Human DEAD-box RNA helicase DDX3X

Target ID:

DDX3XA

Aliases:

DBXDDX14DDX3DDX3XHLP2

Deposition Date:

Monday 21st August 2006

Families:

DExD-DExH RNA helicases

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

2I4I

Authors:

M.HOGBOM,T.KARLBERG,C.ARROWSMITH,H.BERGLUND,R.D.BUSAM,R.COLLINS,A.EDWARDS,M.EHN,S.FLODIN,A.FLORES,S.GRASLUND,B.M.HALLBERG,M.HAMMARSTROM,I.JOHANSSON,T.KOTENYOVA,A.MAGNUSDOTTIR,P.NILSSON-EHLE,P.NORDLUND,T.NYMAN,D.OGG,C.PERSSON,J.SAGEMARK,P.STENMARK,M.SUNDSTROM,A.G.THORSELL,J.UPPENBERG,S.VAN DEN BERG,K.WALLDEN,J.WEIGELT,M.WELIN,L.HOLMBERG-SCHIAVONE,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Stockholm

ICM Datapack:

ICM Datapack

2I4I

tabs

Structure Details

The DExD/H family of RNA-binding helicases consists of a large group of proteins that are involved in general cellular RNA-metabolism such as transcription, splicing, RNA nucleocytoplasmatic transport, translation and ribosome biogenesis. All DExD/H helicases are defined by their ability to bind and hydrolyse ATP and are generally 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, based on primary sequence alignments that are involved in coordination and hydrolysis of ATP and binding of RNA (Cordin, Banroques et al. 2006).

DDX3 is a human DEAD-box helicase encoded by two closely related genes: DDX3X and DDX3Y that are 92% identical in sequence. The cellular function of DDX3X, which is found in all tissues is unclear. DDX3X has been linked to the propagation of hepatitis C and HIV viruses (Mamiya and Worman 1999; Owsianka and Patel 1999; You, Chen et al. 1999; Yedavalli, Neuveut et al. 2004). Recently it was shown that DDX3X is essential for the transport of unspliced/partially spliced HIV-mRNAs from the nucleus to the cytoplasm together with the HIV protein REV(Yedavalli, Neuveut et al. 2004) and it was proposed that DDX3X should facilitate the threading of HIV-mRNAs through the nuclear pore complex. The involvement of DDX3X in HIV-mRNA transport makes it an interesting candidate for antiviral therapy (Kwong, Rao et al. 2005).

We have solved the structure of the conserved core of DDX3X, containing the DEAD-domain and the helicase domain in complex with AMP at 2.2 Ã… resolution. DDX3X has a fold that is typical of a DEAD-box helicase with two α-β RecA-like domains that are connected via a flexible linker and the nucleotide bound in a pocket between the two domains. The DDX3X-specific insertion of 12 amino acids between the WalkerA and the RNA-binding motif Ia in the DEAD-domain is a helix which could form interactions with a putative RNA-substrate.

Materials and Methods

DDX3X

PDB:2I4I

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:
SGC Clone Accession:
Tag:
Host:

Construct

Prelude:
Sequence:SMVEATGNNCPPHIESFSDVEMGEIIMGNIELTRYTRPTPVQKHAIPIIKEKRDLMACAQTGSGKTAAFLLPILSQIYSDGPGEALRAMKENGRYGRRKQYPISLVLAPTRELAVQIYEEARKFSYRSRVRPCVVYGGADIGQQIRDLERGCHLLVATPGRLVDMMERGKIGLDFCKYLVLDEADRMLDMGFEPQIRRIVEQDTMPPKGVRHTMMFSATFPKEIQMLARDFLDEYIFLAVGRVGSTSENITQKVVWVEESDKRSFLLDLLNATGKDSLTLVFVETKKGADSLEDFLYHEGYACTSIHGDRSQRDREEALHQFRSGKSPILVATAVAARGLDISNVKHVINFDLPSDIEEYVHRIGRTGRVGNLGLATSFFNERNINITKDLLDLLVEAKQEVPSWLENMAYEHHYKG
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:30 µl competent BL-21 (DE3) cells were transformed with 2 µl plasmid miniprep for 30 min on ice followed by heatshock at 42°C for 45 sec. SOC, 125 µl, was added to the cellsuspension which was then incubated for 1 hour at 37°C and plated on LB-plates containing kanamycin (50 µg/mL). 20 mL TB with 100 µg kanamycin/mL was inoculated with cells and grown overnight (ON) at 30°C. The inoculation culture was added to 1.5 L TB (supplemented with 50 µg kanamycin/mL) in 2 L bottles. The flask was incubated in the LEX system-water bath at 37°C until OD600 reached 1. At this time the flask was transferred to an 18°C water bath in the LEX-system. Expression of protein was induced by addition of 0.5 mM IPTG and continued for approximately 18 hours.
Cells were harvested by centrifugation in a SLC-6000 rotor for 10 minutes at 5000 rpm. Pellets were suspended in 50 mM Nafosfate pH 7.5, 10 % glycerole, 0.5 mM TCEP, 500 mM NaCl, 10mM imidazole and Complete EDTA-free protease inhibitor (Roche Biosciences) was added (1 tablet per 100 ml, final volume 116 ml). Suspended cells were stored at -80°C until further use.

Purification

Procedure
The cell lysate was loaded onto a HisTrap FF crude column with a peristaltic pump and purified on an Äkta purifier system. The column was washed with IMAC-wash buffer before bound protein was eluted with 500 mM imidazole. Affinity-purification was followed by gel-filtration on a Superdex 200 16/60 gel filtration column in gel-filtration buffer. DDX3X migrated as a nice single peak corresponding to a monomer form of the protein. Fractions containing protein were pooled, the TCEP-concentration adjusted to 2 mM, and concentrated to 2.44 mg/ml in AmiconUltra MWCO 30000 concentrators. The correct mass of the DDX3X construct was verified by mass spectrometry.

TEV-cleavage: The N-terminal His-tag was removed by incubating 20 mg of DDX3X with a His-tagged TEV-protease at a molar ratio of 1:50 in a final volume of 10 ml overnight at 4? C. The reaction mixture was loaded onto a HisTrap FF crude column and cleaved DDX3X was detected in the flow-through and concentrated to 23.8 mg/ml before setting up crystallization drops. SDS-PAGE analysis showed that the purified protein was more than 95% pure (results not shown).

Extraction

Procedure
Before lysis, 8 µl of 250U/µl benzonase (Novagen) was added and lysis was performed by sonication (3Â 4ÂÂ on/4ÂÂ off, amplitude 70%). Cell debris was pelleted by centrifugation (30 min at 20500 rpm in a Sorvall SA-800 rotor). The soluble fraction was filtered through a 0.45 µm syringe filter.
Concentration:
Ligand
MassSpec:
Crystallization:DDX3X crystals were grown using vapor diffusion at 4 °C by mixing equal amounts of protein solution at a concentration of 21 mg/ml containing 20mM ATP?S or 20 mM ADP and 20mM MgCl2 and reservoir solution containing 1.75M Sodium Formate and 100mM Tris-HCl pH 7.5. Crystals appeared after one day and had reached their maximal size in twelve days (approximately 300 µm × 100 µm × 50 µm).
NMR Spectroscopy:
Data Collection:
Data Processing:The structure was solved by Molecular Replacement using the two domains separately. As search model for the DEAD domain, the N-terminal RecA-like domain of DjVLGB (PDB entry: 1WRB) was used and similarly for the helicase domain the ATP-dependent RNA helicase vasa (PDB entry: 2DB3) was used. The asymmetric unit contains one protein monomer. Refmac was used for refinement and Coot for model building. TLS refinement using 4 TLS groups as suggested by tlsmd (http://skuld.bmsc.washington.edu/~tlsmd/) was used with Refmac. In the refinement, data in the interval 15-2.20Å resolution was used and the progress of refinement was followed by decreasing R and Rfree values. At the end of the refinement the values for R=0.1887 and Rfree=0.2182. The final model starts at methionine 167 and ends at tyrosine 580. Residues 407-410, 535-536 and 581-582 are disordered and not visible in the electron density. A Ramachandran plot shows 98% of all residues in favored regions and no outliers in the disallowed regions. Coordinates for the crystal structure were deposited in the Protein Data Bank, accession code 2I4I.

References

Cordin, O., J. Banroques, et al. (2006). "The DEAD-box protein family of RNA helicases." Gene 367: 17-37.
Kwong, A. D., B. G. Rao, et al. (2005). "Viral and cellular RNA helicases as antiviral targets." Nat Rev Drug Discov 4(10): 845-53.
Mamiya, N. and H. J. Worman (1999). "Hepatitis C virus core protein binds to a DEAD box RNA helicase." J Biol Chem 274(22): 15751-6.
Owsianka, A. M. and A. H. Patel (1999). "Hepatitis C virus core protein interacts with a human DEAD box protein DDX3." Virology 257(2): 330-40.
Yedavalli, V. S., C. Neuveut, et al. (2004). "Requirement of DDX3 DEAD box RNA helicase for HIV-1 Rev-RRE export function." Cell 119(3): 381-92.
You, L. R., C. M. Chen, et al. (1999). "Hepatitis C virus core protein interacts with cellular putative RNA helicase." J Virol 73(4): 2841-53.