Human Np95-like Ring Finger Protein Isoform B

Target ID:

UBH14

Aliases:

DKFZp434B0920DKFZp686G0837MGC33463NIRFRNF107RP11-472F14.2URF2

Deposition Date:

Wednesday 23rd March 2005

Families:

Ubiquitin Related BiologyUbiquitylation - E3 Ligases

Related Diseases:

Chromatin and epigenetics

Structure type:

apo

Download Coordinates:

1Z6U

Authors:

J.R.WALKER,G.V.AVVAKUMOV,S.XUE,E.M.NEWMAN,F.MACKENZIE,M.SUNDSTROM,C.ARROWSMITH,A.EDWARDS,A.BOCHKAREV,S.DHE-PAGNON,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

1Z6U

tabs

Structure Details

NIRF (UHRF2) is a multi-domain, nuclear protein containing ubiquitin-like, plant homeobox (PHD), SET- and RING-associated (SRA) and RING finger domains. These domains suggest a biological role in ubiquitination and protein degradation.

Protein levels of NIRF have been found to correlate with the proliferative phase of various cell types, which suggests involvement of this protein in cell cycle regulation. In addition, cultured cell studies showed a stable and phosphorylation-dependent interaction between NIRF and the Cdk2-cyclin E complex. Furthermore, NIRF expression is also significantly elevated in cancer cells, consistent with a potential role in the G1/S phase transition. Its chromosomal location, at 9p23-24.1, also implies clinical importance. Auto-ubiquitination activity confirms that NIRF is an E3 ligase, with a ring finger that probably interacts with ubiquitin-E2 conjugates.

We have determined the high resolution crystal structure of the ring finger of human NIRF, revealing the zinc-finger fold as well as a novel associated domain. The zinc ligation system is of the C3HC4-type, referred to as the 'cross-brace' motif, with metal ligand pairs one (C733 and C735) and three (C753 and C756) coordinating to bind one zinc ion, while pairs two (C748 and H750) and four (C768 and C771) binding the second.

A 60-residue, four helical bundle, formed from three amino terminal helical elements and an additional carboxy terminal helical element, is closely associated with the zinc finger sub-domain. This unique helical bundle could mediate binding with proteins targeted for ubiquitinated. In contrast to c-cbl, the potential E2-binding surface of the NIRF ring finger is hydrophilic, with arginines and small amino acids forming the bulk of the surface. Because NIRF has been implicated in cell cycle regulation, NIRF could mediate ubiquitination of cyclins and cdk's via typical cell cycle related E2's: ubc-4, ubc-h3, ubc-h5, and ubc-h10.

Materials and Methods

Np95-like ring finger (NIRF) protein isoform b

PDB:1Z6U

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:MGC
SGC Clone Accession:ubh14.672.802; plate SDC013:C4
Tag:N-terminal His-tag with integrated thrombin-cleavage site MGSSHHHHHHSSGLVPRGS.
Host:E.coli BL21 (DE3)

Construct

Prelude:
Sequence:gsPSASKVYKASDSAEAIEAFQLTPQQQHLIREDCQNQKLWDEVLSHLVEGPNFLKKLEQSFMCVCCQELVYQPVTTECFHNVCKDCLQRSFKAQVFSCPACRHDLGQNYIMIPNEILQTLLDLFFPGYSKGR
Vector:p28a-LIC

Growth

Medium:TB
Antibiotics:
Procedure:Using the SGC LEX bubbling system, Np95-like ring finger 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. Cells were then induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 0.05 mM, and incubated overnight at 15ºC. The culture was centrifuged and the cell pellets were collected and stored at -80ºC.

Purification

Procedure
The cleared lysate was loaded onto a TALON metal-affinity resin column from BD Biosciences at 4ºC. The column was washed with wash buffer A (10mM Tris-HCl pH 8.0, 0.5 M NaCl, 5% glycerol, 10 mM imidazole, 1 mM ß-mercaptoethanol), wash buffer B (same as wash buffer A but containing 0.05% Tween 20) and again wash buffer A, and the protein was eluted with 10 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5% glycerol, 200 mM imidazole, 1 mM ß-mercaptoethanol. The protein was further purified by gel filtration on a HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham) equilibrated with 20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5% glycerol, 2mM dithiothreitol and concentrated by ultrafiltration.

Extraction

Procedure
The cell pellet was resuspended in lysis buffer (10 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5% glycerol, 2 mM imidazole, 1 mM ß-mercaptoethanol) with protease inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF) and lysed using Microfluidizer. The lysate was cleared by centrifugation.
Concentration:68 mg/mL
Ligand
MassSpec:
Crystallization:Purified Np95-like ring finger protein 3 was crystallized using the hanging drop vapor diffusion method. Crystals grew when the protein (34 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 0.9M sodium citrate, 0.1M Tris-HCl, pH 8.5.
NMR Spectroscopy:
Data Collection:
Data Processing: