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Crystal Structure of the Sulfotransferase Domain of Human Heparan SulfateN-Deacetylase/N-Sulfotransferase 1

1999; Elsevier BV; Volume: 274; Issue: 16 Linguagem: Inglês

10.1074/jbc.274.16.10673

1083-351X

Yoshimitsu Kakuta, Tatsuya Sueyoshi, Masahiko Negishi, Lars C. Pedersen,

Protein Kinase Regulation and GTPase Signaling

Heparan sulfateN-deacetylase/N-sulfotransferase (HSNST) catalyzes the first and obligatory step in the biosynthesis of heparan sulfates and heparin. The crystal structure of the sulfotransferase domain (NST1) of human HSNST-1 has been determined at 2.3-Å resolution in a binary complex with 3′-phosphoadenosine 5′-phosphate (PAP). NST1 is approximately spherical with an open cleft, and consists of a single α/β fold with a central five-stranded parallel β-sheet and a three-stranded anti-parallel β-sheet bearing an interstrand disulfide bond. The structural regions α1, α6, β1, β7, 5′-phosphosulfate binding loop (between β1 and α1), and a random coil (between β8 and α13) constitute the PAP binding site of NST1. The α6 and random coil (between β2 and α2), which form an open cleft near the 5′-phosphate of the PAP molecule, may provide interactions for substrate binding. The conserved residue Lys-614 is in position to form a hydrogen bond with the bridge oxygen of the 5′-phosphate. Heparan sulfateN-deacetylase/N-sulfotransferase (HSNST) catalyzes the first and obligatory step in the biosynthesis of heparan sulfates and heparin. The crystal structure of the sulfotransferase domain (NST1) of human HSNST-1 has been determined at 2.3-Å resolution in a binary complex with 3′-phosphoadenosine 5′-phosphate (PAP). NST1 is approximately spherical with an open cleft, and consists of a single α/β fold with a central five-stranded parallel β-sheet and a three-stranded anti-parallel β-sheet bearing an interstrand disulfide bond. The structural regions α1, α6, β1, β7, 5′-phosphosulfate binding loop (between β1 and α1), and a random coil (between β8 and α13) constitute the PAP binding site of NST1. The α6 and random coil (between β2 and α2), which form an open cleft near the 5′-phosphate of the PAP molecule, may provide interactions for substrate binding. The conserved residue Lys-614 is in position to form a hydrogen bond with the bridge oxygen of the 5′-phosphate. Heparan sulfate chains are ubiquitous as proteoglycans on cell surfaces and in the extracellular matrix. They have been increasingly implicated in various biological processes including cell growth, cell differentiation, blood coagulation, and viral and bacterial infections (1Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (396) Google Scholar, 2Rosenberg R.D. Shworak N.W. Liu J. Schwartz R.J. Zhang L. J. Clin. Invest. 1997; 99: 2062-2070Crossref PubMed Scopus (256) Google Scholar). Reduced biosynthesis of heparan sulfates, for instance, results in defective WINGLESS/WNT signaling in Drosophila (3Hacker U. Lin X. Perrimon Development. 1997; 124: 3565-3573Crossref PubMed Google Scholar, 4Cumberledge S. Reichsman F. Trends Genet. 1997; 13: 421-423Abstract Full Text PDF PubMed Scopus (48) Google Scholar). Mice lacking the heparan sulfate 2-O-sulfotransferase gene die neonatally from defective kidney development (5Bullock S.L. Fletcher J.M. Beddington R.S. Wilson V.A. Genes Dev. 1998; 12: 1894-1906Crossref PubMed Scopus (400) Google Scholar). The recent crystal structures of the fibroblast growth factor-heparin and antithrombin-heparin complexes have shown specific protein-sulfate interactions (6DiGabriele D.A. Lax I. Chen I.D. Svahn M.C. Jaye M. Schessinger J. Henderickson A.W. Nature. 1998; 393: 812-817Crossref PubMed Scopus (327) Google Scholar, 7Jin L. Abrahams P.J. Skinner R. Petitou M. Pike N.R. Carrell W.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14683-14688Crossref PubMed Scopus (638) Google Scholar). Modification by sulfation thus, can alter functional specificity and diversity of heparans and heparins. Heparan sulfation is catalyzed by a group of the Golgi-membrane enzymes called heparan sulfate sulfotransferases. The superfamily includes also a large number of cytosolic sulfotransferases that sulfate low molecular weight substrates such as steroids, bioamines, pharmaceutical drugs, and environmental chemicals. The membrane and cytosolic sulfotransferases share little overall sequence similarity, whereas all sulfotransferases use 3′-phosphoadenosine 5′-phosphosulfate (PAPS) 1The abbreviations used are: PAPS, 3′-phosphoadenosine 5′-phosphosulfate; PAP, 3′-phosphoadenosine 5′-phosphate; PSB-loop, 5′-phosphosulfate binding loop; 5′PSB, 5′-phosphosulfate binding motif; 3′PB, 3′-phosphate binding motif; NST1, the sulfotransferase domain of heparan sulfate N-deacetylase/N-sulfotransferase; EST, estrogen sulfotransferase. as the ubiquitous sulfate donor. The crystal structure of the estrogen sulfotransferase (EST)-PAP-estradiol (E2) complex has revealed the structural motifs for the 5′- and 3′-phosphate binding of PAP (8Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (232) Google Scholar, 9Kakuta Y. Pedersen L.G. Pedersen L.C. Negishi M. Trends Biochem. Sci. 1998; 23: 129-130Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). It remains to be structurally determined whether these motifs are also conserved in heparan sulfate sulfotransferases. Multiple sequence alignments have suggested that these motifs may be conserved (9Kakuta Y. Pedersen L.G. Pedersen L.C. Negishi M. Trends Biochem. Sci. 1998; 23: 129-130Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The reaction mechanisms and specific substrate binding that lead to the diverse heparan sulfations are poorly understood. The bi-functional enzyme HSNST sequentially deacetylates and sulfates the amino group of the disaccharide glucuronic acid-N-acetylglucosamine (GlcA-GlcNAc) unit of heparan sulfate (10Eriksson I. Sandback C.B. Ek B. Lindahl U. Kjellen L. J. Biol. Chem. 1994; 269: 8044-8049PubMed Google Scholar, 11Wei Z. Swiedler S.J. Ishihara M. Orellaana A. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 90: 3885-3888Crossref Scopus (67) Google Scholar). Amino acid sequence alignment of the human HSNST1 with EST identified the N-sulfotransferase domain (NST1) (12Sueyoshi T. Kakuta Y. Pedersen L.C. Wall F.E. Pedersen L.G. Negishi M. FEBS Lett. 1998; 433: 211-214Crossref PubMed Scopus (47) Google Scholar). Human NST1 is similar to the corresponding domain of mouse HSNST reported by Berninsone and Hirschberg (13Berninsone P. Hirschberg C.B. J. Biol. Chem. 1998; 273: 25556-25559Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Subsequently, site-directed mutagenesis has shown that Lys-614 is a critical residue for NST1 catalysis (12Sueyoshi T. Kakuta Y. Pedersen L.C. Wall F.E. Pedersen L.G. Negishi M. FEBS Lett. 1998; 433: 211-214Crossref PubMed Scopus (47) Google Scholar). Using NST1 crystals grown as previously reported (12Sueyoshi T. Kakuta Y. Pedersen L.C. Wall F.E. Pedersen L.G. Negishi M. FEBS Lett. 1998; 433: 211-214Crossref PubMed Scopus (47) Google Scholar), we now describe herein the crystallographic structure NST1. This structure displays the conserved nature of the structure of the PAP-binding site and identifies possible catalytic residues. In addition, a substrate binding site is suggested from the structure. Selenomethionyl NST1, using a pGEX-4T3-NST1 plasmid, was expressed in the methionine auxotrophic Escherichia colistrain B834 (DE3) with a defined minimal essential medium (without methionine) containing 50 mg of selenomethionine per 1 liter of culture. The NST1 was then purified, and crystals (P21212 or P21) were grown under the same conditions as described previously (14Otwinowski Z. Minor W. Methods Enzymol. 1996; 276: 307-326Crossref Scopus (38573) Google Scholar). Heparan sulfate sulfotransferase activity of NST1 was also measured according to the previously described procedure (12Sueyoshi T. Kakuta Y. Pedersen L.C. Wall F.E. Pedersen L.G. Negishi M. FEBS Lett. 1998; 433: 211-214Crossref PubMed Scopus (47) Google Scholar). Two MAD data sets of selenomethionyl NST1 were collected at −180 °C from two separate single crystals (both P21212) on a MAR detector at beamline X9B of the NSLS, Brookhaven National Laboratory. Three wavelengths were selected from the fluorescence spectra: f1 (0.97163 Å: remote), f2 (0.97907 Å: peak), and f3 (0.97940 Å: edge) (Table I). Native data of an NST1 crystal (P21) were collected at −180 °C on an R-axis IV with an RU300 rotating anode generator.Table IData collection and refinement statisticsData collectionData setf1 (set 1)f2 (set 1)f3 (set 1)f1 (set 2)f2 (set 2)f3 (set 2)nativeNSLSNSLSNSLSNSLSNSLSNSLSRaxis IVSpace groupP21212P21212P21212P21212P21212P21212P21Unit cell parameters (Å)a = 89.13a = 89.50a = 89.44a = 89.24a = 88.73a = 88.46a = 45.42b = 55.36b = 55.60b = 55.55b = 55.42b = 55.25b = 55.15b = 54.50c = 66.41c = 67.01c = 66.92c = 66.68c = 66.44c = 66.36c = 68.94beta = 100.053No. of crystals1111111Resolution (Å)2.852.852.853.23.23.22.3No. of unique reflections64396897661358005731570513992Redundancy6.56.36.44.56.76.72.6% Completeness (total)79.583.780.510010099.993.7(last shell)99.690.285.710010010080R sym(total)1R sym = ∑‖I i− 〈I〉‖/∑I i, whereI i is the intensity of the ith observation and 〈I〉 is the mean intensity of the reflection.9.811.19.312.115.49.49.8(last shell)25.029.425.320.626.916.124.1Refinement statisticsData setnativeResolution (Å)50–2.3R cryst/R free(%)1R = ∑∥F o‖ − ‖F c∥/∑‖F o‖, whereR cryst is calculated using 95% of the reflections in refinement and R free is calculated using the remaining 5%.21.0/25.7No. of waters73r.m.s. deviations from idealityBond lengths (Å)0.007Bond angles (deg)1.3Dihetral angles (deg)25.0Improper angles (deg)0.63a R sym = ∑‖I i− 〈I〉‖/∑I i, whereI i is the intensity of the ith observation and 〈I〉 is the mean intensity of the reflection.b R = ∑∥F o‖ − ‖F c∥/∑‖F o‖, whereR cryst is calculated using 95% of the reflections in refinement and R free is calculated using the remaining 5%. Open table in a new tab All data were processed using SCALEPACK and DENZO (14Otwinowski Z. Minor W. Methods Enzymol. 1996; 276: 307-326Crossref Scopus (38573) Google Scholar). Because of heavy ice rings in data set 1 and data set 2 being weak, F(A)s were calculated with CCP4 (15Collaborative Computational Project Number 4 Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar) using data between 20 and 4 Å of data set 1. Positions for four of the six selenium atoms were determined using SHELX96 (16Sheldrick G.M. Dauter Z. Wilson K.S. Hope H. Sieker L.C. Acta Crystallogr. Sec. D. 1993; 49: 18-23Crossref PubMed Google Scholar). Data set 1 was reprocessed to eliminate all reflection near the ice rings between 3.95 and 3.1 Å. Subsequently, the reprocessed data set 1 was merged with data set 2 to obtain a complete data set to 2.85-Å resolution. SHARP (17delaFortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar) was then used for refinement of the selenium sites. Solvent flattening and histogram matching were carried out using DM and Solomon from CCP4 (15Collaborative Computational Project Number 4 Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar). In the model building process using O (18Jones T.A. Zou J.Y. Cowan S.W. Kjeledgaard M. Acta Crystallogr. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar), SigmaA maps were generated by combining the phases from polyalanine fragments with the MAD phases (15Collaborative Computational Project Number 4 Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar). After multiple cycles of positional, torsion angle, and b factor refinements using X-PLOR (19Brunger A.T. Kuriyan J. Karplus M. Science. 1987; 235: 458-460Crossref PubMed Scopus (2126) Google Scholar), the R-factor and R free were 23.8 and 31.9%, respectively. Because some of the loop regions still lacked interpretable density, molecular replacement was employed to determine phases for the data from the P21 crystal. Multiple cycles of manual rebuilding and refinement using the P21 data at 2.3 Å reduced the R andR free factors to 20.9 and 25.8%, respectively. The stereochemistry of the refined model was verified using PROCHECK (15Collaborative Computational Project Number 4 Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar). Data collection and refinement statistics are summarized in TableI. The coordinates have been deposited in the Protein Data Bank with code 1NST. The overall structure of NST1 is roughly spherical with an open cleft (Fig. 1 A). This structure is composed of a five-stranded parallel β-sheet (β1, β2, β3, β4, and β5) with α helices on both sides of the β-sheet (Fig. 1 B). This fold is similar to EST (the 1.9-Å r.m.s. deviation for 97 Cαs in β1, β3, β4, β5, α1, α6, α11, α12, and α13) as well as to the nucleotide binding motif observed in nucleotide kinases (20Murzin A.G. Brenner S.K. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5595) Google Scholar). The loop between β1 and α1 adopts the same PSB-loop configuration as the 5′-phosphate binding site of PAPS. A cavity formed between the PSB-loop, and α6 defines the PAP binding site. Three β strands (β6, β7, and β8) near the C terminus form an anti-parallel β-sheet with a single disulfide bond between β7 and β8. An open cleft that runs perpendicular to the PAP binding cavity is large enough to contain a hexasaccharide chain. The α6 and random coil between β2 and α2 constitute the cleft near the 5′-phosphate of PAP and thus may constitute part of the substrate binding site. The secondary structural elements that comprise the PAP binding site in NST1 and residues forming specific interactions to the PAP molecule are depicted (Fig. 2), respectively. The PSB-loop (residues 612–617) and α1 of NST1 constitute the 5′PSB motif and provide the major binding sites for the 5′-phosphate of the PAP molecule. Backbone amide nitrogens from PSB-loop residues 614–618 are all within hydrogen bonding distance of the 5′-phosphate. The side-chain Nζ of Lys-614 and the Oγs of both Thr-617 and Thr-618 are also hydrogen-bonded to the 5′-phosphate. α6 and β4 are the key elements of the 3′PB motif, and the Oγ of Ser-712 from this helix forms a hydrogen bond to the 3′-phosphate of the PAP molecule. The PAP molecule in NST1 is bound in the same orientation (relative to the PSB-loop) as seen in EST. The PAP binding site, found in the EST structure determined previously, is conserved. The r.m.s. deviation (with EST) for 47 Cαs in α1, α6, β1, β4, and PSB-loop is 1.16 Å. The anti-parallel β-sheet (β6, β7, and β8) and the following random coil provide the remaining interactions for the PAP binding site (Fig. 2). These interactions reveal diversity in the binding site of NST1. The side-chains of Lys-833 and Tyr-837 from this random coil are within hydrogen bonding distance to two oxygen atoms of the 5′-phosphate and the oxygen atom of the 3′-phosphate, respectively. Besides these side-chain interactions, the backbone nitrogens of Gly-834 and Arg-835 are also within hydrogen bonding distance of a 3′-phosphate oxygen of the PAP molecule. The adenine ring from the PAP molecule is in position to form a parallel ring stacking interaction with Phe-816 of β7. Moreover, the backbone oxygen of Trp-817 is within hydrogen bonding distance to the N-6 of the adenine. The interactions of these residues with the PAP molecule are unique features in NST1 that are not present in the crystal structure of the EST-PAP complex (8Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (232) Google Scholar). Lys-614 of NST1 is known to be conserved in other heparan sulfate sulfotransferases as well as in all cytosolic sulfotransferase (9Kakuta Y. Pedersen L.G. Pedersen L.C. Negishi M. Trends Biochem. Sci. 1998; 23: 129-130Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 12Sueyoshi T. Kakuta Y. Pedersen L.C. Wall F.E. Pedersen L.G. Negishi M. FEBS Lett. 1998; 433: 211-214Crossref PubMed Scopus (47) Google Scholar). Although this residue plays a critical role in NST1 activity (12Sueyoshi T. Kakuta Y. Pedersen L.C. Wall F.E. Pedersen L.G. Negishi M. FEBS Lett. 1998; 433: 211-214Crossref PubMed Scopus (47) Google Scholar), the structural basis of its role in catalysis has remained unresolved. The crystal structure of the EST-PAP-vanadate complex (Fig.3) has recently been solved and has provided a possible transition state template for the sulfuryl transfer reaction (21Kakuta 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). Superimposition of NST1 on this structure indicates that the side-chains of Lys-614 (in NST1) and Lys-48 (in EST) exhibit a similar orientation and conformation (Fig. 3). Nζ of Lys-614 is found to be directly coordinated to an oxygen of the PAP molecule in NST1. This oxygen is also coordinated to Lys-48 (Nζ) in EST and is implicated as the bridge oxygen of the leaving phosphate group of PAP (21Kakuta 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). Moreover, the mutation of Lys-614 to Arg gives a variant with a significant level of NST1 activity (63 ± 7.0 and 9.4 ± 2.4 nmol of sulfate/min/mg of protein in the wild-type and K614R mutant, respectively), whereas the K614A mutation abolishes activity completely (12Sueyoshi T. Kakuta Y. Pedersen L.C. Wall F.E. Pedersen L.G. Negishi M. FEBS Lett. 1998; 433: 211-214Crossref PubMed Scopus (47) Google Scholar). These structural and mutational data suggest that Lys-614 may act as a possible proton donor in catalysis, similar to Lys-48 in EST (21Kakuta 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). Lys-833 of NST1 is also coordinated with the bridge oxygen (Fig. 3). Lys-833 is, in fact, conserved not only in Caenorhabditis elegans HSNST but also in human heparan sulfate 3-O-sulfotransferase (see the sequence alignments in Shworaket al. (22Shworak W.N. Liu J. Fritze M.L Schwartz J.J. Zhang L. Logeart D. Rosenberg D.R. J. Biol. Chem. 1997; 272: 28008-28019Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar)). Thus, Lys-833 and its counterparts may play a significant role in catalysis. In sharp contrast to the hydrophobic pocket of estrogen binding site in EST, the putative substrate binding site of NST1 appears to be a large open cleft with a hydrophilic surface, with a random coil (residues 640–647, approximately 12 Å in length) and α6 forming the center of the cleft near the 5′-phosphate of the PAP molecule. This amphipathic random coil positions negatively charged side-chains (Glu-641, Glu-642, Gln-644, and Asn-647) toward the center, whereas the hydrophobic side-chains (Ile-643, Phe-645, and Phe-656) are buried in the hydrophobic core of NST1. The side-chains of residues (Trp-713, His-716, Gln-717, and His-720) in α6 constitute the opposing face of the cleft. The center of this cleft (approximate dimensions: 12 Å in length, 8 Å in width, and 8 Å in depth) is large enough to accommodate a trisaccharide unit of polysaccharide chain. Further studies, such as the determination of the complex structure of NST1 complexed with polysaccharide, are needed to conclude whether this center portion of the cleft is, in fact, the substrate binding site. The striking similarities between the PAP binding orientation in NST1 and EST provide structural evidence that the Golgi membrane and cytosolic enzymes belong to the same family of enzymes. The similar topology and function of Lys-614 to Lys-48 of EST suggest a common reaction mechanism in all sulfotransferases. Lys-833 may be an additional catalytic residue not present in the cytosolic enzymes. The NST1 structure provides an excellent model for investigating the substrate specificity of heparan sulfate sulfotransferases so that we may better understand sulfation at specific positions of glucuronic acid-N-acetylglucosamine.