Download Identification of an estrogen receptor a non covalent ubiquitin

Short Report
2577
Identification of an estrogen receptor a non covalent
ubiquitin-binding surface: role in 17b-estradiolinduced transcriptional activity
Valeria Pesiri, Piergiorgio La Rosa, Pasquale Stano and Filippo Acconcia*
Department of Science, Section Biomedical Science and Technologies, University Roma Tre, Viale Guglielmo Marconi, 446, 00146, Rome, Italy
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 11 March 2013
Journal of Cell Science 126, 2577–2582
ß 2013. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.123307
Summary
Ubiquitin (Ub)-binding domains (UBDs) located in Ub receptors decode the ubiquitination signal by non-covalently engaging the Ub
modification on their binding partners and transduce the Ub signalling through Ub-based molecular interactions. In this way, inducible
protein ubiquitination regulates diverse biological processes. The estrogen receptor alpha (ERa) is a ligand-activated transcription factor
that mediates the pleiotropic effects of the sex hormone 17b-estradiol (E2). Fine regulation of E2 pleiotropic actions depends on E2dependent ERa association with a plethora of binding partners and/or on the E2 modulation of receptor ubiquitination. Indeed, E2induced ERa polyubiquitination triggers receptor degradation and transcriptional activity, and E2-dependent reduction in ERa
monoubiquitination is crucial for E2 signalling. Monoubiquitinated proteins often contain UBDs, but whether non-covalent Ub–ERa
binding could occur and play a role in E2–ERa signalling is unknown. Here, we report an Ub-binding surface within the ERa ligand
binding domain that directs in vitro the receptor interaction with both ubiquitinated proteins and recombinant Ub chains. Mutational
analysis reveals that ERa residues leucine 429 and alanine 430 are involved in Ub binding. Moreover, impairment of ERa association to
ubiquitinated species strongly affects E2-induced ERa transcriptional activity. Considering the importance of UBDs in the Ub-based
signalling network and the central role of different ERa binding partners in the modulation of E2-dependent effects, our discoveries
provide novel insights into ERa activity that could also be relevant for ERa-dependent diseases.
Key words: 17b-estradiol, Estrogen receptor, Ubiquitin, Ubiquitin binding domains, Signal transduction
Introduction
Ubiquitination influences the functions of the target proteins by
either affecting their stability (i.e. Ub proteolytic functions) or
endowing them with additionally signalling properties as well as
by creating new surfaces for intra- and intermolecular
interactions (i.e. Ub non-proteolytic functions). Thus, Ub is an
intracellular messenger, whose nature as a signal resides in the
Ub modification. Single Ub moieties (i.e. monoubiquitination) or
polyubiquitin chains (e.g. K63-linked chains) attached to the
substrate create structural determinants that can be further
exploited for molecular interactions. Consequently, cells have
evolved Ub receptors that bind to the ubiquitinated protein by
non-covalently contacting the Ub modification through specific
UBDs and in this way decode, transduce and amplify the
ubiquitination signal diversity into regulation of physiological
functions (e.g. cell proliferation and migration) (Acconcia et al.,
2009; Husnjak and Dikic, 2012; Woelk et al., 2007).
The ERa is a ligand-activated transcription factor that
mediates the cellular effects of the steroid hormone E2 through
modifications in the pattern of gene expression. Regulation of
ERa gene transcription requires the receptor recruitment to target
gene promoters either directly through interaction with estrogen
responsive elements (i.e. ERE sequences) or indirectly through
ERa interaction with other transcriptional factors (e.g. AP-1; Sp1) (Ascenzi et al., 2006; Smith and O’Malley, 2004). Mounting
evidence indicates that ERa activities are modulated by Ub (La
Rosa and Acconcia, 2011). Polyubiquitination controls the E2dependent regulation of 26S-proteasome ERa degradation, which
is interlaced with ERa transcriptional activity (La Rosa and
Acconcia, 2011; La Rosa et al., 2012). Non-proteolytic Ub
functions in E2:ERa signalling also occur and depend on ERa
monoubiquitination, which is negatively modulated by E2 and
controls E2-dependent cell proliferation (Eakin et al., 2007; La
Rosa et al., 2011a; La Rosa et al., 2011b; Ma et al., 2010).
Monoubiquitinated proteins are often Ub receptors that possess
at least one UBD (Woelk et al., 2006). Although many different
UBDs exist, no specific conservation in terms of UBDs 3Dstructure has been recognized. In fact, UBDs appear to share a
common ‘shape’: structural folds that have a regular secondary
structure (e.g. a-helix and/or Zn2+-finger) are thought to be the
only UBDs common features (Dikic et al., 2009; Husnjak and
Dikic, 2012; Woelk et al., 2007). Interestingly, ERa biochemical
anatomy consists in six modular domains (A to F). The A/B, D
and F domains do not display a folded structure, the C domain
(i.e. the DNA-binding domain, DBD) consists in two Zn2+-finger
motifs and the E domain (i.e. the ligand binding domain, LBD) is
composed of 12 a-helices (Ascenzi et al., 2006). On this basis, we
speculated that an UBD could be present in ERa. Here, we report
the identification of an ERa Ub-binding surface (ERa-UBS) that
is required for E2-dependent ERa transcriptional activity.
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Journal of Cell Science 126 (12)
Results and Discussion
Journal of Cell Science
Identification of an Ub-binding surface on ERa
In vitro pull-down experiments were done to investigate the
possibility that ERa binds Ub. We found that the A/B and the E
domains but not the C domain were able to pull-down
ubiquitinated species from total cellular lysates (Fig. 1A).
Because the A/B domain is non-structured and displays weak
association to ubiquitinated species when compared to the 12 ahelix-containing E domain, we focused on the E domain Ubbinding ability. Fig. 1B shows that this receptor portion is also
able to pull-down recombinant K63-linked polyubiquitin chains,
thus demonstrating that the ERa E domain non-covalently
contacts Ub on the Ub-modified proteins. The E domain also
bound recombinant K48-linked polyubiquitin chains but not
purified mono-Ub (supplementary material Fig. S1A). Thus, as in
the case of other UBDs (Woelk et al., 2007), the ERa Ub-binding
surface (ERa-UBS) in the E domain displays preferential Ubbinding ability and could possess a low Ub-binding affinity.
We next investigated which portion of the ERa E domain is
necessary for Ub-binding. The N-terminal part of the E domain
(i.e. amino acids 301–439) but not the E domain C-terminus (i.e.
amino acids 439–547) was able to pull-down ubiquitinated species
from total cellular lysates (Fig. 1C) and recombinant K63-linked
polyubiquitin chains (Fig. 1D) although with different amount of
bound materials with respect to the intact E domain (Fig. 1A,B).
This evidence indicates that the region of the ERa E domain that
non-covalently associates in vitro with Ub is located within the
protein region encompassing the amino acids 301–439.
Interestingly, our data indicate that disruption of the ERa E
domain reduces the ERa Ub binding abilities. Unfortunately, we
could not narrow down a smaller section of the E domain that
Fig. 1. Ub-binding ability of ERa. (A,C) Schematic of ERa (A, top) and
ERa E (C, left) domain deletions are depicted. Amino acid positions and
domains are indicated. In vitro pull-down assay using the indicated GSTtagged ERa constructs. GST-fusion proteins were incubated with total cellular
lysates extracted from growing HeLa cells and analyzed by immunoblot as
indicated. (B,D) As for A and C except that fusion proteins were incubated
with synthetic polyUb2-7 linked by Lys63.
associated to ubiquitinated species by using rational deletions of
the N-terminal protein portion (i.e. 301–439) (data not shown).
Thus, it is most likely that the ERa-UBS requires an intact E
domain 3D-folding. In turn, in silico molecular modelling
experiments would help rationalize the biochemical structural
requirements for ERa E domain:Ub interaction.
For these reasons, we decided to introduce specific point
mutations within the minimal Ub-binding region (i.e. 301–439) in
the context of the full-length E domain in order to find the critical
residues required for Ub-binding. The residues I358 and the Q375
are important for in vitro ERa monoubiquitination (Eakin et al.,
2007), thus they may be part of the ERa-UBS (Woelk et al., 2006).
