Human SKI-like

Target ID:

SKILA

Aliases:

SKILSNOSnoASnoN

Deposition Date:

Tuesday 30th September 2008

Families:

Signalling proteins

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

3EQ5

Authors:

L.TRESAUGUES,M.WISNIEWSKA,J.ANDERSSON,C.H.ARROWSMITH,H.BERGLUND,C.BOUNTRA,R.COLLINS,L.G.DAHLGREN,A.M.EDWARDS,S.FLODIN,A.FLORES,S.GRASLUND,M.HAMMARSTROM,A.JOHANSSON,I.JOHANSSON,T.KARLBERG,T.KOTENYOVA,L.LEHTIO,M.MOCHE,M.E.NILSSON,T.NYMAN,K.OLESEN,C.PERSSON,J.SAGEMARK,H.SCHUELER,A.G.THORSELL,S.VAN DER BERG,M.WELIN,M.WIKSTROM,J.WEIGELT,P.NORDLUND.

Site:

Stockholm

ICM Datapack:

ICM Datapack

3EQ5

tabs

Structure Details

Members of the TGF-β family of extracellular growth factors regulate cell proliferation, recognition, differentiation, apoptosis, and developmental fate in metazoans (1). One effect of the TGF-β proteins is to inhibit cell proliferation. Loss of cell responsiveness to TGF-β is thought to contribute to the pathogenesis of several epithelial tumors.

TGF-β ligands act by forming an activated heteromeric complex with specific transmembrane TGF-β serine/threonine receptors. The activated receptor catalyzes the phosphorylation of receptor-regulated Smad proteins (R-Smads) (2). Phosphorylated R-Smads then associate with the common partner Smad4 (co-Smad4). The thus formed Smad complex accumulates in the nucleus, where it binds smad-binding elements within promoters of TGF-β responsive genes to regulate their transcription, either by activation or repression, thereby affecting TGF-β dependent cellular responses.
Additional transcriptional regulators modulate the ability of Smad protins to control the expression of TGF-β-responsive genes. One such regulator, the Smad-interacting ski-like protein (SKIL, or SnoN) has emerged as an important regulator of the TGF-β signaling pathway.
SKIL is a member of the ski/sno/dac gene family, which share two regions of high homology. One referred to as the Dachshund homology domain, and a SAND domain. SAND domains are DNA binding domains found in a number of chromatin remodeling proteins. But in SKI and SKILA the DNA binding is absent and instead the SAND domain here interacts with Smads and other regulatory proteins.

Here, the structure of the Dachshund homology domain of SKIL is presented. The structure was solved to a resolution of 2.45 Å (Rfree=27.3%) by molecular replacement using the coordinates of the Dachshund-homology domain of human SKI (PDB-code : 1SBX) (3). The asymmetric unit (space-group C2) is composed of 12 monomers which are packed as a trimer of tetramers. This organization seems to be purely due to crystal packing as revealed by both gel-filtration profile and results from PISA server (4).This domain of SKIL adopts the fold described previously in the structure of the N-terminal domain of Dachshund (5). It consists in a four-stranded anti-parallel β-sheet flanked by three α-helices on one side and one on the other side. The 12 monomers are similar with the exception of helix α2 and the loop connecting α3 to β4 which are responsible for the overall rmsd value of 1.5 Å during superimposition of chain I and C for 94 superimposed residues. Superposition of SKIL and the Dachshund-homology domain of human SKI led to an rmsd of 0.74 Å for 96 superimposed residues in the case of monomer C and 1.30 Å for 84 superimposed residues in the case of monomer F. The increased rmsd value is mainly due to the same regions as described previously. The non-conserved residues between these two proteins are solvent exposed, a feature that could attribute to them a fundamental role during protein-protein interactions.

Materials and Methods

SKIL

PDB:3EQ5

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC059386
Entry Clone Source:Mammalian Gene Collection
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*sm
Host: E.coli BL21(DE3) R3 pRARE, where R3 denotes a derivative of BL21(DE3) resistant to a strain of T1 bacteriophage (SGC Oxford) and the pRARE plasmid originating from the Rosetta strain (Novagen) supplies tRNAs for rare codons.

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*smPSDSSTELTQTVLEGESISCFQVGGEKRLCLPQVLNSVLREFTLQQINTVCDELYIYCSRCTSDQLHILKVLGILPFNAPSCGLITLTDAQRLCNALLRPRT
Vector:pNIC-BSA4

Growth

Medium:
Antibiotics:
Procedure:Cells from a glycerol stock were grown in 10 ml TB supplemented with 8 g/l glycerol, 100 µg/ml kanamycin and 34 µg/ml chloramphenicol at 30 °C overnight. The overnight culture (10 ml) was used to inoculate 0.75 l TB supplemented with 8 g/l glycerol, 50 µg/ml kanamycin and approximately 100 µl PPG P2,000 81380 anti-foam solution (Fluka). The culture was grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 °C until OD600 reached ~2. The bottle was down-tempered to 18 °C over a period of 1 hour before target expression was induced by addition of 0.5 mM IPTG. Expression was allowed to continue overnight and cells were harvested the following morning by centrifugation (4,400 x g, 10 min, 4 °C). The resulting cell pellet (9.0 g wet cell weight) was resuspended in lysis buffer (3 ml/g cell pellet), supplemented with 1000 U Benzonase (Merck) and 0.5 tablet of Complete EDTA-free protease inhibitor (Roche Applied Science). The cell suspension was stored at -80 °C.

Purification

Procedure
Columns
IMAC: Ni-charged 1 ml HiTrap Chelating HP (GE Healthcare)
Gel filtration column: HiLoad 16/60 Superdex 75 Prep Grade (GE Healthcare)

Procedure
Purification of the protein was performed as a two step process on an ÄKTAxpress system (GE Healthcare). Prior to purification, columns were equilibrated with IMAC wash1 buffer and gel filtration buffer, respectively. The filtered lysate was loaded onto the Ni-charged HiTrap Chelating column and washed with IMAC wash1 buffer followed by IMAC wash2 buffer. Bound protein was eluted from the IMAC column with IMAC elution buffer and automatically loaded onto the gel filtration column. Fractions containing the target protein were pooled and fresh TCEP was added to a final concentration of 2 mM. The protein was subsequently concentrated using a Amicon Ultra-15 centrifugal filter device with 5,000 NMWL (Millipore) to 42.7 mg/ml in a volume of 1.2 ml.

Tag removal
The N-terminal histidine tag was proteolytically removed by incubating the target protein with His-tagged TEV protease (van den Berg, S., J. Biotech 121, 291-298 (2006)) at a molar ratio of 30:1 at 4 ºC overnight. The proteolytic reaction went to completion, as judged by SDS-PAGE. Target protein was purified from tag and protease by passing the reaction mixture over a Ni-charged 1 ml HiTrap Chelating HP column (GE Healthcare) pre-equilibrated with IMAC wash1 buffer. The cleaved protein was concentrated and the buffer was changed to GF buffer using a centrifugal filter device with 5,000 NMWL. The final protein concentration was determined to 19.6 mg/ml in a volume of 0.7 ml. The identity of the protein was confirmed by mass spectrometry.

Extraction

Procedure
The cell suspension was quickly thawed in water and diluted to 68 ml with lysis buffer. Cells were disrupted by sonication (Vibra-Cell, Sonics) at 80% amplitude for 3 min effective time (pulsed 4s on, 4s off) and cell debris was removed by centrifugation (49,000 x g, 20 min, 4 ºC). The supernatant was decanted and filtered through a 0.45 µm flask filter.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were obtained by the sitting drop vapour diffusion method in a 96-well plate. 0.1 µl of the protein solution (19.6 mg/ml) was mixed with 0.2 µl of well solution consisting of 0.2 M ammonium nitrate and 20% (w/v) PEG 3350. The plate was incubated at 4 ºC and rhombohedral crystals appeared after 15 to 29 days. After 73 days, the crystal was transferred to cryo solution consisting of 20 mM HEPES, 300 mM NaCl, 20% glycerol, 0.2 M ammonium nitrate and 20% (w/v) PEG 3350, pH 7.5, and flash-frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:125° was collected by frame of 0.5° at ESRF (ID29) (0.97623 Å).
Data Processing:The crystal belonged to space group C2 with the cell parameters a=210.820, b=70.120, c=116.280, α=γ=90.00 and β=100.95. Data was processed and scaled using XDS and XSCALE. PHASER found 7 correct monomers in the asymmetric unit using the structure of the Dachshund-homology domain of human SKI (PDB-code: 1SBX) as a probe. This solution was refined in CNS using simulated annealing. The resulting model was used as a probe in a new query in PHASER and resulted in the discovery of two more monomers. Analysis of the crystallographic contacts allowed to build 3 additional monomers in the density map with Coot, resulting in the final asymmetric unit of 12 monomers. Data in the interval 19.95 - 2.45 Å resolution was used for refinement. Iterative cycles of manual building in both Coot and O and restrained refinement in REFMAC5 were performed. Final TLS refinement using 3 TLS groups for 11 monomer and 4 for monomer E led to the values of 23.2% and 27.3% for R and Rfree respectively.

References

Massagué, J. (1998). TGF-β-signal transduction. Annu. Rev. Biochem. 67, 753-791.
Heldin,CH, Miyazomo, K. and ten Dijke, P. (1997) TGF-β signaling from cell membrane to nucleus through SMAD proteins. Nature 390, 465-471
Wilson JJ, Malakhova M, Zhang R, Joachimiak A and Hegde RS. Crystal structure of the dachshund homology domain of human SKI (2004) Structure 12(5): p. 785-92.
Krissinel E and Henrick K. Inference of macromolecular assemblies from crystalline state (2007) J Mol Biol,. 372(3): p. 774-97.
Kim SS, Zhang RG, Braunstein SE, Joachimiak A, Cvekl A and Hegde RS. Structure of the retinal determination protein Dachshund reveals a DNA binding motif (2002) Structure 10(6): p. 787-95.