Human Proviral Integration Site for MuLV

Target ID:

PIM2A

Deposition Date:

Friday 30th June 2006

Families:

Protein Kinase

Related Diseases:

SignallingCancer

Structure type:

apo

Download Coordinates:

2IWI

Authors:

S.RUSSO,J.E.DEBRECZENI,A.AMOS,A.BULLOCK,O.FEDOROV,F.NIESEN,F.SOBOTT,A.TURNBULL,A.C.W.PIKE,E.UGOCHUKWU,E.PAPAGRIGORIOU,G.BUNKOCZI,F.GORREC,A.EDWARDS,C.ARROWSMITH,J.WEIGELT,M.SUNDSTROM,F.VON DELFT,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2IWI

tabs

Structure Details

The Pim kinase family includes three constitutively active serine/threonine kinases (Pim-1, -2, and -3) that function as important mediators of cytokine signaling pathways in hematopoietic cells. Pim-2 plays an important role in tumour progression and is overexpressed in human chronic lymphocytic leukemia and non-Hodgkin lymphomas. Pim-2 prosurvival signaling can protect cancer cells from apotosis and confers resistance to rapamycin[1]. Substrates include the pro-apoptotic protein BAD, which is phosphorylated on serine 112, thus preventing BAD-Bcl-x L interaction and reversing Bad-induced cell death[2]. Pim-2 also phosphorylates SOCS-1 which stabilizes the protein enabling negative feedback of the Jak/STAT pathway[3].

The Pim kinases are distinguished by a unique hinge-region sequence ERPXPX which removes the +3 hydrogen bond donor and provides potential for the design of specific inhibitors. Pim-2 was crystallized with the ruthenium half-sandwich complex (R)- 5 which is a kinetically-inert metal complex based on the inhibitor staurosporine[4]. Such metal complexes extend the small molecule chemical space previously explored by purely organic scaffolds and produces highly potent inhibitors of the Pim kinases.

Materials and Methods

PIM2: Human Proviral Integration Site for MuLV

PDB:2IWI

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:42821111
Entry Clone Source:Ben Turk (pEBG-Pim2)
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*sm (* TEV protease cleavage)
Host:E.coli BL21(DE3)R3

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsmLTKPLQ GPPAPPGTPTPPPGGKDREAFEAEYRLGP LLGKGGFGTVFAGHRLTDRLQVAIKVIPR NRVLGWSPLSDSVTCPLEVALLWKVGAGG GHPGVIRLLDWFETQEGFMLVLERPLPAQ DLFDYITEKGPLGEGPSRCFFGQVVAAIQ HCHSRGVVHRDIKDENILIDLRRGCAKLI DFGSGALLHDEPYTDFDGTRVYSPPEWIS RHQYHALPATVWSLGILLYDMVCGDIPFE RDQEILEAELHFPAHVSPDCCALIRRCLA PKPSSRPSLEEILLDPWMQTPAEDVPLNP SKGGPAPLAWSLLP
Vector:pLIC

Growth

Medium:
Antibiotics:
Procedure:10 ml from a 50 ml overnight culture in LB, 100µg/ml ampicillin was used to inoculate 1 litre of LB medium containing 100µg/ml ampicillin. Cultures were grown at 37°C until they reached an OD600 of 0.4 and then cooled to 18°C. Expression was induced for 4 hours using 0.15 mM IPTG. The cells were harvested by centrifugation, transferred to 50 ml tubes, resuspended in 30 ml binding buffer, and frozen.

Purification

Procedure
Column 1 : Ion exchange - Nucleic acid removal . DEAE cellulose (DE52, Whatmann), 10 g of resin in 2.5 x 20 cm column. The resin was hydrated in 2.5M NaCl, then washed with 50 ml binding buffer prior to loading the sample.
Buffers: Binding buffer
Procedure: Supernatant was applied at gravity flow, followed by a wash with 50 ml binding buffer. The column flow-through was collected.

Column 2 : Ni-affinity Ni-sepharose, 5 ml of 50% slurry in 1.5 x 10 cm column, equilibrated with binding buffer.
Buffers : Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol; Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 20 mM Imidazole, 5% glycerol; Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 50-250 mM Imidazole, 5% Glycerol.
Procedure: The flowthrough from column 1 was loaded by gravity flow on the Ni-sepharose column. The column was then washed with 50 ml wash buffer at gravity flow. The protein was eluted by gravity flow by applying 5-ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 and 250 mM); fractions were collected until essentially all protein was eluted. After elution DTT was added to a final concentration of 10 mM.