Because the E domain double-mutated in I358 and Q375 to A
(IQAA) is native-like (Eakin et al., 2007) but did not display any
significant differences in Ub-binding when compared with the wt
Fig. 2. Characterization of the ERa-UBS. (A,C,D) Top: schematic of ERa
E domain. Amino acid positions are indicated. Bottom: In vitro pull-down
assay using the indicated GST-tagged ERa constructs. GST-fusion proteins
were incubated with total cellular lysates extracted from growing HeLa cells
(A and C) or with synthetic polyUb2-7 linked by Lys63 (D) and analyzed by
immunoblot as indicated. * indicates significant differences with respect to
the relative wild-type sample. (B) Top: ClustalW (http://www.ebi.ac.uk/
Tools/msa/clustalw2/) alignment of ERa E domain with RNF168 UMI
domain (Pinato et al., 2011). Middle: Projected helices using the Helical
Wheel Projections software tool (http://rzlab.ucr.edu/scripts/wheel/wheel.
cgi). Bottom: Three-dimensional structure of the human ERa LBD-E2
complex (1ERE) (Brzozowski et al., 1997); E2 has been removed. The
structure is drawn in green; conserved amino acids between the ERa E
domain and RNF168 UMI domain are space-filled and stained as indicated.
Journal of Cell Science
ERa ubiquitin-binding surface
E domain (Fig. 2A), I358 and Q375 are not necessary for ERaUBS. Interestingly, bioinformatic analysis identified L428, L429
and A430 in the E domain to have a spatial distribution reminiscent
of the UBD called UMI (Pinato et al., 2011), with L428 buried in
the E domain core protein matrix and L429 and A430 at least
partially exposed to solvent (Fig. 2B). Consistently, while single
substitution of L429 or A430 barely affected the Ub-binding
abilities of the E domain (Fig. 2C), double mutation of L429 and
A430 (L429A,A430G; LAAG) strongly prevented the E domain
Ub-binding (Fig. 2C,D). Notably, introduction of the LAAG
mutation in ERa did not significantly affect proper receptor
folding, as indicated by circular dichroism (CD) on the E-domain
GST-fusion proteins (supplementary material Fig. S1B).
Based on this evidence, we report that two Ub-binding surfaces
could exist within ERa and that the residues L429 and A430
within the E domain are necessary for Ub-binding. Interestingly,
as the A/B and the E domains (Ascenzi et al., 2006) are located
at the N-terminus and at the C-terminus of the ERa
monoubiquitination sites (K302 and K303) (Eakin et al., 2007)
respectively, it is tempting to speculate that avidity-based Ubbinding interactions could direct intra-molecular communications
between distant ERa domains. In this respect, structural crosstalk between distant ERa A/B and E domains has been reported
to be required for the modulation of ERa activities (Métivier
et al., 2002).
Role of ERa-UBS in E2-induced ERa gene transcription
Next, we sought to determine the role of ERa non-covalent Ubbinding on receptor transcriptional activity. Thus, the LAAG
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mutation was generated in the context of the full length receptor
(wt) (Figs 3, 4) and transiently transfected in HeLa or HEK293
cells. Initially, we verified if the LAAG mutation in the context
of the full length ERa reduces Ub-binding also in cells by
evaluating if wt ERa was able to associate to ubiquitinated
proteins in cells. The anti-Ub blot performed after wt ERa
immunoprecipitation resulted in a clear smear only in HEK293
transfected with wt ERa but not in non-transfected HEK293
cells (Fig. 3A). Because ubiquitinated ERa interactors can in
principle associate to the receptor both directly through the Ubbased modification and through a different protein surface
(Woelk et al., 2006) and the anti-Ub antibody cannot
distinguish between these two possibilities, we also performed
wt ERa immunoprecipitation by treating the lysates with SDS in
order to reduce the amount of receptor interactors (Woelk et al.,
2006). Under these conditions, wt ERa was still able to associate
with ubiquitinated proteins although less efficiently than in the
non-treated lysates (Fig. 3A), thus demonstrating that ERa binds
ubiquitinated proteins in cells. Prompted by these results, we next
tested the ability of the LAAG mutant receptor to associate with
ubiquitinated proteins in cells under the above-mentioned
stringent conditions. Immunoprecipitation analysis revealed that
introduction of the double mutation L429A,A430G in ERa
strongly prevents the receptor ability to associate with
ubiquitinated proteins (Fig. 3B). These data demonstrate that
the ERa-UBS directs Ub-binding also in cells.
Next, we determined whether the reduction in ERa Ub-binding
could affect basal ERa transcriptional activity. As shown in
Fig. 3C, equal expression of either wt ERa or of ERa LAAG
Fig. 3. Characterization of the full length ERa-UBS
mutant. (A,B) Ubiquitin and ERa western blot analysis
of ERa immunoprecipitation in non-transfected (Nt) and
pcDNA FLAG wild-type ERa (Wt) HEK293 cells
(A) and in pcDNA FLAG-ERa L429A, A430G (LAAG)
mutant transfected HEK293 cells (B). SDS (0.05%) was
added to the lysates in A where indicated (+) and in B
before immunoprecipitation and the immunoprecipitated
complexes were washed in RIPA buffer containing 0.1%
SDS. (C) Luciferase assay on the HeLa cells transiently
transfected with pcDNA FLAG, pcDNA FLAG-ERa
(Wt) or the pcDNA FLAG-ERa L429A, A430G (LAAG)
mutant together with the 36ERE TATA reporter plasmid.
Insets show Wt and mutant FLAG-ERa expression
normalized on vinculin. (D) ERa dimerization analysis in
growing HEK293 cells transiently co-transfected with
pcDNA FLAG-ERa (Wt) and pEGFP-ERa (Wt), and
with pcDNA FLAG-ERa (LAAG) and pEGFP-ERa
(LAAG) mutants, as indicated. After transfection, cell
lysates were immunoprecipitated with anti-FLAG
antibody, followed by immunoblot with anti-ERa
antibody (rabbit). (E) Confocal miscrocopy analysis of
the HeLa cells transiently transfected with pEGFP-ERa
(Wt) or the pEGFP-ERa (LAAG) mutant kept in
growing conditions.
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Journal of Cell Science 126 (12)
Journal of Cell Science
Fig. 4. Effect of ERa Ub-binding ability on E2-induced
receptor transcriptional activity. (A,C,D) Luciferase
assay on the HeLa cells transiently transfected with
pcDNA FLAG-ERa (wt) or the pcDNA FLAG-ERa
(LAAG) mutant together with either the 36ERE TATA
reporter plasmid (A), with pXP2-D1-2966 cyclin D1
reporter plasmid (pD1) (C) or with the protein complement
3 (pC3) reporter plasmid (D) and then treated for 24 hours
with different doses of E2 (A) or with 10-9 M E2
(C,D). (B) Western blot analysis of the Bcl-2 protein levels
in the HeLa cells transiently transfected with pcDNA
FLAG-ERa (wt) or the pcDNA FLAG-ERa (LAAG)
mutant and then treated for 24 hours with different
doses of E2. Loading control was done by evaluating
vinculin expression in the same filter. * indicates
significant differences with respect to the relative C
sample; ˚ indicates significant differences with respect to
the relative wt ERa E2-treated sample.
mutant triggered the activation of the artificial ERE-containing
reporter constructs (i.e. 3xERE-TATA, pERE) (La Rosa et al.,
2012) to comparable levels. ERa binds DNA to ERE sequences as
a homodimer (Ascenzi et al., 2006) and co-immunoprecipitation
analysis revealed that both wt and mutant receptors were equally
able to dimerize (Fig. 3D), thus further suggesting that receptor
dimerization is not affected by the LAAG mutation. Moreover,
ERa-based signalling is a function of receptor intracellular
localization (i.e. nuclear and extra-nuclear) (Ascenzi et al., 2006;
La Rosa et al., 2012). Thus, we also tested the effect of LAAG
mutation on ERa sub-cellular distribution. Immunofluorescence
analysis showed that both GFP-ERa and GFP-ERa LAAG
mutant have the same nuclear, cytoplasmic and membranetethered localization (Fig. 3E). Thus, we conclude that mutation
of L429 and A430 residues does not influence basal ERa
transcriptional activity (e.g. ERE binding), receptor dimerization
and sub-cellular localization.
Analysis of the ability of E2 to trigger ERa transcriptional
activity revealed that the introduction of the LAAG mutation in
ERa strongly reduced, with respect to the wt ERa, the receptor
ability to activate the 3xERE-TATA reporter constructs as a
dose-dependent function of E2 (Fig. 4A) without affecting E2
binding affinity (Table 1). This effect was still evident when the
E2-induced ERa LAAG mutant transcriptional activity was
tested in the presence of a reporter construct containing the ERE
sequence in the context of the protein complement 3 (pC3)
natural promoter (La Rosa et al., 2011b) (Fig. 4B). Besides the
direct association of ERa to ERE located in the target gene
promoters (Reid et al., 2003), the E2-activated receptor can
regulate the transcription of non-ERE containing genes (e.g.
cyclin D1) through indirect association with other transcription
factors (e.g. AP-1, Sp-1) (Ascenzi et al., 2006; Marino et al.,
2002; Marino et al., 2003). Even in this case, mutation of the
L429 and A430 residues within ERa prevented the E2-induced
wt ERa mediated cyclin D1 promoter activation (pD1) (Fig. 4C).
