PDZ domain of human protein tyrosine phosphatase, non-receptor type 4

Target ID:

PTPN4A

Aliases:

PTPMEGPTPMEG1PTPN4

Deposition Date:

Friday 29th February 2008

Families:

Linkers

Related Diseases:

SignallingNeurobiology

Structure type:

apo

Download Coordinates:

2VPH

Authors:

A.K.Roos,J.Wang,J.M.Elkins,K.Kavanagh,A.C.W.Pike,P.Filippakopoulos,C.H.Arrowsmith,J.Weigelt,A.Edwards,F.vonDelft,C.Bountra,S.Knapp

Site:

Oxford

2VPH

tabs

Structure Details

PTPN4 belongs together with PTPN3 (PTPH1) to the NT5 (non-transmembrane PTP) subfamily of PTPs. PTPN4 contains an N-terminal noncatalytic FERM domain (Band 4.1/ezrin/radixin/moesin homology) followed by a PDZ domain and a C-terminal catalytic domain. The precise function of PTPN4 is not yet known but it has been suggested that this phosphatase plays a role in regulation of the membrane cytoskeleton and signaling downstream of glutamate receptors (GluR). In addition, a role in spermatogenesis and inhibiting cell proliferation has been proposed.

PTPN4 is mainly expressed in the brain with particular high expression levels in the thalamus and cerebellar Purkinje cell layer, moderate expression in olfactory bulb, cerebral cortex, and hippocampus and weak expression in all other brain areas. Furthermore high expression levels have been reported for platelets, the skeletal muscle as well as for testis. In addition the PTPN4 PDZ domain has been shown to interact with the C-terminal PDZ target sequence of the receptors GluRδ2 and a similar interaction has been suggested for GluRε 1. The tyrosine kinases Src and Fyn have been shown to be dephosphorylated by PTPN4. To understand the molecular mechanisms of substrate recruitment we present here the structure of the PDZ domain of PTPN4.

Materials and Methods

PTPN4

PDB:2VPH

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:IMAGE:3853914
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal, TEV cleavable hexahistidine tag
Host:BL21(DE3)-R3-pRARE2 (previously known as Rosetta)

Construct

Prelude:
Sequence:smDNLVLIRMKPDENGRFGFNVKGGYDQKMPVIVSRVAPGTPADLCVPRLNEGDQVVLINGRDIAEHTHDQVVLFIKASCERHSGELMLLVRPNAVESTV
Vector: pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Transformation: The construct DNA was transformed into homemade chemically competent cells of the expression strain by a standard heat shock procedure.Glycerol stock preparation: A number of colonies from the transformation were used to innoculate 1 ml of LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day glycerol stocks were prepared from this overnight culture.Expression: A glycerol stock was used to innoculate 50 ml of TB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day this starter culture was used to innoculate 2x 1L of TB media (18 ml starter culture into each) containing 50 µg/ml kanamycin. After 4.5 hours the temperature was reduced to 20°C. After a further 1.5 hours the cells were induced by the addition of 1.0 mM IPTG. The expression was continued overnight.Cell harvest: Cells were spun at 6238x g for 10 mins at 4°C. The cells were resuspended in 50 ml of Lysis Buffer with the addition of 0.2 mM PMSF. The resuspended cell pellet was placed in a -80°C freezer. Lysis Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 5 mM Imidazole pH 7.4, 0.5 mM TCEP.Cell Lysis: The resuspended cell pellet was lysed by sonication. PEI (polyethyleneimine) was added to a final concentration of 0.25 % and the cell debris and precipitated DNA were spun down (18000 rpm, JA18 rotor, 90 min).

Purification

Procedure
Column 1: Ni-NTA (2 ml volume in a gravity-flow column).Buffers: Binding Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 5 mM Imidazole pH 7.4, 0.5 mM TCEP; Wash Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 25 mM Imidazole pH 7.4, 0.5 mM TCEP; Elution Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.4, 0.5 mM TCEP.Procedure: The clarified cell extract was passed through the column. The column was then washed with Binding Buffer (25 ml) and Wash Buffer (25 ml). The protein was eluted with 10 ml of Elution Buffer.TEV protease digestion: The PTPN4A protein in the elute fraction was exchanged into storage buffer by concentration and dilution, and digested over 48 hours with TEV protease at 4°C.Storage Buffer: 50 mM Hepes pH 7.4, 150 mM NaCl, 0.5 mM TCEPRebinding of impurities to Ni-NTA: The protein was passed through a gravity-flow column containing Ni-NTA resin (2 ml resin volume, pre-equilibrated into Storage Buffer).

Extraction

Procedure
Transformation: The construct DNA was transformed into homemade chemically competent cells of the expression strain by a standard heat shock procedure.Glycerol stock preparation: A number of colonies from the transformation were used to innoculate 1 ml of LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day glycerol stocks were prepared from this overnight culture.Expression: A glycerol stock was used to innoculate 50 ml of TB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day this starter culture was used to innoculate 2x 1L of TB media (18 ml starter culture into each) containing 50 µg/ml kanamycin. After 4.5 hours the temperature was reduced to 20°C. After a further 1.5 hours the cells were induced by the addition of 1.0 mM IPTG. The expression was continued overnight.Cell harvest: Cells were spun at 6238x g for 10 mins at 4°C. The cells were resuspended in 50 ml of Lysis Buffer with the addition of 0.2 mM PMSF. The resuspended cell pellet was placed in a -80°C freezer. Lysis Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 5 mM Imidazole pH 7.4, 0.5 mM TCEP.Cell Lysis: The resuspended cell pellet was lysed by sonication. PEI (polyethyleneimine) was added to a final concentration of 0.25 % and the cell debris and precipitated DNA were spun down (18000 rpm, JA18 rotor, 90 min).
Concentration:The TEV protease cleaved PTPN4A PDZ domain was concentrated to 7.9 mg/ml (measured by 280nm absorbance), distributed into aliquots and frozen at -80°C.
Ligand
MassSpec: Measured: 11026; Expected: 11027.
Crystallization:Crystals grew from a 1:1 ratio of protein to precipitant solution (20 % PEG 3350, 0.2 M (NH4)2H(citrate)), using the vapour diffusion method.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected by equilibration into precipitant solution containing 20% PEG400, and then flash frozen in liquid nitrogen. Data was collected at the Swiss Light Source, beamline X10.
Data Processing:

References

Heneberg, P., and Draber, P. (2002). Nonreceptor protein tyrosine and lipid phosphatases in type I fc(epsilon) receptor-mediated activation of mast cells and basophils. Int Arch Allergy Immunol 128, 253-263.

Moller, N. P., Moller, K. B., Lammers, R., Kharitonenkov, A., Sures, I., and Ullrich, A. (1994). Src kinase associates with a member of a distinct subfamily of protein-tyrosine phosphatases containing an ezrin-like domain. Proc Natl Acad Sci U S A 91, 7477-7481.

Opas, E. E., Rutledge, S. J., Golub, E., Stern, A., Zimolo, Z., Rodan, G. A., and Schmidt, A. (1997). Alendronate inhibition of protein-tyrosine-phosphatase-meg1. Biochem Pharmacol 54, 721-727.