Human cyclophilin G

Target ID:

PPI64

Aliases:

CARS-CypCYPMGC133241SRCyp

Deposition Date:

Wednesday 3rd May 2006

Families:

Isomerases

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2GW2

Authors:

G.BERNSTEIN,W.TEMPEL,T.DAVIS,E.M.NEWMAN,P.J.FINERTY JR.,F.MACKENZIE,J.WEIGELT,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,S.DHE-PAGANON,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2GW2

tabs

Structure Details

PPIG (cyclophilin G, SRCyp, CARS-Cyp) and NKTR (p104, MGC90527) are cyclophilin-containing proteins with long carboxy terminal regions that, like many splicing factors, are rich in serines, arginines and lysines. Although their precise biological roles are unknown, both proteins colocalize with spliceosome components at nuclear speckles (1). The RS (arginine, serine) region of PPIG mediates hetero-multimerization with another RS-containing, splicing factor, pinin (2); RS-mediated hetero-multimerization being a common feature of the spliceosome. And, the phosphorylation pattern and localization of PPIG appears to be regulated along with the cell cycle (3), consistent with direction interaction observed between PPIG and the cell-cycle kinase, CDC2 (4).

The cyclophilin domain of PPIG catalyzes prolyl isomerization of tetrapeptides with a preference for large hydrophobic, non-aromatic residues at the P1 position, but at a maximal rate about 1000 fold slower than rates seen for cyclophilin-A (5). Of thirteen active site residues that are conserved among cyclophilins, H133 and R115 of PPIG are not conserved and may account for lower observed catalytic rates (6). We have solved the high resolution structure of PPIG (2GW2), revealing the configuration of the catalytic residues H133 and R115 as well as the variant loop (158-166), which is implicated in substrate selectivity. Additional experiments are needed to identify the molecular function of the cyclophilin domain and potential substrates. PPIGâ€™s poor affinity for cyclosporin (5) further suggests that there is room for development of PPIG-specific ligands that could be used to treat associated diseases.

Materials and Methods

PPIG

PDB:2GW2

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:66933018
Entry Clone Source:MGC
SGC Clone Accession:ppi64.001.179:G10; plate SDC022 G10
Tag:
Host:BL21 (DE3) Codon Plus RIL

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsMGIKVQRPRCFFDIAINNQPAGRVVFELFSDVCPKTCENFRCLCTGEKGTGKSTQKPLHYKSCLFHRVVKDFMVQGGDFSEGNGRGGESIYGGFFEDESFAVKHNAAFLLSMANRGKDTNGSQFFITTKPTPHLDGHHVVFGQVISGQEVVREIENQKTDAASKPFAEVRILSCGELIP
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:PPIG mutant K125A/E126A was expressed in E.coli BL21 (DE3) codon plus RIL in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin at 37oC to an OD600 of 1.0. Cells were then induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 15oC.

Purification

Procedure
The crude extract was cleared by centrifugation. The clarified lysate was loaded onto 3 ml Ni-NTA column (Qiagen). The column was washed with 5 CV of 50 mM HEPES buffer, pH 7.5, containing 500 mM NaCl, 5% glycerol and 25 mM imidazole, and the protein was eluted with elution buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 250 mM imidazole, 5 % glycerol). The protein was loaded on Superdex200 column (26x60) (Amersham Biosciences), equilibrated with 50 mM HEPES buffer, pH 7.5, and 500 mM NaCl, 5% glycerol, 1 mM DTT, at flow rate 3.5 ml/min. Combined fractions containing ppi64.001.179 mutant K125A/E126A were concentrated to 15.5 mg/ml. The purification yield was 55 mg of purified protein per 1 L of culture.

Extraction

Procedure
Cells were harvested by centrifugation at 8,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80°C. For the purification the cell paste was thawed and resuspended in lysis buffer (50 mM HEPES, pH 7.5, 0.5 M NaCl, 5 mM imidazol, 2 mM ß-mercaptoethanol, 5% glycerol) with protease inhibitor (1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:15 mg/mL
Ligand
MassSpec:xpected MW is 21808.6 Da, measured MW is 21677.9 Da
Crystallization:Purified ppiG K125A/E126A was and crystallized using the sitting drop vapor diffusion method at 18 °C by mixing 0.2 µl of the protein solution with 0.2 µl of the reservoir solution containing 2M ammonium sulfate, 0.2M Sodium chloride, 0.1M Hepes, pH 7.5.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Bourquin, J. P., Stagljar, I., Meier, P., Moosman, P., Silke, J., Baechi, T., Georgiev, O., and Schaffner, W. (1997) Nucleic Acids Research 25, 2055-2061
Lin, C. L., Leu, S., Lu, M. C., and Ouyang, P. (2004) Biochemical and Biophysical Research Communications 321, 638-647
Dubourg, B., Kamphausen, T., Weiwad, M., Jahreis, G., Feunteun, J., Fischer, G., and Modjtahedi, N. (2004) J.Biol.Chem. 279, 22322-22330
Nestel, F. P., Colwill, K., Harper, S., Pawson, T., and Anderson, S. K. (1996) Gene 180, 151-155
Cavarec, L., Kamphausen, T., Dubourg, B., Callebaut, I., Lemeunier, F., Metivier, D., Feunteun, J., Fischer, G., and Modjtahedi, N. (2002) J.Biol.Chem. 277, 41171-41182
Liu, J., Chen, C. M., and Walsh, C. T. (1991) Biochemistry 30, 2306-2310