Crystal Structure of an alpha 1,4-N-Acetylhexosaminyltransferase (EXTL2), a Member of the Exostosin Gene Family Involved in Heparan Sulfate Biosynthesis*

Lars C. PedersenDagger §, Jian DongDagger , Fumiyasu Taniguchi, Hiroshi Kitagawa, Joe M. KrahnDagger , Lee G. Pedersen§, Kazuyuki Sugahara||, and Masahiko NegishiDagger **

From the Dagger  Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology and § Laboratory of Structural Biology and NIEHS, National Institute of Health, Research Triangle Park, North Carolina 27709 and  Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 685-8858, Japan

Received for publication, October 15, 2002, and in revised form, January 14, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

EXTL2, an alpha 1,4-N-acetylhexosaminyltransferase, catalyzes the transfer reaction of N-acetylglucosamine and N-acetylgalactosamine from the respective UDP-sugars to the non-reducing end of [glucuronic acid]beta 1-3[galactose]beta 1-O-naphthalenemethanol, an acceptor substrate analog of the natural common linker of various glycosylaminoglycans. We have solved the x-ray crystal structure of the catalytic domain of mouse EXTL2 in the apo-form and with donor substrates UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine. In addition, a structure of the ternary complex with UDP and the acceptor substrate analog [glucuronic acid]beta 1-3[galactose]beta 1-O-naphthalenemethanol has been determined. These structures reveal three highly conserved residues, Asn-243, Asp-246, and Arg-293, located at the active site. Mutation of these residues greatly decreases the activity. In the ternary complex, an interaction exists between the beta -phosphate of the UDP leaving group and the acceptor hydroxyl of the substrate that may play a functional role in catalysis. These structures represent the first structures from the exostosin gene family and provide important insight into the mechanisms of alpha 1,4-N-acetylhexosaminyl transfer in heparan biosynthesis.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Proteoglycans are composed of core proteins and covalently linked glycosaminoglycan side chains. Proteoglycans are distributed at cell surfaces and in the extracellular matrices of most animal tissues. Heparan sulfate and heparin are uronic acid-containing sulfated glycosaminoglycan chains that are comprised of repeat disaccharide units consisting of alternating GlcNAc and GlcUA or iduronic acid. Heparan sulfates and heparin have been implicated in blood coagulation, cell proliferation and differentiation, tissue morphogenesis, and viral and bacterial infection (1, 2). Accordingly, disruption of enzymes in heparan sulfate biosynthesis can have severe biological consequences in mammals (3). Human hereditary multiple exostoses disorder, characterized by benign tumors of bony outgrowths, has been attributed to mutations in EXT1 and EXT2 of the EXT (exostosin) gene family that encode various enzymes in heparan biosynthesis (4-6). EXT1 and EXT2 are bifunctional enzymes with beta 1,4-glucuronyltransferase and alpha 1,4-N-acetylglucosaminyltransferase activities. Known as heparan polymerases, EXT1 and EXT2 elongate the heparan sulfate chain by alternative additions of GlcUA and GlcNAc to the non-reducing ends. Although a better understanding of how these EXT enzymes function is critical for elucidating their roles in heparan biosynthesis, no protein structure from the EXT family of enzymes is available, and the reaction mechanisms still remains elusive.

In addition to the EXT enzymes, the EXT family also includes so-called EXT-like enzymes EXTL1, EXTL2, and EXTL3 (7, 8). Purified recombinant EXTL2 displays activity for transfer of GlcNAc and GalNAc from the respective UDP-sugars to an acceptor analog, [glucuronic acid]beta 1-3[galactose]beta 1-O-naphthalenemethanol (GlcUAbeta 1-3Galbeta 1-O-naphthalenemethanol) (7). Because of the alpha 1,4-N-acetylglucosaminyltransferase activity, EXTL2 is suggested to act as an enzyme that can transfer GlcNAc to the common linker tetrasaccharide of a core protein (8). On the other hand, the alpha 1,4-N-acetylgalactosaminyltransferase activity is speculated as an activity that could provide a way to terminate glycosaminoglycan biosynthesis all together (9), although it is not known whether EXTL2 is this termination enzyme. EXTL2, the smallest of the known EXT homologues, consists of 330 amino acid residues and shows extensive amino acid sequence homology to the C-terminal region of EXT1 and EXT2 that presumably constitutes the alpha 1,4-N-acetylglucosaminyltransferase domain. Thus, EXTL2 provides an excellent model for investigating the structure-based mechanism of the alpha 1,4-N-acetylglucosaminyltransferase reaction catalyzed by the EXT gene family.

Here we have determined the ternary complex structure of mouse EXTL2 (mEXTL2)1 with UDP and the acceptor substrate analog GlcUAbeta 1-3Galbeta 1-O-naphthalenemethanol, the binary complex structures with UDP-GlcNAc and UDP-GalNAc, and the structure of the apo-enzyme. These structures reveal conserved residues within the EXT gene family that play a role in substrate binding and/or catalysis. A site-directed mutagenesis study was performed to provide functional evidence to support their roles in catalysis. EXTL2 catalyzes a transfer reaction in which the alpha -linkage of the C1-O1 bond in the donor UDP-sugar is retained in the reaction product. Two different mechanisms for the retaining transfer reaction have been proposed, a SN2-like double-displacement mechanism (10) and a SNi-like reaction mechanism (11, 12). The SN2-like double-displacement mechanism would likely involve a direct nucleophilic attack on the UDP-sugar at the C1 atom of the sugar from the opposite side of the UDP moiety. This would invert the stereochemistry at the C1, releasing the UDP moiety, to form a covalent intermediate. The acceptor hydroxyl could then attack the C1 carbon in the same manner releasing the protein side chain inverting the stereochemistry at the C1 atom back to its original state to form the final product. The SNi-like mechanism would involve a dissociation of the UDP and attack on the transition state by the acceptor from the same side as the leaving UDP to maintain the stereochemistry at the C1 carbon. Our present mEXTL2 structures are consistent with the SNi-like reaction mechanism for the retaining glycosyltransferases. The ternary complex also suggests that the UDP leaving group may facilitate sugar transfer by deprotonating the acceptor substrate. These structures represent the first from the EXT gene family and therefore provide important new information for enzymes involved in heparan biosynthesis and provide additional insight into glycosyltransferases in general.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

Protein Expression and Purification-- The upstream primer 5'-CGCGGATCCCATCATCATCATCATCATCATCATCATCATACCAACTTACTCCCCAACATCAAAG and the downstream primer 3'-CCCTAGTCTAGATCACATTTTACTTTTATCGTT were used to amplify the coding region (T38-M330) of EXTL2 from mouse (liver) cDNA by PCR. The PCR product was cloned into the pMAL-2c vector using the BamHI and XbaI restriction sites. The corresponding construct was used to transform Top10 cells followed by retransformation into BL21 (DE3) cells. To express the soluble protein, 100 µl of overnight cell stock was added to 1 liter of 2YT medium plus 200 µg/ml ampicillin in a 2-liter flask and placed on a shaker at 250 rpm at 37 °C for 14 h. 20 ml of culture was added to each of 20 2-liter flasks containing 1 liter of 2YT plus 200 µg/ml ampicillin. These flasks were placed at 37 °C on a shaker set at 250 rpm. When the A600 reached 0.8, the temperature was set to 23 °C, and isopropyl-1-thio-beta -D-galactopyranoside was added to a final concentration of 0.2 mM. Flasks were allowed to shake overnight. Cells were pelleted at 4000 rpm for 10 min and then resuspended in the sonication buffer containing 50 mM Hepes, pH 7.5, 350 mM NaCl, 0.5 mM dithiothreitol, 10 µg/ml 1-chloro-3-tosylamido-7-amino-L-2-heptanone (TLCK), and 10 µg/ml leupeptin. Cells were disrupted by sonication on ice and then spun down at 38,000 rpm for 30 min. The ~300 ml of soluble faction was allowed to bind to amylose resin (New England Biolabs) in batch at 4 °C for 1 h with gentle agitation. Resin was spun down at 4 °C for 10 min at 500 × g. The supernatant was poured off, and the resin was washed with 30 mM Hepes, pH 7.5, 100 mM NaCl at a volume ratio of 10:1 (buffer:resin). Resin was pelleted and washed a total of five times. Protein was then eluted from the resin with a buffer containing 25 mM Hepes, pH 7.5, 100 mM NaCl, 20 mM maltose. Protein was concentrated and dialyzed overnight in 25 mM Hepes, pH 7.5, 100 mM NaCl. Because treatment with factor Xa did not cleave the fusion protein, subtilisin was used to digest the fusion protein at a 1 to 10,000 subtilisin to EXTL2 mass to mass ratio. This cleavage was performed in 25 mM Hepes, pH 7.5, 100 mM NaCl, 1 mM CaCl2 at room temperature for 1 h followed by overnight cleavage at 4 °C. The cleavage resulted in producing EXTL2 fragments with molecular masses of ~30 kDa as determined by SDS-PAGE. The protein was then loaded on to a Ni2+-agarose column and eluted with a gradient elution of imidazole from 0.0 to 0.3 M. The protein was eluted from the Ni2+-agarose with ~45 mM imidazole, dialyzed into 20 mM Hepes, pH 7.5, 100 mM NaCl, and concentrated to 10 mg/ml. Protein concentration was determined using Bradford protein assay reagent (Bio-Rad). Selenomethionine-labeled protein was obtained by transforming the plasmid containing the fusion protein into B834 (DE3) cells. For expression, 1 ml of overnight culture was added to 1 liter of expression medium in a 2-liter flask containing 10 g of M9 minimal salts, 0.5 g of NaCl, 1.0% glucose, 2 mM MgSO4, 0.1 mM CaCl2, 1 µg/ml thiamine, 0.5 g of all essential amino acids (with the exception of methionine), 7.5 mg of FeSO4, and 50 mg of selenomethionine. 50 ml of 2YT was added to the flask for the starter culture. This was allowed to shake at 37 °C at 250 rpm overnight. 200 ml of the starter culture was spun down at 4000 rpm for 10 min. The cells were washed with the expression medium (containing no 2YT) and spun down again. Then the cells were resuspended in 25 ml of expression medium, and 5 ml was added to each of 18 2-liter flasks, containing 1 liter of expression medium. These flasks were placed on a shaker at 37 °C and 250 rpm. When the A600 reached 0.6 the temperature was changed to 23 °C, and isopropyl-1-thio-beta -D-galactopyranoside was added to a final concentration of 0.2 mM. Flasks were allowed to shake overnight. The expressed selenium-labeled protein was purified using the same protocol as the wild-type enzyme with the exception of an addition of 0.5-1 mM dithiothreitol present at all steps.

For mammalian expression of mutated EXTL2 enzymes, each expression plasmid (6 mg) was transfected into COS-1 cells in a 100-mm plate using FuGENETM 6 (Roche Molecular Biochemicals) according to the manufacturer's instructions. Two days after transfection, 1 ml of the culture medium was collected and incubated with 10 ml of IgG-Sepharose (Amersham Biosciences) for 1 h at 4 °C. The beads recovered by centrifugation were washed with and resuspended in 50 mM MES buffer, pH 6.5, containing 20 mM MnCl2 and 171 mM the sodium salt of ATP.

Protein Crystallization, Data Collection, and Refinement-- Crystals were obtained using the hanging drop method by mixing 4 µl of the digested protein in 25 mM Hepes, pH 7.5, 100 mM NaCl with 4 µl of the reservoir solution containing 0.1 M sodium cacodylate, pH 7.5, 10-12% polyethylene glycol 3000 (PEG3000), and 200 mM MgCl2. These crystals diffract at 2.4-Å resolution and were suitable for structural studies. For data collection of the apoprotein, crystals were transferred to a cryo-solution containing 0.1 M sodium cacodylate, pH 7.5, 17% PEG3000, 200 mM MgCl2, and 12.5% ethylene glycol and mounted in a cryo-loop and flash-frozen on the goniometer in a stream of nitrogen gas cooled to -177 °C by a Molecular Structure Corp. X-stream device. Oscillation data for this and all subsequent crystals were collected using phi scans on a rotating anode RUH3R generator equipped with a RAXIS IV area detector and MSC confocal blue mirrors. Data were processed using the programs Denzo and Scalepack (13) (Table I).


                              
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Table I
Crystallographic data statistics
Data statistics are provided for the x-ray crystal structures of mEXTL2 with no substrate present (mEXTL2 apo), in the presence of UDP and GlcUAbeta 1-3Galbeta 1-O-naphthalenemethanol (GlcUAbeta 1-3Galbeta 1-n), UDP-GlcNAc, and UDP-GalNAc. Also shown are the data statistics for the derivative data sets used to phase the apo native data (seleno-methionine, CH3HgCl, and HgCl2).

Crystals of selenomethionine-labeled protein were obtained using the same protocol as the native crystals but with an addition of 1 mM dithiothreitol present in the protein solution. Crystals of the CH3HgCl and HgCl2 derivative data sets were obtained by soaking native crystals in the reservoir solution containing saturated CH3HgCl or HgCl2 for ~15 min and then back-soaking into the cryo-solution and flash-frozen (Table I). The isomorphous difference data between the selenomethionine data set and the native data set were used to find 16 selenium sites (eight sites per monomer with two monomers in the asymmetric unit) with the real space heavy atom search protocol in the program CNS (14). Phases produced from these data were then used to find six mercury sites in both the CH3HgCl and HgCl2 data sets. Using the isomorphous difference data from all three derivatives and anomalous difference data from the mercury derivatives initial phases were calculated to 2.9 Å. The phases were extended to 2.4 Å using the solvent-flipping and phase-extension options in the density modify protocol of CNS (14). The original model of the apo-enzyme of mEXTL2 was built into electron density calculated from these phases using the program O (15). The model was subsequently refined using iterative cycles of model building and refinement in CNS utilizing the minimization, torsion angle refinement, and individual B-factor refinement protocols at each step. When all of the protein density had been accounted for with model building, water molecules were added using the water_pick protocol in CNS. Criteria for water selection were based on a 2.5-sigma cutoff in peak height of a Fo - Fc density map and a hydrogen bond distance criteria between 2.3 and 3.5 Å to an oxygen or nitrogen in the protein model. Additional water molecules were added manually in the program O based on Fo - Fc density maps and the presence of hydrogen bonding partners. Additional cycles of refinement and model building were carried out to obtain the final model. The crystal used for the UDP plus GlcUAbeta 1-3Galbeta 1-O-naphthalenemethanol data set was obtained by soaking an apo-crystal for 20 min in a solution containing three parts of 11% PEG3000, 0.1 M sodium cacodylate, pH 7.5, 0.2 M MgCl2 to one part 12.5% monomethyl ether PEG2000, 9% PEG3000, 0.1 M sodium cacodylate, 0.2 M MgCl2, 50 mM MES, pH 6.5, 5.0 mM UDP, 2.5 mM MnCl2, and 10 mM GlcUAbeta 1-3Galbeta 1-O-naphthalenemethanol. The crystal was then transferred in two steps into the final cryo-solution containing 12.5% monomethyl ether PEG2000, 9% PEG3000, 13% ethylene glycol, 0.1 M sodium cacodylate, pH 7.5, 0.2 M MgCl2, 50 mM MES, pH 6.5, 5 mM UDP, 2.5 mM MnCl2, and 10 mM GlcUAbeta 1-3Galbeta 1-O-naphthalenemethanol. The crystal was flash-frozen, and data were collected. For the UDP-GlcNAc data set, a crystal was soaked in three parts mother liquor to one part cryo-solution containing 10 mM MnCl2, 10 mM UDP-GlcNAc for 20 min. The crystal was transferred in two additional steps into the cryo-solution containing 10 mM MnCl2, 10 mM UDP-GlcNAc and flash-frozen, and data were collected. For the UDP-GalNAc data set, the same procedure was used as for the UDP-GlcNAc data set with the substitution of 20 mM UDP-GalNAc for 10 mM UDP-GlcNAc. For refinement of the data sets containing bound substrates and analogs, the apo-model was refined against the data in CNS using the same methods as for the apo-protein. Substrates and analogs were manually built into Fo - Fc density maps followed by additional rounds of refinement in CNS. Water molecules for the substrate structures were then added using the same criteria as for the apo-structure followed by additional rounds of model building and refinement. All data were always refined at the maximum resolution reported except for the UDP-GlcNAc structure, which was completely refined to 2.3 Å and then the data were extended to 2.1 Å for additional rounds of model building and refinement. The quality of the models was analyzed with the program Procheck (16). The Ramachandran statistics of the model are as follows: apo (87% most favored region, 12.3% additional, 0.7% generous, and no disallowed), UDP plus acceptor (88.6% most favored, 11.2% additional, 0.2% generous, and no disallowed), UDP-GlcNAc (89.9% most favored, 11.2% additional, and no generous or disallowed), and UDP-GalNAc (87.4% most favored, 12.6% additional, and no generous or disallowed).

Site-directed Mutagenesis-- A mouse EXTL2 cDNA fragment that lacks the first 53 N-terminal amino acids of the EXTL2 was amplified by PCR with the full-length cDNA template and oligonucleotides 5'-CGGGATCCGAGATCAAATCCCCGAGCAAG-3' and 5'-CGGGATCCACACAGCATGGAGAAGCCACTG-3' for 5'- and 3'-primers, respectively. The 5'-primer contained an in-frame BamHI site, whereas the intrinsic BamHI site was located 41 bp downstream of the stop codon. A two-stage PCR mutagenesis method was used to construct EXTL2 mutants. Two separate PCR reactions were performed to generate two overlapping gene fragments using the full-length mouse EXTL2 cDNA as a template. In the first PCR, the sense 5'-primer described above and one of the antisense internal mutagenic primers listed below were used: N243A, 5'-GCGATGTCGTCACAGGCCTGCGTCTCATC-3'; D246A, 5'-CATAGCGATGGCGTCACAGTTCTGCGTCTC-3'; H289A, 5'-CTCTGCAGAAAGGCCTCCGCCCGGTGCC-3'; and R293A, 5'-CTTATTTATACAGTAGGATGCCTGCAGAAA-3'. In the second round of PCR, the respective sense internal mutagenic primer (complementary to the antisense internal mutagenic primer) and the antisense 3'-primer described above were used. These two PCR products were gel-purified and then used as a template for a third PCR reaction containing the sense 5'-primer and the antisense 3'-primer described above. This produced the full-length version of the gene with the mutation incorporated. All PCR reactions were carried out with KOD DNA polymerase (TOYOBO). Each final PCR fragment of the mutants, as well as the wild-type EXTL2, was subcloned into the BamHI site of pGIR201protA (17), resulting in the fusion of the mutants, as well as the wild-type EXTL2, to the insulin signal sequence and the protein A sequence present in the vector. An NheI fragment containing each of the above fusion protein sequences was inserted into the XbaI site of the expression vector pEF-BOS (19). The nucleotide sequence of the amplified cDNA was determined with a 377 DNA sequencer (PerkinElmer Life Sciences).

Enzyme Assay-- GlcNAc transferase activities using 0.1 µM UDP-[3H]GlcNAc (39.7 Ci/mmol; PerkinElmer Life Sciences) and GalNAc transferase activities using 0.1 µM UDP-[3H]GalNAc (7 Ci/mmol; Sigma) were tested as described below. The assay mixture (25 µl) for GlcNAc transferase and GalNAc transferase contained 25 mM PIPES, pH 6.5, 15 mM MnCl2, 0.1% (v/v) Triton X-100, a radioactive donor substrate, 1 mM GlcUAbeta 1-3Galbeta 1-O-naphthalenemethanol (custom-synthesized by the Peptide Institute, Inc., Osaka, Japan), 100 µM phenylmethylsulfonyl fluoride, 1 µg/ml each leupeptin and pepstatin A, and 0.5 µg of recombinant EXTL2 protein. The reaction mixtures were incubated at 37 °C for 0.5 h. Radiolabeled products were then separated from UDP-[3H]GlcNAc or UDP-[3H]GalNAc by passage of the reaction mixture through a Sep-Pak® Vac RC C18 cartridge (Waters), as described previously (18). The recovered labeled products were quantified by liquid scintillation counting. To determine Km of an acceptor substrate for GalNAc and GlcNAc transferase activities, increasing concentrations (0, 0.03, 0.1, 0.3, 1, and 3 mM) of GlcUAbeta 1-3Galbeta 1-O-naphthalenemethanol were added to the reaction mixtures. Data were fitted by a s/v - s plot to calculate Km value.

To measure the activity of the mutants, the assay mixture contained 10 µl of the resuspended beads (enzyme source), 250 nmol of GlcUAbeta 1-3Galbeta 1-O-naphthalenemethanol (250 nmol), 250 mM UDP-[3H]GlcNAc (8.21 × 105 dpm), 100 mM MES buffer, pH 6.5, 10 mM MnCl2, and 171 mM the sodium salt of ATP in a total volume of 30 µl. Radiolabeled products were then separated from UDP-[3H]GlcNAc by passage of the reaction mixture through HPLC on a Nova-Pak® C18 column (3.9 × 150 mm; Waters), respectively. The recovered labeled products were quantified by liquid scintillation counting. In addition, the specific activities of all mutants, along with wild-type EXTL2, were calculated based on the transferase activities and the amounts of the purified fusion enzymes that were quantified using the BCA protein assay reagent (Pierce).

    RESULTS AND DISCUSSION
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ABSTRACT
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Enzyme Expression and Characterization-- EXTL2 is a type II Golgi membrane protein that contains a hydrophobic membrane binding domain at the N terminus, followed by a stem region that connects the membrane binding domain to the C-terminal catalytic domain. Protease digestion of the recombinant EXTL2 generated the catalytic domain with an apparent molecular mass of ~30 kDa on an SDS-gel electrophoresis (data not shown). This catalytic domain lacks, at most, 62 N-terminal residues based on the first residue with significant electron density. Using GlcUAbeta 1-3Galbeta 1-O-naphthalenmethanol as an acceptor substrate and the method established previously (18), we characterized the alpha 1,4-N-acetylhexosaminyltransferase activity of the catalytic domain. The domain increased the activity as a function of the acceptor concentration and reached a plateau at ~3 mM concentration. As reported previously (7), this domain catalyzed both UDP-GalNAc and UDP-GlcNA transferase activities. The specific activity (7.55 ± 0.71 nmol/mg EXTL2/h) of the UDP-GalNAc transfer reaction was more than 10-fold higher than that (0.62 ± 0.03 nmol/mg EXTL2/h) of the UDP-GlcNAc transfer reaction. On the other hand, the Km values for the acceptor substrate were similar between the two reactions: 320 and 560 µM when UDP-GalNAc and UDP-GlcNAc were used as the donor substrates, respectively. These results indicated that the bacterially expressed 30-kDa recombinant domain retains the functional characteristics of EXTL2.

Overall Fold-- The catalytic domain of mEXTL2 crystallized in this study is a globular domain consisting of residues Ala-63 to Lys-327. This domain can be divided into two subdomains, referred to as the UDP binding subdomain, comprised of residues Ala-63 to Val-150, and the acceptor binding subdomain, comprised of residues Thr-154 to Lys-327 (Fig. 1, A and B). These two subdomains are connected by the signature sequence motif for UDP-sugar-dependent glycosyltransferases, the DXD motif (Asp-151-Asp-153). This motif interacts with the Mn2+ metal ion and UDP-sugar donor and thus helps position them in the proper orientation for catalysis. The UDP binding subdomain consists of a Rossmann-like fold with four parallel beta -strands, ordered beta 3-beta 2-beta 1-beta 4, each separated in sequence by an alpha -helix. The central core of the acceptor binding subdomain is comprised of two beta -sheets with the planes of the sheets oriented relative to each other by ~90°. The first beta -sheet is a mixed beta -sheet composed of strands beta 10-beta 6-beta 11-beta 13. These strands form a continuous beta -sheet from the UDP binding subdomain resulting in a mixed eight-stranded beta -sheet. The second beta -sheet in the acceptor binding subdomain is a twisted anti-parallel sheet comprised of strands beta 8-beta 7-beta 9-beta 13. Residues at the interface of the two sheets from the acceptor binding subdomain form a hydrophobic core. These sheets are flanked by alpha -helix 4 on one side and an alpha -helix cluster consisting of alpha 5, alpha 6, alpha 7, and alpha 8 on the other side. A disulfide bond formed by the conserved residues Cys-244 and Cys-296 tethers alpha -helices 7 and 8. In addition to these structural elements, a small two-stranded anti-parallel beta -sheet exists between beta -strands 5 and 12. In the apo-form of the enzyme the majority of residues between beta -strand 12 and alpha -helix 8 are disordered.


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Fig. 1.   A, ribbon diagram of the catalytic domain (Ala-63-Lys-327) of mEXTL2 with bound UDP (orange) and GlcUAbeta 1-3Galbeta 1-O-naphthalenemethanol acceptor substrate analog (blue). The UDP-donor binding subdomain is colored lavender, and the acceptor binding subdomain is colored green. The catalytic Mn2+ ion (green) is pictured with water molecules (red), Asp-153 (of the DXD motif), and phosphate oxygens from UDP that define the inner coordination sphere. B, the same figure as Fig. 2A but rotated ~90° about a horizontal axis. These figures were created using Molscript and Raster3D (24, 25).

Donor UDP-Sugar Binding-- Upon binding donor substrate certain residues in the active site undergo conformational changes. In the apo-structure atom OE1 of Glu-288 is within hydrogen bonding distance of OH of Tyr-193 (2.7 Å), and the side chain of His-289 is directed toward the surface of the protein. The position of Glu-288 in the apo-structure is in direct conflict with the position of the donor sugars and the acceptor substrate in the UDP-GlcNAc, UDP-GalNAc, and UDP plus acceptor substrate structures. In the holo-structures these residues take on different conformations such that His-289 is directed toward the position of the donor sugar, and Glu-288 is disordered on the surface of the protein.

The positions of the UDP portions of the UDP-GalNAc and UDP-GlcNAc molecules are very similar. The UDP-sugars bind at the C terminus of beta -strands 1 and 4 of the UDP subdomain (Fig. 1, A and B). The orientation of the uridine ring is dictated by a number of interactions (Fig. 2, A and B). The position of the uridine ring is determined by hydrogen bonding interactions with Gln-72, Asn-101, and Asn-130, as well as through a ring stacking interaction with Tyr-74 (Table II). The ribose ring forms hydrogen bonds with Gln-72 and Asp-152, the middle residue of the conserved DXD motif. The third Asp of the DXD motif, Asp-153, forms an indirect interaction with the alpha - and beta -phosphates through its interaction with the catalytic Mn2+ ion. This Mn2+ ion is also coordinated to oxygen atoms from both the alpha - and beta -phosphates. The remaining ligands to the octahedral coordinated Mn2+ are three water molecules. In the donor-bound structures, Arg-76 forms the only direct protein phosphate interactions.


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Fig. 2.   Stereo diagram of the donor binding site of mEXTL2 with bound UDP-GalNAc (orange). A, residues that form direct interactions with the donor substrate are pictured (khaki). An Fo - Fc annealed omit map of the UDP-GalNAc molecule was calculated at 2.1 Å resolution and is pictured contoured at 3sigma . B, stereo diagram of the donor binding site of mEXTL2 with bound UDP-GlcNAc (orange). Residues that form direct interactions with the donor substrate are pictured (khaki). An annealed Fo - Fc omit map of the UDP-GlcNAc molecule was calculated at 2.3 Å and is pictured contoured at 2.5sigma . These figure were created using Molscript and Raster3D (24, 25).


                              
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Table II
Hydrogen bonding partners of donor and acceptor substrates
Shown are of the potential hydrogen bonding partners to the entire donor substrate UDP-GalNAc, the donor sugar of the UDP-GlcNAc donor substrate (the UDP interactions are very similar to that of UDP-GalNAc), and the acceptor substrate analog when bound to the active site of mEXTL2. Also listed are the additional interactions between the protein and the UDP molecule that occur upon acceptor substrate binding.

Although the UDP portion of the donor substrates UDP-GalNAc and UDP-GlcNAc form the same interactions with the protein, the donor sugars bind in slightly different orientations. As with other glycosyltransferase structures, the first Asp in the DXD motif, Asp-151, forms interactions with both a Mn2+ coordinated water, as well as direct interactions with the donor sugar. For UDP-GalNAc binding, other residues such as Arg-135, Asp-245, and Arg-293 are all in position to form hydrogen bonds with the donor sugar (see Fig. 2A and Table II). In the UDP-GlcNAc structure, the GlcNAc binds in an orientation such that the 4-hydroxyl and 6-hydroxyl groups occupy similar locations as the 3-hydroxyl and 4-hydroxyl groups of the GalNAc moiety of UDP-GalNAc, respectively (Fig. 2B and Table II). In this conformation, Arg-135, Asp-245, and Arg-293 can form different interactions with the donor sugar than seen for UDP-GalNAc. In addition, Asp-246 is within hydrogen bonding distance of the 3-hydroxyl group of the donor sugar. Despite the different interactions between the GlcNAc and GalNAc moieties, the C1 carbons of the UDP-GalNAc and UDP-GlcNAc molecules only differ in position by 0.8 Å in the superposition, suggesting that both molecules could be binding in catalytically relevant conformations.

The residues that form specific interactions with the donor sugar moieties are highly conserved (Fig. 3). Interestingly, many of the residues are found clustered around the totally conserved cysteines, Cys-244 and Cys-296. These residues form a disulfide bond linking alpha -helices 7 and 8. In mouse and human EXT1, EXT2, EXTL1, EXTL2, EXTL3, and EXTL2, as well as Tout-Velu from Drosophila, Arg-135, Asp-151, and Asp-246 are invariant. Residues Asp-153 and Arg-293 only differ in the mouse and human EXTL1 sequence. This is also true for His-289, which is oriented such that atom NE2 is only 4.0 and 4.4 Å from the C1 carbons of the GlcNAc and GalNAc moieties, respectively. In addition to these highly conserved amino acids, the amino acid equivalent to Asp-245 in mEXTL2 is either an Asp or Glu in all the above sequences except the EXTL1s. This suggests that the donor binding pocket for UDP-sugar in other members of the EXT family should be very similar to that of mEXTL2.


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Fig. 3.   Alignment of the amino acid sequence of mEXTL2 with the sequences of the C-terminal domains of the human heparan polymerases hEXT1 (GenBankTM accession number Q16394) and hEXT2 (GenBankTM accession number AAB07008) and the Drosophila EXT homolog Tout-Velu (GenBankTM accession number NP034292). Secondary structure elements from the mEXTL2 structures are displayed above the corresponding residues with beta -strands represented by green rectangles and alpha -helices represented by purple cylinders. Residues involved in binding UDP based on the crystal structure are shown in orange, and those that interact with the donor sugar of the UDP-donor are in red. Residues that line the acceptor substrate binding site are displayed in light blue. The two conserved cysteines involved in a disulfide bond are colored green whereas Asp-246, a proposed catalytic residue, is colored purple. The loop that becomes ordered upon acceptor substrate binding is highlighted in yellow. This alignment was created using ClustalW (26).

UDP Plus Acceptor Substrate Binding-- Binding of the acceptor substrate analog, GlcUAbeta 1-3Galbeta 1-O-naphthalenemethanol, results in the ordering of residues in a loop from Glu-275 to Glu-288. Residues Glu-275 to Thr-277 and Ser-281 to Glu-288 become ordered upon binding of the acceptor substrate analog. Residues from Gly-282 to His-285, along with Trp-284, essentially bury the active site and thus may serve to exclude excess water from the active site during catalysis. Binding of the acceptor results in two new interactions between the protein and the UDP molecule. Backbone amide nitrogens, from Trp-284 and Met-283 that become ordered upon acceptor substrate binding, form interactions with the beta - and alpha -phosphates of UDP, respectively (see Fig. 4 and Table II). Although there are no typical hydrogen bonds between residues in this loop and the acceptor, Trp-284 is oriented so that the plane of the side chain is parallel to the imaginary plane of the two sugars in the acceptor. The positions of the C1 carbon of the acceptor GlcUA and the C3 of the Gal moiety are such that the hydrogens from these carbons point directly into the center of the five- and six-member aromatic rings (3.9 Å). This orientation allows for hydrogen bonding between the aliphatic carbons of the sugar and the pi orbitals of the side chain Trp-284 forming what are known as pi-CH hydrogen bonds (20).


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Fig. 4.   Stereo diagram of the acceptor binding site of mEXTL2 with GlcUAbeta 1-3Galbeta 1 of the GlcUAbeta 1-3Galbeta 1-O-naphthalenemethanol substrate analog (blue) and UDP (orange) pictured. A Fo - Fc omit map of the analog was calculated at 2.7-Å resolution and is pictured contoured at 3sigma (light blue). Possible hydrogen bonds are displayed as dashed black lines. Interactions to the catalytic Mn2+ (green) are displayed as solid black lines. This figure was created using Molscript and Raster3D (24, 25).

A number of residues other than Trp-284 appear to be involved in binding the acceptor analog. Residues His-289, Tyr-193, and Arg-181 are in position to form interactions with the carboxylate group of the GlcUA moiety whereas Arg-181 is the only residue that is in position to form a typical hydrogen bound with the Gal moiety (Table II). In addition to these interactions, the side chains from totally conserved Lys-213 and partially conserved Phe-290 and Trp-284 help orient the acceptor through Van der Waals contacts. The most interesting interactions with the acceptor are those between the acceptor and the beta -phosphate of the UDP product. In this structure, the 3- and 4-hydroxyls of GlcUA are in position to form hydrogen bonds with the beta -phosphate. This suggests that the UDP portion of the donor may participate in acceptor binding and possibly aid in catalysis.

Although there is clear electron density for the GlcUAbeta 1-3Gal portion of the GlcUAbeta 1-3Galbeta 1-O-naphthalenemethanol analog, there is no interpretable density for the naphthalenemethanol moiety. This suggests that this moiety is disordered and forms no specific interactions with the protein. The 1-hydroxyl group of the galactose sugar that is connected to the naphthalenemethanol moiety is directed away from the protein toward the solvent. Thus, based on position of the analog, it is likely that the acceptor binding is determined by the interactions between mEXTL2 and the disaccharide GlcUAbeta 1-3Gal portion. The recognition by mEXTL2 of the disaccharide portion of the acceptor analog is reminiscent of that observed in acceptor binding to GlcAT-1, the beta 1,3-glucuronyltransferase that completes the synthesis of the common glycosaminoglycan linker region. In the crystal structure of GlcAT-1, electron density was found only for the Gal1beta -3Gal portion of the acceptor substrate Gal1beta -3Gal1beta -4Xyl (21). This suggests that for glycosyltransferases similar to EXTL2 and GlcAT-1, only the disaccharide portion of the non-reducing end of a given acceptor substrate may need to be accommodated for catalytic recognition.

Similarities to Other Structures-- Despite little to no sequence homology, the crystal structure of mEXTL2 reveals strong similarity to two other retaining glycosyltransferases for which structures have been determined, galactosyltransferases LgtC from Neisseria meningitidis and bovine alpha -1,3 galactosyltransferase, alpha 3GT (10-12). Unfortunately, one report of the alpha 3GT structure does not include an acceptor substrate, whereas coordinates are not available at the present time from the other. The structure of LgtC provides an ideal comparison, because both a complete donor substrate analog UDP-2-deoxy-2-fluoro-galactose and an acceptor analog 4'-deoxylactose are present in the crystal structure. The root mean square deviation on 171 Calpha s between residue Phe-67 to Leu-273 of mEXTL2 and 1 to Tyr-245 of LgtC is 3.1 Å as determined by the program Dali (22). The superposition of the mEXTL2 structure, with UDP-GalNAc bound to the LgtC structures, reveals that the orientation of the donor sugar molecules with respect to the UDP are quite similar (Fig. 5). In both cases the sugars are not in an extended conformation but rather tucked under the diphosphates of the UDP. Many of the interactions with the galactose moiety are conserved, as well. The first Asp in the DXD motif forms a hydrogen bond with the 3-hydroxyl, as well as Arg-86 from LgtC and Arg-135 from EXTL2. In addition, the 4-hydroxyl group is within hydrogen bonding distance to Asp-188 in LgtC and Asp-245 in EXTL2. Although the UDP-sugar donors bind in a similar orientation, the acceptor substrate in LgtC is oriented so that the planes of the sugars are rotated 90° and thus perpendicular with respect to those of mEXTL2 structure. The substrate in LgtC is translated ~4.5 Å out of the plane of the acceptor in the EXTL2 structure, as well. Despite this large difference in overall position, the position of the two acceptor hydroxyls (when O4 is modeled onto the acceptor for LgtC) with respect to the C1 carbon of the respective donor sugars, is very similar. These positions in the retaining enzymes differ from that observed in the structure of GlcAT-1, an inverting glycosyltransferases where the leaving group and acceptor are found on directly opposite sides of the C1 of the donor sugar (23). In the EXTL2 ternary complex, the acceptor 4-hydroxyl is within hydrogen bonding distance of the UDP molecule. This interaction has also been reported for alpha 3GT (12). In all three structures, LgtC, alpha 3GT, and mEXTL2 the acceptors appear to be located on the same side of the donor sugar as the leaving group suggesting these enzymes catalyze reactions that may proceed by a similar mechanism.


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Fig. 5.   Stereo diagram of the superposition of the active sites of mEXTL2 with UDP-GalNAc (orange) modeled onto the UDP in the UDP/ GlcUAbeta 1-3Galbeta 1-O-naphthalenemethanol (blue)/mEXTL2 structure (khaki) to the crystal structure of the retaining glycosyltransferase LgtC (green) with bound donor and acceptor analogs UDP-2-deoxy-2-fluoro-galactose and 4'-deoxylactose, respectively. The position of the 4'-hydroxyl of the acceptor lactose has been modeled for this figure. Residues with similar interactions with the donor sugar are displayed. This figure was created using Molscript and Raster3D (24, 25).

Catalytic Mechanism-- EXTL2 catalyzes a retaining transfer reaction in which the alpha -linkage of the C1-O1 bond of the donor UDP-sugar is maintained in the acceptor product. The retaining reaction has been suggested to proceed via either a double-displacement or SNi-like reaction mechanism (10-12). The double-displacement mechanism involves a SN2 in-line attack on the C1 carbon of the donor to create a beta -configuration intermediate either covalently linked to the protein or non-covalently-bound, followed by a subsequent SN2 attack on the C1 carbon by the acceptor, converting back to an alpha -linkage. The crystal structures of mEXTL2 do not appear to be consistent with a double-displacement mechanism. In the donor-bound structures, only atom NH2 of Arg-293 is within 4 Å of the C1 carbon, in line with the leaving group, so that, based on position, it could act as a nucleophile and attack the C1 carbon to form a covalent intermediate. Despite interactions with Asp-246 that could neutralize the positive charge, Arg-293 is still unlikely to be a good nucleophile. In addition we could find no examples in the literature where an arginine served as a nucleophile. Another highly conserved residue His-289 is also on the correct side of the donor sugar for nucleophilic attack. However, NE2 of His-289 is 4.0 and 4.4 Å, respectively, from the C1 carbon in the UDP-GlcNAc and UDP-GalNAc structures. In addition, the site-directed mutant H289A maintained full GlcNAc transferase activity (74.4 pmol/mg protein/h) as compared with the wild-type (73.1 pmol/mg protein/h). This suggests His-289 is not required for catalysis and therefore unlikely to function as a catalytic nucleophile. A third possibility for a double-displacement reaction scheme would involve a non-covalent intermediate whereby a water molecule serves as the nucleophile in the first SN2 reaction. Asp-246 could serve as a base in such a reaction. However, Arg-293 would likely require a conformational change to accommodate the water for attack on the C1 carbon of the donor, and the salt bridges between Asp-246 and Arg-293 would have to be broken for Asp-246 to be a good base. In the apo, donor substrate-bound, and acceptor substrate-bound structures, there is no evidence of a water molecule at this position or a conformational change of Arg-293. Therefore the structural and mutagenesis data combined suggest that mEXTL2 is unlikely to proceed via the double-displacement mechanism.

For the SNi-like reaction mechanism, the approach of the acceptor hydroxyl would be from the same side of the C1 carbon as the leaving group UDP, and the reaction would most likely proceed through an oxocarbenium ion-like transition state (see Figs. 6 and 7). The SNi-like reaction is mainly dissociative in nature; therefore increasing the leaving group character of the UDP would help initiate the reaction. Three new interactions exist with the beta -phosphate in the UDP plus acceptor structures that do not exist in the UDP-GlcNAc or UPD-GalNAc structures. They are the hydrogen bond with the amide nitrogen of Trp-284 and between the 3- and 4-hydroxyls of the acceptor (Fig. 4). These interactions may help stabilize the increase in negative charge on the leaving oxygen in the transition state. Superpositions of the UDP-sugar donor structures with the UDP plus acceptor place the beta -phosphate oxygens in slightly different positions. In these superpositions the 4-hydroxyl of the acceptor is not within hydrogen bonding distance of the oxygen in the phosphate-C1 bond. Thus it is unclear whether this interaction exists in the pre-catalytic state with UDP-sugar plus acceptor-bound. If, however, the interaction does exist, it would not only stabilize the increase in charge on the leaving group but would also prime the 4-hydroxyl of the acceptor for a same-side attack on the C1. Otherwise this interaction could form after dissociation of the UDP leaving group in which case the beta -phosphate could still act as a base to deprotonate the acceptor hydroxyl through the phosphate 4-hydroxyl interaction seen in the UDP plus acceptor structure. The similar catalytic implications whereby the UDP can act as a base to deprotonate the acceptor substrate were suggested previously (12) to interpret the active site structure of alpha 3GT. If acceptor binding stabilizes the leaving group and primes the acceptor for catalysis, it is possible that the main role of the hydrogen bonding network between conserved residues Asn-243, Asp-246, and Arg-293 is to orient the substrates in the proper position for catalysis.


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Fig. 6.   Stereo diagram of the superposition of the UDP-GalNAc donor substrate (orange) onto the active site of the UDP/GlcUAbeta 1-3Galbeta 1-O-naphthalenemethanol (blue)/mEXTL2 structure. Residues for which site-directed mutagenesis were attempted are shown in khaki. This figure was created using Molscript and Raster3D (24, 25).


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Fig. 7.   Possible catalytic mechanism for mEXTL2. The question mark denotes the possible interaction of Arg-293 with the C1 carbon that could help stabilize the transition state.

It is also possible these residues may work together to help stabilize the transition state. In this scenario, Arg-293 could stabilize the partial positive charge increase on the oxocarbenium transition state by either interactions with the O5' or C1 of the donor. Under normal circumstances arginine is not a good electron donor. However, both NH1 and NH2 are within hydrogen bonding distances to OD1 of Asp-246, and atom OD1 of Asn-243 is 3.2 Å from NE of Arg-293. These interactions might be capable of neutralizing the positive charge normally associated with arginine. This probably would not be enough to make it a good nucleophile (as discussed in the double-displacement mechanism) but may allow Arg-293 to delocalize the increase in positive charge on the C1 atom in the transition state. This concept is also supported by the site-directed mutagenesis studies. In the D246A mutant a positive charge would exist on Arg-293 thus actually inhibiting the reaction. This would explain why the D246A mutant has undetectable activity, and the R293A mutant that only removes the arginine side chain maintains minimal GlcNAc transferase activity (0.2 pmol/mg protein/h). The N243A mutant, which displays only 1.4 pmol/mg of protein/h of GlcNAc transferase activity could disrupt the hydrogen bond network, greatly reducing the effectiveness of Arg-293. In addition to these conserved interactions with Arg-293, in mEXTL2 Tyr-193 forms a hydrogen bond with the carboxylate group from the acceptor and is 3.1 Å from NH1 of Arg-293. Thus for EXTL2 this hydrogen bonding network, which might include the acceptor carboxylate group, could contribute to stabilization of the transition state.

Conclusions-- The x-ray crystal structures of mEXTL2 represent the first structures from the EXT gene family. These structures reveal residues important for substrate donor and acceptor bindings for the alpha 1,4-N-acetylhexosaminyltransferase activity performed by this enzyme. Based on the data presented here, the most likely mechanism for the alpha 1,4-N-acetylhexosaminyltransfer reaction appears to be the SNi-like mechanism in which interactions between the conserved residues Asn-243, Asp-246, and Arg-293 could help correctly position the substrates for catalysis, as well as stabilize the transition state. However, further analysis and more structures of other retaining glycosyltransferases will likely be required for conclusive determination of the mechanism. Nevertheless, our present study sheds light on the mechanism by which members of the EXT gene family carry out the transfer of GlcNAc from UDP-GlcNAc to an acceptor polysaccharide chain in heparan sulfate biosynthesis and provides additional data for determining how this and other retaining glycosyltransferases function.

    ACKNOWLEDGEMENTS

We thank Drs. Thomas A. Darden and Traci M. T. Hall for critical reading of the manuscript and for many thoughtful discussions on the analysis of the data.

    FOOTNOTES

* The work at Kobe Pharmaceutical University was supported in part by a grant from the Science Research Promotion Fund of the Japan Private School Promotion Foundation, a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan, and by the Human Frontier Science Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1OMX, 1OMZ, 1ON6, 1ON8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

|| To whom correspondence may be addressed. Tel.: 81-0-78-441-7570; Fax: 81-0-78-441-7569; E-mail: k-sugar@kobepharma-u.ac.jp.

** To whom correspondence may be addressed. Tel.: 919-541-2404; Fax: 919-541-0696; E-mail: negishi@niehs.nih.gov.

Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M210532200

    ABBREVIATIONS

The abbreviations used are: m, mouse; MES, 4-morpholineethanesulfonic acid; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); PEG, polyethylene glycol; alpha 3GT, alpha -1,3 galactosyltransferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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