Human sphingosine kinase 1 [isoform 2]

Target ID:

SPHK1B

Deposition Date:

Sunday 5th October 2014

Families:

Non-protein Kinase

Related Diseases:

Cancer

Structure type:

protein-ligand

Download Coordinates:

4V24

Authors:

J.M.ELKINS,J.WANG,F.SORRELL,C.TALLANT,D.WANG,L.SHRESTHA,C.BOUNTRA,F.VONDELFT,S.KNAPP,A.EDWARDS

Site:

Oxford

4V24

tabs

Structure Details

Sphingosine kinases (SPHKs) catalyse the production of sphingosine-1-phosphate (S1P) from sphingosine, the final step in S1P biosynthesis (Kohama et al., 1998; Liu et al., 2000; Olivera et al., 1998; Pitson et al., 2000). SPHKs are not related to the protein kinases, but have homology to other lipid kinases such as the diacylglycerol kinases. Humans have two SPHKs, SPHK1 and SPHK2 with different localisation patterns within the cell (Ding et al., 2007; Hait et al., 2009; Maceyka et al., 2005).
Excess S1P levels are implicated in a variety of diseases such as cancers and inflammation (Kunkel et al., 2013; Pyne and Pyne, 2013). For this reason inhibitors of SPHKs have been sought. The most potent SPHK1 inhibitor yet developed is PF-543 (Schnute et al., 2012).
Here we have determined the crystal structure of human sphingosine kinase 1 (SPHK1) in complex with PF-543 to 1.8 Â resolution. The structure reveals that PF-543 binds in the lipid-binding substrate pocket of SPHK1 and forms a bent 'J' shape similar to that expected for a bound lipid substrate. The (R)-2-(hydroxymethyl)-pyrrolidine head group of PF-543 is rotated 180° compared to what would be expected for a lipid substrate, which explains why PF-543 is only a weak substrate, and a potent inhibitor, for SPHK1.

Materials and Methods

SPHK1

PDB:4V24

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:Mammalian Gene Collection (IMAGE consortium clone ID 4871343).
SGC Clone Accession:
Tag:N-terminal, TEV protease cleavable hexahistidine tag
Host:

Construct

Prelude:
Sequence:SMEPRVEVMDPAGGPRGVLPRPCRVLVLLNPRGGKGKALQLFRSHVQPLLAEAEISFTLMLTERRNHARELVRSEELGRWDALVVMSGDGLMHEVVNGLMERPDWETAIQKPLCSLPAGSGNALAASLNHYAGYEQVTNEDLLTNCTLLLCRRLLSPMNLLSLHTASGLRLFSVLSLAWGFIADVDLESEKYRRLGEMRFTLGTFLRLAALRTYRGRLAYLPVGRVGSKTPASPVVVQQGPVDAHLVPLEEPVPSHWTVVPDEDFVLVLALLHSHLGSEMFAAPMGRCAAGVMHLFYVRAGVSRAMLLRLFLAMEKGRHMEYECPYLVYVPVVAFRLEPKDGKGVFAVDGELMVSEAVQGQVHPNYFWMVS

Glu81 - Ser449 of SPHK1B isoform 2
The N-terminal residues,SM, derive from the vector.
Vector:pFB-LIC-Bse

Growth

Medium:
Antibiotics:
Procedure:

Purification

Buffers
Procedure
Cell Lysis: The resuspended cell pellet was thawed and lysed by sonication on ice. PEI (polyethyleneimine) was added to a final concentration of 0.15 %. The cell debris and precipitated DNA were spun down.Lysis Buffer:50 mM Tris pH 7.8, 200 mM NaCl, 20 mM Imidazole, 0.5 mM TCEP, Sigma protease inhibitor cocktail. Purification: Column 1: 5 ml of Ni-Sepharose in a 2.5 cm diameter gravity flow column. Column 1 Buffers: Binding Buffer: 50 mM Tris pH 7.8, 200 mM NaCl, 20 mM Imidazole, 0.5 mM TCEP, Sigma protease inhibitor cocktail Wash Buffer 1:As Binding Buffer except 40 mM imidazole and 1M NaCl.Wash Buffer 2:As Binding Buffer except 60 mM imidazole.Elution Buffer:As Binding Buffer except 250 mM imidazole. Column 1 Procedure:The clarified supernatant was passed through the column. The column was washed with 100 mL of Binding Buffer, 50 mL each of Wash Buffer 1 and Wash Buffer 2, and 25 mL of Elution Buffer was passed through to elute the protein. Column 2 Procedure: GF Buffer:20 mM Tris pH 7.8, 200 mM NaCl, 0.5 mM TCEP, TEV protease was added and the sample dialysed overnight against GF Buffer.The sample was passed through 1 mL of Ni-Sepharose (pre-equilibrated into GF Buffer) followed by 5 ml of additional GF Buffer. The resin was eluted with 5 ml of GF Buffer containing 10 mM, 20 mM, 40 mM and then 250 mM imidazole. Column 3 Procedure: The flow-through and 10 mM imidazole fraction from Column 2 were combined and concentrated to 5 mL volume. The sample was injected onto an S200 16/60 GF Column (GE Healthcare).

Extraction

Buffers
Procedure
Expression: The protein was expressed using baculovirus infected Sf9 cells. Cells were infected at a density of 2,000,000 cells/ml for 48h. Cell harvest:Cells were spun at 800x g for 20 mins and the pellets resuspended in Lysis Buffer and then frozen at -20°C.
Concentration:Compound PF-543 and ADP were added to the pooled fractions from gel filtration and the sample left overnight. The sample was concentrated to 10 mg/ml (measured by 280 nm absorbance).
Ligand
MassSpec:Mass Spec Characterisation: Expected Observed41120 41120.1
Crystallization:Crystals grew from a mixture of 100 nL SPHK1 protein (10 mg/ml with 1 mM PF-543 and 1 mM ADP), and 50 nL of a well solution containing 37.5% MPD, 0. 1 M BisTris pH 5.5 and 0.1 M ammonium acetate.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Ding, G., Sonoda, H., Yu, H., Kajimoto, T., Goparaju, S.K., Jahangeer, S., Okada, T., and Nakamura, S. (2007). Protein kinase D-mediated phosphorylation and nuclear export of sphingosine kinase 2. J. Biol. Chem. 282, 27493-27502.

Hait, N.C., Allegood, J., Maceyka, M., Strub, G.M., Harikumar, K.B., Singh, S.K., Luo, C., Marmorstein, R., Kordula, T., Milstien, S., et al. (2009). Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate. Science (80-. ). 325, 1254-1257.

Kohama, T., Olivera, A., Edsall, L., Nagiec, M.M., Dickson, R., and Spiegel, S. (1998). Molecular cloning and functional characterization of murine sphingosine kinase. J. Biol. Chem. 273, 23722-23728.

Kunkel, G.T., Maceyka, M., Milstien, S., and Spiegel, S. (2013). Targeting the sphingosine-1-phosphate axis in cancer, inflammation and beyond. Nat. Rev. Drug Discov. 12, 688-702.

Liu, H., Sugiura, M., Nava, V.E., Edsall, L.C., Kono, K., Poulton, S., Milstien, S., Kohama, T., and Spiegel, S. (2000). Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform. J. Biol. Chem. 275, 19513-19520.

Maceyka, M., Sankala, H., Hait, N.C., Le Stunff, H., Liu, H., Toman, R., Collier, C., Zhang, M., Satin, L.S., Merrill, A.H., et al. (2005). SphK1 and SphK2, sphingosine kinase isoenzymes with opposing functions in sphingolipid metabolism. J. Biol. Chem. 280, 37118-37129.

Olivera, A., Kohama, T., Tu, Z., Milstien, S., and Spiegel, S. (1998). Purification and characterization of rat kidney sphingosine kinase. J. Biol. Chem. 273, 12576-12583.

Pitson, S.M., D'andrea, R.J., Vandeleur, L., Moretti, P.A., Xia, P., Gamble, J.R., Vadas, M.A., and Wattenberg, B.W. (2000). Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes. Biochem. J. 350 Pt 2, 429-441.

Pyne, N.J., and Pyne, S. (2013). Sphingosine 1-phosphate is a missing link between chronic inflammation and colon cancer. Cancer Cell 23, 5-7.

Schnute, M.E., McReynolds, M.D., Kasten, T., Yates, M., Jerome, G., Rains, J.W., Hall, T., Chrencik, J., Kraus, M., Cronin, C.N., et al. (2012). Modulation of cellular S1P levels with a novel, potent and specific inhibitor of sphingosine kinase-1. Biochem. J. 444, 79-88.