expression / purification er er / tif2 complex formation gr construct tif2 construct er construct...
TRANSCRIPT
EXPRESSION / PURIFICATION EREXPRESSION / PURIFICATION ER
ER / TIF2 COMPLEX FORMATION
GR CONSTRUCTGR CONSTRUCTTIF2 CONSTRUCTTIF2 CONSTRUCTER CONSTRUCTER CONSTRUCT
EXPRESSION / PURIFICATION TIF2EXPRESSION / PURIFICATION TIF2 CO-EXPRESSION GR / TIF2CO-EXPRESSION GR / TIF2
PURIFICATION GR / TIF2PURIFICATION GR / TIF2
MASS SPEC ANALYSIS ANALYTICAL ULTRACENTRIFUGATION ANALYSIS
thioredoxinHIS6
TEV cleavage site
hER [255-509]HIS6
thrombin cleavage site
hTIF2 [623-772]HIS6
thrombin cleavage site
hGR [524-777] C638A, W557T, W712SNusA
Preparation and characterization Preparation and characterization of complexes between ER and GR of complexes between ER and GR
nuclear receptor LBDs and TIF2 domainnuclear receptor LBDs and TIF2 domain Eiler, S., Granger, F., *Chevreux, G., *Van Dorsselaer, A., Moras, D. and Eiler, S., Granger, F., *Chevreux, G., *Van Dorsselaer, A., Moras, D. and Ruff, M.Ruff, M.
Structural Biology and Genomics Department (CNRS – UMR 7104), IGBMC, Illkirch 67404, FranceStructural Biology and Genomics Department (CNRS – UMR 7104), IGBMC, Illkirch 67404, France
**Laboratoire de Spectrométrie de Masse Bio-Organique (UMR 7509), Strasbourg, FranceLaboratoire de Spectrométrie de Masse Bio-Organique (UMR 7509), Strasbourg, France
Acknowledgements: We thank all members of the Structural Biology and Genomics Department. This work was supported by funds from SPINE EEC QLG2-CT-2002-00988, FNS through the Genopole program, CNRS, INSERM, ULP and local authorities (Region of Alsace, Department of Bas-Rhin and city of Strasbourg).
Nuclear receptors (NRs) are ligand-regulated transcription factors that regulate crucial gene networks responsible for cell growth, differentiation, and homeostasis. They form a superfamily of phylogenetically related proteins and control functions associated with major diseases (diabetes, osteoporosis and cancer). This superfamily has been partitioned into two classes related to they oligomeric behaviour (class I for homodimers and class II for heterodimers). NR-mediated transcription depends on coactivators, proteins that affect the transcriptional machinery in a variety of ways (via associated proteins as histone acetyltransferases, methyltransferases, ubiquitin ligases or like agents that integrate signalling via kinase-signaling pathways). We are interested in the steroid family that belongs to class I. The structural behaviour of these proteins depends heavily on the presence or absence of ligand together with the interaction with partner proteins. In the unliganded form they interact with HSP90, dissociate upon ligand binding and interact with DNA and co activators or co repressors. Preparation of large amount of soluble and functional NRs needed for structural studies is a challenge for the steroid NRs subgroup. Here we describe the preparation and the characterization of the complexes of the glucocorticoid and estrogen receptor ligand binding domains (LBDs) in complex with a TIF2 domain containing the three NR binding motifs.
SUMMARYSUMMARY
Functional complexes of the glucocorticoid and estrogen nuclear receptor LBDs in complex with a TIF2 fragment containing three NR binding motifs have been purified and characterized. We found that one dimer of estrogen or glucocorticoid ligand binding domain binds one molecule of TIF2. Interestingly, we have evidences that the structure of the TIF2 fragment alone is disordered in solution (ESMS and NMR (not schown)) and that the binding of the nuclear receptor induces a partial folding of this molecule.Mild proteolysis assays are under way to define a smaller TIF2 fragment for the interaction with LBDs. Larger TIF2 fragments are tested in expression and co-expression for the interaction with ER and GR LBDs (see poster : Developments of protocols for the preparation of glucocorticoid nuclear receptor complexes, S. Eiler et al.)
CONCLUSIONS & PERSPECTIVES
TIF2
GR monomer + 1 βM
GR monomer + 1 βM + TIF2 monomer
GR dimer + 2 βM + TIF2 monomer
GR / TIF2ER / TIF2
EXPRESSION PARAMETERS
Cells: BL21(DE3) electro-competent Starter for culture: plates (starting from a single colony) Medium: LB + 10% sucrose + 10M dexamethasone Antibiotics: ampicillin + kanamycin Induction: 0.5 mM IPTG Expression: o/n at 18°C (1 liter medium in 5 liters flask)
YIELD = 5 g of cells / liter of culture
HIS-TIF218.5 kD
NUS-GR87.5 kD
Gel filtration: Superdex S200
Thrombin cleavage o/n at 4°C
YIELD = 0.5 mg GR/TIF2 complex for 15g of cells
Lysis of 15g cells
HIS-TIF218.5 kD
NUS-GR87.5 kD
TIF2 16.5 kD
NUS 57 kD
GR 30.5 kD
HIS-TIF2
NUS-GR
Affinity: Hitrap Chelating ZnCl2
gradient from 10mM 500mM imidazole
HIS-TIF2
NUS-GR
Affinity: Hitrap Chelating NiSO4
Lysis of 6 liters of culture
gradient from 10mM 500mM imidazole
YIELD = 2.5 mg ER for 6 liters of culture YIELD = 2.5 mg TIF2 for 3 liters of culture
TRX-ER
o/n at 18°CLB / 10% sucrose / 10M estradiol
Expression
Affinity: Hitrap Chelating NiSO4
Lysis of 6 liters of culture
gradient from 10mM 500mM imidazole
HIS-TIF2
o/n at 18°CLB / 10% sucrose
Expression
2 mg of ER/TIF2 complex
TRX-HIS-TEV-ERlbd HIS-tb-TIF2
DIALYSIS500mM 0mM SB201
TEV cleavage o/n at 4°C+ EDTA + DTT
Gel filtration: Superdex S200ERlbd
TIF2
thioredoxin
Elution profil of gel filtration
TIF2 ER ER+TIF2+TEV
ERlbd
TIF2
thioredoxin
ER / TIF2 GR / TIF2
2 ER+ 1 TIF2
ER monomer
TIF2(non structured)
TIF2 non structured
TIF2
dexamethasone
GR/dexamethasone
ER TIF2 ER+TIF2
NATIVE - PAGE NATIVE - PAGE
GR
TIF
2
GR
+T
IF2
Gel filtration: Superdex S200 Gel filtration: Superdex S200
Ion exchange: Hitrap Q
gradient from 10mM 1000mM NaCl
TIF2
NUS
GR
1 2 3