Human cytokine-like nuclear factor n-pac

Target ID:

NPACA

Aliases:

BM045HIBDLN-PACNP60

Deposition Date:

Friday 20th April 2007

Families:

Oxidoreductases

Related Diseases:

Signalling

Structure type:

protein-ligand

Download Coordinates:

2UYY

Authors:

TICKLE, J., PILKA, E., BUNKOCZI, G., BERRIDGE, G., SMEE, C., KAVANAGH, K.L., HOZJAN, V., NIESEN, F.H., PAPAGRIGORIOU, E., PIKE, A.C.W., TURNBULL, A., ARROWSMITH, C.H., EDWARDS, A., SUNDSTROM, M., WEIGELT, J., VON DELFT, F., OPPERMANN, U.

Site:

Oxford

2UYY

tabs

Structure Details

A recent study identified a nuclear protein (NPAC) composed of three distinguishable domains to be involved in the sorbitol stress induced activation of the MAP kinase p38α. NPAC has an N-terminal PWWP and an AT hook domain which mediate association to the kinase, and an NAD(H) binding/catalytic domain with unknown substrate specificity which shows homologies to hydroxyacid dehydrogenases such as hydroxyisobutyrate dehydrogenase.

The structure of the catalytic domain was solved and shows the typical hydroxyacid dehydrogenase folding pattern.

Materials and Methods

NPAC

PDB:2UYY

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|40556376
Entry Clone Source:Origene
SGC Clone Accession:NPACA-c000
Tag:N-terminal, TEV cleavable hexahistidine tag. Tag sequence: mhhhhhhssgvdlgtenlyfq(*)sm
Host:BL21(DE3)-R3-pRARE2

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsmGSITPTDKKIGFLGLGLMGSGIVSNLLKMGHTVTVWNRTAEKCDLFIQEGARLGRTPAEVVSTCDITFACVSDPKAAKDLVLGPSGVLQGIRPGKCYVDMSTVDADTVTELAQVIVSRGGRFLEAPVSGNQQLSNDGMLVILAAGDRGLYEDCSSCFQAMGKTSFFLGEVGNAAKMMLIVNMVQGSFMATIAEGLTLAQVTGQSQQTLLDILNQGQLASIFLDQKCQNILQGNFKPDFYLKYIQKDLRLAIALGDAVNHPTPMAAAANEVYKRAKALDQSDNDMSAVYRAYIH
Vector:pNIC28-Bsa4.

Growth

Medium:TB
Antibiotics:
Procedure:10µl of a BL21(DE3)-R3-pRARE2 glycerol stock carrying the expression plasmid were inoculated into 5ml of TB with 50µg/ml of kanamycin and 34µg/ml chloramphenicol and grown overnight at 37°C, 200rpm. 10ml of overnight culture were added to 3 x 1L of TB with 50µg/ml kanamycin and incubated at 37°C, 160rpm. After the OD600 reached 1.0, the temperature was dropped to 18°C and 500ul of 1M IPTG was added to the final concentration of ~0.5mM. The culture was then incubated with shaking overnight at 18°C, 160rpm. The following morning the 3L culture was harvested and centrifuged for 10min at 4000rpm. Supernatant was discarded and cell pellets were resuspended in 60ml of a lysis buffer and frozen at -80°C.

Purification

Procedure
The cell extract was loaded on the AKTA Express system The extinction at 280nm was monitored and fractions were collected and analyzed by SDS-PAGE. Positive fractions were pooled and concentrated.

Extraction

Procedure
The thawed cells were broken by 5 passes at 16.000 psi through a high pressure homogeniser followed by centrifugation for 45min at 15.000rpm.
Concentration:Using VivaSpin-15 concentrators with 30kDa cutoff, the sample was concentrated to 10mg/ml. Concentrations were determined from the absorbance at 280nm using NanoDrop.
Ligand
MassSpec:Calculated mass of the construct was 34053. The mass of 34052 was confirmed by mass spectrometry.
Crystallization:Crystals were grown by vapor diffusion at 20°C in 300nl sitting drops. Prior to the crystallisation 5mM NAD+ was added to the protein. The drops were prepared by mixing 200nl of protein solution and 100nl of precipitant consisting of 20% PEG 3350 and 0.2M KSCN. Crystals were transferred to a cryo-protectant consisting of 25% glycerol and 75% well solution with 5mM NAD+ before flash-cooling in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Resolution: 2.6Å; X-ray source: SLS beam X10SA.
Data Processing: