Human eukaryotic translation initiation factor 4E, in complex with a 4EBP1 peptide complex, without cap

Target ID:

EIF4E

Deposition Date:

Tuesday 28th June 2011

Families:

G-Protein Regulators

Related Diseases:

Cancer

Structure type:

protein-protein

Download Coordinates:

Superceded by 3U7X

Authors:

Siddiqui N, Tempel W, Nedyalkova L, Wernimont AK, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Borden KLB, Park H

Site:

Toronto

3SMU

tabs

Structure Details

The eukaryotic translation initiation factor (eIF4E) modulates the expression of genes involved in cell growth, proliferation, and apoptosis via two major pathways: cap-dependent translation and mRNA export. In both nuclear and cytoplasmic compartments, eIF4E associates with the 7- methylguanosine moiety (5′cap) on the 5′ end of mRNA, which is a prerequisite for its function. In the nucleus, eIF4E plays an important role in the export of a subset of growth-promoting mRNAs containing the eIF4E-sensitivity element. eIF4E is regulated by proteins such as eIF4G and eIF4E binding proteins (4EBPs) that bind the dorsal surface of eIF4E, distal to the cap binding site, and modulate cap binding activity. Both proteins increase the affinity of eIF4E for 5′cap.

Here we solved the first crystal structure of cap-free eIF4E in complex with a 4EBP1 peptide. We observed chemical shift perturbation at the dorsal binding site leading to alterations in the core of the protein, which were ultimately communicated to the unoccupied cap binding site of eIF4E. There were notable similarities between the routes taken by 4EBP1 and eIF4G. Thus, binding of 4EBP1 or eIF4G allosterically drives alterations throughout the protein that increase the affinity of eIF4E for the 5′cap.

Materials and Methods

EIF4E

PDB:3SMU

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:The plasmid containing the full-length cDNA for human eIF4E was a gift from Dr N. Shimma, Chugai Pharmaceutical Co., Japan.
SGC Clone Accession:
Tag:
Host:E.coli BL21 (DE3)

Construct

Prelude:
Sequence:EIF4E:MATVEPETTPTPNPPTTEEEKTESNQEVANPEHYIKHPLQNRWALWFFKNDKSKTWQANLRLISKFDTVEDFWALYNHIQLSSNLMPGCDYSLFKDGIEPMWEDEKNKRGGRWLITLNKQQRRSDLDRFWLETLLCLIGESFDDYSDDVCGAVVNVRAKGDKIAIWTTECENREAVTHIGRVYKERLGLPPKIVIGYQSHADTATKSGSTTKNRFVV

4EBP1:PGGTRIIYDRKFLMECRNSP
Vector:pT7 (gift from Dr N. Shimma, Chugai Pharmaceutical Co., Japan)

Growth

Medium:M9 minimal medium
Antibiotics:ampicillin (100µg/L)
Procedure:Luria Bertani (LB) or M9 minimal medium, containing ampicillin (100µg/L), were inoculated and grown at 37 °C until an OD₆₀₀ = 0.6-0.8. For protein expression, cells were induced at 30 °C for 18 h by addition of 0.5 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). Cultures were harvested by centrifugation (7,000 rpm, 15 min., 4 °C) and frozen at -20 °C.

Purification

Procedure
The lysate was cleared by centrifugation (18,000 rpm, 30 min., 4 °C) and the supernatant was clarified by filtration on a 0.45 mum membrane and loaded onto m7GDP-sepharose (cap column). After four washes (30 ml each; 1. lysis buffer, 2. wash buffer, 3. wash buffer containing 0.1 mM GTP and 4. wash buffer), protein was eluted with ~ 60 ml of elution buffer. The eluate was concentrated using an Amicon ultra centrifugal filter device (10 K MWCO, Millipore) to ~4 mL, and then applied to a size-exclusion column (Superdex 75, Amersham) using gel filtration buffer. FPLC fractions were pooled for further analysis and the purity of eIF4E was greater than 95% judging from SDS-PAGE gel. The protein concentration was measured at 280 nm using an extinction coefficient of 53440 M-1 cm-1. The final yield of soluble protein was 6-10mg grown in either LB medium or M9 minimal medium.

Extraction

Procedure
The cell pellet was then resuspended in 30 ml of lysis buffer and cells were disrupted by sonication on ice.
Concentration:eIF4E protein samples for crystallization trials were exchanged into 20mM HEPES, 100mM KCl, 1mM TCEP, pH 7.3 using an Amicon ultra centrifugal filter device (10 K MWCO, Millipore) and concentrated to 8-9mg/mL.
Ligand
MassSpec:
Crystallization:eif4e stock solution in the presence of 0.001 M 4ebp1 peptide and 0.02 M ribavirin was mixed in a 1:1 ratio with 2 M Ammonium Sulfate, 2% PEG400, 0.1 M Na HEPES, pH 7.5. Crystals grew to mountable size within 10-14 days.
NMR Spectroscopy:
Data Collection:
Data Processing: