1. EXPRESSION / PURIFICATION ER ER / TIF2 COMPLEX FORMATION GR CONSTRUCT TIF2 CONSTRUCT ER CONSTRUCT EXPRESSION / PURIFICATION TIF2 CO-EXPRESSION GR / TIF2 PURIFICATION GR / TIF2 MASS SPEC ANALYSISANALYTICAL ULTRACENTRIFUGATION ANALYSIS thioredoxin HIS 6 TEV cleavage site hER  [255-509] HIS 6 thrombin cleavage site hTIF2 [623-772] HIS 6 thrombin cleavage site hGR [524-777] C638A, W557T, W712S NusA Preparation and characterization of complexes between ER and GR nuclear receptor LBDs and TIF2 domain Eiler, S., Granger, F., *Chevreux, G., *Van Dorsselaer, A., Moras, D. and Ruff, M. Structural Biology and Genomics Department (CNRS – UMR 7104), IGBMC, Illkirch 67404, France Structural Biology and Genomics Department (CNRS – UMR 7104), IGBMC, Illkirch 67404, France * Laboratoire 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. SUMMARY 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 / TIF2 ER  / TIF2 EXPRESSION PARAMETERS  Cells: BL21(DE3) electro-competent  Starter for culture: plates (starting from a single colony)  Medium: LB + 10% sucrose + 10  M 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-TIF2 18.5 kD NUS-GR 87.5 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-TIF2 18.5 kD NUS-GR 87.5 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 cultureYIELD = 2.5 mg TIF2 for 3 liters of culture TRX-ER o/n at 18°C LB / 10% sucrose / 10  M estradiol Expression Affinity: Hitrap Chelating NiSO4 Lysis of 6 liters of culture gradient from 10mM  500mM imidazole HIS-TIF2 o/n at 18°C LB / 10% sucrose Expression 2 mg of ER/TIF2 complex TRX-HIS-TEV-ER  lbd HIS-tb-TIF2 DIALYSIS 500mM  0mM SB201 TEV cleavage o/n at 4°C + EDTA + DTT Gel filtration: Superdex S200 ER  lbd TIF2 thioredoxin Elution profil of gel filtration TIF2ERER+TIF2+TEV ER  lbd TIF2 thioredoxin ER  / TIF2 GR / TIF2 2 ER+ 1 TIF2 ER monomer TIF2 (non structured) TIF2 non structured TIF2 dexamethasone GR/dexamethasone ERTIF2ER+TIF2 NATIVE - PAGE GR TIF2 GR+TIF2 Gel filtration: Superdex S200 Ion exchange: Hitrap Q gradient from 10mM  1000mM NaCl TIF2 NUS GR 123