Human Protein Phosphatase 2A Regulatory Subunit PPP2R4

Target ID:

PPP2R4A

Aliases:

MGC2184PP2APPP2R4PR53PTPA

Deposition Date:

Friday 24th February 2006

Families:

Phosphatases

Related Diseases:

CancerNeurobiologySignalling

Structure type:

apo

Download Coordinates:

2G62

Authors:

A.MAGNUSDOTTIR,P.STENMARK,C.ARROWSMITH,H.BERGLUND,R.COLLINS,A.EDWARDS,M.EHN,S.FLODIN,A.FLORES,S.GRASLUND,M.HAMMARSTROM,B.M.HALLBERG,M.HOGBOM,L.HOLMBERG SCHIAVONE,T.KOTENYOVA,P.NILSSON-EHLE,T.NYMAN,D.OGG,C.PERSSON,J.SAGEMARK,M.SUNDSTROM,A.G.THORSELL,S.VAN DEN BERG,K.WALLDEN,J.WEIGELT,P.NORDLUND

Site:

Stockholm

ICM Datapack:

ICM Datapack

2G62

tabs

Structure Details

PP2A is a heterotrimeric Ser/Thr phosphatase that is involved in regulating a plethora of signaling pathways in the cell.
The core enzyme is the heterodimer of the 36 kDa catalytic subunit and the 65 kDa scaffolding subunit that bind one regulatory subunit belonging to one of three non related families.
PP2A is regulated by e.g. post-translational modifications, holoenzyme composition and interaction with cellular and viral proteins (for reviews, see Janssens and Goris 2001; Janssens et al. 2005).
One such interacting protein is PTPA that is highly conserved from humans to yeast and has been shown essential for a fully functional PP2A in yeast (Fellner et al. 2003).
Its biological function is still unclear but recently it was suggested to work as an ATP dependant cis-trans peptidyl prolyl isomerase.
It has also been shown to bind the conserved Tap42 protein (Zheng and Jiang 2005) that is critical for mediating the signal through the TOR (target of rapamycin ) pathway, a nutrient responsive signaling pathway.
Tap42 also interacts with type 2A phosphatases (Inoki et al. 2005).
Zeng et al. have proposed that PTPA is a novel regulatory subunit of type 2A phosphatases in a heterotrimer of Tap42, PTPA and a catalytic subunit of a type 2 phosphatase.
To provide the molecular basis for proper assignment of PTPA´s function, we have solved the crystal structure of human PTPA to 1.6Å.
It is a novel alpha-helical fold of two domains; a small domain made of 4 short helices and a large domain made of 9 long helices.
We have identified a highly conserved cleft as a likely site for peptide binding.

Materials and Methods

PTPA (PPP2R4)

PDB:2G62

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC011605
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*sr. (note that the tag has a point mutation in the last position, M to R))
Host:

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsrNFIIPKKEIHTVPDMGKWKRSQAYADYIGFILTLNEGVKGKKLTFEYRVSEAIEKLVALLNTLDRWIDETPPVDQPSRFGNKAYRTWYAKLDEEAENLVATVVPTHLAAAVPEVAVYLKESVGNSTRIDYGTGHEAAFAAFLCCLCKIGVLRVDDQIAIVFKVFNRYLEVMRKLQKTYRMEPAGSQGVWGLDDFQFLPFIWGSSQLIDHPYLEPRHFVDEKAVNENHKDYMFLECILFITEMKTGPFAEHSNQLWNISAVPSWSKVNQGLIRMYKAECLEKFPVIQHFKFGSLLPIHPVTSG
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Residues 22-323 of hPTPA were expressed in BL21 (DE3) from the pET based vector pNIC-Bsa4 as N-terminal 6xhis fusion. A Tev protease site is present between the his tag and the protein. Colonies were grown in 20 mL of TB supplemented with 8 g/L glycerol, 50 µg/mL Kanamycin at 37°C over night. The following morning 2×10 ml of the over night cultures inoculated 2x750 mL Terrific Broth with 8 g/L glycerol in TunAir flasks. Cells were grown at 37°C until OD600 of 1.2 Â 2 then down-tempering to 18 °C for 1h. Expression of target protein was induced by addition of 0.5 mM IPTG at OD600 of 2- 2.5, and was allowed to continue over night.

Purification

Procedure
The protein was purified using a 1mL Hitrap chealating HP column and a Superdex 75 gelfiltration column (Amersham, UK). The 98+% pure protein (as estimated from SDS page) was concentrated to 35.4mg/mL in 20 mM HEPES, 300 mM NaCl, 10% glycerol, 0.5 mM TCEP. Same methods were used to purify the selenomethionine labeled protein.

Extraction

Procedure
Cells were harvested by centrifugation and pellets were in 50mM Sodium-Fosfate pH 7.5, 500mM NaCl, 10% glycerol (IMAC Bind/Wash1 Buffer) supplemented with one tablet Complete EDTA-free protease inhibitor tablet per cell pellet and freezed at -80 ºC. The cells were briefly thawed in warm water and 1000 U of Benzonase was added. Cells were disrupted by High Pressure Homogenization at 10 000 PSI and samples were centrifuged for 20 minutes at 40 000×g. The soluble fraction was decanted and filtered through 0.45µm prior to loading onto the ÄKTAxpress for furher purification.
Concentration:35 mg/ml
Ligand
MassSpec:The mass spectrometry analysis showed a difference between expected and obtained mass that could be explained by a mutation of a methionine to an arginine. In addition, after selenomethionine labeling the mass increase corresponded to seven and not eight methionines being present in the protein. The construct was sequenced and the position of the methionine to arginine mutation was found at the second methionine in the N-terminal tag.
Crystallization:
NMR Spectroscopy:
Data Collection:For data collection, the crystals were flash frozen in liquid nitrogen after adding cryo solution of 20%glycerol, 2.1 M (NH4)2SO4, 0.3 M NaCl and 0.1 M sodium cacodylate (pH of drop) directly to the drop. The native x-ray data was collected at station PXI at SLS. The selenomethionine data was collected at Bessy-14.1 on a marccd 225 detector.
Data Processing:All data was processed using the programs xds and xscale. The structure of hPTPA was solved by MAD phasing using Solve that was able to identify 5 of the 7 selenium sites present in the asymmetric unit. ARP/wARP was used to build the initial model and to place solvent molecules. Refinement using refmac and manual model building using coot gave the final model consiting of residues 22-323 of hPTPA, 7 residues of the Tev-fusion, 4 sulfates and one glycerol molecules. The N-terminus was built in two conformations. The structure was refined to an R and Rfree of 15.3 % and 18 %, respectively. All residues of the model are within the most favoured or additionally allowed regions of the ramachandran plot.

References

Fellner, T., Lackner, D. H., Hombauer, H., Piribauer, P., Mudrak, I., Zaragoza, K., Juno, C., and Ogris, E. (2003). "A novel and essential mechanism determining specificity and activity of protein phosphatase 2A (PP2A) in vivo." Genes & Development 17(17): 2138-50.

Inoki, K., Ouyang, H., Li, Y., and Guan, K. L. (2005). "Signaling by target of rapamycin proteins in cell growth control." Microbiology and Molecular Biology Reviews 69(1): 79-100.

Janssens, V., and Goris, J. (2001). "Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling." Biochemical Journal 353: 417-39.

Janssens, V., Goris, J., and Van Hoof, C. (2005). "PP2A: the expected tumor suppressor." Current Opinion in Genetics & Development 15(1): 34-41.

Jordens, J., Janssens, V., Longin, S., Stevens, I., Martens, E., Bultynck, G., Engelborghs, Y., Lescrinier, E., Waelkens, E., Goris, J., and Van Hoof, C. (2006). "The Protein Phosphatase 2A Phosphatase Activator Is a Novel Peptidyl-Prolyl cis/trans-Isomerase." J. Biol. Chem. 281(10): 6349-57.

Laskowski, R. A., Watson, J. D., and Thornton, J. M. (2005). "Protein function prediction using local 3D templates." Journal of Molecular Biology 351(3): 614-26.

Zheng, Y., and Jiang, Y. (2005). "The yeast phosphotyrosyl phosphatase activator is part of the tap42-phosphatase complexes." Molecular Biology of the Cell 16(4): 2119-27.