C2 domain of human Itchy homolog E3 ubiquitin protein ligase

Target ID:

ITCH

Aliases:

AIF4AIP4dJ468O1.1NAPP1

Deposition Date:

Monday 30th October 2006

Families:

Ubiquitin Related BiologyUbiquitylation - E3 Ligases

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2NQ3

Authors:

J.R.WALKER,G.V.AVVAKUMOV,S.XUE,C.BUTLER-COLE,P.J.FINERTYJR.,J.WEIGELT,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,S.DHE-PAGANON,STRUCTURAL GENOMICS CONSORTIUM(SGC)

Site:

Toronto

2NQ3

tabs

Structure Details

Substrate specificity of the ubiquitylation system is carried out by E3 ubiquitin-protein ligases, subdivided into two major classifications: those with a HECT (homologous to E6-AP carboxyl terminus) catalytic domain, and those with a RING-finger (really interesting novel gene) domain. The HECT E3 ligases each contain an approximately 350 amino acid C-terminal catalytic domain with a conserved active site cysteine that, unlike the RING-finger E3 ligases, forms a thiolester intermediate with ubiquitin, transferring it from an E2 ligase to the protein substrate. The highly variable N-terminal regions of the HECT-domain E3s interact with specific protein substrates while the catalytic domain interacts with its cognate E2 and carries out the catalytic reaction.

The Itch E3 ligase is a member of the Nedd4/Rsp5p HECT-family that is characterized by an N-terminal C2 domain, 2-4 WW domains, and the C-terminal HECT domain. Itch is implicated in the regulation of the immune response as its absence in non-agouti lethal 18H or Itchy mice leads to immunological and inflammatory diseases. Itch has been shown to mediate a variety of cellular processes such as cellular differentiation, proliferation and apotosis; targets include the membrane bound Notch1 receptor; the transcription factors c-Jun, JunB, p63, the RING-finger E3 ligase Cbl, and the apoptosis inhibitor c-FLIP.

Itch substrate specificity is determined by its four WW domains, which recognize the PPXY motif of its targets. Itch demonstrates cross-talk between the post-transcriptional regulation of proteins by phosphoryation and ubiquitination, as phosphorylation of its third WW domain by the Src kinase Fyn leads to Itch activation, and subsequent increased degradation of JunB, while phosphorylation of the PPXY consensus sequence of c-Jun by the tyrosine kinase c-Abl results in its protection from ubuitination and degradation by Itch. We have initiated our structural investigations into Itch by solving the high-resolution structure of its C2 domain. C2 domains are 130 amino acid protein modules that often bind phospholipids in a Ca2+ dependent manner, although they have evolved into Ca2+ dependent and Ca2+ independent forms. Sequence and structural comparisons suggest that the C2 domain of Itch might be Ca2+ independent.

Materials and Methods

ITCH

PDB:2NQ3
Entry Clone Accession:gi:61676206; SGC clone ID: Itch.001.155, plate SDC091, well B7
Entry Clone Source:MGC
SGC Clone Accession:itch.001.155; plate SDC091:B7
Tag:mhhhhhhhssgrenlyfqg
Host:E.coli BL21 (DE3) Gold
Vector:p28a-LIC

Sequence: mhhhhhhhssgrenlyfqgMSDSGSQLGSMGSLTMKSQLQITVISAKLKENKKNWFGPSPYVEVTVDGQSKKTEKCNNTNSPKWKQPLTVIVTPVSKLHFRVWSHQTLKSDVLLGTAALDIYETLKSNNMKLEEVVVTLQLGGDKEPTETIGDLSICLDGLQLESEVVTNGETT

Growth

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. Cells were then induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 0.1 mM, and incubated overnight at 15°C. The culture was centrifuged and the cell pellets were collected and stored at -80°C.

Purification

Procedure: IMAC purification: 4 microL of clarified supernatant is reserved for later analysis by SDS-PAGE. The rest of the clarified supernatant is then diluted 1:2 in lysis buffer, and loaded at approximately 1mL/min by gravity onto 5 mL of Ni-NTA resin (Qiagen 30450). 5 column volumes of lysis buffer are used to wash the column at approximately 3 mL/min, followed by 5 column volumes of low imidazole buffer (lysis buffer + 10 mM Imidazole (VWR EM-5720) pH 8) at approximately 3 mL/min. A 4 µL sample of the low imidazole wash is saved for later analysis by SDS-PAGE. Samples are eluted from the Ni-NTA resin by exposure to 10 mL elution buffer (lysis buffer + 250 mM imidazole and 10% glycerol (EMD GX0185-5)) at 1mL/min flow rate. A 10 µL sample of the eluate is saved for SDS-PAGE analysis. 10 µL of each eluate is saved for measurement of protein concentration using Bradford reagent (BioRad 500-0202).

Extraction

Procedure: Frozen cell pellets contained in bags (Beckman 369256) obtained from 2L liters of culture are thawed by soaking in warm water for 5 minutes. Each cell pellet is resuspended in 20 mL lysis buffer, 1mM phenylmethanesulfonyl fluoride (Sigma P7626), and 1mL Sigma general protease inhibitor (Sigma P2714-1BTL, resuspended according to manufacturer\'s instructions) and then homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds per pellet. Cell lysis is accomplished by sonication (Virtis408912, Virsonic) on ice: the sonication protocol is 10 sec pulse at half-maximal frequency (5.0), 10 second rest, for 6 minutes total sonication time per pellet. Lysed cells are placed into centrifuge tubes (363647, Beckman Coulter) and centrifuged in a JA25.50 rotor in an Avanti J-20 XPI centrifuge (Beckman Coulter) for 20 minutes at 69,673 x g. The supernatant is decanted into a beaker, and the insoluble pellet discarded

Structure Determination

Crystallization:Protein concentration: 20 mg/mL

Protein buffer: 20 mm TRIS-HCl, pH 8.0, 0.15 M NaCl, 5% glycerol, 2 mm DTT

Protein was mixed in 1:1 ratio with 20% PEG3350, 0.1 M BIS-TRIS, pH 6.0, 0.2 M NH4OAc, 1 mm DTT in a hanging drop plate in a temperature of 298K.
Data Collection:Resolution: 2.0Å, X-ray source: The Industrial Macromolecular Crystallography Association 17-ID beamline at the Advanced Photon Source.