Human UHRF1, RING domain

Target ID:

UHRF1

Aliases:

FLJ21925hNP95ICBP90MGC138707Np95RNF106

Deposition Date:

Thursday 18th December 2008

Families:

Ubiquitin Related Biology

Structure type:

apo

Download Coordinates:

3FL2

Authors:

Walker JR, Avvakumov GV, Xue S, Li Y, Bountra C, Weigelt J, Arrowsmith CH, Edwards AM, Bochkarev A, Dhe-Paganon S

Site:

Toronto

3FL2

tabs

Structure Details

Convergence of the ubiquitylation, acetylation, and methylation systems occurs with UHRF1, a nuclear protein containing domains that bind modified histone H3 (via tandem Tudor domain), modified (hemimethylated) DNA (via Set and RING- associated domain, SRA)[1], and catalyze ubiquitylation (via RING domain). This protein is involved in a number of biological events including transcriptional regulation, DNA repair, and epigenetic code inheritance.

It is known that UHRF1 acts as E3 ligase to catalyze ubiquitylation of histone H3[2]. However, the full spectrum of the ubiquitylation substrates of UHRF1 is not known. As a part of the studies to address this issue, we have solved the crystal structure of the RING domain.

The structure of the UHRF1 RING domain reveals the canonical RING fold tightly associated with a helical bundle. Residues 675-722 and 775-786 of UHRF1 form a four-helix antiparallel bundle that flanks the central RING motif. The UHRF1 RING motif is characterized by a central alpha-helix adjacent to a short antiparallel three-stranded beta-sheet with two Zn2+ atoms bound by three zinc-fingers. Identity and conformation of putative E2-binding human UHRF1 side chains are completely conserved with those of human UHRF2 (PDB 1Z6U); this suggests that both UHRF1 and UHRF2 bind to the same set of E2s.

Materials and Methods

UHRF1

PDB:3FL2

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:ubh12.672.793.133H12 (SDC133H12)
Tag:N-terminal: MGSSHHHHHHSSGLVPRGS
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsEPYSLTAQQSSLIREDKSNAKLWNEVLASLKDRPASGSPFQLFLSKVEETFQCICCQELVFRPITTVCQHNVCKDCLDRSFRAQVFSCPACRYDLGRSYAMQVNQPLQTVLNQLFPGYGNGR
Vector:pET28a-LIC

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 approx. 7. Protein expression was induced with 0.1 mM isopropyl-1-thio-D-galactopyranoside overnight at 15 °C. The culture was centrifuged (12,000 x g, 15 minutes) and cell pellet was collected and stored at -80 °C.

Purification

Procedure
The cleared lysate was loaded onto a 3 mL TALON metal-affinity resin (Clontech) column 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 His-tag was removed by overnight incubation of the protein with thrombin at 4ºC.

The protein was further purified by gel filtration on a HighLoad 16/60 Superdex 200 column (GE Healthcare) equilibrated with Gel Filtration buffer. Fractions containing protein were pooled and concentrated by ultrafiltration to a final protein concentration of 120 mg/ml.

The yield of the protein was 4 mg per liter bacterial culture.

Extraction

Procedure
The cell pellet from a 2 L culture was resuspended in 50 ml Lysis buffer, lysed using a Microfluidizer at 18,000 p.s.i., 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 RING domain with N-terminal addition of glycine and serine residues from the tag.
Crystallization:Crystals of the UHRF1 RING domain were grown at 293K using the hanging drop method by mixing 1 volume of protein solution (60 mg/ml in 20 mM HEPES, pH 7.0, 300 mM NaCl, 1 mM TCEP) with 1 volume of well solution consisting of 27% PEG MME 2000, 0.1 M Tris-HCl, pH 8.5. The crystals were cryoprotected by immersion in Paratone N diluted (1 : 1) with paraffin oil.
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.
Karagianni, P., Amazit, L., Qin, J.and Wong, J. (2008) ICBP90, a novel methyl K9 H3 binding protein linking protein ubiquitination with heterochromatin formation. Mol. Cell. Biol., 28, 705-717.