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Proc Natl Acad Sci U S A. May 6, 2008; 105(18): 6560–6565.
Published online Apr 28, 2008. doi:  10.1073/pnas.0711491105
PMCID: PMC2373330
Biochemistry

Structural and mechanistic insight into the basis of mucopolysaccharidosis IIIB

Abstract

Mucopolysaccharidosis III (MPS III) has four forms (A–D) that result from buildup of an improperly degraded glycosaminoglycan in lysosomes. MPS IIIB is attributable to the decreased activity of a lysosomal α-N-acetylglucosaminidase (NAGLU). Here, we describe the structure, catalytic mechanism, and inhibition of CpGH89 from Clostridium perfringens, a close bacterial homolog of NAGLU. The structure enables the generation of a homology model of NAGLU, an enzyme that has resisted structural studies despite having been studied for >20 years. This model reveals which mutations giving rise to MPS IIIB map to the active site and which map to regions distant from the active site. The identification of potent inhibitors of CpGH89 and the structures of these inhibitors in complex with the enzyme suggest small-molecule candidates for use as chemical chaperones. These studies therefore illuminate the genetic basis of MPS IIIB, provide a clear biochemical rationale for the necessary sequential action of heparan-degrading enzymes, and open the door to the design and optimization of chemical chaperones for treating MPS IIIB.

Keywords: Clostridium perfringens, crystal structure, Sanfilippo, hexosaminidase, NAGLU

Mucopolysaccharidosis (MPS) is one group of autosomal recessive lysosomal storage disorders caused by the accumulation of undegraded glycosaminoglycans in lysosomes. MPS III, or Sanfilippo syndrome, is characterized by the impaired function of one of four lysosomal enzymes involved in the sequential degradation of heparan sulfate. MPS IIIA is caused by the reduced activity of heparan sulfate sulfamidase, MPS IIIB is caused by the reduced activity of an α-N-acetylglucosaminidase (NAGLU), MPS IIIC is caused by the reduced activity of acetylCoA:α-glucosaminide-N-acetyltransferase, and MPS IIID is caused by the reduced activity of N-acetyl-d-glucosamine 6-sulfatase (1). This tragic disease initially manifests as aggressiveness and hyperactivity, later progressing to mental retardation and, ultimately, CNS degeneration. There is currently no cure or effective treatment for this disease, and patients generally only survive into early adulthood.

NAGLU, a family 89 α-N-acetylglucosaminidase (www.cazy.org), is responsible for cleaving the glycosidic bond found in the backbone of heparan and thus plays a key role in heparan recycling. More than 100 different mutations in the naglu gene have been associated with the MPS IIIB phenotype, and biochemical studies have confirmed the deleterious effects of many of these mutations (19). Despite having been cloned >10 years ago, no structural or mechanistic data for NAGLU have been obtained, hindering the development of potential therapeutic strategies to treat patients suffering from MPS IIIB. To gain insight into the structure and function of this medically important enzyme, we cloned CpGH89, a bacterial family 89 glycoside hydrolase from Clostridium perfringens, to use as a model for NAGLU because they share a significant level of amino acid sequence identity (≈30%). Here, we detail the structure of this enzyme and a homology model of the human enzyme. These structures enable us to map the positions of the known mutations causing MPS IIIB onto the structure. Further, we identify potent inhibitors of this enzyme, obtain structures of the enzyme in complex with these inhibitors, and elucidate the catalytic mechanism of this enzyme. These studies should significantly facilitate the rational design of chemical chaperones that could be used to treat this deleterious genetic illness.

Results and Discussion

Activity and Structure of CpGH89.

The genome of C. perfringens, ATCC 13124, contains an ORF (CPF_0859) that encodes a putative 2,095-aa protein. This protein is classified into glycoside hydrolase family 89 and shares significant amino acid sequence similarity with the related family 89 glycoside hydrolase from Homo sapiens known as NAGLU. After molecular dissection of the modular structure of the C. perfringens GH89, we were able to overproduce a truncated construct in Escherichia coli comprising residues 26–916 and purify the resulting polypeptide, which, for simplicity, is referred to here as CpGH89.

CpGH89 was screened for catalytic activity against a panel of synthetic substrates, and activity was detected only toward para-nitrophenyl α-N-acetyl-d-glucosaminide (pNP-α-GlcNAc), consistent with the known activity of NAGLU. This substrate produced a bell-shaped activity/pH profile, suggesting that two residues with kinetic pKa values of ≈6.3 and ≈8.0 must be maintained in appropriate ionization states [see supporting information (SI) Fig. S1]. Further kinetic characterization at the optimum pH of 7.3 was carried out to determine the values of Km (1.1 ± 0.1 mM), kcat (2.6 ± 0.1 × 10−1 s−1), and kcat/Km (2.5 ± 0.3 × 10−1 mM−1s−1) of the enzyme toward pNP-α-GlcNAc. The substrate dNP-α-GlcNAc (2,4-dinitrophenyl α-N-acetyl-d-glucosaminide) (10) is turned over significantly more quickly with corresponding values for Km, kcat, and kcat/Km of 7.4 ± 1.1 × 10−1 mM, 8.5 ± 0.4 s−1, and 11.6 ± 2.3 mM−1s−1, respectively.

The structure of CpGH89 was solved by SIR with a HoCl3 derivative (see Materials and Methods). A single molecule of CpGH89, which is composed of four distinct domains, was present in the asymmetric unit. The N-terminal domain (residues 26–155) is a putative family 32 carbohydrate-binding module (CBM) having the typical β-sandwich fold adopted by many CBMs (11). The catalytic region comprises a small α/β domain (residues 170–280), an elaborated (α/β)8 barrel (residues 280–620), and an all α-helical domain that packs against the first three domains (residues 621–916) (Fig. 1A). The elaborations on the (α/β)8 barrel comprise extensions of defined secondary structure that pile onto one face of the (α/β)8 core (Fig. 1A, green).

Fig. 1.
The structure and mechanism of CpGH89. (A) A cartoon representation of CpGH89 showing its overall fold. Its constituent domains are colored as follows: light blue, N-terminal domain with amino acid sequence similarity to family 32 carbohydrate-binding ...

Family 89 Glycoside Hydrolases Use a Retaining Catalytic Mechanism.

Soaking crystals of CpGH89 with pNP-α-GlcNAc produced a product complex with a single molecule of β-d-GlcNAc in the active site (Fig. 1B). The active site of CpGH89 is a “sock”-shaped cavity lined primarily with aromatic amino acid side chains (Fig. 1 B and C). The bottom of this pocket accommodates the terminal nonreducing sugar unit of the substrate glycoside. The plane formed by the pyranose ring of the sugar sits roughly perpendicular to the entrance path into the active site. Also, the active site architecture limits the enzyme to act strictly as an exo-glycosidase. The positioning of the Glu-483 and Glu-601 side chains and their ≈6.5-Å separation in this complex suggests that these are the catalytic residues (Fig. 1 A–C). Glu-601 resides below the A face of the sugar ring with Oε ≈2.8–3.1 Å from the C1, whereas Glu-483 is positioned directly above the B face of the pyranose ring at what would be a distance of ≈2.5–3.0 Å from the oxygen in an α-glycosidic bond. Glu-601 is therefore suitably poised for nucleophilic attack of the anomeric carbon of a bound substrate, whereas Glu-483 could act as the acid/base. This architecture suggested a two-step catalytic mechanism, in which the stereochemistry at C1 of the glycon is retained. NMR analysis of the enzyme-catalyzed hydrolysis of dNP-α-GlcNAc (Fig. 1D) clearly revealed the rapid appearance of the α-anomer of GlcNAc (α-d-GlcNAc), illuminating a catalytic mechanism proceeding via retention of stereochemistry. The gradual appearance of the β-anomer of GlcNAc was due to the slow mutarotation of α-d-GlcNAc.

Mutation of Glu-601 to alanine abolished activity as far as could be evaluated by our enzyme assays using pNP-α-GlcNAc. The analogous substitution at Glu-483 reduced the value of kcat 10-fold (3.2 ± 0.4 × 10−2 s−1) and increased the value of Km 2-fold (2.3 ± 0.4 mM), thus decreasing kcat/Km by ≈20-fold (1.4 ± 0.3 × 10−2 mM−1s−1) and confirming these residues' key roles in catalysis. This apparent lack of activity of the Glu601Ala CpGH89 mutant also supports assignment of this residue as the catalytic nucleophile. This finding is consistent with previous studies of other retaining glycoside hydrolases that reveal deletion of the catalytic nucleophile results in massive impairments in catalysis (typically >103-fold) (12, 13). Mutagenesis of Glu-483, in contrast, results in a much smaller impairment in catalysis than the wild-type enzyme. Modest changes in the Michaelis–Menten parameters resulting from the mutation of acid/base residues are consistent with the cleavage of the glycosidic linkage of these activated aryl glycosides substrates not greatly benefiting from general acid catalysis (12, 13). Furthermore, Glu-483 is further from C1 (≈3.6 Å) but is appropriately positioned to the hydrogen bond with the axially oriented glycosidic oxygen of a bound substrate, suggesting that this group is the general acid/base catalytic residue. Given that the enzyme is a retaining α-glycosidase, there is no precedent, and thus it does not appear likely that the 2-acetamido group could be productively involved in catalysis in a direct manner as a catalytic group. Indeed, there is no structural evidence to imply participation of the N-acetyl group. Therefore, it is most likely that CpGH89 and, by extension, all members of GH89 use a double-displacement retaining mechanism (see Fig. S2) (14, 15). In CpGH89, we propose Glu-483 as the general acid/base catalytic residue and Glu-601 as the catalytic nucleophile, residues that are conserved across all known members of the GH89 family (data not shown).

Insights into MPS IIIB.

There is substantial genetic information describing mutations in the naglu gene that give rise to MPS IIIB and some biochemical characterization of these mutant NAGLU enzymes (7, 16). Further progress, however, has been hindered because no structure of NAGLU or any homologue is available that can be used to link this genetic information to the enzyme structure. CpGH89 has ≈30% overall amino acid sequence identity to NAGLU (see Fig. S3), allowing for the construction of a complete model of NAGLU. This model shows the overall structural conservation (Fig. 2A), whereas an examination of the active site residues of CpGH89 overlaid with the predicted NAGLU active site shows that the architecture of the active sites is almost entirely conserved (Fig. 2B and Fig. S3). The only residue that is not conserved within the active site is His-482 in CpGH89, which is substituted by an asparagine in NAGLU (Fig. 2B). Accordingly, CpGH89 acts as an excellent model of NAGLU in terms of detailed substrate recognition and catalysis, as well as overall fold.

Fig. 2.
Structural location of naturally occurring mutations in NAGLU. (A) A cartoon representation of the homology model of NAGLU showing its overall fold. The coloring of the domains is as for CpGH89 in Fig. 1A. (B) A structural overlay of the active site of ...

CpGH89 and, by extension, NAGLU are obligate exo-acting enzymes with active site architectures that appear to be unable to accommodate substrates in which the saccharides are modified. For example, processing of a sulfated terminal N-acetyl-d-glucosamine residue would not be possible, a prediction that, to the best of our knowledge, has not been experimentally demonstrated (17). This strict specificity necessitates a route to generate nonreducing terminal α-N-acetylglucosamine residues on heparan. Thus, the degradation and recycling of heparan in the human body occurs via the sequential action of three additional enzymes: heparan sulfate sulfamidase, acetylCoA:α-glucosaminidase-N-acetyltransferase, and N-acetylglucosamine 6-sulfatase (17). These enzymes are responsible for generating nonreducing terminal N-acetyl-d-glucosamine residues, and deficiency in the activity of each one of these three enzymes results in MPS IIIA, IIIC, or IIID, respectively. Given the strict structural requirement of the α-N-acetylglucosamidase for unmodified nonreducing α-linked N-acetyl-d-glucosamine residues, it becomes clear how a deficiency in any of these four enzymes could stall heparan sulfate depolymerization.

The MPS IIIB phenotype is associated with numerous missense, nonsense, and deletion mutations, with the missense mutations far outnumbering the rest. Mapping the positions of these known missense mutations onto the model of NAGLU is revealing. These mutations are randomly scattered throughout the protein, with only four occurring at residues within the active site (Fig. 2C and Fig. S3). Remarkably, there are no mutations of the catalytic residues perhaps because these mutations are so deleterious as to be prenatally lethal. By analogy to other lysosomal storage disorders, such as Tay-Sachs disease (18), these mutations likely influence the protein by reducing its stability and resulting in less functional enzyme reaching the lysosome. Indeed, Yogalingam et al. (16) recently demonstrated that mutations in NAGLU do indeed affect the stability and transiting of the enzyme through the secretory pathway. It is a well described phenomenon that many point mutations in proteins cause changes in folding that abolish proper trafficking (19, 20). Ironically, in many cases, these physiologically disastrous mutations do not destroy the catalytic activity and may only cause slight folding anomalies (21).

One proposed approach to treating these diseases that stem from decreased protein stability involves the use of small molecules (22). Compounds called chemical chaperones have been described: They bind to the newly biosynthesized protein and thereby increase its stability. These “chaperoned” mutant proteins are more stable and can therefore be transported out of the endoplasmic reticulum, avoiding intracellular degradation (2228). With this point in mind, we sought potential inhibitors that may aid in identifying, designing, and developing efficient chemical chaperones to treat MPS IIIB.

GH89 Inhibitors: Potential Chemical Chaperones?

We assessed the binding of three known N-acetylglucosaminidase inhibitors: 2-acetamido-1,2-dideoxynojirimycin (2AcDNJ) (29), O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) (30), and 6-acetamido-6-deoxycastanospermine (6AcCAS) (31) to CpGH89 by isothermal titration calorimetry (ITC) and kinetic methods (Fig. 3 A and B; see Fig. S4 for 6AcCAS data). The Ki for PUGNAc was determined by Dixon plot analysis to be 6.2 ± 0.1 μM (Fig. 3A Inset), which agreed with the dissociation constant (Kd) of 5.5 ± 0.3 μM determined by ITC (Fig. 3A). The Ki- and ITC-determined Kd values for 6AcCAS were likewise in good agreement at 92 ± 5 nM and 90 ± 20 nM, respectively (Fig. S4). Because the artificial substrates available for CpGH89 are turned over quite slowly, high concentrations of enzyme (67 nM) were required for kinetic assays, and this requirement defined the lower limit at which Ki values could be measured. However, the Kd for the tight binding inhibitor 2AcDNJ was determined by ITC to be 3.6 ± 0.3 nM, indicating ≈1,000-fold tighter binding of this inhibitor to CpGH89 relative to PUGNAc. Further, a pattern of competitive inhibition is observed for PUGNAc (Fig. 3A Inset) and 6AcCAS (Fig. S4B); because PUGNAc and 2AcDNJ inhibitors bind to the same location (Fig. 3), it seems most likely that all three inhibitors bind in a competitive manner.

Fig. 3.
Inhibitor binding by CpGH89. (A and B) Isotherms of CpGH89 binding to PUGNAc (A) and 2AcDNJ (B) produced by ITC. (Upper) Raw heat measurements. (Lower) Integrated heats. The solid lines show the fit of a one site-binding model to the data. (A Inset) Dixon ...

Diffraction data from crystals soaked with 2AcDNJ or PUGNAc yielded excellent electron density for the sugar rings in the active site pocket (Fig. 3 C and D). However, the phenyl ring substituent of PUGNAc was so disordered as to render its electron density undetectable (Fig. 3C). Numerous polar interactions with the equatorial substituents at C3–C5 likely confer high specificity for the gluco-configured sugars (Fig. 3 E and F). The basis for recognition of the 2-acetamido group also is clearly revealed in these complexes. A tight nonpolar pocket formed by the side chains of Trp-366, Trp-685, Tyr-825, and Tyr-305 accommodates the N-acetyl group (Fig. 3 C and D). Selectivity for the N-acetyl group of substrate and these inhibitors is likely imparted by the close van der Waals interactions between these aromatic residues and the substituent, as well as a favorably oriented 2.6-Å hydrogen bond between the carbonyl and Tyr-305 (Fig. 3 C–F). The structural basis for the comparatively weak binding of PUGNAc is unclear, although it may result from difficulties of the active site in accommodating the phenylcarbamate group.

The structure of CpGH89 and the model of NAGLU clearly reveal that mutations leading to MPS IIIB are scattered throughout the tertiary structure of the enzyme, in keeping with the notion that the instability of mutated NAGLU can result in the MPS IIIB phenotype (16). Mutant lysosomal enzymes that are unstable, including point mutants of NAGLU, can be retained in the endoplasmic reticulum by quality control mechanisms and thereby fail to traffic to lysosomes (16, 20, 27). When used at low concentrations, inhibitors of lysosomal glycosidases can be used as “chemical chaperones” to bind to and stabilize these mutant enzymes, thereby facilitating their maturation and passage to lysosomes (2328). Once in the lysosome, these mutant enzymes have catalytic activities that support normal cellular functioning (2328). Accordingly, a chemical chaperone approach to treating MPS IIIB is likely viable, and our results indicate that 2AcDNJ and 6AcCAS are effective inhibitors that may provide an initial lead for developing such chemical chaperones. Indeed, both of these inhibitors also have been shown to inhibit NAGLU (32) in vitro with nanomolar potencies (Ki for 2AcDNJ = 450 nM and Ki for 6AcCAS = 87 nM), in keeping with the results we observe here for CpGH89. These data, in combination with the near-complete conservation of active site residues, strongly support the validity of using the structure of CpGH89 as a guide for inhibitor design. However, the use of these particular compounds may be limited because they are known to be potent inhibitors of several other human enzymes. Nevertheless, there remains the potential to generate more selective inhibitors (28, 33, 34).

In summary, the data presented here reveal the structural basis underlying the specificity of NAGLU for terminal nonreducing N-acetyl-d-glucosamine residues as well as its place within the glycosaminoglycan catabolic pathway. The extensive stereochemically specific interactions with the sugar hydroxyl groups, coupled with the sock-shaped architecture of the active site, strictly defines selectivity for the α-configured glycosidic linkage of unornamented terminal N-acetyl-d-glucosamine residues. Further, the collective structural, mechanistic, and inhibition data presented here provide a clear template that will facilitate the design of effective chemical chaperones that specifically target NAGLU.

Materials and Methods

All reagents are from Sigma–Aldrich unless otherwise stated.

Cloning, Protein Production, and Purification.

The gene fragment comprising nucleotides 76–2748 of the cpgh89 gene (locus tag CPF_0859), which encodes a polypeptide with a putative N-terminal family 32 carbohydrate-binding module followed by the catalytic module, was PCR amplified from C. perfringens ATCC 13124 genomic DNA (Sigma) and cloned into pET-28a(+) (Novagen) via 5′ and 3′ NheI and XhoI restriction endonuclease sites to generate pCBM1GH89 by using standard PCR-cloning procedures. The polypeptide encoded by this recombinant gene comprises an N-terminal H6 tag, followed by a thrombin protease cleavage site fused to the N terminus of the CBM32/GH89 modules, and is referred to as CpGH89. “Mega-primer” PCR site-directed mutagenesis procedures were used to introduce the E483A and E601A substitutions. The DNA sequence fidelity of all constructs was verified by bidirectional sequencing. CpGH89 was produced in BL21 star (DE3) E. coli cells (Invitrogen) harboring the pCBM1GH89 and purified by immobilized metal affinity chromatography by using methods described previously (35).

