Structural and biochemical studies of the C-terminaldomain of mouse peptide-N-glycanase identifyit as a mannose-binding moduleXiaoke Zhou*†, Gang Zhao*†, James J. Truglio*†, Liqun Wang*†, Guangtao Li†, William J. Lennarz†,and Hermann Schindelin*†‡§

*Center for Structural Biology and †Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215; and ‡Rudolf VirchowCenter for Experimental Biomedicine and Institute of Structural Biology, University of Wurzburg, Versbacher Strasse 9, 97078 Wurzburg, Germany

Edited by John Kuriyan, University of California, Berkeley, CA, and approved September 15, 2006 (received for review April 11, 2006)

The inability of certain N-linked glycoproteins to adopt their nativeconformation in the endoplasmic reticulum (ER) leads to theirretrotranslocation into the cytosol and subsequent degradation bythe proteasome. In this pathway the cytosolic peptide-N-glycanase(PNGase) cleaves the N-linked glycan chains off denatured glyco-proteins. PNGase is highly conserved in eukaryotes and plays animportant role in ER-associated protein degradation. In highereukaryotes, PNGase has an N-terminal and a C-terminal extensionin addition to its central catalytic domain, which is structurally andfunctionally related to transglutaminases. Although the N-terminaldomain of PNGase is involved in protein–protein interactions, thefunction of the C-terminal domain has not previously been char-acterized. Here, we describe biophysical, biochemical, and crystal-lographic studies of the mouse PNGase C-terminal domain, includ-ing visualization of a complex between this domain andmannopentaose. These studies demonstrate that the C-terminaldomain binds to the mannose moieties of N-linked oligosaccharidechains, and we further show that it enhances the activity of themouse PNGase core domain, presumably by increasing the affinityof mouse PNGase for the glycan chains of misfolded glycoproteins.

endoplasmic reticulum � N-linked glycoproteins � proteasome �protein degradation � deglycosylation

Proteins need to acquire their native conformation afterprotein synthesis to carry out their biological functions.

Protein folding in the endoplasmic reticulum (ER) is assisted bymolecular chaperones, such as calnexin and calreticulin (1), thatuse N-linked oligosaccharides attached to newly synthesizedproteins as tags to detect their folding status. The oligosaccha-ride chains are attached via N-glycosidic bonds to the side-chainamide groups of Asn residues and initially consist of a tetra-decamer with the composition (GlcNAc)2(Man)9(Glc)3. Dy-namic processing of the terminal glucose residues is essential forproper folding. Correctly folded proteins are transported to theGolgi complex for further carbohydrate modification, whereasaberrantly folded proteins are retro-translocated to the cytosolfor degradation, which involves their ubiquitination, deglycosy-lation, and proteolytic digestion by the proteasome.

Peptide-N-glycanase (PNGase) catalyzes the deglycosylationof several misfolded N-linked glycoproteins (2, 3) by cleaving thebulky glycan chain before the proteins are degraded by theproteasome (4). PNGase is highly conserved in eukaryotes andpossesses a catalytic Cys, His, and Asp triad embedded in atransglutaminase fold. Both mouse and yeast PNGase have beenreported to interact with HR23B�Rad23, a protein that is alsoinvolved in DNA damage recognition (5–7). Recently the struc-tures of yeast and mouse PNGase in complex with Rad23�HR23B and an inhibitor (8), carbobenzyloxy-Val-Ala-Asp-�-f luoromethyl ketone (Z-VAD-fmk), have been solved (9, 10).The yeast protein corresponds to the central region of mousePNGase. The Z-VAD-fmk inhibitor covalently attaches to theactive site Cys, which otherwise carries out a nucleophilic attack

on the �-N-glycosidic bond linking the Asn side chain and thefirst GlcNAc residue, thereby cleaving the glycan chain from theprotein.

Early studies (11) revealed that mouse PNGase binds to freeglycan chains derived from its glycoprotein substrates, and thatthis binding inhibits the activity of PNGase, thus suggesting thatmouse PNGase has a carbohydrate-binding activity. Moreover,recent studies revealed that PNGase specifically acts on theunfolded form of high-mannose type N-glycosylated proteins (4,12, 13). However, how PNGase binds to glycan chains and howit recognizes the high-mannose type substrates is unknown. Inthis study, we present the structure of the C-terminal domain ofmouse PNGase, which is present in higher eukaryotes rangingfrom Caenorhabditis elegans to humans with 30% sequenceidentity between these two species. Biochemical, biophysical,and crystallographic studies reveal that it contains a mannose-binding domain, which presumably contributes to the oligosac-charide-binding specificity of mouse PNGase. These findingssuggest that the C-terminal domain increases the binding affinitybetween mouse PNGase and its substrates.

Results and DiscussionOverall Structure of the Mouse PNGase C-Terminal Domain. Due todifficulties in obtaining large single crystals, this domain (resi-dues 451–651) was expressed as either an intein fusion or aHis-tagged protein. Both purified proteins yielded crystals thatdiffracted to �2 Å; however, the space groups differed (P3221and C2, respectively). The structure of the intein-tagged mousePNGase C-terminal domain was solved by using single isomor-phous replacement and anomalous scattering with the aid of aHg derivative (Table 2, which is published as supporting infor-mation on the PNAS web site) and was refined at 1.9-Åresolution (Table 1) to an R factor of 0.167 (Rfree � 0.217). TheN-terminal residues 451–471 are disordered in this structure.Subsequently, the structure of His-tagged form of the proteinwas refined at 2-Å resolution (Table 1) to an R factor of 0.15(Rfree � 0.207). The two structures are very similar as reflectedin a rms deviation in the C� positions of 0.24 Å. However, in theHis-tagged protein model, residues 454–463 could be visualized,

Author contributions: X.Z., W.J.L., and H.S. designed research; X.Z., G.Z., L.W., and H.S.performed research; G.Z. and G.L. contributed new reagents�analytic tools; J.J.T. and H.S.analyzed data; and X.Z., W.J.L., and H.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Abbreviations: PNGase, peptide-N-glycanase; Z-VAD-fmk, carbobenzyloxy-Val-Ala-Asp-�-fluoromethyl ketone; ER, endoplasmic reticulum; ITC, isothermal titration calorimetry.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org [PDB ID codes 2G9F (intein-tagged protein), 2G9G(His-tagged protein), and 2I74 (complex)].

§To whom correspondence should be addressed. E-mail: hermann.schindelin@virchow.uni-wuerzburg.de.

© 2006 by The National Academy of Sciences of the USA

17214–17219 � PNAS � November 14, 2006 � vol. 103 � no. 46 www.pnas.org�cgi�doi�10.1073�pnas.0602954103

Dow

nloa

ded

by g

uest

on

Dec

embe

r 19

, 202

1

thus leaving only residues 451–453 and 464–472 as unassignedin the electron density maps. Due to the presence of theseadditional residues, discussion in this article focuses on theHis-tagged model, which is shown in Figs. 1 and 4.

The mouse PNGase C-terminal domain is a slightly elongatedmolecule and displays a �-sandwich architecture, which is com-posed of two layers, containing nine and eight antiparallel�-strands, respectively, and three additional short helices (Fig. 1).One of the layers, which will be referred to as the front layer (basedon the orientation shown in Fig. 1A), deviates strongly from astandard �-sheet and consists of an antiparallel six-stranded �-sheet(�-strands 4–6, 11, 14, and 17), including a very long strand (�11)that is also involved in the formation of a second four-strandedantiparallel �-sheet (�-strands 8, 9, 11, and 16). This sheet is rotatedby �90° relative to the six-stranded �-sheet and is located at one endof the molecule. �-Strands 16 and 17, which reside in the four-stranded and six-stranded �-sheets, respectively, are separated by anine-residue-long loop, whereas the loops connecting �4 and �5 inthe six-stranded sheet, as well as �8 and �9 in the four-strandedsheet, completely disrupt this front layer. The back layer (Fig. 1A)displays a more traditional architecture, with a five-stranded anti-parallel �-sheet (�-strands 7, 10, 12, 13, and 15) that contains a shortedge strand (�7), which leaves sufficient room to allow a shortthree-stranded �-sheet (�1–3) also to hydrogen bond in a parallelfashion with �10. The three helices are distributed throughout thestructure, with the longest helix (�1) at one end of the molecule,which will be referred to as the proximal end because it is adjacentto the N and C termini, and the shortest helix (310-2) at the distalend of the molecule. The first 310 helix (310-1) links �-strands 6 and7 and is located between the proximal and distal ends.

An analysis of the degree of sequence conservation in the contextof the three-dimensional structure of the protein reveals that adepression between two loop regions and the adjacent �-strands(�8 and �9 and the �15 and �16 junctions) is one of the two mosthighly conserved regions (Fig. 1 B and C). The residues decoratingthe saddle-shaped depression at the distal end include two trypto-phans, Trp-532 and Trp-624, which sit on opposite sides on theridges flanking the saddle and are separated by 11 Å. Threeadditional residues, Tyr-536, Phe-525, and Lys-527, are located onthe concave side of the saddle.

The Mouse PNGase C-Terminal Domain Is Similar to the Sugar-BindingDomain of Fbs1. The structure of the C-terminal domain wascompared with a nonredundant set of proteins from the ProteinData Bank by using the Dali server (14), which identified thesugar-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 theSCF E3 ubiquitin ligase, which is composed of the Skp1, Cul1,Roc1�Rbx1, and F-box proteins (15). The F-box proteins are thesubstrate-binding components of this E3 complex, and in the caseof Fbs1, it was shown to recognize N-linked glycans, especially thechitobiose core via its sugar-binding domain (16, 17).

Although the C-terminal domain of mouse PNGase shares only10% sequence identity with the sugar-binding domain of Fbs1, thetwo proteins are very similar in their tertiary structures and can besuperimposed with an rms deviation of 3.7 Å for 128 alignedresidues of 184 present in Fbs1 (Fig. 5, which is published assupporting information on the PNAS web site). Like the C-terminaldomain of mouse PNGase, the sugar-binding domain of Fbs1features a �-sandwich architecture, and its front sheet also displaysthe strong curvature. The chitobiose-binding region of Fbs1 hasbeen mapped to two loop regions of its sugar-binding domain,which connect the two �-sheets at the distal end of the elongatedmolecule (15). The surrounding loops in this area adopt dissimilarconformations between the mouse PNGase C-terminal domain andthe sugar-binding domain of Fbs1, which result in completelydifferent 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-terminaldomain of mouse PNGase may also be involved in carbohydratebinding, with the saddle-shaped depression being the most likelybinding site based on sequence conservation. The types of residueslocated in this putative binding pocket are entirely consistent withthose commonly observed in carbohydrate binding (18, 19). Inaddition to having structural similarities, the two proteins also sharebiochemical properties. Very recently, Yoshida et al. (20) reportedthat Fbs1 interacts with N-glycoproteins, especially denaturedglycoproteins, in agreement with the fact that PNGase acts onmisfolded N-glycosylated proteins (12, 13, 20). In the same study,SCFFbs1 was shown to coimmunoprecipitate with p97, a protein thatalso interacts with mouse PNGase (21). p97 is an AAA ATPasethat functions as an extractor for misfolded proteins from the ERto the cytosol (22, 23). That both SCFFbs1 and PNGase interact withp97 suggests that p97 may form a platform for SCFFbs1 and PNGase,which may sequentially bind to the same N-glycoprotein immedi-ately after its extraction from the ER by the retrotranslocon. In thismodel, the denatured glycoprotein is immediately recognized bySCFFbs1, ubiquitinated by the SCF E3 ligase once it emerges fromthe ER lumen, and then forwarded to PNGase for deglycosylation,which suggests that the ER-associated degradation pathway ishighly cooperative and more processive than previously realized.

Table 1. PNGase refinement statistics

His-tagged native Intein-fused nativeMannopentaose

complex

Resolution limits, Å 20–2.0 20–1.9 20–1.75Number of reflections 12,453 14,827 33,995Number of protein�solvent atoms 1,543�142 1,475�111 2,921�453R (Rfree) 0.150 (0.207) 0.167 (0.213) 0.172 (0.208)Deviations from ideality

Bond distances, Å 0.014 0.017 0.015Bond angles, ° 1.495 1.413 1.543Chiral volumes, Å3 0.096 0.094 0.125Planar groups, Å 0.006 0.007 0.007Torsion 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�0Average B factor, Å2 29.4 44.3 18.6

Rcryst � ���Fo� � �Fc�����Fo�, where Fo and Fc are the observed and calculated structure factor amplitudes. Rfree isthe same as Rcryst for 5% of the data randomly omitted from refinement. Ramachandran statistics indicate thefraction of residues in the most favored, additionally allowed, generously allowed, and disallowed regions of theRamachandran diagram, as defined by PROCHECK [CCP4 suite (35)].

Zhou et al. PNAS � November 14, 2006 � vol. 103 � no. 46 � 17215

BIO

CHEM

ISTR

Y

Dow

nloa

ded

by g

uest

on

Dec

embe

r 19

, 202

1

The Mouse PNGase C-Terminal Domain Binds to OligomannoseCarbohydrates. To investigate whether the C-terminal domain ofmouse PNGase indeed binds to carbohydrates, isothermal titrationcalorimetry (ITC) experiments were performed with two oligosac-charides (�3,�6-mannopentaose and N,N�-diacetylchitobiose) aspossible ligands, both of which are part of the N-linked glycan chain(Fig. 2). The C-terminal domain was shown to bind to mannopen-taose with a dissociation constant (Kd) of �67 �M. Full-lengthmouse PNGase was found to have a binding affinity very similar tothat of the C-terminal fragment, whereas the mouse PNGasefragment without the C-terminal domain (1–450) displayed nodetectable binding to mannopentaose (data not shown). Therefore,

the C-terminal domain of mouse PNGase is at least primarilyresponsible for the binding of mouse PNGase to mannopentaose.

