Human DEAD-box RNA helicase DDX20, conserved domain I in complex with AMPPNP

Target ID:

DDX20A

Aliases:

DDX20DKFZp434H052DP103GEMIN3

Deposition Date:

Wednesday 31st October 2007

Families:

ATPases

Related Diseases:

Neurobiology

Structure type:

protein-ligand

Download Coordinates:

3B7G

Authors:

T.KARLBERG,L.TRESAUGUES,C.H.ARROWSMITH,H.BERGLUND,R.D.BUSAM,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,S.VAN DEN BERG,J.WEIGELT,M.WELIN,L.HOLMBERG-SCHIAVONE

Site:

Stockholm

3B7G

tabs

Structure Details

The ATP-dependent DEAD-box RNA helicase 20 (EC 3.6.1.-, DDX20, DEAD
box protein DP 103,Gemin-3) belongs to the RNA-binding DExD-box family
of helicases that are involved in all aspects of cellular
RNA-metabolism such as transcription, splicing, RNA
nucleocytoplasmatic transport, translation and ribosome
biogenesis. All proteins in this family are believed to bind and
hydrolyze ATP using the energy to assist in the folding or unfolding
of RNA or in the assembly/disassembly of RNA-protein
complexes. Members of the family normally consist of two conserved
domains which contain 9 signature motives that are involved in
ATP-binding and hydrolysis, substrate binding and helicase activity
(Cordin et al. 2006). Additional domains that are unique to a
particular helicase usually flank the two conserved domains.

DDX20 was initially identified as a protein that associates with
Epstein-Barr virus nuclear proteins EBNA2 and EBNA3C that regulate
transcription of several genes (Grundhoff et al. 1999). Recent results
suggest that DDX20 itself is a transcriptional regulator and it is
speculated that the protein represses transcription through
recruitment of histone deacetylases (Fuller-Pace, 2006). DDX20 is also
suggested to be involved in snRNP assembly (Charroux et
al. 1999). Conserved domain 1 of DDX20, containing the signature
DEAD-box motif was crystallized in the presence of AMPPNP and the
three-dimensional structure determined at 1.9 Å. The structure
shows that domain 1 of the protein folds into an α-β
RecA-like domain.

We have also solved the human DEAD-box RNA helicase DDX20, conserved
domain I in complex with ADP (PDB code 2OXC).

Materials and Methods

DDX20 + AMPPNP

PDB:3B7G

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|23270929
Entry Clone Source:Mammalian Gene Collection
SGC Clone Accession:DDX20A-s001
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*smRTAQDLSSPRTRTGDVLLAEPADFESLLLSRPVLEGLRAAGFERPSPVQLKAIPLGRCGLDLIVQAKSGTGKTCVFSTIALDSLVLENLSTQILILAPTREIAVQIHSVITAIGIKMEGLECHVFIGGTPLSQDKTRLKKCHIAVGSPGRIKQLIELDYLNPGSIRLFILDEADKLLEEGSFQEQINWIYSSLPASKQMLAVSATYPEFLANALTKYMRDPTFVRLNS
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Cells from a glycerol stock were grown in 20 ml TB supplemented with 8 g/l glycerol, 100 µg/ml kanamycin and 34 µg/ml chloramphenicol 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 and approximately 200 µl BREOX FMT 30 anti-foam solution (Cognis Performance Chemicals UK Ltd). The culture was grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 ºC until OD600 reached ~1.7. 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, 10 min, 4 ºC). The resulting cell pellet (31.8 g wet cell weight) was resuspended in lysis buffer (1.5 ml/g cell pellet), supplemented with one 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.

Tag removal
The N-terminal histidine tag was proteolytically removed by incubating the target protein with His-tagged TEV protease at a molar ratio of 50:1 at 4 ºC overnight. The proteolytic reaction did not reach completion, as judged by SDS-PAGE. Target protein was purified from uncleaved protein, tag and protease by passing the reaction mixture over a Ni-charged 1 ml HisTrap FF column (GE Healthcare) pre-equilibrated with GF buffer (500 mM NaCl and 10 mM imidazole). The cleaved protein was concentrated and the buffer was exchanged to 20 mM HEPES, 500 mM NaCl, 10% glycerol, 2 mM TCEP, pH 7.5, using a Vivaspin 20 centrifugal filter device with 10,000 MWCO (Sartorius). The final protein concentration was determined to 33.8 mg/ml in a volume of 0.17 ml and the identity of the protein was confirmed by mass spectrometry.

Extraction

Procedure
The cell suspension was quickly thawed in water and 2000 U Benzonase (Merck) were added. 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 (49,100 x g, 20 min, 4 ºC). The supernatant was decanted and filtered through a 0.45 µm flask filter.
Concentration:
Ligand
AMPPNPMassSpec:
Crystallization:The crystal were obtained by the sitting drop vapour diffusion method in a 96-well plate. 0.1 µl protein solution (diluted to 20 mg/ml) including 20 mM ADP and 10 mM MgCl2 was mixed with 0.1 µl of well solution consisting of 0.1 M bis-Tris, pH 5.5, 0.2 M NaCl and 12% PEG 3350. The plate was incubated at 4 ºC and crystals appeared after one day and continued to grow for one more week. The crystal was briefly transferred to a cryo solution consisting of well solution complemented with 28% glycerol, and flash frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Cordin O., Banroques J., Tanner N.K., and Linder P. (2005) The
DEAD-box protein family of RNA helicases. Gene 367: 17-37.
PubMed 16337753
Fuller-Pace F.P. (2006) DExD/H box RNA helicases: multifunctional
proteins with important roles in transcriptional regulation. Nucleic
Acids Res. 34:4206-15.
PubMed 16935882
Grundhoff A.T., Kremmer E., Tureci O., Glieden A., Gindorf C., Atz J.,
Mueller-Lantzsch N., Schubach W.H. and Grasser F.A. (1999)
Characterization of DP103, a novel DEAD box protein that binds to the
Epstein-Barr virus nuclear proteins EBNA2 and
EBNA3C. J. Biol. Chem. 274:19136-44.
PubMed 10383418
Charroux B., Pellizzoni L., Perkinson R.A., Shevchenko A., Mann M. and
Dreyfuss G. (1999) Gemin3: A novel DEAD box protein that interacts
with SMN, the spinal muscular atrophy gene product, and is a component
of gems. J. Cell Biol., 147:1181-1194.
PubMed 10601333