Human natural killer-tumor recognition sequence; cyclophilin like protein

Target ID:

PPI63

Aliases:

DKFZp686F1754DKFZp686G0426DKFZp686J06106DKFZp686N24126MGC90527p104

Deposition Date:

Wednesday 21st June 2006

Families:

Isomerases

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2HE9

Authors:

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

Site:

Toronto

ICM Datapack:

ICM Datapack

2HE9

tabs

Structure Details

NKTR (p104, MGC90527) and PPIG (cyclophilin G, SRCyp, CARS-Cyp) are human cyclophilin-containing proteins with unique long carboxy-terminal regions rich in serines, arginines and lysines. Their biological roles are unknown, being implicated in both nuclear activity and antigen recognition. Although highly expressed in cardiac, digestive, and muscular tissues, NKTR was initially isolated from the surface of human natural killer cells 1-3 and was shown to be important for natural killer-like activity and recognition of tumors by T cells 2,4. Fly, worm, and parasitic orthologs show conserved carboxyl-terminal sequences, suggesting involvement in the splicing and processing of mRNA5-7.

The cyclophilin domain of NKTR catalyzes prolyl isomerization of tetrapeptides with a preference for large hydrophobic, non-aromatic residues at the P1 position, but at a maximal rate about 100 fold slower than rates seen for cyclophilin-A5,8. Of thirteen active site residues that are conserved among cyclophilins, H132 and R114 of NKTR (and its paralog) are divergent and may account for lower observed catalytic rates when compared with cyclophilin A. We have solved the high resolution structure of NKTR (2HE9) [scene 1], revealing the configuration of the active site residues [scene 2] as well as the loop that is unique to these cyclophilins (47-57) [scene 3]. Additional experiments are needed to identify the molecular function of the cyclophilin domain and potential substrates. NKTRâ€™s poor affinity for cyclosporin 8 but well-define active site pocket [scene 4] further suggests that there is room for development of NKTR-specific ligands that could be used to treat associated diseases.

Materials and Methods

NKTR: human natural killer-tumor recognition sequence

PDB:2HE9

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi:61676206
Entry Clone Source:Genscript
SGC Clone Accession:ppi63.0007.0179:B4; plate SDC042 B4
Tag:
Host:E.coli BL21 (DE3) Gold

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsPQCHFDIEINREPVGRIMFQLFSDICPKTCKNFLCLCSGEKGLGKTTGKKLCYKGSTFHRVVKNFMIQGGDFSEGNGKGGESIYGGYFKDENFILKHDRAFLLSMANRGKHTNGSQFFITTKPAPHLDGVHVVFGLVISGFEVIEQIENLKTDAASRPYADVRVIDCGVLATK
Vector:p28a-LIC

Growth

Medium:TB
Antibiotics:
Procedure:The protein was expressed in E. coli BL21 (DE3) grown in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin at 37°C to an OD600 of 7.5. Cells were then induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 0.1 mM, and incubated overnight at 15°C. The culture was centrifuged and the cell pellets were collected and stored at -80°C.Extraction buffer, extraction method : Lysis buffer: 50 mM Tris pH 8.0, 500 mM NaCl 2mM CaCl2. Frozen cell pellets contained in bags (Beckman 369256) obtained from 2L liters of culture are thawed by soaking in warm water for 5 minutes. Each cell pellet is resuspended in 20 mL lysis buffer, 1mM phenylmethanesulfonyl fluoride (Sigma P7626), and 1mL Sigma general protease inhibitor (Sigma P2714-1BTL, resuspended according to manufacturer's instructions) and then homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds per pellet. Cell lysis is accomplished by sonication (Virtis408912, Virsonic) on ice: the sonication protocol is 10 sec pulse at half-maximal frequency (5.0), 10 second rest, for 6 minutes total sonication time per pellet. Lysed cells are placed into centrifuge tubes (363647, Beckman Coulter) and centrifuged in a JA25.50 rotor in an Avanti J-20 XPI centrifuge (Beckman Coulter) for 20 minutes at 69,673 x g. The supernatant is decanted into a beaker, and the insoluble pellet discarded

Purification

Procedure
Column 1: 3 mL Ni-NTA column (Qiagen)
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 (lysis buffer + 10 mM Imidazole (VWR EM-5720) pH 8) at approximately 3 mL/min. A 4 microL 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 (lysis buffer + 250 mM imidazole and 10% glycerol (EMD GX0185-5)) at 1mL/min flow rate. A 10 µL sample of the eluate is saved for SDS-PAGE analysis. 10 µL of each eluate is saved for measurement of protein concentration using Bradford reagent (BioRad 500-0202).

Column 2: XK 16x65 column (GE Healthcare)
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 2mL 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.

Concentration: Purified proteins are concentrated using either 4 mL or 15 mL concentrators with an appropriate molecular weight cut-off (Amicon Ultra-15 10,000 MWCO, UFC901024 or 5,000 MWCO, UFC900524, as appropriate, Millipore) to a final concentration of 20 mg/mL for crystallographic screening or other biophysical studies

Extraction

Procedure

Concentration:
Ligand
MassSpec:
Crystallization:Crystallization trials were set up using the hanging drop vapor diffusion method. The protein drop was equilibrated against a reservoir solution (1:1 volume ratio) for the final concentration at 21% Peg 3350, 0.25M potassium sulfate; Protein solution: 50 mM Tris pH 7.5, 100 mM NaCl, 1 mM DTT, 15 mg/mL protein, VAPOR DIFFUSION, HANGING DROP, temperature 298.0K.
NMR Spectroscopy:
Data Collection:Resolution: 2.0Å, X-ray source: The Industrial Macromolecular Crystallography Association 17-ID beamline at the Advanced Photon Source.
Data Processing:

References

Frey,J.L. et al. Mechanism of target cell recognition by natural killer cells: characterization of a novel triggering molecule restricted to CD3- large granular lymphocytes. J. Exp. Med. 174, 1527-1536 (1991).
Anderson,S.K. et al. A cyclophilin-related protein involved in the function of natural killer cells. Proc. Natl. Acad. Sci. U. S. A 90, 542-546 (1993).
Alkhatib,G., Murata,K. & Roder,J.C. Cellular distribution of a natural killer cell tumour recognition-related surface antigen in purified human lymphocytes. Immunology 92, 173-179 (1997).
Chambers,C.A. et al. Expression of the NK-TR gene is required for NK-like activity in human T cells. J. Immunol. 152, 2669-2674 (1994).
Cavarec,L. et al. Identification and characterization of Moca-cyp - A Drosophilia melanogaster nuclear cyclophilin. J. Biol. Chem. 277, 41171-41182 (2002).
Ma,D., Nelson,L.S., LeCoz,K., Poole,C. & Carlow,C.K. A novel cyclophilin from parasitic and free-living nematodes with a unique substrate- and drug-binding domain. J. Biol. Chem. 277, 14925-14932 (2002).
Page,A.P., Kumar,S. & Carlow,C.K. Parasite cyclophilins and antiparasite activity of cyclosporin A. Parasitol. Today 11, 385-388 (1995).
Rinfret,A., Collins,C., Menard,R. & Anderson,S.K. The N-terminal cyclophilin-homologous domain of a 150-kilodalton tumor recognition molecule exhibits both peptidylprolyl cis-trans-isomerase and chaperone activities. Biochemistry 33, 1668-1673 (1994).