Human itchy E3 ubiquitin protein ligase, WW domains, in complex with a TXNIP peptide

Target ID:

ITCH

Aliases:

AIF4AIP4dJ468O1.1NAPP1

Deposition Date:

Tuesday 28th October 2014

Families:

Ubiquitin Related BiologyUbiquitylation - E3 Ligases

Related Diseases:

Signalling

Structure type:

protein-protein

Download Coordinates:

Superceded by 5CQ2

Authors:

Liu Y, Tempel W, Bountra C, Arrowsmith CH, Edwards AM, Min J

Site:

Toronto

4ROH

tabs

Structure Details

In the complex structure, the PPCY peptide has the sequence N-TPEAPPCYMDVI-C, corresponding to residues 327′-338′ of TXNIP (hereafter, TXNIP residue numbers are marked with a prime (′) to distinguish them from the Itch residue numbers). The residues P331' and P332' of the PPCY peptide form a polyproline type II helix, whereas residues 334′-337′ adopt a helical fold. The binding site of the peptide is located on one face of the WW sheet and consists of two canonical grooves, namely the XP groove and tyrosine-binding groove (Tyr groove). P331′ and P332′ insert into the XP groove by stacking against the conserved W466 and Y455 residues of WW3, respectively. The side chain of Y334′ is accommodated in the Tyr groove formed by the conserved residues V457, H459 and R462 of WW3. In addition to the hydrophobic packing, some hydrogen bonds between the peptide and WW domains contribute to the stabilization of the complex, for example between the carbonyl of P332′ and the hydroxyl of T464, along with the phenolic hydroxyl of Y334′ and Nπ of H459.

Materials and Methods

ITCH

PDB:4ROH
Entry Clone Accession:NP_001244066.1.
Entry Clone Source:Open Biosystems
SGC Clone Accession:ITCH (ITCH:YTC002-B06): M139-E298
Tag:N-terminal tag: MKIEEHHHHHHSSGKLMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLSSGLVPRGSGTAAQPAENLYFQGRRASVEL
Host:Escherichiacoli BL21 (DE3)
Vector:pET28GST-lic (modified)

Construct Sequence: grrasvelEFDPLGPLPPGWEKRTDSNGRVYFVNHNTRITQWEDPRSQGQLNEKPLPEGWEMRFTVDGIPYFVDHNRRTTTYIDPRTGKSALDNGPQ

Growth

Procedure:Lex system, 37 Â°C 3-4h, 15 Â°C 18-24h

Purification

Procedure: IMAC: Unclarified lysate was mixed with 3-4 mL of Ni-NTA superflow Resin (Qiagen) per 200 mL lysate. The mixture was incubated with mixing for at least 30 minutes at 4°C. The mixture was then loaded onto an empty comLum (BioRad) and washed with 40 mL wash buffer. Samples were eluted from the resin by exposure to 2-3 column volumes (approx. 10-15 mL) of elution buffer. Concentration of eluted protein was estimated by OD280

Gel filtration chromatography: An XK 16x60 column (GE Healthcare) packed with HighLoad Superdex 75 resin (GE Healthcare) was pre-equilibrated with gel filtration buffer for 1.5 column volumes using an AKTA explorer (GE Healthcare) at a flow rate of 1.0 mL/min. The concentrate sample (approx. 3 mL, concentrated by using 15 mL concentrators with a 3,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm) from the IMAC step was loaded onto the column at 1 mL/min, and 2mL fractions were collected into 96-well plates (VWR 40002-012) using peak fractionation protocols. Fractions observed by a UV absorption chromatogram to contain the protein were pooled.

Extraction

Procedure: Frozen cell pellet contained in bags (Beckman 369256) obtained from 2L of culture were thawed by soaking in warm water. Each cell pellet was resuspended in 200 mL lysis buffer and homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds per pellet. Cell lysis was accomplished by sonication (Virtis408912, Virsonic) on ice: the sonication protocol was 10 seconds pulse at 80% of maximal frequency (8.0), 10 seconds rest, for 10 minutes total sonication time per pellet.
Concentration:Purified proteins were concentrated using 15 mL concentrators with a 5,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm, typically resulting in a final concentration around 10 mg/mL.

Structure Determination

Crystallization:Recombinant human ITCH ww3-ww4 domain was mixed with PPCY peptide of TXNIP at molar ratio of 1:3 and co-crystallized using the sitting drop vapour diffusion method at 18 °C. The crystals were obtained in a buffer containing 30% PEG 4K, 0.2 M MgCl2, 0.1 M Tris HCI, pH 8.5. Crystals were soaked in a cryoprotectant consisting of 100% reservoir solution and 15% glycerol.