Enzyme Kinetics.

All steady-state kinetic studies were carried out at 25°C in a Cary/Varian 300 Bio UV-Visible Spectrophotometer. The pH dependence of CpGH89 was determined by using 133 nM enzyme and a pNP-α-GlcNAc concentration of 1 mM. Then, 0.1 M sodium acetate was used to buffer within the pH range 4.6–6.0, 0.1 M sodium phosphate for the pH range 6.0–8.0, and 0.1 M glycine-glycine for the pH range 8.0–9.8. At the transition between buffers, one or two pHs were overlapped to control for the effects of the buffer on the enzyme activity. Standard-reaction mixtures for the determination of kinetic constants were done at the optimal pH (0.1 M sodium phosphate, pH 7.3) in 750-μl volumes containing 15.2 nM CpGH89 or 390 nM Glu483Ala, 10% BSA, and 0–2.5 mM pNP-α-GlcNAc. The 4-nitrophenolate production was measured at 400 nm over a 5-min time period. Rate of release was determined by linear regression over the linear period of release on the curve. All experiments were performed in triplicate. Michealis–Menten parameters were determined by nonlinear curve fitting by using Origin7. Similarly, determination of the kinetic constants for dNP-α-GlcNAc, synthesized as described previously (10), was done in 0.1 M sodium phosphate (pH 7.3) in 750-μl volumes containing 240 nM CpGH89, 10% BSA, and 0–3.7 mM dNP-α-GlcNAc.

NMR Experiments.

1H NMR spectroscopy (600 MHz Bruker AMX spectrometer) was used to identify the products of the enzyme-catalyzed reaction. The reaction was carried out in ≈0.6 ml [25°C, 0.1 M Gly-Gly (pH 7.7)] containing 3 mM dNP-α-GlcNAc. Initiation of the reaction was done by the addition of 30 μl of a 70 μM stock of the enzyme CpGH89. The hydrolysis of the dNP-α-GlcNAc was monitored until the reaction reached equilibrium. Spectral data were collected with 64,000 data points (64 k) over a spectral width of 7,211 Hz (12.01 ppm), with a relaxation delay of 1 s and 32 scans. An initial spectrum (referred to as time 0) containing substrate and buffer was acquired before the addition of enzyme.

Inhibitor-Binding Studies.

The Ki value for CpGH89 binding to PUGNAc and 6AcCAS was determined by linear regression of Dixon plots for inhibitor concentrations ranging from 1/3 to 3 times Ki.

ITC was performed with a VP-ITC (MicroCal) as described previously (35). CpGH89 was dialyzed extensively against 0.1 M sodium phosphate (pH 7.3). Solid inhibitor was resuspended in buffer saved from the dialysis. Twenty-five 10-μl injections of 80–100 μM 2AcDNJ or 6AcCAS were titrated into 6–8 μM protein. Similarly, 25 5-μl injections of 1,700 μM PUGNAc were titrated into 50 μM CpGH89. The C values (36) were maintained between 10 and 1,000. Binding stoichiometry, enthalpy, and the equilibrium-association constant were determined by fitting the heat of dilution-corrected data to a one site-binding model. All values reported were averages, and standard deviations of experiments were performed in triplicate.

Crystallization, Data Collection, Structure Solution, and Refinement.

CpGH89 in 20 mM Tris·HCl (pH 8.0) was digested with thrombin overnight at room temperature. Digested protein was applied to an S-200 Sephacryl high-resolution size-exclusion column, and fractions containing pure CpGH89 (as assessed by SDS/PAGE) were pooled and concentrated. Crystal trials were set up by using the hanging drop vapor diffusion method with both thrombin-treated and untreated CpGH89. Diffraction-quality crystals were obtained in 2.0 M (NH4)2SO4 with 3% glycerol (no buffer). Both thrombin-treated and untreated CpGH89 yielded crystals.

The crystals were cryoprotected by a 10-s immersion in a solution containing 2 M ammonium sulfate and 30% glycerol, followed by freezing in a nitrogen Cryostream. Diffraction data were collected with a Rigaku R-AXIS IV++ area detector coupled to an MM-002 x-ray generator with Osmic “blue” optics and an Oxford Cryostream 700. Data were processed with Crystal Clear/d*trek (37).

A holmium (III) derivative was obtained by soaking native CpGH89 crystals in mother liquor supplemented with 1 mM HoCl3 for 3 days. This crystal was cryoprotected as for native crystals. The heavy atom substructure of two holmium sites was determined with the programs ShelxC/D (38) by using the isomorphous differences between the derivative and a native dataset using data to a resolution of 4.5 Å. Refinement of heavy atom parameters and initial phasing to a resolution of 3.0 Å was performed with SHARP (39). Density modification with DM (40) was used to improve and extend the phases to 2.4 Å and ultimately yielded interpretable electron density maps. ARP/wARP (41) was able to build the backbone of ≈40% of the molecule. Manual model building in COOT (42) allowed the backbone of the initial model to be corrected and extended to ≈65% completeness with some side chains. This initial model was used as input into ARP/wARP, which was then able to build a model of ≈90% completeness with ≈80% of the side chains correctly docked. The model was manually completed in COOT, followed by refinement with REFMAC (43).

Inhibitor complexes (2-acetamido-1,2-dideoxynojirimycin and PUGNAc) and product complexes were obtained by adding excess inhibitor or substrate (pNP-α-GlcNAc) to mother liquor and soaking for a period of 1 h to up to 1 week. Crystals were cryoprotected as for the native data. These complexes were solved by using the native CpGH89 model as a starting point. Ligands were built into the models by using COOT. When necessary, appropriate REFMAC library files containing the restraints for the ligands were obtained by using the PRODRG Server (44).

In all datasets, the same 5% of the observations were flagged as “free” (45) and used to monitor refinement procedures. Water molecules were added by using the REFMAC implementation of ARP/wARP and inspected visually before deposition. See Table S1 for all data collection, refinement, and model validation statistics.

Homology Modeling.

A homology model of NAGLU was constructed from the CpGH89-product complex coordinates by using methods essentially identical to those described previously (46) with DeepView and the Swiss-Model Automated Comparative Protein Modeling Server (47). The resulting model was subjected to 10 rounds of model idealization with REFMAC, and the output model was manually examined residue by residue. The model was validated by tools within COOT and PROCHECK (48).

Accession Codes.

Coordinates and structure factors have been deposited with the PDB codes of 2VCC for the native structure, 2VCA for the GlcNAc complex, 2VC9 for the 2AcDNJ complex, and 2VCB for the PUGNAc complex.

Supplementary Material

Supporting Information:

Acknowledgments.

This work was supported by Canadian Institutes of Health Research Grants MOP 68913, ABB, and NIP79924 (to D.J.V.), Michael Smith Foundation for Health Research and Natural Sciences and Engineering Research Council of Canada Doctoral Fellowships (to E.F.-B.), a Canada Research Chair award in Chemical Glycobiology and a Michael Smith Foundation for Health Research Scholar award (to D.J.V.), and a Canada Research Chair award in Molecular Interactions and a Michael Smith Foundation for Health Research Scholar award (to A.B.B.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 2VCC (for the native structure), 2VCA (for the GLcNAc complex), 2VC9 (for the 2AcDNJ complex), and 2VCB (for the PUGNAc complex)].

This article contains supporting information online at www.pnas.org/cgi/content/full/0711491105/DCSupplemental.

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