Human ubiquitin-like containing PHD and RING finger domains 1 (UHRF1/ICBP90/NP95) Set and Ring Associated (SRA) domain

Target ID:

ubh12

Aliases:

FLJ21925hNP95ICBP90MGC138707Np95RNF106

Deposition Date:

Thursday 29th November 2007

Families:

Ubiquitin Related Biology

Related Diseases:

CancerChromatin and epigenetics

Structure type:

apo

Download Coordinates:

3BI7

Authors:

Avvakumov G.V., Xue S., Walker J.R., Butler-Cole C., Arrowsmith C.H, Weigelt J., Edwards A., Dhe-Paganon, S.

Site:

Toronto

3BI7

tabs

Structure Details

This gene encodes a member of a subfamily of RING-finger type E3 ubiquitin ligases. The protein binds to specific DNA sequences, and recruits a histone deacetylase to regulate gene expression. Its expression peaks at late G1 phase and continues during G2 and M phases of the cell cycle. It plays a major role in the G1/S transition by regulating topoisomerase IIalpha and retinoblastoma gene expression, and functions in the p53-dependent DNA damage checkpoint. Multiple transcript variants encoding different isoforms have been found for this gene.[1]

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 IIa and the retinoblastoma protein), but also hemimethylated CpG islands downstream of the replication fork. 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 begun studying these proteins with the structural biology of component domains, and present here the first high-resolution crystal structure of the SET and Ring finger Associated domain of a UHRF protein. An 8-stranded β-barrel cupping a short α-helix forms the globular domain, potentially representing a new DNA binding fold. Further studies are underway.

Materials and Methods

UHRF1

PDB:3BI7

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:ubh12.414.617.125B05 (SDC125B05)
Tag:N-terminal: M
C-terminal: AHHHHHH
Host:BL21 (DE3)

Construct

Prelude:
Sequence:MPSNHYGPIPGIPVGTMWRFRVQVSESGVHRPHVAGIHGRSNDGAYSLVLAGGYEDDVDHGNFFTYTGSGGRDLSGNKRTAEQSCDQKLTNTNRALALNCFAPINDQEGAEAKDWRSGKPVRVVRNVKGGKNSKYAPAEGNRYDGIYKVVKYWPEKGKSGFLVWRYLLRRDDDEPGPWTKEGKDRIKKLGLTMQYPEGYLEALANAHHHHHH
Vector:pNIC-CH

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 37ºC to an OD600 of 7.5. Protein expression was induced 0.05 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15ºC. The culture was centrifuged (12,000 x g, 15 minutes) and cell pellets collected and stored at -80ºC. 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 manufacturerÂs instructuion and the protein was purified as below.

Purification

Procedure
Column 1: TALON metal-affinity resin column (BD Biosciences)
Column 2: HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham)

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 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 and concentrated by ultrafiltration using an Amicon Ultra centrifugal filter with 10 kD cutoff to a final concentration of 34 mg/ml.The yields of the protein and its selenomethionyl derivatives were approx. 4 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:34 mg/ml.
Ligand
MassSpec:Mass-spectroscopy by LCMS showed pure product of correct molecular weight corresponding the SRA selenomethionyl derivative lacking N-terminal methionine residue (removed by bacterial aminopeptidase in the course of protein expression) and two other methionine residues substituted with selenomethionine
Crystallization:Crystals of the UHRF1 SRA domain selenomethionyl derivative were grown at 298 K using the hanging drop method by mixing 1 volume of 34 mg/ml protein with 1 volume of well solution consisting of 1.4 M ammonium sulfate, 0.1 M bis-Tris, pH 6.0, 0.2 M NaCl 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: