Structural and biochemical studies of the C-terminal domain of mouse peptide-N-glycanase identify it as a mannose-binding module

Abstract

The inability of certain N-linked glycoproteins to adopt their native conformation in the endoplasmic reticulum (ER) leads to their retrotranslocation into the cytosol and subsequent degradation by the proteasome. In this pathway the cytosolic peptide-N-glycanase (PNGase) cleaves the N-linked glycan chains off denatured glycoproteins. PNGase is highly conserved in eukaryotes and plays an important role in ER-associated protein degradation. In higher eukaryotes, PNGase has an N-terminal and a C-terminal extension in addition to its central catalytic domain, which is structurally and functionally related to transglutaminases. Although the N-terminal domain of PNGase is involved in protein–protein interactions, the function of the C-terminal domain has not previously been characterized. Here, we describe biophysical, biochemical, and crystallographic studies of the mouse PNGase C-terminal domain, including visualization of a complex between this domain and mannopentaose. These studies demonstrate that the C-terminal domain binds to the mannose moieties of N-linked oligosaccharide chains, and we further show that it enhances the activity of the mouse PNGase core domain, presumably by increasing the affinity of mouse PNGase for the glycan chains of misfolded glycoproteins.

Proteins need to acquire their native conformation after protein synthesis to carry out their biological functions. Protein folding in the endoplasmic reticulum (ER) is assisted by molecular chaperones, such as calnexin and calreticulin (1), that use N-linked oligosaccharides attached to newly synthesized proteins as tags to detect their folding status. The oligosaccharide chains are attached via N-glycosidic bonds to the side-chain amide groups of Asn residues and initially consist of a tetradecamer with the composition (GlcNAc)₂(Man)₉(Glc)₃. Dynamic processing of the terminal glucose residues is essential for proper folding. Correctly folded proteins are transported to the Golgi complex for further carbohydrate modification, whereas aberrantly folded proteins are retro-translocated to the cytosol for degradation, which involves their ubiquitination, deglycosylation, and proteolytic digestion by the proteasome.

Peptide-N-glycanase (PNGase) catalyzes the deglycosylation of several misfolded N-linked glycoproteins (2, 3) by cleaving the bulky glycan chain before the proteins are degraded by the proteasome (4). PNGase is highly conserved in eukaryotes and possesses a catalytic Cys, His, and Asp triad embedded in a transglutaminase fold. Both mouse and yeast PNGase have been reported to interact with HR23B/Rad23, a protein that is also involved in DNA damage recognition (5–7). Recently the structures of yeast and mouse PNGase in complex with Rad23/HR23B and an inhibitor (8), carbobenzyloxy-Val-Ala-Asp-α-fluoromethyl ketone (Z-VAD-fmk), have been solved (9, 10). The yeast protein corresponds to the central region of mouse PNGase. The Z-VAD-fmk inhibitor covalently attaches to the active site Cys, which otherwise carries out a nucleophilic attack on the β-N-glycosidic bond linking the Asn side chain and the first GlcNAc residue, thereby cleaving the glycan chain from the protein.

Early studies (11) revealed that mouse PNGase binds to free glycan chains derived from its glycoprotein substrates, and that this binding inhibits the activity of PNGase, thus suggesting that mouse PNGase has a carbohydrate-binding activity. Moreover, recent studies revealed that PNGase specifically acts on the unfolded form of high-mannose type N-glycosylated proteins (4, 12, 13). However, how PNGase binds to glycan chains and how it recognizes the high-mannose type substrates is unknown. In this study, we present the structure of the C-terminal domain of mouse PNGase, which is present in higher eukaryotes ranging from Caenorhabditis elegans to humans with 30% sequence identity between these two species. Biochemical, biophysical, and crystallographic studies reveal that it contains a mannose-binding domain, which presumably contributes to the oligosaccharide-binding specificity of mouse PNGase. These findings suggest that the C-terminal domain increases the binding affinity between mouse PNGase and its substrates.

Results and Discussion

Overall Structure of the Mouse PNGase C-Terminal Domain.

Due to difficulties in obtaining large single crystals, this domain (residues 451–651) was expressed as either an intein fusion or a His-tagged protein. Both purified proteins yielded crystals that diffracted to ≈2 Å; however, the space groups differed (P3₂21 and C2, respectively). The structure of the intein-tagged mouse PNGase C-terminal domain was solved by using single isomorphous replacement and anomalous scattering with the aid of a Hg derivative (Table 2, which is published as supporting information on the PNAS web site) and was refined at 1.9-Å resolution (Table 1) to an R factor of 0.167 (R_free = 0.217). The N-terminal residues 451–471 are disordered in this structure. Subsequently, the structure of His-tagged form of the protein was refined at 2-Å resolution (Table 1) to an R factor of 0.15 (R_free = 0.207). The two structures are very similar as reflected in a rms deviation in the C^α positions of 0.24 Å. However, in the His-tagged protein model, residues 454–463 could be visualized, thus leaving only residues 451–453 and 464–472 as unassigned in the electron density maps. Due to the presence of these additional residues, discussion in this article focuses on the His-tagged model, which is shown in Figs. 1 and 4.

Table 1.

PNGase refinement statistics

	His-tagged native	Intein-fused native	Mannopentaose complex
Resolution limits, Å	20–2.0	20–1.9	20–1.75
Number of reflections	12,453	14,827	33,995
Number of protein/solvent atoms	1,543/142	1,475/111	2,921/453
R (Rfree)	0.150 (0.207)	0.167 (0.213)	0.172 (0.208)
Deviations from ideality
Bond distances, Å	0.014	0.017	0.015
Bond angles, °	1.495	1.413	1.543
Chiral volumes, Å3	0.096	0.094	0.125
Planar groups, Å	0.006	0.007	0.007
Torsion angles, °	7.12, 32.44, 15.34	6.95, 36.77, 13.56	6.59, 36.55, 12.53
Ramachandran statistics	0.901/0.082/0.018/0	0.927/0.061/0.012/0	0.905/0.082/0.012/0
Average B factor, Å2	29.4	44.3	18.6

R_cryst = Σ||F_o| − |F_c||/Σ|F_o|, where F_o and F_care the observed and calculated structure factor amplitudes. R_free is the same as R_crystfor 5% of the data randomly omitted from refinement. Ramachandran statistics indicate the fraction of residues in the most favored, additionally allowed, generously allowed, and disallowed regions of the Ramachandran diagram, as defined by PROCHECK [CCP4 suite (35)].

Fig. 1.

Structure and multiple sequence alignment of the mouse PNGase C-terminal domain. (A) Ribbon representation of the crystal structure. The front layer of the β-sandwich is colored in cyan, and the back sheet and loops are in gray with helices in orange. β-strands have been labeled. Figs. 1 A and B, 2C, 3 A and B, and 4 have been generated with PyMOL (36). (B) Sequence conservation of the C-terminal domain in the context of its three-dimensional structure. Strictly conserved residues have been mapped in red and conserved residues in light orange onto a surface representation of the molecule. A and B differ by a rotation of ≈90° around the vertical axis. (C) Multiple sequence alignment of PNGase C-terminal domains (Homo sapiens, human; Pan troglodytes, chimpanzee; Canis familiaris, dog; Mus musculus, mouse; Rattus norvegicus, rat; Gallus gallus, chicken; Drosophila melanogaster, fruit fly; Anopheles gambiae, mosquito; C. elegans, nematode). Strictly conserved residues are displayed in white on a red background, and type-conserved residues are shown in red. Secondary structure elements and residue numbers refer to the mouse protein. This figure was generated with the program ESPript (37). Blue stars, Ala substitutions of these residues abolish mannopentaose binding.

Fig. 4.

Hypothetical model of the relative arrangements of the core and C-terminal domains of mouse PNGase. (A) Ribbon diagram of the core domain in green and the C-terminal domain in cyan. In the core domain, residues of the catalytic triad are colored in red, and other conserved residues in the binding pocket are colored in light orange. The mannose-binding residues in the C-terminal domain are displayed in yellow. The C terminus of the core domain is labeled C, and N terminus of the C-terminal domain is labeled N. (B) Close-up view in the same orientation as in A, with the protein in a surface representation. The carbon atoms of the chitobiose are colored in yellow, and those of the mannotetraose are colored in green. The carbon atoms of the PNGase inhibitor Z-VAD-fmk are shown in magenta.

The mouse PNGase C-terminal domain is a slightly elongated molecule and displays a β-sandwich architecture, which is composed of two layers, containing nine and eight antiparallel β-strands, respectively, and three additional short helices (Fig. 1). One of the layers, which will be referred to as the front layer (based on the orientation shown in Fig. 1A), deviates strongly from a standard β-sheet and consists of an antiparallel six-stranded β-sheet (β-strands 4–6, 11, 14, and 17), including a very long strand (β11) that is also involved in the formation of a second four-stranded antiparallel β-sheet (β-strands 8, 9, 11, and 16). This sheet is rotated by ≈90° relative to the six-stranded β-sheet and is located at one end of the molecule. β-Strands 16 and 17, which reside in the four-stranded and six-stranded β-sheets, respectively, are separated by a nine-residue-long loop, whereas the loops connecting β4 and β5 in the six-stranded sheet, as well as β8 and β9 in the four-stranded sheet, completely disrupt this front layer. The back layer (Fig. 1A) displays a more traditional architecture, with a five-stranded antiparallel β-sheet (β-strands 7, 10, 12, 13, and 15) that contains a short edge strand (β7), which leaves sufficient room to allow a short three-stranded β-sheet (β1–3) also to hydrogen bond in a parallel fashion with β10. The three helices are distributed throughout the structure, with the longest helix (α1) at one end of the molecule, which will be referred to as the proximal end because it is adjacent to the N and C termini, and the shortest helix (3₁₀-2) at the distal end of the molecule. The first 3₁₀ helix (3₁₀-1) links β-strands 6 and 7 and is located between the proximal and distal ends.

An analysis of the degree of sequence conservation in the context of the three-dimensional structure of the protein reveals that a depression between two loop regions and the adjacent β-strands (β8 and β9 and the β15 and β16 junctions) is one of the two most highly conserved regions (Fig. 1 B and C). The residues decorating the saddle-shaped depression at the distal end include two tryptophans, Trp-532 and Trp-624, which sit on opposite sides on the ridges flanking the saddle and are separated by 11 Å. Three additional residues, Tyr-536, Phe-525, and Lys-527, are located on the concave side of the saddle.

The Mouse PNGase C-Terminal Domain Is Similar to the Sugar-Binding Domain of Fbs1.

The structure of the C-terminal domain was compared with a nonredundant set of proteins from the Protein Data Bank by using the Dali server (14), which identified the sugar-binding domain of Fbs1 (15) as its closest structural homolog with a Z score of 9.1. Fbs1 is an F-box protein, a component of the SCF E3 ubiquitin ligase, which is composed of the Skp1, Cul1, Roc1/Rbx1, and F-box proteins (15). The F-box proteins are the substrate-binding components of this E3 complex, and in the case of Fbs1, it was shown to recognize N-linked glycans, especially the chitobiose core via its sugar-binding domain (16, 17).

Although the C-terminal domain of mouse PNGase shares only 10% sequence identity with the sugar-binding domain of Fbs1, the two proteins are very similar in their tertiary structures and can be superimposed with an rms deviation of 3.7 Å for 128 aligned residues of 184 present in Fbs1 (Fig. 5, which is published as supporting information on the PNAS web site). Like the C-terminal domain of mouse PNGase, the sugar-binding domain of Fbs1 features a β-sandwich architecture, and its front sheet also displays the strong curvature. The chitobiose-binding region of Fbs1 has been mapped to two loop regions of its sugar-binding domain, which connect the two β-sheets at the distal end of the elongated molecule (15). The surrounding loops in this area adopt dissimilar conformations between the mouse PNGase C-terminal domain and the sugar-binding domain of Fbs1, which result in completely different surface models of the two proteins (data not shown).

The structural similarity between the two proteins suggested that, despite the differences in the region encompassing the ligand-binding site of the sugar-binding domain of Fbs1, the C-terminal domain of mouse PNGase may also be involved in carbohydrate binding, with the saddle-shaped depression being the most likely binding site based on sequence conservation. The types of residues located in this putative binding pocket are entirely consistent with those commonly observed in carbohydrate binding (18, 19). In addition to having structural similarities, the two proteins also share biochemical properties. Very recently, Yoshida et al. (20) reported that Fbs1 interacts with N-glycoproteins, especially denatured glycoproteins, in agreement with the fact that PNGase acts on misfolded N-glycosylated proteins (12, 13, 20). In the same study, SCF^Fbs1 was shown to coimmunoprecipitate with p97, a protein that also interacts with mouse PNGase (21). p97 is an AAA ATPase that functions as an extractor for misfolded proteins from the ER to the cytosol (22, 23). That both SCF^Fbs1 and PNGase interact with p97 suggests that p97 may form a platform for SCF^Fbs1 and PNGase, which may sequentially bind to the same N-glycoprotein immediately after its extraction from the ER by the retrotranslocon. In this model, the denatured glycoprotein is immediately recognized by SCF^Fbs1, ubiquitinated by the SCF E3 ligase once it emerges from the ER lumen, and then forwarded to PNGase for deglycosylation, which suggests that the ER-associated degradation pathway is highly cooperative and more processive than previously realized.

The Mouse PNGase C-Terminal Domain Binds to Oligomannose Carbohydrates.

To investigate whether the C-terminal domain of mouse PNGase indeed binds to carbohydrates, isothermal titration calorimetry (ITC) experiments were performed with two oligosaccharides (α3,α6-mannopentaose and N,N′-diacetylchitobiose) as possible ligands, both of which are part of the N-linked glycan chain (Fig. 2). The C-terminal domain was shown to bind to mannopentaose with a dissociation constant (K_d) of ≈67 μM. Full-length mouse PNGase was found to have a binding affinity very similar to that of the C-terminal fragment, whereas the mouse PNGase fragment without the C-terminal domain (1–450) displayed no detectable binding to mannopentaose (data not shown). Therefore, the C-terminal domain of mouse PNGase is at least primarily responsible for the binding of mouse PNGase to mannopentaose.

Fig. 2.

Ligand binding to the C-terminal domain of mouse PNGase. (A) (Upper) Raw ITC data showing the binding of mannopentaose to the C-terminal domain. (Lower) Fit of the experimental data (black squares) with a one-site binding model (thin line). (B) Schematic representation of the first seven residues of the N-linked oligosaccharide. Orange squares, N-acetylglucosamine; blue circles, mannose residues with numbers. Mannoses 1 and 2 and mannoses 2 and 3 are linked by α-1,6-glycosidic linkages, whereas mannoses 2 and 4 and mannoses 1 and 5 are connected by α-1,3-glycosidic bonds. (C) Close-up view into the putative oligomannose binding pocket. Residues altered by site-directed mutagenesis are shown in stick representation. Carbon atoms of residues that retain no binding affinity to mannopentaose after mutation to Ala are colored in yellow, whereas those of residues that retain partial binding affinity are colored in blue. Carbon atoms of residues that have no effect after substitution with Ala are shown in cyan.

On the other hand, neither the C-terminal domain of mouse PNGase nor the full-length enzyme showed any affinity toward chitobiose in ITC experiments. This unexpected result indicates that mouse PNGase does not engage in high-affinity interactions with the first two acetylglucosamine residues of the glycan chain, but instead may require the peptide part of the substrate for a high-affinity interaction. For comparison, the same ITC experiments were also carried out with yeast PNGase (data not shown). The yeast enzyme neither binds to mannopentaose, which is consistent with the absence of the C-terminal domain in yeast PNGase, nor interacts with chitobiose as one would expect based on the close structural relationship between the core domain of mouse PNGase and full-length yeast PNGase (9, 10).

Because α3,α6-mannotriose has been reported to inhibit the function of mouse PNGase (11), it was also used as substrate in the ITC binding assay. Mannotriose showed a binding affinity identical to that of the C-terminal domain of mouse PNGase as mannopentaose (data not shown), indicating that this branched structure may be the minimal binding unit required for interactions with this domain. Mouse PNGase may mainly recognize the high-mannose type of oligosaccharide substrates at the second branch site, which consists of mannose residues 2, 3, and 4 (Fig. 2B), in agreement with the complex structure described below.

Mannose-Binding Site of the Mouse PNGase C-Terminal Domain.

Based on the conservation of solvent-exposed residues in the C-terminal domain and its structural similarity with the sugar-binding domain of Fbs1, site-directed mutagenesis was carried out to probe the role of selected residues in oligomannose binding. These residues were individually replaced with Ala, and their mannopentaose-binding affinities were investigated by using ITC (Table 3, which is published as supporting information on the PNAS web site). These studies revealed that the K527A, E529A, W532A, Y536A, W624A, Q625A, Q628A, and R631A substitutions abolished binding of the mouse PNGase C-terminal domain to mannopentaose, whereas the F525A and E541A mutants resulted in slightly reduced binding affinities. Two additional substitutions, K530A and K540A, at residues that are not highly conserved revealed no detectable effects on binding. To confirm that the substitutions do not affect the overall structure of the C-terminal domain, the corresponding variants were analyzed by CD spectroscopy (Fig. 6A, which is published as supporting information on the PNAS web site).

From an analysis of the crystal structure, it became clear that all of these residues are located on the concave part of the saddle, except Lys-530 and Lys-540, which are pointing away from the groove (Fig. 2C). The oligomannose-binding site is apparently formed by β-strands 8, 9, and 16, which provide the concave part of the saddle, and the loops between β8 and β9, as well as β15, β16, and the second 3₁₀ helix, which form the ridges on either side of the saddle. Trp-532 resides in the loop between β-strands 8 and 9 and Trp-624 in the 3₁₀-2 helix, and these residues are on either side of the binding site.

Structure of the Mouse PNGase C-Terminal Domain in Complex with Mannopentaose.

The crystal structure of this domain in complex with mannopentaose in space group P2₁ containing two molecules (A and B) in the asymmetric unit was refined at 1.75-Å resolution to an R factor of 0.172 (R_free of 0.208). Three of the five mannoses (Man2–4) are well defined in the electron density maps (Fig. 3A), whereas Man1 is rather flexible, and Man5 is completely disordered. Man1 represents the reducing end of the mannopentaose and is connected to the chitobiose core; however, it does not engage in hydrogen bonds with the C-terminal domain (Fig. 3B). Man2, Man3, and Man4 lie within the binding groove and form several hydrogen bonds with the protein. Man2 is located at the center of the binding groove and hydrogen-bonds to the side chains of Asp-531, Trp-532, and Gln-625. Man3 has the best defined density and hydrogen-bonds to Glu-529, Gln-625, Gln-628, and Arg-631. Man4 interacts only with Lys-527. In addition to these direct protein–substrate interactions, there are water-mediated interactions (Fig. 3B), which differ in number between the two complexes present in the asymmetric unit. Compared with the apo structure, the loop between β8 and β9 moved ≈1.4 Å toward the binding pocket. At the same time, rotations of the side chains of Asp-531 and Trp-532 in this loop allow interactions with the substrate. Overall there is an excellent agreement between the cocrystal structure and the binding studies involving altered residues (compare Figs. 2C and 3B).

Fig. 3.

Mannopentaose binding activity of the C-terminal domain. (A) Stereoview of a 2F_o − F_c omit map contoured at 1 times the rms deviation, with the mannose residues omitted from molecule B. The carbon atoms of the mannotetraose are colored in green, and their numbering is the same as in Fig. 2B. (B) Stereoview of the hydrogen-bonded interactions between the C-terminal domain and mannotetraose as observed in molecule B. Carbon atoms of interacting residues are shown in yellow, potential hydrogen bonds are indicated as dashed lines in magenta, and water molecules are shown as red spheres. (C) RNase B digestion by PNGase. Time course of the reactions involving full-length mouse PNGase, mouse PNGase ΔC (residues 1–450), and the K527A variant of full-length mouse PNGase, with RNase B as the substrate. The curves were obtained by densitometric analysis of SDS/PAGE gels and represent the average of two experiments. The error bars indicate the resulting standard deviations; however, in some instances, the standard deviations are smaller than the geometric symbols that represent the data points.

The C-Terminal Domain Enhances the Catalytic Activity of Mouse PNGase.

To investigate whether the C-terminal domain contributes to the deglycosylation activity of mouse PNGase (Fig. 3C and Fig. 6B), we compared the enzymatic activity of full-length mouse PNGase, mouse PNGase ΔC (residues 1–450), and the mouse PNGase K527A variant, a mutant in which the binding of the C-terminal domain to mannopentaose is abolished according to our ITC studies. RNase B, a high-mannose type N-glycosylated protein and well characterized PNGase substrate, was used as a substrate (12). The assay showed that the half life (t_½) of glycosylated RNase B in the presence of full-length mouse PNGase is ≈2.5 min, whereas it is ≈80 min in the presence of mouse PNGase ΔC. This dramatic difference between the full-length protein and the mouse PNGase ΔC truncation confirmed that the C-terminal domain is very important for mouse PNGase activity. The mouse PNGase K527A mutant displayed an intermediate activity in this assay with a t_½ of ≈5 min. This finding indicates that although no binding to mannopentaose of the corresponding mouse PNGase C-terminal domain variant could be detected in our ITC experiments, a residual affinity presumably remains, which increases the catalytic activity of the core domain. In conclusion, although the catalytic core domain of mouse PNGase exhibits de-N-glycosylation activity in the absence of the C-terminal domain, the presence of this domain greatly accelerates substrate turnover.

Relative Arrangement of the C-Terminal and Catalytic Domains of PNGase.

Recently, the structure of the core domain of mouse PNGase in complex with the XPC binding domain of HR23B has been determined (10). This structure, especially the complex with the inhibitor Z-VAD-fmk, provides additional information on how PNGase recognizes its substrate. As demonstrated here, substrate binding by mouse PNGase involves not only the core domain, but also the C-terminal domain. Because the relative arrangement of the two domains is important in understanding the substrate specificity of PNGase from higher eukaryotes, possible domain orientations were investigated by manually positioning the structures of the mouse PNGase core and C-terminal domains while at the same time fulfilling two conditions: (i) The C terminus of the core domain and the N terminus of the C-terminal domain have to be in proximity because there are only four residues in between. Nevertheless, certain movements within this region appear possible because both ends are flexible. (ii) Because the active site cysteine (Cys-306) in the core domain has to be in close proximity to the N-glycosidic bond of the substrate, the distance between its side chain and the center of the mannose-binding motif in the C-terminal domain was required to be within 25 Å, the maximum length of four pyranoses.

In the resulting model (Fig. 4), a continuous cleft is visible that on one end accommodates the peptide represented by the Z-VAD-fmk inhibitor, followed by the chitobiose moiety in the center, and finally, the mannotetraose in the C-terminal domain. The peptide inhibitor and the chitobiose are adjacent to the catalytic triad in the core domain, whereas additional conserved residues in the core domain may be involved in chitobiose and peptide binding because of their close spatial proximity. The chitobiose orientation is based on a docking calculation with the core domain (24). Although we did not detect binding of chitobiose to the core domain in ITC experiments, Suzuki et al. (25) recently used mass spectrometry to detect binding of affinity-labeled chitobiose to the catalytic cysteine of yeast PNGase. The presence of the oligomannose binding site in the C-terminal domain extends the substrate-binding cleft of mouse PNGase and therefore presumably enhances the catalytic efficiency of the enzyme. Moreover, because both the SCF^Fbs1 E3 ligase and mouse PNGase bind to p97, the degradation efficiency of misfolded protein may be increased as p97 could function as a common platform for the ubiquitination of misfolded glycoproteins and removal of N-glycans.

Materials and Methods

Crystallization and Structure Determination.

Protein expression and purification are described in Supporting Materials and Methods, which is published as supporting information on the PNAS web site. Crystals of the mouse PNGase C-terminal domain derived from an intein-fusion protein were grown by using the hanging-drop vapor diffusion method against a reservoir solution containing 14–16% PEG 8000, 0.1 M Tris·HCl (pH 8.5), 0.12 M MgCl₂, and 10 mM DTT. Larger and better diffracting crystals were obtained by using seeding techniques. The heavy atom derivative was prepared by soaking with 10 mM sodium ethylmercurithiosalicylate for 10 min. Crystals of the His₆-tagged C-terminal domain were obtained with a reservoir solution containing 1.6 M Li₂SO₄ and 0.1 M Hepes (pH 7.0). Crystals of the complex were grown in a solution containing 19% PEG 4000, 11.5% 2-propanol, 0.1 M Tris·HCl (pH 7.5), and 0.2 M calcium acetate. Diffraction data of the apo structures were collected on beam line X26C and that of the complex structure on beam line X25 of the National Synchrotron Light Source at Brookhaven National Laboratory at 100 K. Diffraction data were indexed, integrated and scaled with HKL2000 (26).

The structure of the mouse PNGase C-terminal domain was determined by single isomorphous replacement and anomalous scattering (SIRAS). One major Hg-site was identified by SHELXD (27), and a minor site was identified by difference Fourier methods. Phase refinement was carried out with SHARP (28) to 3.3 Å, followed by solvent flattening with SOLOMON (29). A preliminary model consisting of ≈80% of the residues without side chains was built with the aid of the program O (30), and starting from this model ARP/wARP (31) was able to build 160 of 201 residues. The protein model was completed manually and was refined with REFMAC5 (32). Water molecules were added automatically with ARP/wARP. Structures of the His₆-tagged protein and complex were solved by molecular replacement by using MOLREP (33). In the complex, there are two molecules (A and B) in the asymmetric unit, which are very similar to each other, except in the flexible loop between β16 and β17. Overall, molecule B is slightly better defined in the electron density maps and is shown in Fig. 3 A and B.

ITC Experiments.

ITC measurements were carried out by using a VP-ITC microcalorimeter (MicroCal, Northampton, MA). Before the experiment, the proteins were dialyzed overnight at 4°C against a buffer containing 10 mM Tris·HCl (pH 8.5) and 0.15 M NaCl. The ligands were dissolved in the same buffer to minimize the heat of dilution. Proteins at concentrations ranging from 15 to 30 μM were titrated with 0.6–1.2 mM α3,α6-mannopentaose, N,N-diacetyl-chitobiose, or α3,α6-mannotriose at 18°C. The binding parameters were calculated with Origin version 7.0 (OriginLab, Northampton, MA) by fitting the data to a single-site binding model.

Activity Assay.

The reaction mixture was prepared at room temperature in a buffer containing 20 mM Tris (pH 8.5), 0.25 M NaCl, and 5 mm DTT. All proteins used in the assay were purified at the same time, following the same protocol. The molar ratio of enzyme to substrate was 1:40 in each reaction. The RNase B substrate (Sigma, St. Louis, MO), at a concentration of 5 μg/μl, was denatured by incubation at 95°C for 15 min before the start of the assay. The reaction was stopped by the addition of SDS sample buffer and heating at 95°C for 10 min. The resulting Coomassie-stained gels were quantitated by densitometry with the ImageJ program (34).

Abbreviations

PNGase

peptide-N-glycanase

Z-VAD-fmk

carbobenzyloxy-Val-Ala-Asp-α-fluoromethyl ketone

ER

endoplasmic reticulum

ITC

isothermal titration calorimetry.