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• W2014549797 abstract "Heparan sulfate interacts with antithrombin, a protease inhibitor, to regulate blood coagulation. Heparan sulfate 3-O-sulfotransferase isoform 1 performs the crucial last step modification in the biosynthesis of anticoagulant heparan sulfate. This enzyme transfers the sulfuryl group (SO3) from 3′-phosphoadenosine 5′-phosphosulfate to the 3-OH position of a glucosamine residue to form the 3-O-sulfo glucosamine, a structural motif critical for binding of heparan sulfate to antithrombin. In this study, we report the crystal structure of 3-O-sulfotransferase isoform 1 at 2.5-Å resolution in a binary complex with 3′-phosphoadenosine 5′-phosphate. This structure reveals residues critical for 3′-phosphoadenosine 5′-phosphosulfate binding and suggests residues required for the binding of heparan sulfate. In addition, site-directed mutagenesis analyses suggest that residues Arg-67, Lys-68, Arg-72, Glu-90, His-92, Asp-95, Lys-123, and Arg-276 are essential for enzymatic activity. Among these essential amino acid residues, we find that residues Arg-67, Arg-72, His-92, and Asp-95 are conserved in heparan sulfate 3-O-sulfotransferases but not in heparan N-deacetylase/N-sulfotransferase, suggesting a role for these residues in conferring substrate specificity. Results from this study provide information essential for understanding the biosynthesis of anticoagulant heparan sulfate and the general mechanism of action of heparan sulfate sulfotransferases. Heparan sulfate interacts with antithrombin, a protease inhibitor, to regulate blood coagulation. Heparan sulfate 3-O-sulfotransferase isoform 1 performs the crucial last step modification in the biosynthesis of anticoagulant heparan sulfate. This enzyme transfers the sulfuryl group (SO3) from 3′-phosphoadenosine 5′-phosphosulfate to the 3-OH position of a glucosamine residue to form the 3-O-sulfo glucosamine, a structural motif critical for binding of heparan sulfate to antithrombin. In this study, we report the crystal structure of 3-O-sulfotransferase isoform 1 at 2.5-Å resolution in a binary complex with 3′-phosphoadenosine 5′-phosphate. This structure reveals residues critical for 3′-phosphoadenosine 5′-phosphosulfate binding and suggests residues required for the binding of heparan sulfate. In addition, site-directed mutagenesis analyses suggest that residues Arg-67, Lys-68, Arg-72, Glu-90, His-92, Asp-95, Lys-123, and Arg-276 are essential for enzymatic activity. Among these essential amino acid residues, we find that residues Arg-67, Arg-72, His-92, and Asp-95 are conserved in heparan sulfate 3-O-sulfotransferases but not in heparan N-deacetylase/N-sulfotransferase, suggesting a role for these residues in conferring substrate specificity. Results from this study provide information essential for understanding the biosynthesis of anticoagulant heparan sulfate and the general mechanism of action of heparan sulfate sulfotransferases. Heparan sulfate (HS) 1The abbreviations used are: HS, heparan sulfate; AT, antithrombin; 3-OST, 3-O-sulfotransferase; PAPS, 3′-phosphoadenosine 5′-phosphosulfate; PAP, 3′-phosphoadenosine 5′-phosphate; PSB, phosphosulfate binding; NST-1, N-deacetylase/N-sulfotransferase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; ITC, isothermal titration calorimetry; MOPS, 4-morpholinepropanesulfonic acid; WT, wild type. is widely expressed in animal and human tissues. It has diverse roles in development, assisting viral infections, and homeostasis (1Esko J.D. Selleck S.B. Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1239) Google Scholar, 2Rosenberg R.D. Showrak N.W. Liu J. Schwartz J.J. Zhang L. J. Clin. Investig. 