Enzymatic treatment : (Dephosphorylation and Histag cleavage) Samples containing PIM-2 were pooled and 200µg GST-lambda phosphatase and 100µg TEV protease added for overnight incubation at 4°C: protein solution contained elution buffer plus 10 mM DTT and 0.05 mM MnCl2.

Column 3: MonoQ ion exchange
Buffers: Buffer A: 50 mM HEPES pH 7.5; Buffer B: 50 mM HEPES pH 7.5, 1 M NaCl.
Procedure: Dephosphorylated PIM-2 was applied to a 1 ml MonoQ column in buffer A and eluted from the column by a linear gradient with buffer B. Homogeneous non-phosphorylated PIM-2 eluted at ~100 mM NaCl.

Column 4: Size Exclusion Chromatography
Buffers: 50 mM HEPES pH 7.5, 250 mM NaCl
Procedure: The monoQ elution was concentrated to ~2 ml and injected onto a HiLoad 16/60 S75 Gel Filtration column and ran at 1 ml/min. 1.8 ml fractions were collected.

Concentration : Samples containing unphosphorylated protein were pooled and concentrated in Centricons (10 kDa cut off) to 11 mg/ml. Dephosphorylation was monitored using LC- ESI MS-Tof.

Extraction

Procedure
The frozen cells were thawed on ice and binding buffer (plus 1 mM PMSF, 0.5 mM TCEP, 50 mM L-Arg, 50 mM L-Glu) added to a final volume of 50 ml. Cells were lysed using a high pressure cell disruptor. The lysate was centrifuged at 16,500 RPM for 30 minutes and the supernatant collected for purification.
Concentration:
Ligand
MassSpec:Masses of purified proteins were confirmed using an Agilent LC/MSD TOF system with reversed-phase HPLC coupled to electrospray ionisation and an orthogonal time-of-flight mass analyser. Proteins were desalted prior to mass spectrometry by rapid elution from a C3 column with a gradient of 5-95% acetonitrile in water with 0.1% formic acid. The purified PIM-2 protein was homogeneous and had an experimental mass of 34277 Da as expected from its primary structure.
Crystallization:The ruthenium half-sandwich complex compound (R)- 5 was added to 0.6 mM (from a 10 mM stock solution in DMSO) prior to protein concentration to 11 mg/ml in 50 mM HEPES pH 7.5, 250 mM NaCl, 10 mM DTT. Crystals were grown at 4°C in 1.5µl sitting drops mixing 0.3µl PIM-2 solution with 1.2µl of mother liquor (90 mM HEPES pH 7.5, 1.44 M Na/KPO 4 ).
NMR Spectroscopy:
Data Collection:PIM-2 complex with (R)- 5 (PDB 2IWI): 2.8 Å , X-ray source: Swiss Light Source synchrotron beamline SLS -X10.
Data Processing:

References

Fox CJ, Hammerman PS, Cinalli RM, Master SR, Chodosh LA, Thompson CB. The serine/threonine kinase Pim-2 is a transcriptionally regulated apoptotic inhibitor. Genes Dev. (2003) 17, 1841-54.
Yan B, Zemskova M, Holder S, Chin V, Kraft A, Koskinen PJ, Lilly M. The PIM-2 kinase phosphorylates BAD on serine 112 and reverses BAD-induced cell death. J Biol Chem. (2003) 278, 45358-67.
Chen XP, Losman JA, Cowan S, Donahue E, Fay S, Vuong BQ, Nawijn MC, Capece D, Cohan VL, Rothman P. Pim serine/threonine kinases regulate the stability of Socs-1 protein. Proc Natl Acad Sci U S A. (2002) 99, 2175-80.
Debreczeni JE, Bullock AN, Atilla GE, Williams DS, Bregman H, Knapp S, Meggers E. Ruthenium Half-Sandwich Complexes Bound to Protein Kinase Pim-1.Angew Chem Int Ed Engl. (2006) 45, 1580-1585.