Human CXXC finger protein 1, CXXC domain, in complex with Non-Methylated CpG islands (DNA-TCGA)

Target ID:

CXXC1

Deposition Date:

Friday 4th February 2011

Families:

Miscellaneous

Related Diseases:

Chromatin and epigenetics

Structure type:

protein-protein

Download Coordinates:

3QMH

Authors:

Xu C, Bian CB, Lam R, Dong A, Min J

Site:

Toronto

3QMH

tabs

Structure Details

Overall, all the CXXC1 CXXC-DNA complex structures are very similar. The CXXC domain of CFP1 consists of two alpha helices and one short 310 helix with two long loops linking them. Eight conserved cysteine residues bind two zinc ions to form two C4-type zinc fingers, with the first three cysteines and the last cysteine binding one zinc ion and the middle four cysteines binding the other zinc ion. The crescent-shaped CFP1 CXXC domain is wedged into the major groove of the CpG DNA and forms extensive interactions between the CXXC domain and DNA. The DNA-binding surface of CFP1 is predominantly positively charged, interacting with the negatively charged DNA. In addition to electrostatic interactions, a network of hydrogen bonds between the CXXC domain and DNA, including several water-mediated interactions, contribute to CFP1-DNA binding. Interestingly, only the middle four nucleotides including the CpG dinucleotide contribute to the CXXC binding.

The overall structure of the CFP1 CXXC domain resembles the recently reported structure of the MLL CXXC domain. The major differences between these two CXXC domain structures are at the amino (N)- and carboxy (C)-termini. Both N- and C-termini of the CXXC domain extend into a minor groove of the CpG DNA in the MLL-DNA complex structure. In contrast, the C-terminus of the CFP1 CXXC domain forms a short 310 helix and interacts only with the major groove of DNA. The first α-helix (α1) of the CFP1 CXXC domain hangs over the DNA backbone with the preceding loop extending into the minor groove but not making direct contact with DNA.

Materials and Methods

CXXC1

PDB:3QMH

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC029922
Entry Clone Source:MGC AT12-F4 (BC029922)
SGC Clone Accession:CXXC1_13; plate JMC022H12
Tag:N-terminal tag: MGSSHHHHHHSSGRENLYFQG
Host:BL21 (DE3) Codon plus RIL (Stratagene)

Construct

Prelude:
Sequence:MHHHHHHSSGRENLYFQGQIKRSARMCGECEACRRTEDCGHCDFCRDMKKFGGPNKIRQKCRLRQCQLRARESYKYFPSS
Vector:pET28a-MHL

Growth

Medium:LB (Sigma L7658)
Antibiotics:50 µg/mL kanamycin (BioShop Canada KAN 201)
Procedure:A 250 mL flask containing LB (Sigma L7658) supplemented with 50 µg/mL kanamycin (BioShop Canada KAN 201) was inoculated from a glycerol stock of the bacteria. The flask was shaken overnight (16 hours) at 250 rpm at 37 °C. Using the Lex system, a 2L bottle (VWR 89000-242) containing 1800 mL of TB (Sigma T0918) supplemented with 1.5% glycerol, 50 ug/ mL kanamycin and 600 µl antifoam 204 (Sigma A-8311) was inoculated with 50 mL overnight LB culture, and incubated at 37 °C. The temperature of the media was reduced to 15 °C one hour prior to induction and induced at OD₆₀₀ = 6 with 100 µM isopropyl-thio-β-D-galactopyranoside (BioShop Canada IPT 001). Cultures were aerated overnight (16 hours) at 15 °C, and cell pellets collected by centrifugation and frozen at -80 °C.

Purification

Procedure
IMAC: Unclarified lysate was mixed with 2-3 mL of Ni-NTA superflow Resin (Qiagen) per 40 mL lysate. The mixture was incubated with mixing for at least 45 minutes at 4 degrees Celsius. The mixture was than loaded onto an empty comLum (BioRad) and washed with 100 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. Concentrated protein sample was further purified by superdex200 Gel filtration column.

Gel filtration chromatography: An XK 26x65 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 dialyzed sample from the IMAC step (approx. 15 mL) was loaded onto the column at 1.5 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 25-40 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 sec pulse at half-maximal frequency (5.0), 10 second 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 15 mg/mL.
Ligand
MassSpec:
Crystallization:Crystals of human CXXC1 CXXC domain in complex with CCGG CpG dsDNA (1:1.2) were grown at 291 K using the hanging drop method by mixing equal volumes of 0.1 M HEPES pH 7.5, 0.2 M CaCl₂, 28% PEG400. Crystals of human CXXC1 CXXC domain in complex with ACGG CpG dsDNA (1:1.2) were grown at 291 K using the hanging drop method by mixing equal volumes of 0.1M HEPES pH 7.5, 0.2 M CaCl₂, 28% PEG400. Crystals of human CXXC1 CXXC domain in complex with TCGA CpG dsDNA (1:1.2) were grown at 291 K using the hanging drop method by mixing equal volumes of 0.1-M Hepes sodium, pH 7.5, 0.1 M MgCl₂, 30% 550 MME. The crystals were cryoprotected by dragging through paratone oil.
NMR Spectroscopy:
Data Collection:
Data Processing: