Human OTU domain, ubiquitin aldehyde binding 1 [Homo sapiens]

Target ID:

OTUB1

Aliases:

FLJ20113FLJ40710HSPC263MGC111158MGC4584OTB1OTU1

Deposition Date:

Wednesday 16th January 2008

Families:

Deubiquitinating Enzymes - Non-USPUbiquitin Related Biology

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2ZFY

Authors:

Akutsu M, Walker JR, Li Y, Weigelt J, Arrowsmith CH, Bountra C, Edwards AM, Bochkarev A, Dhe-Paganon S

Site:

Toronto

ICM Datapack:

ICM Datapack

2ZFY

tabs

Structure Details

The product of this gene is a member of the OTU (ovarian tumor) superfamily of predicted cysteine proteases. The encoded protein is a highly specific ubiquitin iso-peptidase, and cleaves ubiquitin from branched poly-ubiquitin chains but not from ubiquitinated substrates. It interacts with another ubiquitin protease and an E3 ubiquitin ligase that inhibits cytokine gene transcription in the immune system. It is proposed to function in specific ubiquitin-dependent pathways, possibly by providing an editing function for polyubiquitin chain growth. Alternative splicing results in multiple transcript variants.[1]

The crystal structure of OTUB1 reveals the canonical OTU fold, composed of a papain-like core (two lobes, one α-helical, the other β-sheet). Like its nearest neighbor (OTUB2), the active site includes p1 and p1' pockets, sites for two ubiquityl units of a chain (hydrolysis occurs at the isopeptide bond between the linkage). The distorted appearance of the catalytic triad, with His267 not within hydrogen-bonding distance of the catalytic cysteine (Cys91), might suggest that the protein was crystallized in an autoinhibited state. The p1' site likely holds structural information concerning the enzyme's substrate specificity, which is in question.

Materials and Methods

OTUB1

PDB:2ZFY
Entry Clone Accession:NP_060140
Entry Clone Source:otub1.BC007519.MGC.AU30F4.pOTB7
SGC Clone Accession:otub1.040.271.72C04 (SDC072)
Tag:N-terminal: MGSSHHHHHHSSGLVPR*GS (removed)
Host:E.coli BL21(DE3)
Vector:pET28a-LIC

Sequence: mgsshhhhhhssglvpr*gsEIAVQNPLVSERLELSVLYKEYAEDDNIYQQKIKDLHKKYSYIRKTRPDGNCFYRAFGFSHLEALLDDSKELQRFKAVSAKSKEDLVSQGFTEFTIEDFHNTFMDLIEQVEKQTSVADLLASFNDQSTSDYLVVYLRLLTSGYLQRESKFFEHFIEGGRTVKEFCQQEVEPMCKESDHIHIIALAQALSVSIQVEYMDRGEGGTTNPHIFPEGSEPKVYLLYRPGHYDILYK

Growth

Medium:TB
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. 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: Imac: The cleared lysate from a 2 L culture was loaded onto 3 ml TALON metal-affinity resin column (BD Biosciences) at 4ºC. The column was washed with 40 ml Wash buffer, and the protein was eluted with 10 ml Elution buffer.
Tag removal: 1 Unit of thrombin (Sigma T9681) per milligram of protein was added to the 10 mL sample, stored overnight without shaking at 4ºC.
Gel-filtration: It was further purified by gel filtration on a HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham) equilibrated with Gel Filtration buffer and concentrated to 68 mg/ml by ultrafiltration using Amicon Ultra centrifugal filter with 10 kD cutoff and stored at -80 ºC. Protein yield was 17 mg per liter of bacterial culture.

Extraction

Procedure: The cell pellet was defrosted and cells were lysed by sonication, 10 s on, 10 s off at 40% amplitude for 10 min. The lysate was cleared by centrifugation for 45 minutes at 15,500 RPM, 4°C.
Concentration:68 mg/mL

Structure Determination

MassSpec:Mass-spectroscopy by LCMS shows that the product was pure and of correct molecular weight
Crystallization:Purified protein was crystallized using the hanging drop vapor diffusion method. Crystals grew when the protein (68 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 30% PEG 8000, 0.2 M Sodium acetate, and 0.1 M Sodium cacodylate at pH 6.5 in 293 Kelvin.