Human DEAD-box RNA-helicase DDX47, conserved domain I in complex with AMP

Target ID:

DDX47A

Aliases:

DDX47DKFZp564O176E4-DBPFLJ30012HQ0256MSTP162

Deposition Date:

Tuesday 20th November 2007

Families:

ATPases

Related Diseases:

Cancer

Structure type:

protein-ligand

Download Coordinates:

3BER

Authors:

T.KARLBERG,R.D.BUSAM,C.H.ARROWSMITH,H.BERGLUND,R.COLLINS,L.G.DAHLGREN,A.EDWARDS,S.FLODIN,A.FLORES,S.GRASLUND,M.HAMMARSTROM,I.JOHANSSON,A.KALLAS,T.KOTENYOVA,L.LEHTIO,M.MOCHE,M.E.NILSSON,P.NORDLUND,T.NYMAN,C.PERSSON,J.SAGEMARK,L.SVENSSON,A.G.THORSELL,L.TRESAUGUES,S.VAN DEN BERG,J.WEIGELT,M.WELIN,L.HOLMBERG-SCHIAVONE

Site:

Stockholm

ICM Datapack:

ICM Datapack

3BER

tabs

Structure Details

The ATP-dependent DEAD-box RNA helicase DDX47 (DEAD box protein 47, EC: 3.6.1.-) belongs to the DExD/H RNA-binding helicase family of proteins that are involved in all aspects of cellular RNA-metabolism such as transcription, splicing, RNA transport, translation and ribosome biogenesis. All proteins in this family are supposed to bind and hydrolyze trinucleotides, mainly ATP, and are believed to function as RNA chaperones that bind RNA and assist in unwinding of RNA secondary structure or folding of RNA complexes. Members of this family normally consist of two domains: an N-terminal domain with the conserved DExD/H motif and a C-terminal helicase domain (Cordin et al. 2006).

DDX47 has been linked to the nucleolar protein NOP132 and hence localized to the nucleolus where it is hypothesized to be involved in the processing of rRNA (Sekiguchi et al. 2006). Further, DDX47 has been identified as a binding partner to the GABAA receptor-associated protein (GABARAP). Co-transfection of GABARAP and DDX47 cDNA into a tumor cell line has induced apoptosis (Lee et al. 2005).

We have determined the three-dimensional structure of the conserved domain 1 (DEAD-domain) of human DDX47 to a resolution of 1.4 Å. The structure was solved with the MR (molecular replacement) method using HERA N-terminal domain as a search template (PDB: 2GXS). The protein's fold resembles an α-β RecA-like domain. Curiously enough, AMP was observed in the electron density, very clearly, despite the fact that ADP and MgCl2 had been added during purification and to the crystallization drops.

Materials and Methods

DDX47

PDB:3BER

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC068009
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-Gold(DE3)pRARE2, where BL21-Gold(DE3) cells (Stratagene) have been transformed with pRARE2 originating from the Rosetta2 strain (Novagen). The pRARE2 plasmid supplies tRNAs for rare codons.

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*smEEHDSPTEASQPIVEEEETKTFKDLGVTDVLCEACDQLGWTKPTKIQIEAIPLALQGRDIIGLAETGSGKTGAFALPILNALLETPQRLFALVLTPTRELAFQISEQFEALGSSIGVQSAVIVGGIDSMSQSLALAKKPHIIIATPGRLIDHLENTKGFNLRALKYLVMDEADRILNMDFETEVDKILKVIPRDRKTFLFSATMTKKVQKLQRAALKNPVKCAVSS
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Cells from a glycerol stock were streaked onto LB-agar plates. 5-10 colonies were used to inoculate 20 ml TB supplemented with 8 g/l glycerol, 100 µg/ml kanamycin and 34 µg/ml chloramphenicol and the cells were grown at 30 ºC overnight. The overnight culture (20 ml) was used to inoculate 1.5 l TB supplemented with 8 g/l glycerol, 50 µg/ml kanamycin, 34 µg/ml chloramphenicol and approximately 250 µl PPG P2,000 81380 anti-foam solution (Fluka). The culture was grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 ºC until OD600 reached ~1.2. The culture was down-tempered to 18 ºC 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 (5,500 x g, 15 min, 4 ºC). The resulting cell pellet (18.9 g wet cell weight) was resuspended in lysis buffer (1 ml/g cell pellet), supplemented with 0.5 tablet of Complete EDTA-free protease inhibitor (Roche Applied Science). The cell suspension was stored at -80 ºC.

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, 10,000 NMWL (Millipore) to 17.6 mg/ml in a volume of 0.13 ml. The identity of the protein was confirmed by mass spectrometry.

Extraction

Procedure
The cell suspension was quickly thawed in water and 1000 U Benzonase (Merck), 2 mM ADP and 2 mM MgCl2 were added to the cell suspension. The cells were disrupted by sonication (Vibra-Cell, Sonics) at 80% amplitude for 3 min effective time (pulsed 4s on, 4s off). Cell debris was removed by centrifugation (49,100 x g, 20 min, 4 ºC) and 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 protein solution (diluted to 17 mg/ml) including 5 mM ADP and 5 mM MgCl2 was mixed with 0.2 µl of well solution consisting of 0.1 M Na/K-phosphate pH 5.2, 0.05 M NaCl and 42.5% (v/v) PEG 200. The plate was incubated at 4 ºC and one pyramidal crystal appeared after three weeks and continued to grow for two more weeks to reach maximal size (approx. 200 µm × 140 µm × 30 µm). The crystal was picked directly from the drop and flash frozen in liquid nitrogen without cryo protectant.
NMR Spectroscopy:
Data Collection:Diffraction data up to 1.4 Å resolution was collected at ESRF beamline ID-29.
Data Processing:The structure was solved by molecular replacement using HERA N-terminal domain as template (PDB: 2GXS). The space group was C2 with cell dimensions a=93.05 Å b=70.37 Å c=35.86 Å. One monomer was located in the asymmetric unit. Automatic model building using Arp/warp was performed; Refmac5 was used for refinement and Coot for model building. Data in the interval 30.0-1.40 Å resolution was used and at the end of the refinement the R values were: R=16.54% and Rfree=18.91%. Coordinates for the crystal structure were deposited in the Protein Data Bank, accession code 3BER.

References

Lee J.H., Rho S.B. and Chun T. (2005) GABAA receptor-associated protein (GABARAP) induces apoptosis by interacting with DEAD (Asp-Glu-Ala-Asp/His) box polypeptide 47 (DDX 47). Biotechnology Letters 27: 623-628.
Cordin O., Banroques J., Tanner N.K., and Linder P. (2006) The DEAD-box protein family of RNA helicases. Gene 367: 17-37.
Sekiguchi T., Hayano T., Yanagida M., Takahashi N. and Nishimoto T. (2006) NOP132 is required for proper nucleolus localization of DEAD-box RNA helicase DDX47. Nucleic Acids Research 34: 4593-4608.