Human Dead-box RNA helicase DDX19, in complex with an ATP-analogue and RNA

Target ID:

DDX19A

Aliases:

DBP5DDX19DDX19BRNAh

Deposition Date:

Wednesday 28th January 2009

Families:

ATPases

Related Diseases:

Cancer

Structure type:

protein-ligand

Download Coordinates:

3G0H

Authors:

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

Site:

Stockholm

3G0H

tabs

Structure Details

The DExD/H family of RNA-binding helicases consists of a large group of proteins involved in general cellular RNA-metabolism such as transcription, splicing, RNA nucleocytoplasmatic transport, translation and ribosome biogenesis (1). All DExD/H helicases bind and hydrolyze ATP, and are believed to unwind RNA-secondary structure or assist in the folding of RNA/RNP complexes thus acting as RNA chaperones. Proteins in this family normally consist of two domains: an N-terminal domain with the conserved DExD/H motif and a C-terminal helicase domain. There are at least eight conserved motifs that are involved in coordination and hydrolysis of ATP and binding of RNA (2,3).

DDX19 is a human DEAD-box helicase that is required for mRNA export through the nuclear pore complex (4). Its yeast ortholog, Dbp5 (5), associates with nascent mRNA already in the nucleus. Previously, we solved the structure of DDX19, containing the DEAD-domain and the helicase domain in complex with ADP, at 2.7 Å resolution (3EWS). Now, DDX19 in complex with MgADPNP and RNA (a decauracil mRNA mimic) has been solved to 2.7Å resolution. DDX19 has the typical DEAD-box helicase fold with two α-β RecA-like domains connected via a flexible linker. The nucleotide binding site is located in a pocket between the two domains. The structure shows that both domains contribute to nucleotide and RNA binding. Six bases of the RNA molecule are visible in the electron density, positioned across the top of the cleft. As in previous helicase complexes, DDX19 introduces a kink in the RNA molecule, but unlike the previous structures, the two bases downstream of the kink do not stack with each other.

Comparing the two DDX19 structures (with and without RNA bound) shows that an α-helical segment of the N-terminal flanking region is wedged between the core domains in the RNA-free protein. This helix prevents cleft closure and positions the N-terminal flanking sequence in the RNA binding cleft, or in its proximity. In the ADPNP-RNA ternary complex, this α-helix has moved out to allow formation of the closed state, enabling RNA binding and nucleotide hydrolysis.

Materials and Methods

DDX19

PDB:3G0H

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC003626
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*smEDRAAQSLLNKLIRSNLVDNTNQVEVLQRDPNSPLYSVKSFEELRLKPQLLQGVYAMGFNRPSKIQENALPLMLAEPPQNLIAQSQSGTGKTAAFVLAMLSQVEPANKYPQCLCLSPTYELALQTGKVIEQMGKFYPELKLAYAVRGNKLERGQKISEQIVIGTPGTVLDWCSKLKFIDPKKIKVFVLDEADVMIATQGHQDQSIRIQRMLPRNCQMLLFSATFEDSVWKFAQKVVPDPNVIKLKREEETLDTIKQYYVLCSSRDEKFQALCNLYGAITIAQAMIFCHTRKTASWLAAELSKEGHQVALLSGEMMVEQRAAVIERFREGKEKVLVTTNVCARGIDVEQVSVVINFDLPVDKDGNPDNETYLHRIGRTGRFGKRGLAVNMVDSKHSMNILNRIQEHFNKKIERLDTDDLDEIE
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Cells from a glycerol stock were grown in 30 ml TB supplemented with 8 g/l glycerol, 100 µg/ml kanamycin and 34 µg/ml chloramphenicol at 30 °C overnight. 10 ml of the overnight culture was used to inoculate 750 ml 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 ~2.2. 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 (14 g wet cell weight) was resuspended in lysis buffer (2 ml/g cell pellet), supplemented with 1250 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 200 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 an Amicon Ultra-15 centrifugal filter device with 30,000 NMWL (Millipore) to 20.6 mg/ml in a volume of 1 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 over the weekend. 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 HisTrap HP column (GE Healthcare) pre-equilibrated with IMAC wash1 buffer. The protein was eluted at 35 mM imidazole.The cleaved protein was concentrated using a Vivaspin 20 centrifugal filter device with 30,000 MWCO (Sartorius) and subsequently dialyzed against GF buffer containing 2 mM TCEP. The final protein concentration was determined to 27.5 mg/ml in a volume of 0.16 ml The identity of the protein was confirmed by mass spectrometry.

Extraction

Procedure
The cell suspension was quickly thawed in water. 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. Protein solution, deca-uracil ssRNA, ADPNP and MgCl2 were mixed and concentrated to yield a final sample composition of 20 mg/ml protein, with a 10 molar excess of decauracil ssRNA, ADPNP and MgCl2 in 30 mM HEPES, 10% glycerol, 50 mM NaCl, pH 7.5. 0.1 µl of the protein sample was then mixed with 0.2 µl of well solution consisting of 0.1 M Tris pH 8, 0.25 M trimethylamine n-oxide, 14% PEG MME 2000. The plate was incubated at 4 ºC and crystals appeared after seven days. Cryo solution containing well solution and 25% glycerol was added directly to the drop. Crystals were mounted and flash-frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Data sets were collected at ESRF (beamline ID-29) to 2.7 Å resolution. The crystal belonged to space group P212121 with cell dimensions a=41.6 Å, b=81.1 Å, c=124.7 Å. One monomer was located in the asymmetric unit.
Data Processing:Data were integrated and scaled using XDS. The structure was solved by Phaser using the DDX19 apo-structure (3ews) as a search model. Domains were searched for separately. The structure was refined with RefMac5. TLS parameters were refined using individual domains as rigid groups. Model building was done using Coot. The structure finally refined to R-factors of 0.220 and 0.275 for R and Rfree, respectively. Coordinates were deposited in the Protein Data Bank with an accession code 3g0h.

References

Linder, P. (2006) Dead-box proteins: a family affair: active and passive players in RNP-remodeling. Nucleic Acids Res. 34:4168-80.
Cordin, O., Banroques, J., et al. (2006) The DEAD-box protein family of RNA helicases. Gene 367: 17-37.
Pyle, A.M. (2008) Translocation and unwinding mechanisms of RNA and DNA helicase. Annu. Rev. Biophys. 37:317-36.
Schmitt, C., von Kobbe, C. et al. (1999) Dbp5, a DEAD-box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p. EMBO J. 18:4332-47.
Snay-Hodge, C.A., Colot, H.V., Goldstein, A.L. & Cole, C.N. (1998) EMBO J. 17, 2663-2676.