Human Proviral Interacting Protein for MulV

Target ID:

PIM1A

Aliases:

PIMPIM1

Deposition Date:

Tuesday 2nd November 2004

Families:

Protein Kinase

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

1XWS

Authors:

S.KNAPP,J.DEBRECZENI,A.BULLOCK,F.VON DELFT,M.SUNDSTROM,C.ARROWSMITH,A.EDWARDS,K.GUO

Site:

Oxford

ICM Datapack:

ICM Datapack

1XWS

tabs

Structure Details

Pim1 is a cytoplasmic highly conserved serine/threonine kinase that was first discovered as a
preferential proviral insertion site in Moloney Murine Leukemia Virus (MoMuLV) induced T-cell lymphomas.
The expression pattern of Pim-1 is widespread and the protein is over-expressed in a series of tumors
but highest expression levels are found in cells of the hematopoietic and lymphoid system.

Pim1 is induced by cytokines like interleukins and GM-CSF through the Jak/STAT pathway
and has been therefore implemented in the proliferation and differentiation of blood cells.
Pim1 has also been implicated directly in the regulation of the Jak/STAT pathway by stabilizing the negative regulator Socs-1.
Recent data like Pim1 also to cell cycle control and apoptosis.

We determined the structure of human Pim1 1 in complex with an inhibitor of the bisindolyl maleimide class.
This inhibitor class has been developed for the inhibition of PKC kinase
and a related compound is currently in phase III clinical trials as a potential drug
against diabetes complications like diabetic retinopathy as well as ventricular hypertrophy in heart failure.

PIM1

PDB:1XWS

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:The expressed protein has the sequence from gi:33304198 which differs from gi:4505811 by a single change R250G (bold in the sequence below)
Entry Clone Source:TSK
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m). N-terminal his6 tag, TEV-protease cleavable (*)
Host:BL21(DE3)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsMLLSKINSLAHLRAAPCNDLHATKLAPGKEKEPLESQYQVGPLLGSGGFGSVYSGIRVSDNLPVAIKHVEKDRISDWGELPNGTRVPMEVVLLKKVSSGFSGVIRLLDWFERPDSFVLILERPEPVQDLFDFITERGALQEELARSFFWQVLEAVRHCHNCGVLHRDIKDENILIDLNRGELKLIDFGSGALLKDTVYTDFDGTRVYSPPEWIRYHRYHGRSAAVWSLGILLYDMVCGDIPFEHDEEIIGGQVFFRQRVSSECQHLIRWCLALRPSDRPTFEEIQNHPWMQDVLLPQETAEIHLHSLSPGPS
Vector:pLIC-SGC

Growth

Medium:
Antibiotics:
Procedure:10 ml overnight cultures in LB, 100 µg/ml ampicillin were centrifuged, resuspended in fresh buffer, and used to inoculate 1 litre of LB medium containing 100 µg/mL ampicillin. Cultures were grown at 37ºC until they reached an OD₆₀₀ of 0.3 and then cooled to 18 ºC. Expression was induced for 4 hours using 1 mM IPTG at an OD600 of 0.8. The cells were collected by centrifugation, transferred to 50 ml tubes, resuspended in 30 ml binding buffer, and frozen. Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol.

Purification

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

Ni-affinity: Ni-NTA (Qiagen), 5 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer. 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 to 250 mM imidazole, 5% Glycerol. The flowthrough from column 1 was loaded by gravity flow on the Ni-NTA column. The column was then washed with 3 x 10 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, 250 mM); fractions were collected until essentially all protein was eluted. After elution DTT was added to a final concentration of 10 mM. Samples containing Pim1 were pooled and alkaline phosphatase was added in the same buffer. Protein sample was treated for 12 h.

Ion exchange Mono Q column: Buffer A : 50 mM Hepes, pH 7.5. Buffer B : 50 mM Hepes, pH 7.5, 1000 mM NaCl. Dephosphorylated Pim1 was applied to MonoQ in buffer A and eluted from the column by a linear gradient.

Pim1 samples containing unphosphorylated protein were pooled and concentrated in Centricons (10 kDa cut off). Dephosphorylation was monitored using LC-ESI MS-Tof.

Dephosphorylation (l-phosphatase): a GST fusion with the lambda phosphatase. TEV protease cleavage. Both treatments carried out simultaneously: protein solution contained 10 mM DTT and 0.05 mM MnCl₂ (higher MnCl₂ concentrations caused precipitation).

Extraction

Procedure
The frozen cells were thawed on ice and lysed using a high pressure cell disruptor. The lysate was centrifuged at 17,000 RPM for 30 minutes. Supernatant was collected and binding buffer was added to 50 mL.
Concentration:
Ligand
BIM-1 inhibitor (2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl) maleimide, HCl) - purchased from Calbiochem and added at a concentration of 1 mM from a 50 mM DMSO stock solution to the protein.MassSpec:The purified protein was homogeneous and had an experimental mass of 35546 Da as expected from its primary structure. Masses of purified proteins were confirmed by LC-MS, 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 off a C3 column with a gradient of 5-95% acetonitrile in water with 0.1% formic acid.
Crystallization:Crystals were grown at 4degC in 3 microL sitting drops mixing 1.5 microL Pim1 (10 mg/mL in 50 mM Hepes, pH 7.5, 280mM NaCl, 5% Glycerol, 10 mM DTT with 1.5 µl of a solution containing 0.2 M Na2SO4, 20% PEG 3350, 10% ethylene glycol and 0.5% DMSO.
NMR Spectroscopy:
Data Collection:
Data Processing: