Human DECH-box RNA Helicase MDA5, DECH-domain

Target ID:

MDA5A

Aliases:

HlcdIDDM19IFIH1MDA-5MDA5MGC133047

Deposition Date:

Monday 29th October 2007

Families:

ATPases

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

3B6E

Authors:

T.KARLBERG,M.WELIN,,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,L.TRESAUGUES,S.VAN DEN BERG,J.WEIGELT,L.HOLMBERG-SCHIAVONE

Site:

Stockholm

ICM Datapack:

ICM Datapack

3B6E

tabs

Structure Details

The ATP-dependent DECH-box RNA helicase MDA5 (Melanoma differentiation-associated protein 5 also known as IFIH1 - Interferon-induced helicase C domain-containing protein 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 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).

MDA5 was discovered as being involved in the regulation of the growth and differentiation of melanoma cells (Kang et al. 2002). More recently MDA5 has been identified as a sensor of double-stranded viral RNA resulting in activation of anti-viral responses (Kato et al. 2006). The full-length protein contains two N-terminal CARD-domains (caspase recruitment domain) in addition to conserved domains 1 and 2 of DExD/H-box helicases. MDA5 triggers activation of a number transcription factors including IRF3, IRF7 and NF-B downstream in the signaling pathway (Unterholzner and Bowie 2007).

We have determined the three-dimensional structure of the conserved domain 1 (DECH-domain) of human MDA5 to a resolution of 1.6 Å. The structure was solved with SAD (single anomalous dispersion) method using selenomethione labeled protein. This is the first human structure of an RNA helicase with DExH-motif signature. The protein folds and resembles an α-β RecA-like domain. The N-terminal loop-helix seems important for packing in the crystal. No nucleotide is seen bound despite high concentrations of ADP and MgCl2 both in purification and during crystallization. Interestingly, the side chain from residue Arg337 is blocking access for binding of a nucleotide in the otherwise conserved binding pocket. The present structure will aid in the understanding of the human defense against viruses.

Materials and Methods

MDA5

PDB:3B6E

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC111750
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*smNSNMGSDSGTMGSDSDEENVAARASPEPELQLRPYQMEVAQPALEGKNIIICLPTGSGKTRVAVYIAKDHLDKKKKASEPGKVIVLVNKVLLVEQLFRKEFQPFLKKWYRVIGLSGDTQLKISFPEVVKSCDIIISTAQILENSLLNLENGEDAGVQLSDFSLIIIDECHHTNKEAVYNNIMRHYLMQKLKNNRLKKENKPVIPLPQILGLTAS
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:

Native protein
Cells from a glycerol stock were streaked onto LB-agar plates. 5-10 colonies were used to inoculate 2 x 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 cultures were used to inoculate 2 x 1.5 l TB supplemented with 8 g/l glycerol, 50 µg/ml kanamycin, 34 µg/ml chloramphenicol and approximately 200 µl PPG P2,000 81380 anti-foam solution (Fluka). The cultures were grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 ºC until OD600 reached ~1.3. The cultures were 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 (81.2 g wet cell weight) was resuspended in lysis buffer (1 ml/g cell pellet), supplemented with 1 tablet of Complete EDTA-free protease inhibitor (Roche Applied Science). The cell suspension was stored at -80 ºC.

SeMet labeled protein
Cells from a glycerol stock were streaked onto LB-agar plates. 5-10 colonies were used to inoculate 2 x 40 ml LB supplemented with 100 µg/ml kanamycin and 34 μg/ml chloramphenicol and the cells were grown at 30 ºC overnight. 60 ml of the overnight culture were used to inoculate 6 flasks with 1.5 l minimal medium (without amino acids) supplemented with 50 µg/ml kanamycin, 34 μg/ml chloramphenicol and approximately 250 µl PPG P2,000 81380 anti-foam solution (Fluka) per bottle. The cultures were grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 ºC until OD600 reached ~1. The flasks were down-tempered to 18 ºC and after 30 minutes, amino acids were added (Van Duyne, G. D., J. Mol. Biol. 229, 105-124 (1993)). About one hour later, expression of target protein was induced by addition of 0.5 mM IPTG. The expression was allowed to continue at 18 ºC overnight. Cells were harvested the following morning by centrifugation (5,000 x g, 13 min, 4 ºC). The resulting cell pellet (54 g from 9 liter culture) was resuspended in lysis buffer (2 ml/g cell pellet) supplemented with two tablets of Complete EDTA-free protease inhibitor (Roche Applied Science) and stored at -80 ºC. Selenomethionine enriched protein was grown in minimal medium containing: 25 mM Na₂HPO₄, 25 mM KH₂PO₄, 50 mM NH4Cl, 5 mM Na₂SO₄, 0.4% (w/v) glucose, 2 mM MgSO4, 0.1 mM CaCl2, 1.0 µM MnCl2, 10 µM FeSO4, pH ~7. Mix of amino acids added (per liter culture): 100 mg each of lysine, threonine, phenylalanine and 50 mg each of leucine, isoleucine, valine, L(+)-selenomethionine.

Purification

Procedure

Columns
IMAC 1: Ni-charged 1 ml HiTrap Chelating HP (GE Healthcare)
IMAC 2: Ni-charged 1 ml HisTrap FF crude (GE Healthcare)
Gel filtration column: HiLoad 16/60 Superdex 75 Prep Grade (GE Healthcare)

Procedure
Purification of the native 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, 10,000 NMWL (Millipore) to 27.2 mg/ml in a volume of 0.3 ml. The samples were stored at -80 ºC. Purification of the SeMet enriched protein was performed in basically the same way. The filtered lysate was purified by IMAC and gel filtration chromatography followed by a concentration step. The final SeMet-protein concentration was 33.5 mg/ml in a volume of 1.5 ml.

Tag removal
The N-terminal histidine tag was proteolytically removed by incubating the target protein (27.2 mg/ml in 0.3 ml) with His-tagged TEV protease at a molar ratio of 50:1 in 4 ºC overnight. The proteolytic reaction went to completion, as judged by SDS-PAGE. The reaction mixture was diluted with GF buffer to reduce the TCEP concentration. The target protein was subsequently purified from tag and protease by passing it over a Ni-charged HisTrap FF crude column (GE Healthcare) pre-equilibrated with GF buffer. The cleaved protein was eluted with IMAC wash2 buffer. The protein was concentrated and the buffer changed to GF buffer (with 2 mM TCEP) using an Amicon Ultra-15 centrifugal filter device, 10,000 NMWL (Millipore). The concentration was measured to 21.9 mg/ml in a volume of 0.35 ml. The identity of the protein was confirmed by mass spectrometry. The histidine tag removal of SeMet labeled protein was achieved in basically the same way as described for the native protein. Purified SeMet labeled protein (33.5 mg/ml) was incubated with TEV protease at 4 ºC overnight, passed over a Ni-charged HisTrap FF crude column and concentrated to a final concentration of 48.1 mg/ml in 0.8 ml.

Extraction

Procedure
The cell suspension was quickly thawed in water and 4000 U Benzonase (Merck) were added. Cells were disrupted by sonication (VibraCell, Sonics) at 70% amplitude for 3 min effective time (puls 4s on, 4s off) and cell debris was removed by centrifugation (49,000 x g, 30 min, 4 ºC). The supernatant was decanted and filtered through a 0.45 µm flask filter. The SeMet pellet was basically treated in the same way.
Concentration:
Ligand
MassSpec:
Crystallization:Native crystals were obtained by the sitting drop vapour diffusion method in a 96-well plate. 0.2 µl of the protein solution (diluted to 19.9 mg/ml) including 20 mM ADP and 10 mM MgCl2 was mixed with 0.1 µl of well solution consisting of 0.1 M Tris pH 8.0 and 1.2 M sodium-citrate. The plates were incubated at 20 ºC and one pyramidal crystal was obtained after one day and continued to grow for two more weeks. The crystal was quickly transferred to cryo solution consisting of well solution complemented with 20% glycerol and flash frozen in liquid nitrogen. SeMet crystals were produced in the same way by mixing 0.2 µl of the SeMet-protein solution (diluted with GF buffer to 20 mg/ml) including 20mM ADP and 10mM MgCl2 with 0.1 µl of well solution consisting of 0.1 M bis-Tris-Propane pH 7.5, and 65% Tacsimate. The plate was incubated at 20 ºC and crystals were obtained after one day and continued to grow for one more week. The crystals were quickly transferred to cryo solution consisting of well solution complemented with 20% glycerol and flash frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Diffraction data up to 2.2 Å resolution was collected at BESSY (BL14-1).
Data Processing:The structure was solved by SAD using Seleno-methionine labeled protein crystals. The space group was P6522 with cell dimensions a=72.8 Å, b=72.8 Å, c=182.3 Å. The asymmetric unit contains one monomer. The obtained model was thereafter used as an initial model for refinement with native data to a resolution of 1.6 Å. The same space group P6522 with cell dimensions a=72.8 Å, b=72.8 Å, c=182.8 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 20.0-1.60 Å resolution was used and at the end of the refinement the R values were: R= 17.9% and Rfree= 20.4%. Coordinates for the crystal structure were deposited in the Protein Data Bank, accession code 3B6E.

References

Unterholzner L. and Bowie A.G. (2007) The interplay between viruses and innate immune signaling: Recent insights and therapeutic opportunities. Biochem. Pharmacol. Epub ahead of print
Cordin O., Banroques J., Tanner N.K., and Linder P. (2006) The DEAD-box protein family of RNA helicases. Gene 367: 17-37.
Kato H., Takeuchi O., Sato S., Yoneyama M., Yamamoto M., Matsui K., Uematsu S., Jung A., Kawai T., Ishii K.J., Yamaguchi O., Otsu K., Tsujimura T., Koh C-S., Reis e Sousa C., Matsuura Y., Fujita T. and Akira S. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441: 101-105.
Kang D-C., Gopalkrishnan R.V., Wu Q., Jankowsky E., Pyle A.M. and Fisher P.B. (2002) mda-5: An interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties. PNAS 99: 637-642.