ETS domain of human ETS2 in complex with DNA

Target ID:

ETS2A

Deposition Date:

Thursday 30th May 2013

Families:

Miscellaneous

Structure type:

protein-protein

Download Coordinates:

4BQA

Authors:

J.A.Newman, C.D.O. Cooper, T. Krojer, L. Sherestha, N. Burgess-Brown, C.H.Arrowsmith, A.Edwards, O.Gileadi

Site:

Oxford

4BQA

tabs

Structure Details

The Ets transcription factor family consists of 28 genes in humans containing the evolutionarily conserved 85 amino acid Ets DNA-binding domain, originally identified as a viral oncogene (E26 Transformation-Specific). Ets proteins exhibit ubiquitous or tissue-specific expression and are particularly involved in differentiation processes and the response to signalling pathways. Ets-2 like its closely related and much more widely studied homologue Ets-1, is subject to multiple levels of regulation of its DNA binding and transactivation properties. Chiefly among these is the presence of an auto-inhibitory module which allosterically inhibits DNA binding.
We have determined the crystal structure of a DNA complex of the Ets-2 Ets domain to 2.5 Å resolution crystal structure. Unexpectedly the Ets domain crystallized with two distinct species in the asymmetric unit, which closely resemble the auto-inhibited and DNA bound forms of Ets-1. Comparisons of the two forms gives insights into how the presence or absence of the auto-inhibitory module may be transmitted to the rest of the Ets domain, with a number of intramolecular salt bridges formed in the auto-inhibited form (presumably stabilized by the presence of the auto-inhibitory module), which are evenly distributed throughout the molecule and may serve to subtly alter the conformation or dynamics of the residues forming the DNA interface.

Materials and Methods

ETS2

PDB:4BQA

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:IMAGE:3852274
Entry Clone Source:
SGC Clone Accession:ETS2A-c061
Tag:Tag TEV-cleavable N-terminal his6 tag.
Host:BL21(DE3)-R3-pRARE2

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq^SMNKPTMSFKDYIQERSDPVEQGKPVIPAAVLAGFTGSGPIQLWQFLLELLSDKSCQSFISWTGDGWEFKLADPDEVARRWGKRKNKPKMNYEKLSRGLRYYYDKNIIHKTSGKRYVYRFVCDLQNLLGFTPEELHAILGVQ
Vector:pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ].

Growth

Medium:
Antibiotics:
Procedure:The expression plasmid was transformed into the host strain and plated on LB-agar containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol. Several colonies were combined to inoculate a 1ml culture in TB (+ 50 µg/ml kanamycin, 34 µg/ml chloramphenicol). The culture was grown overnight, glycerol was added to 15% v/v (from a 50% stock), and the resulting glycerol stock was frozen at -80°C in 100 µl aliquots. A loopful of cells from the glycerol stock was inoculated into 10-ml of TB medium containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol and grown overnight at 37°C. 1L of TB medium (containing 50 µg/ml kanamycin) was inoculated with 10 ml of the overnight culture and grown in 2.5L UltraYield baffled flasks until OD600 of 2.0. Cells were cooled to 18°C, IPTG added to 0.1 mM and growth continued at 18°C overnight. The cells were collected by centrifugation then the pellets were scraped out and transferred to 50-ml Falcon tubes and frozen at -80°C.

