Human striatal enriched protein tyrosine phosphatase (STEP)

Target ID:

PTPN5A

Aliases:

FLJ14427PTPSTEPSTEP

Deposition Date:

Monday 10th April 2006

Families:

Phosphatases

Related Diseases:

NeurobiologySignalling

Structure type:

apo

Download Coordinates:

2CJZ

Authors:

J.E.DEBRECZENI,A.BARR,J.ESWARAN,C.SMEE,N.BURGESS,O.GILEADI,P.SAVITSKY,M.SUNDSTROM,C.ARROWSMITH,A.EDWARDS,J.WEIGELT,S.KNAPP,F.VON DELFT

Site:

Oxford

ICM Datapack:

ICM Datapack

2CJZ

tabs

Structure Details

PTPN5 is a member of the KIM (kinase interaction motif) domain containing phosphatases and the R7 family of receptor tyrosine phosphatases (PTP's). In cells, PTPN5 counter balances the activity of MAP kinases in the cytosol and is therefore critical regulators of the cellular response to extracellular stimuli. It is a brain-specific phosphatase expressed preferentially in neurons of the striatum, hippocampus and cortex. Alternative splicing of PTPN5 produces a number of isoforms which are either membrane-associated (e.g. STEP61) or shorter cytosolic variants (STEP46). In addition, a recent study links PTPN5 to disease progression of Alzheimer's disease suggesting this phosphatase as an interesting drug target.

The structure of PTPN5 is characterized by an atypical WPD-loop conformation. This catalytically important loop has been shown to switch between a closed and an open conformation upon substrate binding. In order to understand how this particular WPD loop conformation influences substrate binding we generated also an inactive mutant in which the active site cysteine (Cys472) has been mutated to a serine residue and co-crystallized this mutant with the a fragment of the ERK2 activation loop (DHTGFLpTEpYVATR), a specific PTPN5 substrate. The structure of the substrate complex was solved at 1.7 Å resolution and refined to an R-factor of 17.5% (R free 20.5%). However, only the phosphotyrosine was visible in the electron density suggesting that the flanking peptide residues are disordered. Despite the unstructured peptide sequence the well resolved phosphotyrosine residue gave still interesting insight in substrate recognition in the active site as well as in rearrangements in the PTPN5 structure in the presence of substrate.

Materials and Methods

PTPN5A

PDB:2CJZ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_116170
Entry Clone Source:Origine
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m); TEV-cleavable (*) N-terminal his6 tag.
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsmSRVLQA EELHEKALDPFLLQAEFFEIPMNFVDPKE YDIPGLVRKNRYKTILPNPHSRVCLTSPD PDDPLSSYINANYIRGYGGEEKVYIATQG PIVSTVADFWRMVWQEHTPIIVMITNIEE MNEKCTEYWPEEQVAYDGVEITVQKVIHT EDYRLRLISLKSGTEERGLKHYWFTSWPD QKTPDRAPPLLHLVREVEEAAQQEGPHCA PIIVHCSAGIGRTGCFIATSICCQQLRQE GVVDILKTTCQLRQDRGGMIQTCEQYQFV HHVMSLYEKQLSHQS
Vector:pLIC- SGC1

Growth

Medium:
Antibiotics:
Procedure:Grow starter cultures from freshly transformed colonies in 10 ml LB , 0.1 mg/ml amp. This started culture was diluted 1:1000 in fresh media and was grown at 37°C to a density of (OD600) 0.3 and than transferred to 18°C. Expression was induced at an OD 600 of 0.8 using 1 mM IPTG. Cells were harvested after 3h by centrifugation, transferred to 50-ml tubes, and frozen in liquid nitrogen.

Purification

Procedure
Column 1 : Ni affinity, HisTrap (1 ml), in AKTA-express.
The cell extract was loaded on the column at 0.8 ml/minute on an AKTA-express system (GE/Amersham). The column was then washed with 10 volumes of Loading buffer, 10 volumes of wash buffer and then eluted with elution buffer at 0.8 ml/min. The eluted peak of A280 was automatically collected.

Column 2: SEC using AKTA-express

Column 3: HiTrap Q
Protein fraction from desalting column loaded at 1 ml/min, the column was then washed with 10 ml of buffer A and eluted with a 20-minute gradient to 50% buffer B, followed by a step to 100% buffer B.

Protein concentration: Centricons 10 kDa cut off

Extraction

Procedure
The cell pellets (20 gr wet wt) were re-suspended in 50 ml extraction buffer, and lysed by a high pressure cell disrupter. Supernatant was centrifuged for 30 minutes at 20 rpm in a JA 20 rotor
Concentration:
Ligand
MassSpec:LC- ESI -MStof confirmed the correct mass expected for this construct.
Crystallization:Crystals were grown at 4°C in 200nl sitting drops mixing 150 nl of PTPN5A (10 mg/ml in 50mM Hepes pH 7.5, 200mM NaCl ,10mM DTT) with 50 nl of a solution containing 25% PEG 3350, 0.2M LiSO4, 100 mM Bis Tris Propane pH 5.5.
Crystals were also grown in the presence of 1 mM substrate peptide ( DHTGFLpTEpYVATR ) using similar conditions.
NMR Spectroscopy:
Data Collection:Diffraction data were collected using a Rigaku FRE rotating anode equipped with Varimax multilayer mirrors and a Rigaku HTC detector to 2.05 Å.
Data collection was performed on flash frozen crystals at 100K, 15% glycerol was used as cryoprotectant.
Data Processing:

References

Pulido, R., Zuniga, A. & Ulrich, A. (1998). PTP-SL and STEP protein tyrosine phosphatases regulate the activation of the extracellular signal-regulated kinases ERK1 and ERK2 by association through a kinase interaction motif. EMBO J. 17 , 7337â€“7350.

Lombroso PJ, Murdoch G, Lerner M. (1991). Molecular characterization of a protein-tyrosine-phosphatase enriched in striatum. Proc Natl Acad Sci 88:7242-7246.

Valjent E, Pascoli V, Svenningsson P, Paul S, Enslen H, Corvol JC, Stipanovich A, Caboche J, Lombroso PJ, Nairn AC, Greengard P, Herve D, Girault JA. (2005). Regulation of a protein phosphatase cascade allows convergent dopamine and glutamate signals to activate ERK in the striatum. Proc Natl Acad Sci 102:491-496.

Eswaran, J. , von Kries JP, Marsden B, Longman E, Debreczeni JE, Ugochukwu E, Turnbull A, Lee WH, Knapp S, Barr AJ. (2006) Crystal structures and inhibitor identification for PTPN5, PTPRR and PTPN7 - a family of human MAP-kinase specific protein tyrosine phosphatases. Biochem J 395, 483-491.

Snyder, E.M. , Nong Y, Almeida CG, Paul S, Moran T, Choi EY, Nairn AC, Salter MW, Lombroso PJ, Gouras GK, Greengard P. (2005) Regulation of NMDA receptor trafficking by amyloid-beta. Nat Neurosci 8, 1051-1058.