Human eukaryotic translation initiation factor 4A isoform 2

Target ID:

EIF4A2

Aliases:

BM-010DDX2BEIF4AEIF4F

Deposition Date:

Monday 17th December 2007

Families:

G-Protein Regulators

Structure type:

apo

Download Coordinates:

3BOR

Authors:

Dimov S., Hong B.,Tempel W., MacKenzie F., Karlberg T., Arrowsmith C.H., Edwards A.M., Weigelt J., Bochkarev A., Park H.

Site:

Toronto

3BOR

tabs

Structure Details

The DEAD-box proteins form a widely dispersed family of proteins found in all eukaryotes and most prokaryotes. The DEAD-box proteins have been found in more than 500 proteins. The name of the family was derived from the amino-acid sequence D-E-A-D (Asp-Glu-Ala-Asp) of its Walker B motif. The DEAD-box proteins are associated with nearly all processes involving RNA, from transcription to RNA decay, but their precise roles, functions and regulation largely remain to be elucidated. Eukaryotic initiation factor 4A (eIF4A, DDX2A) is the prototype of all DEAD-box helicases. It is essential for the initiation step of protein synthesis where it unwinds mRNA secondary structure to allow for the localization of AUG-codon on the mRNA by the ribosome. eIF4A is a potential anti proliferative target: I) natural compounds have been identified that inhibit initiation of translation by interacting with eIF4A and its protein partner eIF4B and II) the tumour suppressor protein Pdcd4 binds to eIF4A and thereby inhibit CAP-dependent translation.
We have determined the three-dimensional structure of the ATP-binding domain of human eIF4A isoform 2 (eIF4A2) at 1.85 Å.

Materials and Methods

EIF4A2

PDB:3BOR

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_001958.2
Entry Clone Source:
SGC Clone Accession:HPC075-H02
Tag:mhhhhhhssgrenlyfq*g
Host:BL21-CodonPlus(DE3)-RIL

Construct

Prelude:
Sequence:mhhhhhhssgrenlyfqgGVIESNWNEIVDNFDDMNLKESLLRGIYAYGFEKPSAIQQRAIIPCIKGYDVIAQAQSGTGKTATFAISILQQLEIEFKETQALVLAPTRELAQQIQKVILALGDYMGATCHACIGGTNVRNEMQKLQAEAPHIVVGTPGRVFDMLNRRYLSPKWIKMFVLDEADEMLSRGFKDQIYEIFQKLNTSIQVVLLSATMPTDVLEVTKKFMRDPIRILVKKE
Vector:pET28-mhl

Growth

Medium:Terrific Broth
Antibiotics:
Procedure:LEX Bubbling. The target protein was expressed in E. coli by inoculating 100 mL of overnight culture grown in Luria-Bertani medium into a 1.8 L of Terrific Broth medium in the presence of 50 μg/mL kanamycin and chloramphenicol at 37 °C. When OD600 was ~3.0, the temperature of the media was lowered to 15 °C and the culutre was induced with 1mM IPTG, and the cells were allowed to grow overnight before harvesting and flash frozen in liquid nitrogen and stored at -80 °C before use.

Purification

Procedure
The lysate was centrigued at 16,000 rpm for 60 minutes and the supernatant was passed through two open columns filled with DE52 and 5 mL 50% Ni-NTA beads at 4 °C. The Ni beads were then washed using washing buffer and the proteins eluted using 16 mL elution buffer. The elutant was loaded onto Superdex-75 gel filtration column. Eluted fractions were pooled and concentrated using amicon centrifugal filter (m.w. cut-off 10,000 ). The purity of the proteins was higher than 95% judged by SDS-PAGE.

Extraction

Procedure
Frozen cells were thawed and suspended in 150 mL the binding buffer and supplemented with protease inhibitor cocktail (SIGMA Catalog # P8849), and 2 μL (Sigma Catalog # E1014, 250U/μL) benzonase, and lysed using Microfluidizer (French press).
Concentration:15 mg/mL, in 20 mM HEPES pH 8.0 150 mM NaCl 1 mM TCEP buffer
Ligand
N/AMassSpec:Measured 26894.2, expected 26893.0
Crystallization:
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.