Purification

Procedure

Extraction

Procedure
Cell extraction : Frozen cell pellets (approx. 80g) were thawed briefly in a bath of warm water (20 - 37°C) then transferred to ice. Three volumes (i.e. 3 ml for every gram of cells) of lysis buffer was added, and the cells were resuspended by agitating and disrupted by pulsed sonication (10 sec on 20 sec off) for 20 minutes on ice. Polyethylenimine was added to the lysate at a final concentration of 0.15 % and lysates were centrifuged for 60 minutes at 50,000xg to remove cellular debris.
Lysis buffer: 50 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 1 mM TCEP, 10 mM Imidazole

Column 1 : 2ml bed volume Ni-IDA
Solutions:
Wash buffer: 50 mM HEPES, pH 7.5, 500 mM NaCl, 30 mM imidazole, 5% Glycerol, 1 mM TCEP.
Elution buffer: 50 mM HEPES, pH 7.5, 500 mM NaCl, 300 mM imidazole, 5% Glycerol, 1 mM TCEP.
Procedure: The cell extract was incubated with 5ml of Ni-IDA resin (GE Healthcare) for 1hr at 4°c with gentle agitation. The resin was pelleted by centrifugation (700g for 6 min), and the supernatant was decanted. The resin was washed initially with 10 column volumes of lysis buffer and pelleted by centrifugation. Ten column volumes of wash buffer1 were added and the resuspended resin was transferred to a gravity flow column and washed with 5 column volumes of wash buffer. Proteins were eluted by addition of 2 x 10 ml of elution buffer and combined with the fractions from the wash buffer.

Enzymatic treatment and Column 2: The N-terminal His6-tag was cleaved by incubating the protein overnight with tobacco etch virus (TEV) protease (1:42 ratio at 4°C) whilst being dialyzed into dialysis buffer (50 mM HEPES, 500 mM NaCl, 5 % Glycerol, 1 mM TCEP) using a 3,500 MWCO snakeskin dialysis membrane.
Cleaved protein was purified by passing over a 5 ml pre-equilibriated 50% Ni-IDA bead solution. Elution was done in GF buffer supplemented with step gradients of 20 mM, 40 mM 100 mM and 300 mM imidazole, with flowthrough and fractions collected.

Column 3 : Gel filtration, Hiload 16/60 Superdex S75 prep grade, 120 ml (GE Healthcare)GF buffer: 50 mM HEPES, pH 7.5, 500 mM NaCl, 5% Glycerol, 1 mM TCEP.
Procedure: The fractions from Ni-IDA column were concentrated to 2ml using a 3 KDa MWCO centrifugal concentrator and loaded on the gel filtration column in GF buffer at 1.2 ml/min. Eluted proteins were collected in 2-ml fractions and analysed on SDS-PAGE.
Concentration:The cleaved purified protein was concentrated using a 3 KDa MWCO centrifugal concentrator to 15 mg/ml and stored at 4°C. The protein concentration was determined spectrophotometrically using ε280 = 32430
Ligand
MassSpec:Observed mass of protein was 16424.4 calculated mass is 16423.9
Crystallization:Crystallisation experiments were performed using the sitting drop vapour diffusion technique. DNA oligonucleotides corresponding to the sequence 5'-ACCGGAAGTG and 5'-CACTTCCGGT were resuspended in 10mM HEPES pH 7.5, 50 mM NaCl to a final DNA concentration of 1 mM. The two oligonucleotides were mixed in a 1:1 ratio and heated to 95°C and allowed to cool slowly to room temperature. Protein and DNA were mixed in a 1:1.1 ratio (protein:DNA) and the protein DNA mixture was concentrated using a 3 KDa MWCO centrifugal concentrator to a final protein concentration of 15 mg/ml. Crystals of the ETS2-DNA complex grew at 20°C from conditions containing 15% PEG 3350, 0.25M NaCl, , 0.1M bis-tris pH 5.5. Crystals transferred to a cryo solution consisting of well solution supplemented with 20% Ethelyene glycol before being loop mounted and plunged into liquid nitrogen.
NMR Spectroscopy:
Data Collection:Data was collected at diamond light source beamline I02. Data were processed using XDS, and the structure was solved by molecular replacement using the model of the FEV DNA complex (PDBid 3zp5) and the program PHASER. Model building was performed with the program COOT and the model refinement was performed using PHENIX_REFINE to a final Rfactor=23.5 % , Rfree =25.5 %.
Data Processing: