Human zinc finger, CW type with PWWP domain 2 (F78R mutant), Znf_CW domain

Target ID:

ZCWPW2

Deposition Date:

Monday 23rd March 2015

Families:

Methyl-lysine readers

Related Diseases:

Chromatin and epigenetics

Structure type:

apo

Download Coordinates:

4Z0R

Authors:

Liu Y, Tempel W, Bountra C, Arrowsmith CH, Edwards AM, Min J

Site:

Toronto

4Z0R

tabs

Structure Details

The zinc finger of ZCWPW2 adopts similar tertiary structure as solved zinc finger of ZCWPW2 (4O62), consisting of two β strands (β1: W30-Q32, β2: W41-L43), one α helix (S45-K51) and one one-turn 310-helice (C60-M62). The four zinc binding cystines are located at the loop between the two β strands (C33 and C38), the 310-helix (C60) and the loop after the 310-helix (C71). The H3K4me3 peptide adopts a β strands and forms a 3-stranded anti-parallel β sheet together with the two β strands of CW zinc finger. The backbone amine group of H3A1 snugly embedded in negative charged pocket formed by the loop after β2 strand and forms a conserved salt bridge with the carboxylate oxygen of H54 and E56. The trimethyllysine K4me3 is bound in a conserved negative charged pocket located in the two β strands and flanked by two aromatic residues W30 (β1 strand) and W41 (β2 strand). There are there molecules of CW zinc finger and one H3K4me3 peptide molecules in the asymmetric unit. Residue Y25 of the other two CW zinc finger molecules disrupts into each other’s K4me3 binding pockets and blocks the binding site for K4me3

Materials and Methods

ZCWPW2

PDB:4Z0R

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_001035522.1.
Entry Clone Source:EHS1001-9390983
SGC Clone Accession:ZCWPW2_9 (JMC01B:H12): V21-F78 with F78R mutation
Tag:N-terminal tag: MHHHHHHSSGRENLYFQG
Host:Escherichiacoli BL21 (DE3)

Construct

Prelude:
Sequence:MHHHHHHSSGRENLYFQGVENMYVNKVWVQCENENCLKWRLLSSEDSAKVDHDEPWYCFMNTDSRYNNCSISEEDD
Vector:pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:Lex system, 37 °C 3-4h, 15 °C 18-24h

Purification

Procedure
IMAC: Unclarified lysate was mixed with 3-4 mL of Ni-NTA superflow Resin (Qiagen) per 200 mL lysate. The mixture was incubated with mixing for at least 30 minutes at 4°C. The mixture was then loaded onto an empty comLum (BioRad) and washed with 40 mL wash buffer. Samples were eluted from the resin by exposure to 2-3 column volumes (approx. 10-15 mL) of elution buffer. Concentration of eluted protein was estimated by OD280

Gel filtration chromatography: An XK 16x60 column (GE Healthcare) packed with HighLoad Superdex 75 resin (GE Healthcare) was pre-equilibrated with gel filtration buffer for 1.5 column volumes using an AKTA explorer (GE Healthcare) at a flow rate of 1.0 mL/min. The concentrate sample (approx. 3 mL, concentrated by using 15 mL concentrators with a 3,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm) from the IMAC step was loaded onto the column at 1 mL/min, and 2mL fractions were collected into 96-well plates (VWR 40002-012) using peak fractionation protocols. Fractions observed by a UV absorption chromatogram to contain the protein were pooled.

Extraction

Procedure
Frozen cell pellet contained in bags (Beckman 369256) obtained from 2L of culture were thawed by soaking in warm water. Each cell pellet was resuspended in 200 mL lysis buffer and homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds per pellet. Cell lysis was accomplished by sonication (Virtis408912, Virsonic) on ice: the sonication protocol was 10 seconds pulse at 80% of maximal frequency (8.0), 10 seconds rest, for 10 minutes total sonication time per pellet.
Concentration:Purified proteins were concentrated using 15 mL concentrators with a 5,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm, typically resulting in a final concentration around 20 mg/mL.
Ligand
MassSpec:
Crystallization:Recombinant human ZCWPW2 CW zinc finger with F78R mutation was mixed with H3K4me3 peptide at molar ratio of 1:3 and co-crystallized using the sitting drop vapour diffusion method at 18 °C. The crystals were obtained in a buffer containing 2 M NH4SO4, 2% PEG 400, 0.1 M Na Hepes, pH 7.5. Crystals were soaked in a cryoprotectant consisting of 100% reservoir solution and 15% glycerol.
NMR Spectroscopy:
Data Collection:
Data Processing: