Cytosolic sulfotransferases (SULTs), often referred as Phase II enzymes of chemical defense, are a superfamily of enzymes that catalyze the transfer of a sulfonate group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to an acceptor group of substrates. This reaction modulates the activities of a large array of small endogenous and foreign chemicals including drugs, toxic compounds, steroid hormones, and neurotransmitters. In some cases, however, SULTs activate certain food and environmental compounds to mutagenenic and carcinogenic metabolites.1
Twelve human SULTs have been identified, which are partitioned into three families: SULT1, SULT2 and SULT4. The SULT1 family is further divided in four subfamilies, A, B, C, and E, and comprises eight members (1A1, 1A2, 1A3, 1B1, 1C1, 1C2, 1C3, and 1E1).2 Despite sequence and structural similarity among the SULTs, the family and subfamily members appear to have different biological function. SULT1 family shows substrate-binding specificity for simple phenols, estradiol, and thyroid hormones, as well as environmental xenobiotics and drugs. Human SULT1B1 is expressed in liver, colon, small intestine, and blood leukocytes, and shows substrate-binding specificity to thyroid hormones and benzylic alcohols.3 Human SULT1C1 is expressed in the adult stomach, kidney, and thyroid, as well as in fetal kidney and liver.4 SULT1C1 catalyzes the sulfonation of p-nitrophenol and N-hydroxy-2-acetylaminofluorene in vitro. However, the in vivo function of the enzyme remains unknown.5
We intend to solve the structures for all of the SULTs for which structural information is not yet available, and compare the structural and functional features of the entire SULT superfamily. Here we report the structures of two members of SULT1 family, SULT1B1 and SULT1C1, both in complex with the product of the PAPS cofactor, adenosine-3′-5′-diphosphate (PAP).
The SULT1B1 and SULT1C1 genes were amplified by PCR from the Mammalian Gene Collection clones (accession codes gi:4507305 and gi:45935387 for SULT1B1 and SULT1C1, respectively) and subcloned into a modified pET-28a vector (details described on http://www.sgc.utoronto.ca/SGC-WebPages/toronto-vectors.php). To improve the solubility of SULT1C1, Cys-293 was mutated to Ser using a Quick-Change kit (Stratagen). The corresponding constructs were transformed into Escherichia coli BL21 (DE3) codon plus RIL (Stratagene) and the cells grown in Terrific Broth to an OD600 of 1.5. SULT1B1 and SULT1C1 expression was induced by the addition of 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG), and the cells incubated with shaking overnight at 15°C. Cells were harvested by centrifugation, frozen in liquid nitrogen, and stored at −80°C. The frozen cell paste was thawed and the cells resuspended in lysis buffer (30 mM Tris-HCl, pH 8.5, 0.5 M NaCl, 5 mM imidazole, 2 mM β-mercaptoethanol, 5% glycerol) with protease inhibitor (0.1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through a Microfluidizer (Microfluidics Corporation) at 20,000 psi. The lysate was clarified by centrifugation and then loaded on a 5 mL HiTrap Chelating column (Amersham Biosciences), charged with Ni2+. The column was washed with 50 mL of 20 mM Tris-HCl buffer, pH 8.5, containing 500 mM NaCl and 50 mM imidazole, and the protein eluted with elution buffer (20 mM Tris-HCl, pH 8.5, 500 mM NaCl, 250 mM imidazole). The protein-containing fractions were loaded at a flow rate of 4 mL/min on a Superdex200 column (26 × 60) (Amersham Biosciences), equilibrated with 20 mM Tris-HCl buffer, pH 8.5, and 150 mM NaCl. Thrombin (Sigma) was added to the combined fractions containing the protein and incubated overnight at 4°C. The protein was then purified to homogeneity by ion-exchange chromatography on a Source 30Q column (10 × 10) (Amersham Biosciences), equilibrated with 20 mM Tris-HCl, pH 8.5, and eluted with a linear gradient up to 500 mM NaCl (30 column volumes).
Purified SULT1B1 and SULT1C1 were incubated with 3′Phosphoadenosine 5′-phosphate (PAP) at a 1:5 molar ratio of protein:PAP and crystallized using the hanging drop method at 20°C by mixing: for SULT1B1, 2 μL of the protein solution with 2 μL of the reservoir solution containing 0.1 M BisTris, pH 6.5, 0.2 M ammonium sulfate, and 16–20% polyethylene glycol 4000; for SULT1C1, 2 μL of the protein solution with 2 μL of the reservoir solution containing 0.1 M K2HPO4 and 12–16% polyethylene glycol 3350.
Diffraction data for SULT1B1 extended to 2.1 Å and were measured on an FR-E/R-axis IV++; Rigaku/MSC. For SULT1C1, a 1.8 Å data set was collected at beam-line 17ID of the APS synchrotron (Argonne National Laboratory). Data were processed and merged with HKL2000.6 The structure of SULT1B1 was solved using the molecular replacement (MR) method with the program MOLREP,7 using the model of human SULT1E1 as a template (PDB code 1G3M).8 The structure of SULT1B1 was used as the MR model for solving the crystal structure of SULT1C1. Cycles of ARP/wARP9 model building, followed by manual adjustments in O,10 reduced the crystallographic R factor to the final value of: SULT1B1, 0.175 (Rfree = 0.235); SULT1C1, 0.184 (Rfree = 0.227). Statistics and data processing and refinement are reported in Table I. Atomic coordinates of SULT1B1 and SULT1C1 have been deposited in the PDB with codes 1XV1 and 2ETG, respectively.
Table I. Statistics on Data Collection and Refinement
Crystals were frozen at 100 K under a nitrogen gas cold stream in the cryoprotectant solution. A Rigaku/MSC FR-E generator with Cu radiation was used. A R-axis IV++ detector was positioned at a distance of 150 mm from the sample. Rotations of 0.5° were performed.
P 21 21 21
Cell parameters, a, c [Å; °]
44.8, 77.1, 189.4;
48.8, 40.5, 154.6, β = 91.8
Number of residues included
Total number of atoms, including ligands and solvent
RMS on bonds length (Å), angles (°)
Results and Discussion.
The monomer structures of SULT1C1 and SULT1B1 are presented in Figure 1A and B, respectively. The general architecture of the two complexes is as would be predicted from the previous studies.11 The overall structure is a central four-stranded parallel β-sheet surrounded by α-helices. The structures superpose very closely with a root-mean-square deviation of 1.23 Å over 250 Cα atoms. A loop centered on residue 90 (Figs. 1 and 2, cyan loop) was excluded from the superposition; the shift between Cα atoms of Gln-90 (SULT1C1) and Arg-90 (SULT1B1) was 5.0 Å. Two more fragments that were ordered in the SULT1B1 structure, fragment 66–78 [Figs. 1(B) and 2, gold loop] and fragment 236–255 (magenta loop), were disordered in SULT1C1. The corresponding loop (fragment 230–259) in the structure of SULT1A3 (PDB IDs: 1CJM or 2A3R) was also disordered (data not shown).12, 13 Consistent with all other SULT structures reported previously, the PAP binding site, its binding mode, and conformation are virtually identical in all the SULT1 family structures. For detailed description, see ref.14.
The SULT1B1 and SULT1C1 structures were compared to the structure of human SULT1A1, which was solved in the presence of its substrate β-estradiol (PDB ID: 2D06).15 The gold and magenta loops in SULT1A1 form the binding pocket for the substrate, and have a conformation similar to that in the apo structure of SULT1B1. The active site lies at the bottom of pocket formed by these loops. The cyan, magenta, and gold loops appear to be flexible in the entire SULT class. It is likely that the open state of the loops would allow PAP/PAPS interchange. The cyan and magenta loops likely determine substrate specificity.
The sequences of eight members of the SULT1 family have been superimposed and compared. From this analysis, the residues contributing to the PAP/PAPS binding are highly conserved (not shown). In contrast, the regions of the paralogous proteins that correspond to the cyan, magenta, and gold loops show significant variation. This supports the notion that the substrate specificity, to large extent, is defined by these structural features.
The Structural Genomics Consortium is a public private charitable partnership that receives funds from Canada Foundation for Innovation, Canadian Institutes for Health Research, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, the Ontario Innovation Trust, the Ontario Research, and Development Challenge Fund and the Wellcome Trust.