Therefore, mutation of the ERa-UBS prevents the E2-dependent
activation of direct and indirect ERa transcriptional activity.
Finally, we investigated the physiological expression of the
endogenous E2-responsive Bcl-2 gene (Acconcia et al., 2005b).
Dose-response analysis revealed that the LAAG mutation
reduced the ability of the E2:ERa complex to increase the Bcl2 cellular levels as compared with the wt ERa (Fig. 4D). Taken
together, these data demonstrate that the reduction in ERa
non-covalent Ub-binding results in an overall lower ERa
transcriptional activity.
In conclusion, we identify here the structural determinants
required for ERa to non-covalently associate to Ub and
further demonstrate the involvement of the ERa-UBS in the E2induced ERa transcriptional activity. E2-dependent productive
gene transcription involves the sequential formation of
macromolecular complexes centred on ERa (Manavathi et al.,
2012; Tarallo et al., 2011). Many of the ERa binding partners are
either ubiquitinated proteins or enzymes of the ubiquitination
cascades (i.e. Ub-ligases) (La Rosa and Acconcia, 2011; Métivier
Table 1. E2 binding to wild-type and mutant ERa
IC50 (M)
Kit
E2
1.0610
ERa Rec
28
ERa wt
28
3.060.24610
ERa LAAG
210
7.060.91610
2.660.40610210
In vitro ERa affinity (as indicated by the IC50 value) was determined using the HitHunter EFC assay (Discoverx) according to manufacturer instructions for E2
towards either recombinant ERa protein (ERa Rec), wild-type or LAAG mutant receptors (ERa wt or ERa LAAG, respectively) extracted from transfected
HEK293 cells. Kit indicates the value of E2 affinity to ERa as reported in the manufacturer’s specification, and has been introduced as a positive control
reference; the value is similar to that measured with ERa Rec (Panvera). Results show that the E2 affinity to ERa wt is the same as that for the mutant ERa LAAG.
Overall affinity of ERa wt and ERa LAAG with respect to ERa Rec can be ascribed to the fact that ERa wt and ERa LAAG E2 binding abilities were measured in
equivalent amounts of total cellular lysates.
ERa ubiquitin-binding surface
et al., 2003; Reid et al., 2003). Thus, our discoveries open the
possibility that the assembly of E2-dependent ERa-based
macromolecular complexes could be due to the presence of
ERa UBS (present results), ERa monoubiquitination (Eakin et al.,
2007; La Rosa et al., 2011a) or both. Remarkably, the ERa extranuclear signalling also integrates and controls receptor
transcriptional activity in the modulation of the E2-dependent
cellular processes (e.g. proliferation) (Ascenzi et al., 2006; La
Rosa et al., 2012). Thus, ERa Ub-binding could play critical roles
in the modulation of ERa nuclear and extra-nuclear activities and
of E2-regulated cellular processes.
Materials and Methods
Cell culture and reagents
HeLa and HEK293 cells were grown as previously described (La Rosa et al.,
2011b). Specific antibodies against FLAG epitope (M2) and vinculin (Sigma, St.
Louis, MO, USA); anti-ERa (HC-20) anti-Bcl-2 (C-2) and anti-ubiquitin (P4D1)
(Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. All other antibodies
were purchased by Cell Signalling Technology Inc. (Beverly, MA, USA). All the
products were from Sigma-Aldrich (St. Louis, MO, USA). Analytical or reagent
grade products, without further purification, were used.
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proteins were measured at the concentration of 0.075 mg/ml. For each sample,
nine sequential spectra were recorded and averaged. Data are shown, without
smoothing, in terms of mean molar ellipticity per residue.
Cellular and biochemical assays
Protein extraction, immunoprecipitation assays and western blots as well as HeLa
and HEK293 cells transfection, luciferase assays and confocal microscopy analysis
were performed as previously described (La Rosa et al., 2011b).
Statistical analysis
Statistical analysis was performed using the ANOVA test with the InStat.3
software system (GraphPad Software Inc., San Diego, CA). In all analyses,
P,0.01 was considered significant, but for densitometric analysis P,0.05 was
considered significant. Data are means6s.d. of three independent experiments.
Author contributions
V.P. performed pull-down, transfection, western blot analysis,
confocal microscopy, binding and circular dicroism experiments;
P.L.R. performed the dimerization assay; P.S. performed circular
dicroism and analyzed the relative data; F.A. conceived the idea and
planned the research, prepared the plamids, performed the
transfection and binding experiments, analyzed the data and wrote
the paper.
Journal of Cell Science
Plasmids and constructs
The reporter plasmid 3xERE TATA, protein complement 3 (pC3) and the pXP2D1-2966 plasmid (pD1) and the pcDNA FLAG-ERa have been described (La Rosa
et al., 2011b). The pEGFP-ERa was obtained by subcloning the ERa ORF from
the pSG5-HE0 (Acconcia et al., 2005a) into the pEGFP-C2 obtained by Dr Simona
Polo, IFOM, Milan.
Site-directed mutagenesis of the ERa L429 and A430 residues were performed
by using the QuikChange kit (Stratagene, La Jolla, CA) and the following
oligonucleotides: 59-GAGATCTTCGACATGCTGGCGGCTACATCATCTCGGTTC-39 (for single L429A mutation) on the wt ERa ORF used as template; 59ATCTTCGACATGCTGCTGGGTACATCATCTCGGTTCCGC-39 (for single
A430G mutation) on the wt ERa ORF used as template; 59-ATCTTCGACATGCTGGCGGGTACATCATCTCGGTTCCGC-39 (for double L429A, A430G
mutation) on the ERa ORF L429A mutant used as template (bold underlined
nucleotides differ from the wt ERa ORF). Plasmids were then sequenced to verify
the introduction of the desired mutations. pGEX-6P-3 ERa E domain I358A,
Q375A was subcloned from pET151D-ERa LBD I358A, Q375A generously
provided by Dr Rachel Klevit (Eakin et al., 2007). To engineer constructs
harboring ERa deletions domains, PCR amplification was performed with the
oligonucleotides in supplementary material Table S1.
PCR products were then cloned into pGEX-6P-3 (obtained by Dr Simona Polo,
IFOM, Milan) using BamHI/XhoI sites. All constructs were sequence verified.
GST pull-down assays and circular dichroism
GST fusion proteins were expressed, purified and as described in (Belenghi et al.,
2003) except that all ERa-E domain encoding constructs were prepared in the
presence of 20 mM E2. Pull-down experiments were performed as previously
reported (La Rosa et al., 2011b; Penengo et al., 2006) by incubating 30 mg of GSTfusion proteins with either 300 mg of growing HeLa cells total lysate for 3 hours at
4 ˚C or with 0.5 mg of polyUb2-7 linked by Lys63 (Boston Biochem) for 1 hour at
4 ˚C in the presence of 20 mM E2. All experiments were normalized by running 1/
10 of the pull-down on an SDS-PAGE gel. Proteins were detected by Comassie
Brilliant Blue staining (Com.). For circular dichroism analysis GST-fusion proteins
were eluted and recovered from the beads by treatment with 20 mM free
glutathione in 50 mM Tris HCl (pH 9) buffer. The elution buffer, which is
unsuitable for spectral measurement in the far UV region, was exchanged by
dialyzing the proteins (two times) against 100 volumes of 25 mM sodium
phosphate (pH 7.5), 150 mM NaF at 4 ˚C, for 10 hours.
UV absorption spectroscopy
The molar extinction coefficients at 280 nm (e280) of GST-WT and GST-LAAG
fusion proteins (476 residues, 54.8 kDa), calculated by the method of Gill and von
Hipple (Gill and von Hippel, 1989), resulted to be 65,110 cm21 M21. The proteins
were centrifuged (16 ˚C, 10 minutes, 15,000 rpm) to remove traces of scattering
particles, and their absorption spectra were recorded on an Agilent HP8453 diode
array spectrophotometer, using 1 cm quartz cell. Samples were perfectly
transparent at all wavelengths greater than 300 nm. The protein concentrations
were measured by reading the absorption at 280 nm.
Circular dichroism measurements
CD spectra in the far-UV region were recorded at 16 ˚C using a Jasco J-600
spectropolarimeter and 0.05 cm quartz cells. GST-WT and GST-LAAG fusion
Funding
This study was supported by grants from Associazione Italiana
Ricerca sul Cancro (AIRC) [grant number MFAG12756] and Ateneo
Roma Tre to F.A.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.123307/-/DC1
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