Human peptidylprolyl isomerase C

Target ID:

PPI66

Aliases:

CYPCMGC3673

Deposition Date:

Wednesday 26th October 2005

Families:

Isomerases

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2ESL

Authors:

J.R.WALKER,T.DAVIS,E.M.NEWMAN,P.J.FINERTY JR.,F.MACKENZIE,J.WEIGELT,M.SUNDSTROM,C.ARROWSMITH,A.EDWARDS,A.BOCHKAREV,S.DHE-PAGANON,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

2ESL

tabs

Structure Details

PPIC is one of two human cyclophilins with a well defined signal sequence.
It has been shown to be secreted from a variety of cells, including cultured leptomeningeal cells1 and adipocytes2. But unlike the nearest paralog, PPIB, a role in endoplasmic reticulum biology, including foldase activity of nascent peptides or chaperone activity of unfolded proteins, has not been shown for PPIC.
Although competent to bind cyclosporin and catalyze petidyl-prolyl isomerization3, the biological roles of PPIC are unknown.

An important hint at function is the strong interaction between PPIC and CyCap [Cyclophilin C associated protein, a.k.a. ppicap (Petidyl Prolyl Isomerase C Associated Protein), Lgals3bp (lectin, galactoside-binding, soluble, 3 binding protein), and Mac-2bp]3,4.
Heavily glycosylated, CyCap is a secreted galectin that mediates cell aggregation 5 and inflammation3,6.
The concentration of CyCap in body fluids has prognostic value, particularly for cancer and immunodeficiencies7,8. But its role in biology and pathophysiology is far from being understood.

We have determined the high resolution structure of human PPIC in complex with Cyclosporin A.
Numerous residues that ring the active site are unique to PPIC and may mediate specific substrate interactions.
As active site residues are conserved between the major forms of PPIâ€™s, the conformation and configuration (all trans) of cyclosporin A bound to PPIC is essentially the same as when bound to PPIA9-11.
Of note, the majority of residues that differ from the murine ortholog of PPIC are clustered to a small patch of surface adjacent to the active site.
This area may be involved in species-specific protein interactions.

Materials and Methods

PPIC

PDB:2ESL

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:4505991
Entry Clone Source:MGC
SGC Clone Accession:ppi66.025.212:D6; plate SDC023 D6
Tag:N-terminal His-tag with integrated thrombin-cleavage site MGSSHHHHHHSSGLVPRGS.
Host:E.coliBL21 (DE3)

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsGAEGFRKRGPSVTAKVFFDVRIGDKDVGRIVIGLFGKVVPKTVENFVALATGEKGYGYKGSKFHRVIKDFMIQGGDITTGDGTGGVSIYGETFPDENFKLKHYGIGWVSMANAGPDTNGSQFFITLTKPTWLDGKHVVFGKVIDGMTVVHSIELQATDGHDRPLTNCSIINSGKIDVKTPFVVEIADW
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:PPIC was expressed in E. coli BL21 (DE3) grown in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin at 37ºC to an OD600 of 7.5. Cells were then induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 0.05 mM, and incubated overnight at 15ºC. The culture was centrifuged and the cell pellets were collected and stored at -80ºC.

Purification

Procedure
IMAC purification: 4 microL of clarified supernatant is reserved for later analysis by SDS-PAGE. The rest of the clarified supernatant is then diluted 1:2 in lysis buffer, and loaded at approximately 1mL/min by gravity onto 5 mL of Ni-NTA resin (Qiagen 30450). 5 column volumes of lysis buffer are used to wash the column at approximately 3 mL/min, followed by 5 column volumes of low imidazole buffer at approximately 3 mL/min. A 4 µL sample of the low imidazole wash is saved for later analysis by SDS-PAGE. Samples are eluted from the Ni-NTA resin by exposure to 10 mL elution buffer at 1mL/min flow rate. A 10 microL sample of the eluate is saved for SDS-PAGE analysis. 10 microL of each eluate is saved for measurement of protein concentration using Bradford reagent (BioRad 500-0202).

Size exclusion chromatography: An XK 16x65 column (part numbers 18-1031-47 and 18-6488-01, GE Healthcare) packed with HighLoad Superdex 200 resin (10-1043-04, GE Healthcare) is pre-equilibrated with gel filtration buffer for 1.5 column volumes using an AKTAxpress (18-6645-05, GE Healthcare) at a flow rate of 3 mL/min. 5 mL of sample is loaded onto the column at 1.5 mL/min, and 2 mL fractions are collected into 96-well plates (VWR 40002-012) using peak fractionation protocols with the following parameters: (Slope; min. peak width 0.833 min; level 0.000 mAU; peak start slope 10.000 AU/min; peak end slope 20.000 AU/min). Peak fractions are analyzed for purity using SDS-PAGE or visual analysis of the chromatogram and pooled.

Extraction

Procedure
The cell pellet was resuspended in lysis buffer + protease inhibitor (0.1µM phenylmethyl sulfonyl fluoride, PMSF) and lysed using Microfluidizer. The lysate was cleared by centrifugation.
Concentration:15 mg/mL
Ligand
Cyclosporin AMassSpec:
Crystallization:Purified PPIC was crystallized using the hanging drop vapor diffusion method. Crystals grew when the protein (15 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 25% PEG MME 550, 0.1 M zinc acetate, 0.1 M MES at pH 6.5 in 293K temperature.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

1. Ohe,Y., Ishikawa,K., Itoh,Z. & Tatemoto,K. Cultured leptomeningeal cells secrete cerebrospinal fluid proteins. J. Neurochem. 67, 964-971 (1996).

2. Wang,P. et al. Profiling of the secreted proteins during 3T3-L1 adipocyte differentiation leads to the identification of novel adipokines. Cellular and Molecular Life Sciences 61, 2405-2417 (2004).

3. Friedman,J. & Weissman,I. Two cytoplasmic candidates for immunophilin action are revealed by affinity for a new cyclophilin: one in the presence and one in the absence of CsA. Cell 66, 799-806 (1991).

4. Friedman,J., Trahey,M. & Weissman,I. Cloning and characterization of cyclophilin C-associated protein: a candidate natural cellular ligand for cyclophilin C. Proc. Natl. Acad. Sci. U. S. A 90, 6815-6819 (1993).

5. Tinari,N. et al. Glycoprotein 90K/MAC-2BP interacts with galectin-1 and mediates galectin-1-induced cell aggregation. Int. J. Cancer 91, 167-172 (2001).

6. Trahey,M. & Weissman,I.L. Cyclophilin C-associated protein: a normal secreted glycoprotein that down-modulates endotoxin and proinflammatory responses in vivo. Proc. Natl. Acad. Sci. U. S. A 96, 3006-3011 (1999).

7. Grassadonia,A. et al. 90K (Mac-2 BP) and galectins in tumor progression and metastasis. Glycoconj. J. 19, 551-556 (2004).

8. D'Ostilio,N. et al. Prognostic value of a novel interferon-inducible 90K tumor antigen. Ann. N. Y. Acad. Sci. 784, 288-293 (1996).

9. Theriault,Y. et al. Solution structure of the cyclosporin A/cyclophilin complex by NMR. Nature 361, 88-91 (1993).

10. Pflugl,G. et al. X-ray structure of a decameric cyclophilin-cyclosporin crystal complex. Nature 361, 91-94 (1993).

11. Ke,H., Zhao,Y., Luo,F., Weissman,I. & Friedman,J. Crystal structure of murine cyclophilin C complexed with immunosuppressive drug cyclosporin A. Proc. Natl. Acad. Sci. U. S. A 90, 11850-11854 (1993).