1997; 99: 2062-2070Crossref PubMed Scopus (255) Google Scholar, 3Liu J. Thorp S.C. Med. Res. Rev. 2002; 22: 1-25Crossref PubMed Scopus (245) Google Scholar). HS is a highly sulfated polysaccharide consisting of 1-4-linked sulfated glucosamine and sulfated glucuronic/iduronic acid residues. The specific sequences of sulfated saccharide in HS determine its various functions. Synthesis of biologically active HS is accomplished through a complex biosynthetic pathway. HS is initially synthesized as a copolymer of glucuronic acid and N-acetylated glucosamine by d-glucuronyl and N-acetyl-d-glucosaminyltransferase followed by various modifications in the Golgi apparatus (4Lindahl U. Kusche-Gullberg M. Kjellen L. J. Biol. Chem. 1998; 273: 24979-24982Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). These modifications include N-deacetylation and N-sulfonation of glucosamine, C5 epimerization of glucuronic acid to form iduronic acid residues, and 2-O-sulfonation of iduronic and glucuronic acid residues as well as 6-O-sulfonation and 3-O-sulfonation of the glucosamine units. The stepwise sulfonation reactions are illustrated in Fig. 1. All of the enzymes that are responsible for the biosynthesis of HS have been cloned and characterized (5Kuberan B. Lech M.Z. Beeler D.L. Wu Z.L. Rosenberg R.D. Nat. Biotechnol. 2003; 21: 1343-1346Crossref PubMed Scopus (134) Google Scholar). The blood coagulation pathway is composed of a cascade of proteolytic reactions ultimately generating fibrin thrombi. The proanticoagulant activity of this cascade is balanced by several natural anticoagulant mechanisms. Binding of HS to antithrombin (AT) represents the most important of these mechanisms. HS achieves its anticoagulant activity by interacting with AT, which undergoes a conformation change, generating the active form of AT to inhibit blood coagulation factors Xa and thrombin. This anticoagulant process prevents the formation of deleterious blood clots. Heparin, a specialized HS found in mast cells, is the most commonly used anticoagulant drug. Anticoagulant HS and heparin contain structurally defined AT binding pentasaccharide sequences with the structure, -GlcNS(or Ac)6S-GlcUA-GlcNS3S±6S-IdoUA2S-GlcNS6S- (GlcUA is glucuronic, and IdoUA is iduronic acid) (Fig. 1) (6Atha D.H. Lormeau J.-C. Petitou M. Rosenberg R.D. Choay J. Biochemistry. 1985; 24: 6723-6729Crossref PubMed Scopus (189) Google Scholar). The 3-O-sulfo glucosamine unit (GlcNS3S±6S) in this binding site is essential for interaction with AT. The lack of a 3-O-sulfo group in this unit decreases AT binding affinity by 18,000-fold (6Atha D.H. Lormeau J.-C. Petitou M. Rosenberg R.D. Choay J. Biochemistry. 1985; 24: 6723-6729Crossref PubMed Scopus (189) Google Scholar). A recently approved anticoagulant drug, Arixtra, prepared by organic synthesis is based on this pentasaccharide scaffold. The recently reported enzymatic synthesis of a similar pentasaccharide utilizes 3-OST-1 in the last modification step (5Kuberan B. Lech M.Z. Beeler D.L. Wu Z.L. Rosenberg R.D. Nat. Biotechnol. 2003; 21: 1343-1346Crossref PubMed Scopus (134) Google Scholar) and opens up a new approach to preparation of such anticoagulants. The essential physiological role of 3-O-sulfo HS (or anticoagulant HS) in blood coagulation is best demonstrated through the study of AT and AT mutants. A severe thrombosis phenotype is observed in mice carrying an AT mutant defective in heparin binding, suggesting a key role for HS-AT interaction in balancing procoagulant and anticoagulant activities in vivo (7Dewerchin M. Herault J.-P. Wallays G. Petitou M. Schaeffer P. Millet L. Weitz J.I. Moons L. Collen D. Carmeliet P. Herbert J.-M. Cir. Res. 2003; 93: 1120-1126Crossref PubMed Scopus (28) Google Scholar). Additionally, patients with AT mutants defective in heparin and HS binding suffer from thrombosis (8Koide T. Odani S. Takahashi K. Ono T. Sakuragawa N. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 289-293Crossref PubMed Scopus (154) Google Scholar, 9van Boven H.H. Lane D.A. Semin. Hematol. 1997; 34: 188-204PubMed Google Scholar, 10Boyer C. Wolf M. Vedrenne J. Meyer D. Larrieu M.J. Thromb. Haemostasis. 1986; 56: 18-22Crossref PubMed Scopus (36) Google Scholar). Furthermore, it appears that complete ablation of AT-HS binding is required to reveal the full physiological role of anticoagulant HS in vivo (11Weitz J.I. J. Clin. Investig. 2003; 111: 952-954Crossref PubMed Scopus (41) Google Scholar). The final step in the biosynthesis of anticoagulant HS can be catalyzed by either 3-OST-1 or 3-OST-5 isoforms (12Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 13Duncan M.B. Chen J. Krise J.P. Liu J. Biochim. Biophys. Acta. 2004; 1671: 34-43Crossref PubMed Scopus (32) Google Scholar). Gene redundancy in the biosynthesis of anticoagulant HS helps to explain why 3-OST-1 knockout mice failed to exhibit a prothrombotic phenotype (14HajMohammadi S. Enjyoji K. Princivalle M. Christi P. Lech M. Beeler D.L. Rayburn H. Schwartz J.J. Barzegar S. de Agostini A.I. Post M.J. Rosenberg R.D. Shworak N.W. J. Clin. Investig. 2003; 111: 989-999Crossref PubMed Scopus (140) Google Scholar). Heparan sulfate 3-O-sulfotransferase (3-OST) is present in at least six different isoforms that have unique expression patterns in human tissues (15Liu J. Rosenberg R.D. Taniguchi N. Fukuda M. Handbook of Glycosyltransferases and Their Related Genes. Springer-Verlag, Tokyo2002: 475-483Google Scholar, 16Xia G. Chen J. Tiwari V. Ju W. Li J.-P. Malmström A. Shukla D. Liu J. J. Biol. Chem. 2002; 277: 37912-37919Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The amino acid sequences of the different isoforms have 50-80% homology in their sulfotransferase domains (15Liu J. Rosenberg R.D. Taniguchi N. Fukuda M. Handbook of Glycosyltransferases and Their Related Genes. Springer-Verlag, Tokyo2002: 475-483Google Scholar). These different 3-OST isoforms transfer sulfuryl groups to the 3-OH position of glucosamine units residing within the context of different saccharide sequences. As a result, the HS products generated by these different isoforms exhibit unique and distinctive biological activities. It is known that HS modified by 3-OST-1 and 3-OST-5 display anticoagulant activity, whereas HS modified by 3-OST-3 and 3-OST-5 serve as entry receptors for herpes simplex virus-1 (12Liu J. Shworak N.W. Fritze L.M.S. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 16Xia G. Chen J. Tiwari V. Ju W. Li J.-P. Malmström A. Shukla D. Liu J. J. Biol. Chem. 2002; 277: 37912-37919Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 17Shukla D. Liu J. Blaiklock P. Shworak N.W. Bai X. Esko J.D. Cohen G.H. Eisenberg R.J. Rosenberg R.D. Spear P.G. Cell. 1999; 99: 13-22Abstract Full Text Full Text PDF PubMed Scopus (868) Google Scholar). The structural features of 3-OST isoforms that dictate substrate specificity are currently unknown. The sulfotransferase family can be organized into two categories based on the sub-cellular localizations; they are cytosolic and Golgi sulfotransferases (18Negishi M. Pedersen L.G. Petrotchenko E. Shevtsov S. Gorokhov A. Kakuta Y. Pedersen L.C. Arch. Biochem. Biophys. 2001; 390: 149-157Crossref PubMed Scopus (267) Google Scholar, 19Grunwell J.R. Bertozzi C.R. Biochemistry. 2002; 41: 13117-13126Crossref PubMed Scopus (29) Google Scholar). The crystal structures of different sulfotransferases reveal structural similarity in their PAPS binding sites between the cytosolic and Golgi sulfotransferases, suggesting that different sulfotransferases probably follow similar mechanisms in the transfer of the sulfuryl group even though they exhibit high selectivity in substrate binding (20Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (232) Google Scholar, 21Kakuta Y. Sueyoshi T. Negishi M. Pedersen L.C. J. Biol. Chem. 1999; 274: 10673-10676Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The