Human MORC family CW-type zinc finger 3, ZnF domain, in complex with H3K4me3 peptide

Target ID:

MORC3

Deposition Date:

Thursday 26th June 2014

Families:

Methyl-lysine readers

Related Diseases:

Chromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

4QQ4

Authors:

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

Site:

Toronto

4QQ4

tabs

Structure Details

The zinc finger of MORC3 adopts similar tertiary structure as solved zinc finger of ZCWPW1 (2RR4) and ZCWPW2 (4O62), consisting of two β strands (β1: Q408-Q412, β2: W419-L422) and two one-turn 310-helices (η1: C435-N437, η2: P441-F443). The four zinc binding cystines are located at the loop between the two β strands (C413 and C416), the first 310-helix (C435) and the loop after the second 310-helix (C446). 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 P430 and E431. The trimethyllysine K4me3 is bound in a conserved negative charged pocket located in the two β strands and flanked by two aromatic residues W410 (β1 strand) and W419 (β2 strand). There are two molecules of CW zinc finger and three zinc molecules in the asymmetric unit. The third zinc molecule is stabilized by two cystines (C416 from the two CW zinc finger molecule) and two Chloride ions from the protein buffer.

Materials and Methods

MORC3

PDB:4QQ4

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_056173.1.
Entry Clone Source:AT100-d5
SGC Clone Accession:MORC3_10 (JMC01B:H03): E400-P460
Tag:N-terminal tag: MHHHHHHSSGRENLYFQG
Host:Escherichiacoli BL21 (DE3)

Construct

Prelude:
Sequence:MHHHHHHSSGRENLYFQGEDIQKRPDQTWVQCDACLKWRKLPDGMDQLPEKWYCSNNPDPQFRNCEVPEEPEDEDLVHP
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 MORC3 CW zinc finger 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 20% PEG 3350, 0.2 M NH4Cl. Crystals were soaked in a cryoprotectant consisting of 100% reservoir solution and 15% glycerol.
NMR Spectroscopy:
Data Collection:
Data Processing: