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Structural Basis for Acceptor Substrate Recognition of a Human Glucuronyltransferase, GlcAT-P, an Enzyme Critical in the Biosynthesis of the Carbohydrate Epitope HNK-1

2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês

10.1074/jbc.m400622200

ISSN

1083-351X

Autores

Shinako Kakuda, T. Shiba, Masji Ishiguro, Hideki Tagawa, Shogo Oka, Yasuhiro Kajihara, Toshisuke Kawasaki, Soichi Wakatsuki, Ryuichi Kato,

Tópico(s)

Monoclonal and Polyclonal Antibodies Research

Resumo

The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules. Its structure is characterized by a terminal sulfated glucuronyl acid. The glucuronyltransferases, GlcAT-P and GlcAT-S, are involved in the biosynthesis of the HNK-1 epitope, GlcAT-P as the major enzyme. We overexpressed and purified the recombinant human GlcAT-P from Escherichia coli. Analysis of its enzymatic activity showed that it catalyzed the transfer reaction for N-acetyllactosamine (Galβ1-4GlcNAc) but not lacto-N-biose (Galβ1-3GlcNAc) as an acceptor substrate. Subsequently, we determined the first x-ray crystal structures of human GlcAT-P, in the absence and presence of a donor substrate product UDP, catalytic Mn2+, and an acceptor substrate analogue N-acetyllactosamine (Galβ1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide. The asymmetric unit contains two independent molecules. Each molecule is an α/β protein with two regions that constitute the donor and acceptor substrate binding sites. The UDP moiety of donor nucleotide sugar is recognized by conserved amino acid residues including a DXD motif (Asp195-Asp196-Asp197). Other conserved amino acid residues interact with the terminal galactose moiety of the acceptor substrate. In addition, Val320 and Asn321, which are located on the C-terminal long loop from a neighboring molecule, and Phe245 contribute to the interaction with GlcNAc moiety. These three residues play a key role in establishing the acceptor substrate specificity. The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules. Its structure is characterized by a terminal sulfated glucuronyl acid. The glucuronyltransferases, GlcAT-P and GlcAT-S, are involved in the biosynthesis of the HNK-1 epitope, GlcAT-P as the major enzyme. We overexpressed and purified the recombinant human GlcAT-P from Escherichia coli. Analysis of its enzymatic activity showed that it catalyzed the transfer reaction for N-acetyllactosamine (Galβ1-4GlcNAc) but not lacto-N-biose (Galβ1-3GlcNAc) as an acceptor substrate. Subsequently, we determined the first x-ray crystal structures of human GlcAT-P, in the absence and presence of a donor substrate product UDP, catalytic Mn2+, and an acceptor substrate analogue N-acetyllactosamine (Galβ1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide. The asymmetric unit contains two independent molecules. Each molecule is an α/β protein with two regions that constitute the donor and acceptor substrate binding sites. The UDP moiety of donor nucleotide sugar is recognized by conserved amino acid residues including a DXD motif (Asp195-Asp196-Asp197). Other conserved amino acid residues interact with the terminal galactose moiety of the acceptor substrate. In addition, Val320 and Asn321, which are located on the C-terminal long loop from a neighboring molecule, and Phe245 contribute to the interaction with GlcNAc moiety. These three residues play a key role in establishing the acceptor substrate specificity. Carbohydrate molecules on the cell surface modulate a variety of cellular functions, including cell-to-cell interactions (1Rutishauser U. Acheson A. Hall A.K. Mann D.M. Sunshine J. Science. 1988; 240: 53-57Crossref PubMed Scopus (673) Google Scholar, 2Jessell T.M. Hynes M.A. Dodd J. Annu. Rev. Neurosci. 1990; 13: 227-255Crossref PubMed Scopus (181) Google Scholar). The HNK-1 carbohydrate epitope, which is recognized by HNK-1 monoclonal antibodies, is found on many neural cell adhesion molecules such as NCAM (3Kruse J. Mailhammer R. Wernecke H. Faissner A. Sommer I. Goridis C. Schachner M. Nature. 1984; 311: 153-155Crossref PubMed Scopus (585) Google Scholar), myelin-associated glycoprotein (4McGarry R.C. Helfand S.L. Quarles R.H. Roder J.C. Nature. 1983; 306: 376-378Crossref PubMed Scopus (370) Google Scholar), L1 (3Kruse J. Mailhammer R. Wernecke H. Faissner A. Sommer I. Goridis C. Schachner M. Nature. 1984; 311: 153-155Crossref PubMed Scopus (585) Google Scholar), transiently expressed axonal glycoprotein-1 (5Dodd J. Morton S.B. Karagogeos D. Yamamoto M. Jessell T.M. Neuron. 1988; 1: 105-116Abstract Full Text PDF PubMed Scopus (641) Google Scholar), P0 (6Bollensen E. Schachner M. Neurosci. Lett. 1987; 82: 77-82Crossref PubMed Scopus (115) Google Scholar), and also on some glycolipids (7Chou D.K. Ilyas A.A. Evans J.E. Costello C. Quarles R.H. Jungalwala F.B. J. Biol. Chem. 1986; 261: 11717-11725Abstract Full Text PDF PubMed Google Scholar, 8Ariga T. Kohriyama T. Freddo L. Latov N. Saito M. Kon K. Ando S. Suzuki M. Hemling M.E. Rinehart K.L. Kusunoki S. Yu R.K. J. Biol. Chem. 1987; 262: 848-853Abstract Full Text PDF PubMed Google Scholar). Expression of the HNK-1 carbohydrate epitope is spatially and temporally regulated during development of the central and peripheral nervous systems (9Bronner-Fraser M. Dev. Biol. 1986; 115: 44-55Crossref PubMed Scopus (367) Google Scholar, 10Schwarting G.A. Jungalwala F.B. Chou D.K. Boyer A.M. Yamamoto M. Dev. Biol. 1987; 120: 65-76Crossref PubMed Scopus (142) Google Scholar, 11Yoshihara Y. Oka S. Watanabe Y. Mori K. J. Cell Biol. 1991; 115: 731-744Crossref PubMed Scopus (57) Google Scholar). In addition, the HNK-1 carbohydrate epitope is presumed to be involved in cell-to-cell interactions such as cell adhesion (12Keilhauer G. Faissner A. Schachner M. Nature. 1985; 316: 728-730Crossref PubMed Scopus (411) Google Scholar), migration (13Bronner-Fraser M. Dev. Biol. 1987; 123: 321-331Crossref PubMed Scopus (147) Google Scholar), and neurite extension (14Martini R. Xin Y. Schmitz B. Schachner M. Eur. J. Neurosci. 1992; 4: 628-639Crossref PubMed Scopus (176) Google Scholar). The structure of HNK-1 carbohydrate epitope is known to be HSO3-3GlcAβ1-3Galβ1-4GlcNAc-R, which is shared by glycolipids and glycoproteins (7Chou D.K. Ilyas A.A. Evans J.E. Costello C. Quarles R.H. Jungalwala F.B. J. Biol. Chem. 1986; 261: 11717-11725Abstract Full Text PDF PubMed Google Scholar, 8Ariga T. Kohriyama T. Freddo L. Latov N. Saito M. Kon K. Ando S. Suzuki M. Hemling M.E. Rinehart K.L. Kusunoki S. Yu R.K. J. Biol. Chem. 1987; 262: 848-853Abstract Full Text PDF PubMed Google Scholar, 15Voshol H. van Zuylen C.W.E.M. Orberger G. Vliegenthart J.F.G. Schachner M. J. Biol. Chem. 1996; 271: 22957-22960Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Because the inner structure, Galβ1-4GlcNAc, is found commonly in various glycoproteins and glycolipids, and the terminal sulfo-3-glucuronyl group is essential for both immunoreactivity with HNK-1 monoclonal antibodies (16Ilyas A.A. Chou D.K.H. Jungalwala F.B. Costello C. Quarles R.H. J. Neurochem. 1988; 55: 594-601Crossref Scopus (63) Google Scholar) and for their functions (17Schmitz B. Schachner M. Ito Y. Nakano T. Ogawa T. Glycoconj. J. 1994; 11: 345-352Crossref PubMed Scopus (43) Google Scholar), the terminal structure is unique and important. Therefore, in order to elucidate the functions of the HNK-1 carbohydrate epitope, it is important to characterize the enzymes in the biosynthesis pathway, which are unique to the HNK-1 epitope. The HNK-1 carbohydrate epitope is synthesized in a stepwise manner through the addition of β-1,3-linked glucuronic acid (GlcA) 1The abbreviations used are: GlcA, glucuronic acid; UDP-GlcA, uridine diphosphoglucuronic acid; r.m.s.d., root mean square deviation; ASOR, asialo-orosomucoid; MES, 4-morpholineethanesulfonic acid. by glucuronyltransferase(s) to precursor N-acetyllactosamine followed by the addition of sulfate group by sulfotransferase(s). It has been reported that there may be two types of glucuronyltransferases associated with the biosynthesis of the HNK-1 carbohydrate epitope in the mammalian brain (18Chou D.K.H. Flores S. Jungalwala F.B. J. Biol. Chem. 1991; 266: 17941-17947Abstract Full Text PDF PubMed Google Scholar, 19Das K.K. Basu M. Basu S. Chou D.K.H. Jungalwala F.B. J. Biol. Chem. 1991; 266: 5238-5243Abstract Full Text PDF PubMed Google Scholar, 20Kawashima C. Terayama K. Ii M. Oka S. Kawasaki T. Glycoconj. J. 1992; 9: 307-314Crossref PubMed Scopus (21) Google Scholar, 21Oka S. Terayama K. Kawashima C. Kawasaki T. J. Biol. Chem. 1992; 267: 22711-22714Abstract Full Text PDF PubMed Google Scholar). We have recently purified and cloned the HNK-1-associated glucuronyltransferase, GlcAT-P, from rat and human (22Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6093-6098Crossref PubMed Scopus (122) Google Scholar, 23Terayama K. Seiki T. Nakamura A. Matsumori K. Ohta S. Oka S. Sugita M. Kawasaki T. J. Biol. Chem. 1998; 273: 30295-30300Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 24Mitsumoto Y. Oka S. Sakuma H. Inazawa J. Kawasaki T. Genomics. 2000; 15: 166-173Crossref Scopus (45) Google Scholar). More recently, we succeeded in generating mice with targeted deletion of the GlcAT-P gene. The GlcAT-P-deficient mice exhibited reduced long-term potentiation at the Schaffer collateral-CA1 synapses and defects in spatial memory formation (25Yamamoto S. Oka S. Inoue M. Shimuta M. Manabe T. Takahashi H. Miyamoto M. Asano M. Sakagami J. Sudo K. Iwakura Y. Ono K. Kawasaki T. J. Biol. Chem. 2002; 277: 27227-27231Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Based on the amino acid sequence information of GlcAT-P cDNA, another HNK-1-associated glucuronyltransferase, GlcAT-S, and a proteoglycan-associated glucuronyltransferase, GlcAT-I, have been cloned and characterized (19Das K.K. Basu M. Basu S. Chou D.K.H. Jungalwala F.B. J. Biol. Chem. 1991; 266: 5238-5243Abstract Full Text PDF PubMed Google Scholar, 26Tone Y. Kitagawa H. Imiya K. Oka S. Kawasaki T. Sugahara K. FEBS Lett. 1999; 459: 415-420Crossref PubMed Scopus (41) Google Scholar, 27Seiki T. Oka S. Terayama K. Imiya K. Kawasaki T. Biochem. Biophys. Res. Commun. 1999; 255: 182-187Crossref PubMed Scopus (70) Google Scholar, 28Kitagawa H. Taoka M. Tone Y. Sugahara K. Biochem. J. 2001; 358: 539-546Crossref PubMed Google Scholar, 29Imiya K. Ishizaki T. Seiki T. Saito F. Inazawa J. Oka S. Kawasaki T. Gene (Amst.). 2002; 296: 29-36Crossref PubMed Scopus (15) Google Scholar, 30Marcos I. Galan J.J. Borrego S. Antinolo G. J. Hum. Genet. 2002; 47: 677-680Crossref PubMed Scopus (16) Google Scholar). These enzymes catalyze the transfer of GlcA from a donor substrate, uridine diphosphoglucuronic acid (UDP-GlcA), to a reducing terminal residue of oligosaccharide chain in the presence of manganese. GlcAT-I transfers GlcA to Galβ1-3Galβ1-4Xylβ1-O-Ser in a biosynthesis pathway of proteoglycan (19Das K.K. Basu M. Basu S. Chou D.K.H. Jungalwala F.B. J. Biol. Chem. 1991; 266: 5238-5243Abstract Full Text PDF PubMed Google Scholar, 26Tone Y. Kitagawa H. Imiya K. Oka S. Kawasaki T. Sugahara K. FEBS Lett. 1999; 459: 415-420Crossref PubMed Scopus (41) Google Scholar). The substrate binding and the reaction mechanisms of GlcAT-I have been discussed at an atomic level based on its crystal structure in complex with donor and acceptor substrate (31Pedersen L.C. Tsuchida K. Kitagawa H. Sugahara K. Darden T.A. Negishi M. J. Biol. Chem. 2000; 275: 34580-34585Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 32Pedersen L.C. Darden T.A. Negishi M. J. Biol. Chem. 2002; 277: 21869-21873Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 33Negishi M. Dong J. Darden T.A. Pedersen L.G. Pedersen L.C. Biochem. Biophys. Res. Commun. 2003; 303: 393-398Crossref PubMed Scopus (51) Google Scholar). On the other hand, GlcAT-P can transfer GlcA to Galβ1-4GlcNAc-R but not to lacto-N-biose (Galβ1-3GlcNAc) (23Terayama K. Seiki T. Nakamura A. Matsumori K. Ohta S. Oka S. Sugita M. Kawasaki T. J. Biol. Chem. 1998; 273: 30295-30300Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 34Oka S. Terayama K. Imiya K. Yamamoto S. Kondo A. Kato I. Kawasaki T. Glycoconj. J. 2000; 17: 877-885Crossref PubMed Scopus (12) Google Scholar). To elucidate acceptor substrate specificity of GlcAT-P, we overexpressed it in Escherichia coli and purified and characterized acceptor substrate specificity in vitro. We then solved crystal structures of GlcAT-P in four different forms: (1) apo-form, (2) with UDP-GlcA and Mn2+, (3) with UDP-GlcA, Mn2+, and an acceptor substrate analogue, N-acetyllactosamine, and (4) with UDP-GlcA, Mn2+, and a natural acceptor substrate, an asparagine-linked biantennary nonasaccharide. Based on the structural and biochemical results, we describe the molecular mechanism of substrate recognition of HNK-1 associated glucuronyltransferase, GlcAT-P. Materials—UDP-[14C] glucuronic acid (12.1 GBq/mmol) was purchased from ICN Radiochemicals. Asialo-orosomucoid (ASOR) was prepared by hydrolysis of orosomucoid in 0.05 m H2SO4 at 80 °C for 1 h. An asparagine-linked biantennary nonasaccharide conjugated with Fmoc (9-fluorenylmethoxycarbonyl group) was prepared according to the method described previously (35Kajihara Y. Suzuki Y. Sasaki K. Juneja L.R. Methods Enzymol. 2003; 362: 44-64Crossref PubMed Scopus (11) Google Scholar). N-Acetyllactosamine and lacto-N-biose were purchased from Sigma-Aldrich. Lacto-N-neotetraose was kindly provided by Dr. Koizumi (Kyowa Hakko Kogyo Co., Ltd.). Protein Expression and Purification—The coding region of the catalytic domain of GlcAT-P was amplified from a cloned human GlcAT-P cDNA (24Mitsumoto Y. Oka S. Sakuma H. Inazawa J. Kawasaki T. Genomics. 2000; 15: 166-173Crossref Scopus (45) Google Scholar) as a template by polymerase chain reaction (PCR). The amplified DNA fragment was cloned into a bacterial expression vector, pET-28a(+) (Novagen). The expressed protein contained the following sequence: Leu83-Ile334 (the amino acid positions of human GlcAT-P are shown by superscript numbers). An E. coli strain BL21(DE3)pLysS (Stratagene), which was transformed with the plasmid, was cultivated in an LB broth containing 0.4% glucose and 20 μg/ml kanamycin at 30 °C by vigorous aeration, and then induced by the addition of 1 mm isopropyl-1-thio-β-d-galactopyranoside. The E. coli cells were harvested by centrifugation and suspended in a buffer containing 0.15 m NaCl, 20 mm Tris-HCl, pH 6.0. After centrifugation of the sonicated cell suspension, a cleared lysate was diluted by addition of an equal volume of the same buffer, and then applied to an open column, which was made from Hi-trap Heparin HP (Amersham Biosciences) resin. The column was washed with a 0.4 m NaCl buffer, 10× column volume, and then eluted by a 0.75 m NaCl buffer. The fractions containing GlcAT-P were collected, diluted with 20 mm Tris-HCl buffer to adjust the NaCl concentration to 0.45 m, and then loaded to the Hi-Trap Heparin HP column. The sample was eluted by NaCl gradient (0.45-0.75 m), and the fractions containing GlcAT-P were collected. The pH of the sample was adjusted to 8.0 by addition of 1 m Tris. Hi-Trap Chelating (Amersham Biosciences) column was charged with bivalent metal ions by flowing 0.1 m CuSO4 and then equilibrated with a buffer containing of 0.5 m NaCl, 20 mm Tris-HCl, pH 8.0. The sample was then loaded onto the column, washed with the same buffer, eluted by glycine gradient (0-50 mm), and the GlcAT-P-containing fractions were collected. Then, the sample was diluted to the NaCl concentration of 0.25 m, loaded to the Hi-Trap Heparin HP column, which had been equilibrated with 0.25 m NaCl, 20 mm Tris-HCl, pH 8.0, washed with 0.25 m NaCl, 50 mm MES, pH 6.0, and eluted with 0.7 m NaCl, 50 mm MES, pH 6.0. The peak fractions were collected and concentrated by ultrafiltration using Millipore UFV4BCC25 at 2,000 rpm. The yield of GlcAT-P protein sample was 1 mg per 2 liters of culture. Enzymatic Assay—The glucuronyltransferase activity for ASOR or oligosaccharides was measured as described previously (21Oka S. Terayama K. Kawashima C. Kawasaki T. J. Biol. Chem. 1992; 267: 22711-22714Abstract Full Text PDF PubMed Google Scholar) with slight modifications. An equivalent amount of GlcAT-P or GlcAT-S enzyme was incubated at 37 °C for 1 h (ASOR) or 3 h (oligosaccharides) in a reaction mixture, with a final volume of 50 μl, containing 100 mm MES (pH 6.5), 0.2% Nonidet P-40, 20 mm MnCl2, 20 μg ASOR, or 200 μm oligosaccharide, 100 μm UDP-[14C]GlcA (200,000 dpm). In the case of ASOR, the assay mixture was spotted onto a 2.5-cm Whatman No.1 disc after incubation of the mixture. The disc was washed with a 10% (w/v) trichloroacetic acid solution three times, followed by washing with ethanol/ether (2:1, v/v) and then with ether. The disc was air-dried, and the radioactivity of [14C]GlcA-ASOR was counted with a liquid scintillation counter (Beckman LS-6000). In the case of oligosaccharides, the reaction was terminated by addition of 1 ml of 5 mm phosphate buffer, pH 6.8. The products were then separated by passing through anion exchange resin AG1-X4 (1 ml), which had been equilibrated with 5 mm phosphate buffer, pH 6.8. The column was washed with 5 ml of the buffer, and the flow-through and washing fractions were collected. The radioactivity was counted with a liquid scintillation counter (Beckman LS-6000). Crystallization—Crystallization conditions for the apo-form of GlcAT-P were screened using the hanging drop vapor diffusion method. The apo-form of GlcAT-P was crystallized in 2.0-μl hanging drops over 0.2-ml reservoirs containing 20% (w/v) PEG2000MME, and 0.24 m di-sodium tartrate. The crystals were grown to 0.3 × 0.1 × 0.1 mm in 2-3 days at 289 K. For data collection, the crystals were transferred to a mother liquor solution containing 15% glycerol, and flash-frozen in liquid nitrogen. To obtain the ternary (and quaternary) complex crystals of the GlcAT-P, the apo-form crystals were soaked into the buffer containing 20% (w/v) PEG2000MME, 0.24 m di-sodium tartrate, 15% glycerol, 10 mm UDP-GlcA, 10 mm MnCl2 (and 10 mmN-acetyllactosamine) for 0.5-6 h, and flash-frozen in liquid nitrogen. To obtain crystals of the GlcAT-P in complex with Mn2+, UDP and asparagine-linked bi-antennary nonasaccharide, 10 mm UDP-GlcA, 10 mm MnCl2, 10 mm asparagine-linked bi-antennary nonasaccharide were added to the protein solution and incubated for 30 min at 4 °C. Then, the mixture was crystallized in 2.0-μl hanging drops over 0.2 ml reservoirs containing 20% (w/v) PEG2000MME, and 0.1 m MES-NaOH (pH 6.0). X-ray Data Collection and Processing—For crystal structure analysis, the data sets were collected at 100 K with synchrotron radiation at PF-AR-NW12 and were processed using HKL2000 (36Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar). All crystals belong to the orthorhombic space group P212121 with similar unit cell parameters, a = 61, b = 86, c = 123 Å, and 57% solvent content. In all crystal structures, there were two monomers in an asymmetric unit. Data collection and processing statistics of the data sets are summarized in Table I.Table IData collection statisticsData setApoComplex 1aComplex 1 contains Mn2+, UDP, and N-acetyllactosamine.Complex 2bComplex 2 contains Mn2+, and UDP.Complex 3cComplex 3 contains Mn2+, UDP, and asparagine-linked bi-antennary nonasaccharide.Crystallographic dataSpace groupP212121P212121P212121P212121Unit cella/b/c (Å)61.5/85.7/123.061.3/85.8/122.861.7/85.7/122.961.1/85.8/122.9α/β/γ (°)90.0/90.0/90.090.0/90.0/90.090.0/90.0/90.090.0/90.0/90.0Data processing statisticsData setApoComplex 1Complex 2Complex 3cComplex 3 contains Mn2+, UDP, and asparagine-linked bi-antennary nonasaccharide.Beam linePF-AR-NW12PF-AR-NW12PF-AR-NW12PF-AR-NW12Wavelength (Å)1.00.9741.01.0Resolution (Å)dValues in parentheses are for the highest resolution shell.40-1.85 (1.92-1.85)50-1.82 (1.89-1.82)50-1.9 (1.97-1.9)50-1.9 (1.97-1.9)Total reflections377,310279,401364,123365,669Unique reflections54,71057,14451,30051,460Completeness (%)dValues in parentheses are for the highest resolution shell.96.6 (79.9)97.1 (88.4)98.2 (87.5)99.4 (96.4)Rmerge (%)dValues in parentheses are for the highest resolution shell.eRmerge = ∑h∑j|Ij(h) - 〈I(h)〉|/∑ h∑jIj(h), where Ij(h) is the jth measurement of reflection indices h and 〈I(h}〉 is the mean intensity.7.5 (41.3)6.7 (37.9)6.0 (31.9)4.4 (26.6)I/σ(I)110.3 (2.5)10.4 (2.8)13.3 (4.3)16.0 (5.8)a Complex 1 contains Mn2+, UDP, and N-acetyllactosamine.b Complex 2 contains Mn2+, and UDP.c Complex 3 contains Mn2+, UDP, and asparagine-linked bi-antennary nonasaccharide.d Values in parentheses are for the highest resolution shell.e Rmerge = ∑h∑j|Ij(h) - 〈I(h)〉|/∑ h∑jIj(h), where Ij(h) is the jth measurement of reflection indices h and 〈I(h}〉 is the mean intensity. Open table in a new tab Structure Determination and Refinement—The crystal structure of the apo-form of GlcAT-P was solved by the molecular replacement method using the human GlcAT-I (PDB code: 1FGG) as a search model. Molecular replacement calculations were carried out using the program MOLREP (37Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4153) Google Scholar). The current model with 381 water molecules and one tartrate ion was refined using CNS (38Brünger 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 (16967) Google Scholar) for the resolution range from 40.0 to 1.85 Å, and has an R-factor of 19.8% and an Rfree of 22.8. In the Ramachandran plot, 88.3% are in the most favored regions and no residues are in the disallowed regions. The following residues have not been modeled because of weak or no associated electron density: in molecule A (chain A), residues 157-161 and in molecule B (chain B), residues 151-162. For refinement of the complexes, the apo-form models were refined first against the complex data using CNS. Substrates and co-factors were then manually built into the FO-FC electron density maps followed by additional rounds of refinement. The final refinement statistics are shown in Table II. Figures were produced using MOLSCRIPT (39Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), RASTER-3D (40Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3875) Google Scholar), CONSCRIPT (41Lawrence M.C. Bourke P. J. Appl. Crystallogr. 2000; 33: 990-991Crossref Scopus (75) Google Scholar), and GRASP (42Nicholls A. Sharp K.A. Honig B. Proteins Struct. Funct. Genet. 1991; 11: 281-296Crossref PubMed Scopus (5316) Google Scholar).Table IIRefinement statisticsData setApoComplex 1aComplex 1 contains Mn2+, UDP, and N-acetyllactosamine.Complex 2bComplex 2 contains Mn2+ and UDP.Complex 3cComplex 3 contains Mn2+, UDP, and asparagine-linked bi-antennary nonasaccharide.Resolution (Å)40-1.8540-1.8240-1.940-1.9No. of reflections54.63957,09351,21051,364Rwork/RfreedR-factor = ∑h∥Fo(h)| -|Fc(h)∥/∑h|Fo(h)|, and Rfree was calculated using 5% of data excluded from refinement.19.8/22.820.5/24.420.1/22.920.2/22.6Number of non-hydrogen atomsProtein atoms3954394439393920UDP atoms505050Mn2+ ions222Acceptor sugars atoms5252Tartrate atoms10101010Water molecules381360414326R.m.s.d. from ideal valuesBond length (Å)0.0050.0050.0050.005Bond angle (°)1.221.441.471.46Ramachandran plot (%)Most favored88.389.387.888.0Additionally allowed10.09.311.010.2Generously allowed1.71.41.31.2Disallowed0000Average B - value (Å2)Protein atoms (chain A/chain B)22.8/23.623.4/23.026.4/25.927.7/27.0UDP atoms24.726.028.0Mn2+ ions18.520.021.0Acceptor sugars atoms26.431.1Tartrate atoms23.823.425.224.9Water molecules30.427.432.431.4a Complex 1 contains Mn2+, UDP, and N-acetyllactosamine.b Complex 2 contains Mn2+ and UDP.c Complex 3 contains Mn2+, UDP, and asparagine-linked bi-antennary nonasaccharide.d R-factor = ∑h∥Fo(h)| -|Fc(h)∥/∑h|Fo(h)|, and Rfree was calculated using 5% of data excluded from refinement. Open table in a new tab Computer-aided Model Building—The structure of the complete substrate-enzyme complex was calculated by adding a GlcA moiety to the crystal structure of the quaternary complex (UDP-GlcA, Mn2+, and N-acetyllactosamine) since the x-ray structure of the quaternary complex did not show electron density for the GlcA moiety of the UDP-GlcA. The UDP moiety in the crystal structure was modified by adding the GlcA residue at the β-phosphate oxygen, and the UDP-GlcA structure was modified by energy minimization at the binding cleft. The initial conformation of the glucuronyl group was selected at the binding cleft by searching a proper position for the SN2-type reaction with the hydroxyl group of the galactose residue of N-acetyllactosamine. Several water molecules overlapped with the newly introduced glucuronyl group were removed, and only the remaining water molecules were energy-minimized, using Discover 3 (version 98.0, Molecular Simulations Inc. San Diego, CA). After capping solvent water provided from the Assembly module of Insight II (version 2000, Molecular Simulations Inc), the complex structure was energy-minimized until the final root mean square deviation (r.m.s.d.) became less than 0.1 kcal/mol/Å. During this minimization, residues longer than 10 Å away from the substrates, UDP-GlcA and N-acetyllactosamine, all Cα atoms, and the Mn2+ atom were fixed. All minimizations and molecular dynamics calculations were carried out under the same conditions, unless otherwise mentioned. The entire system was then covered by solvent water (20 Å thick) and these solvent water molecules were fixed in the following molecular dynamics calculation. The energy-minimized complex structure was further optimized with molecular dynamics calculations at 298 K with the cell multipole method, a distance-dependent dielectric constant, and a time step of 1 fs for 100 ps by sampling conformations every 1 ps using Discover 3. One hundred conformations were minimized in this way until the final r.m.s.d. became less than 0.1 kcal/mol/Å, and the lowest energy conformation was selected as an optimized structure. To generate a model for lacto-N-biose in the acceptor binding site of GlcAT-P, the 1,4-glycosidic bond of N-acetyllactosamine was changed to a 1,3-glycosidic bond maintaining the position of the galactose residue in the substrate-binding cleft. Water molecules, which collide with the N-acetylglucosamine residue, were removed. Following an energy minimization of the remaining water molecules using Discover 3, the cap water molecules were added at the acceptor binding site within 20 Å from the reaction center at C1 of the glucuronic acid moiety, and the complex structure was optimized by the minimization/molecular dynamics procedures described above. A Lewis X structure was generated by connecting α-l-fucose residue to C-3-OH of N-acetyllactosamine in the substrate-binding cleft. After removal of water molecules that collide with the fucose residue, the complex structure was optimized by the minimization/molecular dynamics procedure within the water shell described above. Overall Structure of GlcAT-P—The recombinant protein was designed as an N-terminal truncated form because the GlcAT-P is a type II membrane protein and its N terminus contains a transmembrane and a stem regions. The truncated GlcAT-P (residues 83-334) was expressed in E. coli and purified as described under "Experimental Procedures." The purified protein showed a glucuronyltransferase activity, transfer of GlcA from UDP-GlcA to a donor substrate (described below). It indicates that the recombinant enzyme purified from E. coli maintains the activity of the natural enzyme (23Terayama K. Seiki T. Nakamura A. Matsumori K. Ohta S. Oka S. Sugita M. Kawasaki T. J. Biol. Chem. 1998; 273: 30295-30300Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 34Oka S. Terayama K. Imiya K. Yamamoto S. Kondo A. Kato I. Kawasaki T. Glycoconj. J. 2000; 17: 877-885Crossref PubMed Scopus (12) Google Scholar). Using the recombinant protein, we succeeded in obtaining crystals of GlcAT-P in a variety of conditions: apo, with a donor substrate and manganese, and another with additional acceptor substrate. We first solved the structure of the apo-form of GlcAT-P by the molecular replacement method using the catalytic domain of GlcAT-I (PDB code: 1FGG) as a search model at 1.85 Å (Tables I and II). The asymmetric unit of the crystal lattice contains one protein dimer as in the GlcAT-I crystal structure (31Pedersen L.C. Tsuchida K. Kitagawa H. Sugahara K. Darden T.A. Negishi M. J. Biol. Chem. 2000; 275: 34580-34585Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). The asymmetric unit contains two independent molecules, related by a non-crystallographic 2-fold axis (Fig. 1). One of the two molecules, molecule B, in the asymmetric unit has a longer disordered region (residues 151-162) than the other molecule A (residues 157-161). Therefore, the structure of molecule A is described here. The overall structure of GlcAT-P is a GT-A fold (33Negishi M. Dong J. Darden T.A. Pedersen L.G. Pedersen L.C. Biochem. Biophys. Res. Commun. 2003; 303: 393-398Crossref PubMed Scopus (51) Google Scholar) with a Rossmann-like fold consisting of twelve β-strands and seven α-helices (Fig. 2). It is divided into two regions as in the case of GlcAT-I. These two regions are connected by the DXD motif (Asp195-Asp196-Asp197), which are conserved in many UDP-sugar-dependent glycosyltransferases (33Negishi M. Dong J. Darden T.A. Pedersen L.G. Pedersen L.C. Biochem. Biophys. Res. Commun. 2003; 303: 393-398Crossref PubMed Scopus (51) Google Scholar). The N-terminal region (residues 83-197), referred to as a UDP-sugar binding region, contains an α/β Rossmann-like fold, β1-α1-β2-α2-β3-α3′-α3-β4, and the β-strands form a parallel β-sheet (order: β4-β1-β2-β3). The C-terminal region (residues 198-334), referred to as an acceptor substrate bind

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