structures of a number of cytosolic sulfotransferases complexed with acceptor substrates have been solved, providing clues that have led to a better understanding of their catalytic mechanism and mode of substrate recognition (22Kakuta Y. Petrotchenko E.V. Pedersen L.C. Negishi M. J. Biol. Chem. 1998; 273: 27325-27330Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 23Lee K.A. Fuda H. Lee Y.C. Negishi M. Strott C.A. Pedersen L.C. J. Biol. Chem. 2003; 278: 44593-44599Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 24Pedersen L.C. Petrotchenko E. Shevtsov S. Negishi M. J. Biol. Chem. 2002; 277: 17928-17932Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). HS sulfotransferases are considered to be Golgi sulfotransferases. The crystal structure of the binary complex of the sulfotransferase domain of the HS N-deacetylase/N-sulfotransferase (NST-1) and 3′-phosphoadenosine 5′-phosphate (PAP) was solved by Kakuta et al. (21Kakuta Y. Sueyoshi T. Negishi M. Pedersen L.C. J. Biol. Chem. 1999; 274: 10673-10676Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). This represents the first and only structure of a HS sulfotransferase previously determined. In this study, we report the crystal structure of a binary complex of mouse 3-OST-1 and PAP. The structure of this enzyme is very similar to NST-1 but with unique structural features in the HS binding cleft. Results from mutational analysis of amino acid residues at and around the active site in 3-OST-1 reveal a series of amino acid residues that is critical for sulfotransferase activity. These results, when combined with a structural alignment to NST-1 and sequence alignments to other isoforms of 3-OST, provide insight into the role certain residues may play in catalysis and dictating substrate specificity for these HS sulfotransferases. Materials—Full-length mouse 3-OST-1 cDNA (m3-OST-1-pcDNA3) was purified from a mouse L-cell cDNA library (25Shworak N.W. Liu J. Fritze L.M.S. Schwartz J.J. Zhang L. Logeart D. Rosenberg R.D. J. Biol. Chem. 1997; 272: 28008-28019Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). [35S]PAPS was prepared by incubating 0.4-2 mCi/ml [35S]Na2SO4 (carrier-free, MP Biomedical) and 16 mm ATP with 5 mg/ml dialyzed yeast extract (Sigma) (26Bame K.J. Esko J.D. J. Biol. Chem. 1989; 264: 8059-8065Abstract Full Text PDF PubMed Google Scholar). HS from bovine kidney was purchased from MP Biomedical (Irvine, CA). PAP was purchased from Sigma. Preparation of 3-OST-1 Bacterial Expression Plasmid (b3-OST-1-pET28)—The cDNA fragment encoding the catalytic domain of 3-OST-1 (G48-H311) was amplified from m3-OST-1-pcDNA3 with a 5′ overhang containing an NdeI site and a 3′ overhang containing an EcoRI site. This construct was inserted into the pET28a vector (Novagen) using the NdeI and EcoRI restriction sites to produce a His6-tagged protein. The resultant plasmid (b3-OST-1-pET28) was sequenced to confirm the reading frame and the lack of mutations within the coding region (University of North Carolina, DNA sequencing core facility). The plasmid, b3-OST-1-pET28, was transformed into BL21(DE3)RIL cells (Stratagene) for the expression of 3-OST-1. Protein Expression and Purification—Cells containing the b3-OST-1-pET28 were grown in 12 2.8-liter Fernbach flasks containing 1 liter of LB media with 50 μg/ml kanamycin at 37 °C. When the A600 reached 0.6-0.8, the temperature was lowered to 22 °C for 15 min. Isopropyl-β-d-thiogalactopyranoside was then added to a final concentration of 200 μm, and the cells were allowed to shake overnight. Cells were pelleted and resuspended in 120 ml of sonication buffer, 25 mm Tris, pH 7.5, 500 mm NaCl, and 10 mm imidazole. Cells were disrupted by sonication then spun down. The supernatant was applied to nickel nitrilotriacetic acid-agarose resin (Qiagen) in batch and washed with sonication buffer. The resin was loaded onto a column, and the protein was eluted with an imidazole gradient from 10 to 250 mm. The protein was dialyzed then concentrated to 16 mg/ml in 20 mm Tris, pH 7.5, 100 mm NaCl, and 4 mm PAP. A total of 28 mg of protein was obtained. Protein Crystallization and Structure Solution—Crystals of 3-OST-1 were grown using the hanging drop method at 4 °C by mixing 2 μl of the protein solution with 2 μl of the reservoir solution containing 0.1 m citrate, pH 5.5, and 11% polyethylene glycol 4000. Before data collection, crystals were transferred to a solution containing 0.1 m citrate, pH 5.5, 20% polyethylene glycol 4000, 0.1 m NaCl, 4 mm PAP, and 12.5% ethylene glycol. The crystals were mounted in a loop and flash-frozen in liquid nitrogen. Data were collected at -180 °C on a RaxisIV area detector for the low resolution data set. A high resolution data set was collected on another crystal at the Advanced Photon Source on SERCAT beamline 22 using a MAR225 area detector. Both data sets were processed using HKL2000 (Table I) (27Otwinowski Z. Minor V. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38526) Google Scholar). A model of NST-1 (21Kakuta Y. Sueyoshi T. Negishi M. Pedersen L.C. J. Biol. Chem. 1999; 274: 10673-10676Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) consisting of residues 603-632, 642-664, 670-736, 744-868, and PAP was used as a search molecule for molecular replacement using Molrep in CCP4 with the low resolution data set (28Bailey S. Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (41) Google Scholar, 29Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4148) Google Scholar). The positions of two (A and B) of the three final molecules in the asymmetric unit were found by molecular replacement. These two molecules were refined in CNS (30Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar) and improved by iterative cycles of model building in O (31Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13009) Google Scholar) and refinement in CNS until an Rfree value of 35% was obtained. At this point, sparse electron density became visible for the third molecule (C). A copy of molecule A was manually inserted into the density and refined. Density for molecule C was very poor, and the B-factors were very high, so refinement was carried out using non-crystallographic restraints between the third molecule and the first molecule. Residues for molecule C, which contained no electron density, were deleted. Refinement of molecule C dropped the Rfree to 31%. The final model was obtained by iterative cycles of model building and refinement of the three molecules against the high resolution data set. The final molecule contains residues 54-311 for molecule A and B and 54-269 and 281-311 for molecule C.Table ICrystallographic data statisticsData set3-OST-1 (high)3-OST-1 (low)Unit cell dimensionsa = b = 300.14 c = 84.20a = b = 298.69 c = 83.82α = β = γ = 90°α = β = γ = 90°Space groupI4 (1) 22I4 (1) 22No. of observations741,873180,101Unique reflections63,92843,945Rsym (%) (last shell)aRsym=∑(|Ii−⟨I⟩|)/∑(Ii) where Ii is the intensity of the Ith observation, and ⟨I⟩ is the mean intensity of the reflection.11.6 (37.1)13.7 (61.5)I/σI (last shell)7.4 (1.7)6.7 (1.4)Mosaicity0.410.62Completeness (%) (last shell)96.7 (83.6)93.9 (82.4)Refinement statisticsResolution (Å)25.0-2.525.0-2.8Rcryst (%)bRcryst=∑||Fo|−|Fo||∑|Fo| calculated from working data set.24.3Rfree (%)cRfree was calculated from 5% of data randomly chosen not to be included in refinement.26.4No. of water178Mean B value (Å)58.3For:Complex AComplex BComplex CProtein43.0639.17100.69PAP31.2333.1869.76Root mean square deviation from ideal valuesdRamachandran results were determined by MolProbity.Bond length (Å)0.009Bond angle (°)1.4Dihedral angle (°)22.4Improper angle (°)0.95Ramachandran statistics94.57% residues are in favored (98%) regions99.74% residues are in allowed (>99.8%) regionseTwo residues from complex C, which lie outside this region, are in allowed regions based on PROCHECK (28).a Rsym=∑(|Ii−⟨I⟩|)/∑(Ii) where Ii is the intensity of the Ith observation, and ⟨I⟩ is the mean intensity of the reflection.b Rcryst=∑||Fo|−|Fo||∑|Fo| calculated from working data set.c Rfree was calculated from 5% of data randomly chosen not to be included in refinement.d Ramachandran results were determined by MolProbity.e Two residues from complex C, which lie outside this region, are in allowed regions based on PROCHECK (28Bailey S. Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (41) Google Scholar). Open table in a new tab Preparation of 3-OST-1 Mutant Plasmids—A total of 28 point mutants of 3-OST-1 were prepared using b3-OST-1-pET28 as the template and the Gene Tailor site-directed mutagenesis kit from Invitrogen. The lengths and sequences of the primers for preparation of those mutants were designed based on the manufacturer's protocol for this mutagenesis kit and were synthesized by Invitrogen. The resultant constructs were sequenced to confirm the anticipated mutation (University of North Carolina, DNA sequencing core facility). Expression and Purification of 3-OST-1 Mutants—The expression plasmids for various 3-OST-1 mutants were transformed individually into BL21(DE3)RIL cells (Stratagene). Each mutant construct was grown in 100 ml of LB broth and induced by isopropyl-β-d-thiogalactopyranoside as described above. The bacteria cells were harvested and solubilized in sonication buffer before sonication. The lysate was subjected to a 400-μl nickel nitrilotriacetic acid-agarose column (0.75 × 1 cm) followed by a 5-ml of wash with sonication buffer. Mutant proteins were eluted with 1 ml of elution buffer containing 25 mm Tris, 500 mm NaCl, and 250 mm imidazole, pH 7.5. Approximately 25 μl of the eluent was subjected to the analysis on a 16.5% Tris-Tricine PAGE gel (Bio-Rad), and the gel was stained by Coomassie Blue. The expression level of the mutant protein was estimated by determining the intensity of the Coomassie-stained protein band near 30 kDa. As a positive control, we expressed wild type 3-OST-1 (b3-OST-1-pET28) along with the mutants. The expression levels and the sulfotransferase activity of mutant proteins were normalized to those of wild type protein. Several clones harboring mutant proteins, including R67E, K68A, R72E, E90Q, K123A, and R276A, were grown in 2-4-liter cultures to obtain sufficient amounts of protein for analysis by isothermal titration calorimetry (ITC). Procedures for expression and purification of these mutants were essentially identical to those for the wild type 3-OST-1. Mutant protein was purified by nickel nitrilotriacetic acid-agarose chromatography, and purity was estimated to be greater than 80% by a 16.5% Tris-Tricine PAGE gel. Determination of the Sulfotransferase Activity—Sulfotransferase activity was determined by incubating ∼5 μl (10-100 ng) of purified mutant or wild type 3-OST-1 proteins with 10 μg of HS (from bovine kidney, ICN) and 5-10 × 104 cpm of [35S]PAPS (∼10 μm) in 50 μl of buffer containing 50 mm MOPS, pH 7.0, 10 mm MnCl2,5mm MgCl2, and 1% Triton X-100. The reaction was incubated at 37 °C for 30 min and quenched by the addition of 6 m urea and 100 mm EDTA. The sample was then subjected to a 200-μl DEAE-Sepharose chromatography to purify the [35S]HS. The quantity of [35S]HS was then determined by liquid scintillation counting. The negative control contained all the components with the exception of 3-OST-1 proteins. Determination of the Binding of AT and 3-O-Sulfated HS—Approximately 5 × 106 cpm of [35S]HS was incubated with 5 μg of human AT (Cutter Biological) in 50 μl of reaction buffer containing 10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm Mn2+, 1 mm Mg2+, 1 mm Ca2+, 10 μm dextran sulfate, 0.02% sodium azide, and 0.0004% Triton X-100 for 30 min at room temperature. 60 μl of 1:1 slurry of pretreated concanavalin A-Sepharose (from Sigma) was added, and the reaction was agitated for 1 h at room temperature on an orbital shaker. The gel was washed three times with the buffer containing 10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm Mn2+, 1 mm Mg2+, 1 mm Ca2+, 10 μm dextran sulfate, 0.02% sodium azide, and 0.0004% Triton X-100, and the bound [35S]HS was eluted with the same buffer containing 1000 mm NaCl. ITC—ITC was performed on a MicroCal VP-ITC. Solutions were cooled to 10 °C and degassed under vacuum before use. Experiments were conducted using 17-21 μm protein in 100 mm phosphate buffer, pH 7.0) and 100 mm NaCl at 10 °C. Titrations were performed by injecting 5 μl of 4 mm PAP in 100 mm phosphate buffer, pH 7.0, and 100 mm NaCl. Data analysis was completed using Origin software. Determination of PAP Binding Affinity Using PAP Chromatography—To determine the binding affinity of the wild type and mutant proteins to PAP, 3′,5′-ADP-agarose chromatography (Sigma) was used. Approximately 200 μg of proteins, including wild type 3-OST-1, K68A, H92F, and D95N and R276A, in 5 ml of a buffer containing 25 mm Tris, pH 7.0, and 100 mm NaCl was loaded onto a 3′,5′-ADP-agarose column (7 × 52 mm) pre-equilibrated with 25 mm Tris, pH 7.0, and 100 mm NaCl at a flow rate of 0.5 ml/min. Unbound material was removed by washing with 5 ml of 25 mm Tris, pH 7.0, and 100 mm NaCl. Bound proteins were eluted with a linear gradient of NaCl from 100 mm to 1 m in 10 ml. The samples from the collected fractions were analyzed by SDS-PAGE followed by staining with Coomassie Blue. Overall Fold—The catalytic domain of mouse 3-OST-1 (G48-H311) was successfully expressed in Escherichia coli. The catalytic domain has higher solubility than the full-length protein, making it more amenable to crystallization. The 3-OST-1 protein crystallized in the space group I4122 with 3 protein molecules (A, B, and C) in the asymmetric unit. Each molecule of 3-OST-1 contains one molecule of PAP bound. Two of the protein molecules (A and B) in the asymmetric unit stack together, forming hollow channels with the long dimension running parallel to the c axis of the unit cell. The rim of the channel is composed of 4 molecules of both A and B. The hollow inner solvent channel has a diameter of ∼62 Å. The third molecule (C) is involved in cross-linking these channels by interacting with molecules B and a molecule C coming from another channel. This packing arrangement creates a cell with an overall solvent content of 76% and a Matthews coefficient of 5.5 (32Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7917) Google Scholar). Molecules A and B are well ordered and have similar overall B-factors (Table I). Molecule C, however, is highly disordered, with overall B-factors ∼2.5 times greater than molecules A and B, suggesting it may bind in slightly different orientations or have only partial occupancy in the crystal lattice. Because molecule C is highly disordered, the discussion of the structure will focus on molecules A and B. The crystal structure of the 3-OST-1·PAP complex (Fig. 2a)is roughly spherical and contains a large open cleft. The structure is centered around an α/β motif common to all sulfotransferases (18Negishi M. Pedersen L.G. Petrotchenko E. Shevtsov S. Gorokhov A. Kakuta Y. Pedersen L.C. Arch. Biochem. Biophys. 2001; 390: 149-157Crossref PubMed Scopus (267) Google Scholar). This motif consists of a five-stranded parallel β-sheet flanked on both sides by α helices. At the heart of the fold is a strand-loop-helix motif (Thr-61-Ser-79) consisting of the first β-strand (The-61-Ile-64) and the first α-helix (Thr-71-Ser-79), which contains the phosphosulfate binding (PSB) loop (Gly-65-Gly-70) (22Kakuta Y. Petrotchenko E.V. Pedersen L.C. Negishi M. J. Biol. Chem. 1998; 273: 27325-27330Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). This loop is very similar in structure to the P-loop found in protein kinases and forms extensive interactions with the 5′-phosphate of the PAP (Fig. 2b). As in other sulfotransferases, the helix that runs across the top of the PAP binding pocket and into the open cleft is also present. The C-terminal portion of the enzyme consists of a short three-stranded anti-parallel β-sheet. Strands 2 and 3 of this sheet are stabilized by a disulfide bond (Cys-260-Cys-269). A coil consisting of residues 270-281 connects this sheet to the C-terminal helix. This coil practically buries the PAP molecule in the active site and may be susceptible to a conformational change since it is ordered in molecules A and B and there is no electron density visible for it in molecule C. The overall fold of 3-OST-1 is most similar to that of the sulfotransferase domain of NST-1 (21Kakuta Y. Sueyoshi T. Negishi M. Pedersen L.C. J. Biol. Chem. 1999; 274: 10673-10676Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) (Fig. 3a). The root mean square of 230 structurally equivalent Cαs is 1.3 Å, as determined by the program O (31Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13009) Google Scholar). All of the significant secondary structural features are conserved between the two enzymes. The major differences occur in the loop regions as described below. PAP Binding Site—A number of hydrogen-bonding interactions are involved in positioning the PAP molecule within the active site (Fig. 2b). Side chains from residues Lys-68 and Thr-71 of the PSB loop and Lys-274 of the coil that buries the PAP both form interactions with the 5′-phosphate (Table II). In addition, backbone amide nitrogens from the PSB loop (Gly-70, Thr-71, and Arg-72) are also within hydrogen-bonding distance of the 5′-phosphate. Although there are no protein interactions with the ribose ring, atom N6 of the adenosine base is" @default.
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• W2014549797 title "Crystal Structure and Mutational Analysis of Heparan Sulfate 3-O-Sulfotransferase Isoform 1" @default.
• W2014549797 cites W1520872047 @default.
• W2014549797 cites W1539796472 @default.
• W2014549797 cites W1964160816 @default.
• W2014549797 cites W1964746058 @default.
• W2014549797 cites W1967627631 @default.
• W2014549797 cites W1971540677 @default.
• W2014549797 cites W1979155909 @default.
• W2014549797 cites W1979360414 @default.
• W2014549797 cites W1981520657 @default.
• W2014549797 cites W1983174721 @default.
• W2014549797 cites W1985863112 @default.
• W2014549797 cites W1987792040 @default.
• W2014549797 cites W1995017064 @default.
• W2014549797 cites W2001371234 @default.
• W2014549797 cites W2007530420 @default.
• W2014549797 cites W2010846012 @default.
• W2014549797 cites W2013083986 @default.
• W2014549797 cites W2014694459 @default.
• W2014549797 cites W2019074842 @default.
• W2014549797 cites W2028231353 @default.
• W2014549797 cites W2044804476 @default.
• W2014549797 cites W2051005823 @default.
• W2014549797 cites W2052953335 @default.
• W2014549797 cites W2058663941 @default.
• W2014549797 cites W2068608069 @default.
• W2014549797 cites W2080528351 @default.
• W2014549797 cites W2080807893 @default.
• W2014549797 cites W2097493124 @default.
• W2014549797 cites W2115409006 @default.
• W2014549797 cites W2123115870 @default.
• W2014549797 cites W2156014566 @default.
• W2014549797 cites W2156496950 @default.
• W2014549797 cites W2171575560 @default.
• W2014549797 cites W2374418898 @default.
• W2014549797 cites W2471776857 @default.
• W2014549797 cites W4242945710 @default.
• W2014549797 cites W4251915841 @default.
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