Human UHRF1, tandem tudor domain

Target ID:

UHRF1

Aliases:

FLJ21925hNP95ICBP90MGC138707Np95RNF106

Deposition Date:

Thursday 29th May 2008

Families:

Miscellaneous

Related Diseases:

CancerChromatin and epigeneticsSignalling

Structure type:

apo

Download Coordinates:

3DB4

Authors:

Walker JR, Avvakumov G, Xue S, Dong A., Li Y, Bountra C, Wikstrom M, Arrowsmith CH, Edwards AM, Bochkarev A, Dhe-Paganon S.

Site:

Toronto

ICM Datapack:

ICM Datapack

3DB4

tabs

Structure Details

Convergence of the ubiquitylation, acetylation, and methylation systems occurs with two human proteins, UHRF1 and UHRF2. This duo is involved in a number of biological roles including transcriptional upregulation, DNA repair, and epigenetic code inheritance, having the ability to simultaneously bind DNA and recruit key nuclear enzymes. Their DNA binding modules recognize not only inverted CAATT boxes upstream of particular genes (topoisomerase IIα and the retinoblastoma protein), but also hemimethylated CpG islands downstream of the replication fork (1). UHRF proteins orchestrate all three major epigenetic enzymes, including histone acetylases (HDAC1), methyl transferases (DNMT1), and E3 ligases (Ring domain), at least in part to maintain and control epigenetic fidelity during cell proliferation and DNA repair.

We have found a previously unrecognized domain in the N-terminal portion of human UHRF1 (residues 126 - 285) and determined its crystal structure. The structure reveals a tandem Tudor domain (TTD) with closest overall similarity to another known structure being the TTD of Fragile X mental retardation protein (PDB entry 2BKD; RMSD of 3.3 Å, over 96 Cα positions). TTDs have two closely associated lobes with variable relative orientations. Individual lobes have a five-stranded β-barrel fold also present in SH3, CPH, Kow, and single-Tudor domains (Dali or Pfam). The two lobes of UHRF1 (referred to here as TTD1 and TTD2), superimpose well with one another (RMSD of 2.5 &ring;) despite a lack of detectable sequence identity. Members of the protein 'Royal Family' including Tudor, chromo, and MBT domains have been shown to specifically bind to methylated histone peptides, and possess a conserved aromatic cage composed of two to four aromatic residues that utilize cation-π interactions to bind to a positively charged methylated lysines and possibly methylated arginines. As a related structure (PDB entry 3DB3) shows, the TTD of human UHRF1 is capable of binding trimethylated lysine, too.

Materials and Methods

UHRF1

PDB:3DB4

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_037414
Entry Clone Source:ubh12.BC113875.OBS.MHS4426-98361361.pCR-BluntIITOPO
SGC Clone Accession:ubh12.126.285.130H02 (SDC130H02)
Tag:N-terminal: MHHHHHHSSGRENLYFQG
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgrenlyfqgMWDETELGLYKVNEYVDARDTNMGAWFEAQVVRVTRKAPSRDEPCSSTSRPALEEDVIYHVKYDDYPENGVVQMNSRDVRARARTIIKWQDLEVGQVVMLNYNPDNPKERGFWYDAEISRKRETRTARELYANVVLGDDSLNDCRIIFVDEVFKIERPGE
Vector:pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:The protein was expressed in E. coli BL21 (DE3) grown in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin at 37°C to an OD600 of 7.5. Protein expression was induced 0.05 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15°C. The culture was centrifuged (12,000 x g, 15 minutes) and cell pellets collected and stored at -80°C. In order to obtain the selenomethionyl derivative of the UHRF1 SRA domain, the cells were grown in M9 medium supplemented with glycerol using a M9 SeMET High-Yield growth media kit package (MD045004-50L, Medicilon) according to manufacturer's instructuion and the protein was purified as below.

Purification

Procedure
The cleared lysate was loaded onto a 3 mL TALON metal-affinity resin column (BD Biosciences) at 4ºC. The column was washed with 10 mL Wash buffer A, 10 mL Wash buffer B, and 10 mL Wash buffer A. The protein was eluted with 6 mL Elution buffer. The N-terminal His-tag was removed by overnight incubation of the protein with TEV protease at 4 C.

The protein was further purified by gel filtration on a HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham) equilibrated with Gel Filtration buffer. Fractions containing protein (analyzed by SDS-PAGE) were pooled.

Final purification was achieved by ion-exchange chromatography on a 5-ml HiTrapQ column using linear 0 - 50 % gradient of Ion-exchange buffer B in Ion-exchange buffer A. The target protein eluted around 30% buffer B. The corresponding fractions were combined and protein was concentrated by ultrafiltration using an Amicon Ultra centrifugal filter with 10 kD cutoff to a final concentration of 28 mg/ml.

The yields of the protein and its selenomethionyl derivatives were approx. 7 and 9 mg per liter of bacterial culture, respectively.

Extraction

Procedure
The cell pellet from a 2 L culture was resuspended in 50 ml Lysis buffer, lysed using a Microfluidizer at 18,000 PSI, and cleared by centrifugation at 40,000 x g for 30 min.
Concentration:
Ligand
MassSpec:Mass-spectroscopy by LC/MS showed pure product of correct molecular weight corresponding selenomethionyl derivative of the tandem-Tudor domain with addition of an N-terminal glycine residue from the tag and all four methionine residues substituted with selenomethionine.
Crystallization:Crystals of the UHRF1 tandem-Tudor domain selenomethionyl derivative were grown at 298 K using the hanging drop method by mixing 1 volume of 23 mg/ml protein with 1 volume of well solution consisting of 10% PEG 8000, 0.2 M ammonium sulfate, 0.1 M sodium cacodylate, pH 6.5, and 1 mM TCEP. The crystals were cryoprotected by immersion in the well solution mixed in 1:1 ratio with a water solution containing 20% (w/v) sucrose, 4% (w/v) glucose, 18% (v/v) glycerol and 18% (v/v) ethylene glycol.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Avvakumov, G. V., Walker, J. R., Xue, S., Li, Y., Duan, S., Bronner, C., Arrowsmith, C. H., and Dhe-Paganon, S. (2008) Structural basis for recognition of hemi-methylated DNA by the SRA domain of human UHRF1, Nature.