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