Human nicotiamide ribose kinase 1 in complex with tiazofurin

Target ID:

C9orf95

Aliases:

bA235O14.2FLJ20559NRK1RP11-235O14.2

Deposition Date:

Wednesday 28th February 2007

Families:

Non-protein Kinase

Related Diseases:

MetabolismDrug metabolism and toxicology

Structure type:

protein-ligand

Download Coordinates:

2P0E

Authors:

W.M.RABEH,W.TEMPEL,L.NEDYALKOVA,R.LANDRY,C.H.ARROWSMITH,A.M.EDWARDS,M.SUNDSTROM,J.WEIGELT,A.BOCHKAREV,C.BRENNER,H.PARK,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2P0E

tabs

Structure Details

Nicotinamide adenine dinucleotide (NAD+) plays important roles in the cell as a redox factor in processes such as oxidative phosphorylation and the TCA cycle and as a substrate for NAD+-consuming enzymes (Belenky et al., 2007). In addition, NAD+ can protect against neurodegeneration through the stimulation of the NRK pathway (Sasaki et al., 2006). In eukaryotes, tryptophan is the precursor of NAD+ but organisms with a low tryptophan intake can synthesize NAD+ by taking up nicotinamide riboside (NR) and converting it to NAD+ through phosphorylation and adenylylation (Bieganowski and Brenner, 2004). Nicotinamide riboside kinase (NRK) catalyzes the phosphorylation of NR, using ATP as a phosphate source, to produce nicotinamide mononucleotide (NMN). NMN is then adenylylated by NMN adenylyltransferases to produce NAD+.

Tiazofurin is an anti-tumor agent that is converted to the NAD+ analog, tiazofurin adenine dinucleotide (TAD+). Tiazofurin, which has overall structural similarity to NR, is phosphorylated by NRK with high specificity (Bieganowski et al., 2004). TAD+ then acts as a potent inhibitor of inosine monophosphate dehydrogenase (Carney et al., 1985). Inhibition of the latter enzyme lowers the levels of intracellular guanosine nucleotides that are essential for tumor cell growth and replication (Carney et al., 1985).

Materials and Methods

NRK1

PDB:2P0E

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC001366
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal Hexahistidine tag and thrombin cleavage sequence site: mgsshhhhhhssglvpr*gs
Host:E.coli BL21 (DE3) codon plus RIL

Construct

Prelude:
Sequence:mgsshhhhhhssglvpr*gsKTFIIGISGVTNSGKTTLAKNLQKHLPNCSVISQDDFFKPESEIETDKNGFLQYDVLEALNMEKMMSAISCWMESARHSVVSTDQESAEEIPILIIEGFLLFNYKPLDTIWNRSYFLTIPYEECKRRRSTRVYQPPDSPGYFDGHVWPMYLKYRQEMQDITWEVVYLDGTKSEEDLFLQVYEDLIQEL
Vector:p28a-LIC

Growth

Medium:TB
Antibiotics:
Procedure:The target was expressed in E. coli by inoculating 100 mL of overnight culture grown in Luria-Bertani medium containing 50 mg/L kanamycin and 50 mg/L chloramphenicol into a 1.8 L of Terrific Broth medium containing 50 mg/L kanamycin and 50 mg/L chloramphenicol. The culture was grown at 37ºC with the LEX bubbling system. When OD600 was ~3.0, the culture was induced with 1mM IPTG and the temperature was reduced to 15ºC, and the cells were allowed to grow overnight. Cultures were harvested by centrifugation and the cell pellets were flash frozen and stored at -80˚C.

Purification

Procedure
Column 1: DE52 column

Column 2: 5 mL Ni-NTA column (Qiagen)

Column 3: Superdex 200 column (26x60, Amersham Biosciences)

The lysate was centrifuged at 19000 xg for 30 min and the supernatant was passed through DE52 (Whatman) column (15mL bed volume) equilibrated with the binding buffer and the flow through was loaded onto 5 mL Ni-NTA column (Qiagen) equilibrated with the same binding buffer at 4 ºC. The Ni-NTA column was washed with 150 mL of the washing buffer and the protein was eluted with 15 mL of the elution buffer. The protein was further purified and desalted using a gel filtration column, Superdex 200 (26/60), which was pre-equilibrated with Gel filtration buffer.

The protein was concentrated using an Amicon Ultra centrifugal filter with 5 kDa cut off to a final concentration of 30 mg/mL. Protein concentrations were measured using Bradford assay and the purity was >95% based on SDS-PAGE analysis. The His tag was cleaved using 1 unit of thrombin (Sigma T9681) per milligram of protein.

Extraction

Procedure
The thawed cell pellets from 4L were resuspended in 100 mL of the Lysis buffer with a protease inhibitor cocktail (0.1 mM M benzamidine-HCl and 0.1 mM phenylmethyl sulfonyl fluoride), and 0.5% CHAPS. The cells were lysed by microfluidizer at 20,000 psi.
Concentration:40 mg/mL
Ligand
MassSpec:
Crystallization:Crystallization trials were set up using the sitting drop vapor diffusion method at 18ºC. The protein (40mg/mL) containing 10mM Tiazofurin, 1mM ADP and 20mM MgCl2 was equilibrated against a reservoir solution (1:1 volume ratio) containing 25% PEG3350, 0.2M sodium phosphate monobasic and 0.1M Bicine pH 9.0. The crystals reached a size of 100 microns within a week and were cryoprotected by washing them with a 1:1 Paratone-N : mineral oil mixture. The crystals then were frozen by directly immersing them into liquid nitrogen.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Belenky,P., Bogan,K.L., and Brenner,C. (2007). NAD+ metabolism in health and disease. Trends Biochem. Sci., 32, 12-19.
Bieganowski,P. and Brenner,C. (2004). Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans. Cell., 117, 495-502.
Carney,D.N., Ahluwalia,G.S., Jayaram,H.N., Cooney,D.A., and Johns,D.G. (1985). Relationships between the cytotoxicity of tiazofurin and its metabolism by cultured human lung cancer cells. J. Clin. Invest., 75, 175-182.
Sasaki,Y., Araki,T., and Milbrandt,J. (2006). Stimulation of nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy. J. Neurosci., 26, 8484-8491.