Human sulfotransferase family, cytosolic, 1C, member 2

Target ID:

SULT1C2

Aliases:

humSULTC2ST1C1ST1C2SULT1C1

Deposition Date:

Thursday 4th May 2006

Families:

Transferases

Related Diseases:

Drug metabolism and toxicologyMetabolism

Structure type:

apo

Download Coordinates:

2GWH

Authors:

W.TEMPEL,P.PAN,L.DOMBROVSKI,A.AL-HASSANI,M.VEDADI,P.LOPPNAU,J.WEIGELT,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,A.N.PLOTNIKOV,STRUCTURAL GENOMICS CONSORTIUM(SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2GWH

tabs

Structure Details

The synthetic biocide pentachlorophenol (PCP) was first introduced as a wood preservative in the 1930s.
Since then, its application has been expanded to fungicide, bactericide, herbicide, molluscicide, algicide, and insecticide.
PCP has been detected in fruits, vegetables, meats, water and soils.
Due to the medical concern of chronic and acute PCP poisoning, its use in agriculture has been restricted in many countries.
Although the range of its biological actions is still being actively explored, it is a well recognized enzyme inhibitor
for cytosolic sulfotransferases.

To address the question of how PCP interacts with cytosolic sulfotransferases, we have determined the crystal structure of human SULT1C2
in complex with the sulfuryl donor product PAP and PCP at 1.8 Å.
In comparison with the previously determined apo-SULT1C2 crystal structure (2AD1),
the ternary complex revealed
a large conformational change of the protein upon PCP and PAP binding.
PCP is found in the substrate binding pocket.
The position of the hydroxyl group of PCP would allow for strong hydrogen bonding interactions with the catalytic Histidine115, accounting for the inhibitory effect of PCP on sulfotransferase activity.
The ternary complex crystal structure provides atomic details of PCP binding and insights for understanding the mechanism of PCP inhibition for cytosolic sulfotransferases.

Materials and Methods

SULT1C2

PDB:2GWH

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi: 28830308
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated thrombin protease site: MGSSHHHHHHSSGLVPRGS
Host:E.coli BL21 (DE3) codon plus RIL (Stratagen).

Construct

Prelude:
Sequence:gsEDFTFDGTKRLSVNYVKGILQPTDTCDIWDKIWNFQAKPDDLLISTYPKAGTTWTQEIVELIQNEGDVEKSKRAPTHQRFPFLEMKIPSLGSGLEQAHAMPSPRILKTHLPFHLLPPSLLEKNCKIIYVARNPKDNMVSYYHFQRMNKALPAPGTWEEYFETFLAGKVCWGSWHEHVKGWWEAKDKHRILYLFYEDMKKNPKHEIQKLAEFIGKKLDDKVLDKIVHYTSFDVMKQNPMANYSSIPAEIMDHSISPFMRKGAVGDWKKHFTVAQNERFDEDYKKKMTDTRLTFHFQF
Vector:pET28a-LIC

Growth

Medium:
Antibiotics:
Procedure:SULT1C2 was expressed in E.coli BL21 (DE3) codon plus RIL in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin at 37oC to an OD600 of 0.8. Cells were then induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 0.5 mM, and incubated overnight at 15oC.

Purification

Procedure
The clarified lysate was loaded onto 5 ml HiTrap Chelating column (Amersham Biosciences), charged with Ni2+. The column was washed with 10 CV of 50 mM HEPES-NaOH, pH 7.4,, containing 500 mM NaCl and 50 mM imidazole, and the protein was eluted with elution buffer (50 mM HEPES-NaOH, pH 7.4,, 500 mM NaCl, 250 mM imidazole). The protein was loaded on Superdex200 column (26x60) (Amersham Biosciences), equilibrated with 20 mM MES-NaOH buffer, pH 6.5, and 250 mM NaCl, at flow rate 4 ml/min. Thrombin (Sigma) was added to combined fractions containing SULT1C2 and incubated overnight at 4oC. The protein was further purified to homogeneity by ion-exchange chromatography on Source 30S column (10x10) (Amersham Biosciences), equilibrated with buffer 20 mM MES-NaOH buffer, pH 6.5, and eluted with linear gradient of NaCl up to 500 mM concentration (30CV). Purification yield was 56 mg of the protein per 1L of culture.

Extraction

Procedure
Cells were harvested by centrifugation at 6,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80°C. For the purification the cell paste was thawed and resuspended in lysis buffer (50 mM HEPES-NaOH, pH 7.4, 0.5 M NaCl, 5 mM imidazol, 2 mM ß-mercaptoethanol, 5% glycerol) with protease inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:11.7 mg/ml
Ligand
MassSpec:expected MW is 34965.1, measured MW is 34965.6
Crystallization:10 mg/mL purified SULT1C2 was mixed with 2mM 3'-phosphoadenosine 5'-phosphate (PAP, Sigma) and 2mM pentachlorophenol (PCP, Sigma) in 20 mM MES-NaOH buffer, pH 6.5, and incubated on ice for 30 mins. SULT1C2-PAP-PCP complex was crystallized using the sitting drop method at 20°C by mixing 0.8 µl of the protein-cofactor-inhibitor mix with 0.8 µl of the reservoir solution containing 25% polyethylene glycol 3350, 0.2 M Li Sulfate, 0.1M Bis-Tris pH 6.5.
NMR Spectroscopy:
Data Collection:
Data Processing: