were added, as indica ted.
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 (%)
In presence of CoRNR1 In presence of CoRNR2
RAR–RXR + CoRNR1 RAR–RXR RAR–RXR + CoRNR2 RAR–RXR
43 57 47 53
Ligand RAR–RXR/L + CoRNR1 RAR–RXR/L RAR–RXR/L + CoRNR2 RAR–RXR/L
AM80 RAR-agonist 0 100 0 100
BMS493 RAR-inverse agonist 75 25 22 78
BMS614 RAR-antagonist 56 44 6 94
HX531 RXR-antagonist 51 49 50 50
4964 S. Sanglier et al.(Eur. J. Biochem. 271) Ó FEBS 2004
If 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 s tability of
HD–corepressor complexes, provides a good support for
a Ôstructurally-specificÕ interaction. To establish that t he
interactions detected by ESI-MS in presence of RXR
ligands are really the result of i n-solution interactions rather
than artefactual nonspecific associations occurring during
the ESI process [ 54], rigorous control e xperiments 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 e xcess of
ligand (a threefold molar excess), no b inding of a second
HX531 molecule is observed on RXRa (data not shown).
At this level, two h ypotheses may be advanced to explain
the binding of a second ligand molecule to the h eterodimer:
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 t he 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 (ele c-
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 an d hydrogen bonds are emphasized, w hile
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 s table 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, i t h as been
13
difficult to link the number of polar
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 to tally surrounds the ligand prevents its ejection from
the pocket. Whether dissociation experiments might be used
to directly describe the type of interactions in volved 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 s pecies pr esent in s olution, 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 E SI 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
relative transactivation
etOH
Am80
BMS493
BMS614
HX531
Gal-NCoR + VP16 - RARα
5xGal4
Gal4

LucβG
NCoR
RAR
VP16
Fig. 5. Enhanced interaction between RARa and NCoR induced by
synthetic retinoids. Mammaliantwo-hybridassaywithGal-NCoRas
bait and VP16-RARa as prey were performed to assess the influence of
indicated syn thetic retinoids on interaction between RARa and NCoR
in a cellular context.
Ó FEBS 2004 ESI-MS to investigate noncovalent complexes (Eur. J. Biochem. 271) 4965
Acknowledgements
We are grateful to Pascal Eberling fo r the synthesis of the CoRNR1
corepression peptid e, to Dr Lazar for the kind gift o f t he CoRNR2
peptide a nd 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. i s supported b y g rants from t he 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 S quibb.
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Ó FEBS 2004 ESI-MS to investigate noncovalent complexes (Eur. J. Biochem. 271) 4967