On the other hand, neither the C-terminal domain of mousePNGase nor the full-length enzyme showed any affinity towardchitobiose in ITC experiments. This unexpected result indicatesthat mouse PNGase does not engage in high-affinity interactionswith the first two acetylglucosamine residues of the glycan chain,but instead may require the peptide part of the substrate for ahigh-affinity interaction. For comparison, the same ITC experi-ments were also carried out with yeast PNGase (data not shown).The yeast enzyme neither binds to mannopentaose, which isconsistent with the absence of the C-terminal domain in yeast

Fig. 1. Structure and multiple sequence alignment of the mouse PNGase C-terminal domain. (A) Ribbon representation of the crystal structure. The front layerof the �-sandwich is colored in cyan, and the back sheet and loops are in gray with helices in orange. �-strands have been labeled. Figs. 1 A and B, 2C, 3 A andB, and 4 have been generated with PyMOL (36). (B) Sequence conservation of the C-terminal domain in the context of its three-dimensional structure. Strictlyconserved 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 rotationof �90° around the vertical axis. (C) Multiple sequence alignment of PNGase C-terminal domains (Homo sapiens, human; Pan troglodytes, chimpanzee; Canisfamiliaris, 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 elementsand residue numbers refer to the mouse protein. This figure was generated with the program ESPript (37). Blue stars, Ala substitutions of these residues abolishmannopentaose binding.

17216 � www.pnas.org�cgi�doi�10.1073�pnas.0602954103 Zhou et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 19

, 202

1

PNGase, nor interacts with chitobiose as one would expect basedon the close structural relationship between the core domain ofmouse PNGase and full-length yeast PNGase (9, 10).

Because �3,�6-mannotriose has been reported to inhibit thefunction of mouse PNGase (11), it was also used as substrate in theITC binding assay. Mannotriose showed a binding affinity identicalto that of the C-terminal domain of mouse PNGase as mannopen-taose (data not shown), indicating that this branched structure maybe the minimal binding unit required for interactions with thisdomain. Mouse PNGase may mainly recognize the high-mannosetype of oligosaccharide substrates at the second branch site, whichconsists of mannose residues 2, 3, and 4 (Fig. 2B), in agreement withthe complex structure described below.

Mannose-Binding Site of the Mouse PNGase C-Terminal Domain.Based on the conservation of solvent-exposed residues in theC-terminal domain and its structural similarity with the sugar-binding domain of Fbs1, site-directed mutagenesis was carried outto probe the role of selected residues in oligomannose binding.These residues were individually replaced with Ala, and theirmannopentaose-binding affinities were investigated by using ITC(Table 3, which is published as supporting information on the PNASweb site). These studies revealed that the K527A, E529A, W532A,Y536A, W624A, Q625A, Q628A, and R631A substitutions abol-ished binding of the mouse PNGase C-terminal domain to man-nopentaose, whereas the F525A and E541A mutants resulted inslightly reduced binding affinities. Two additional substitutions,K530A and K540A, at residues that are not highly conservedrevealed no detectable effects on binding. To confirm that thesubstitutions do not affect the overall structure of the C-terminaldomain, the corresponding variants were analyzed by CD spectros-copy (Fig. 6A, which is published as supporting information on thePNAS web site).

From an analysis of the crystal structure, it became clear that allof these residues are located on the concave part of the saddle,except Lys-530 and Lys-540, which are pointing away from thegroove (Fig. 2C). The oligomannose-binding site is apparentlyformed by �-strands 8, 9, and 16, which provide the concave part

of the saddle, and the loops between �8 and �9, as well as �15, �16,and the second 310 helix, which form the ridges on either side of thesaddle. Trp-532 resides in the loop between �-strands 8 and 9 andTrp-624 in the 310-2 helix, and these residues are on either side ofthe binding site.

Structure of the Mouse PNGase C-Terminal Domain in Complex withMannopentaose. The crystal structure of this domain in complexwith mannopentaose in space group P21 containing two molecules(A and B) in the asymmetric unit was refined at 1.75-Å resolutionto an R factor of 0.172 (Rfree 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 disor-dered. Man1 represents the reducing end of the mannopentaoseand is connected to the chitobiose core; however, it does not engagein hydrogen bonds with the C-terminal domain (Fig. 3B). Man2,Man3, and Man4 lie within the binding groove and form severalhydrogen bonds with the protein. Man2 is located at the center ofthe binding groove and hydrogen-bonds to the side chains ofAsp-531, Trp-532, and Gln-625. Man3 has the best defined densityand hydrogen-bonds to Glu-529, Gln-625, Gln-628, and Arg-631.Man4 interacts only with Lys-527. In addition to these directprotein–substrate interactions, there are water-mediated interac-tions (Fig. 3B), which differ in number between the two complexespresent in the asymmetric unit. Compared with the apo structure,the loop between �8 and �9 moved �1.4 Å toward the bindingpocket. At the same time, rotations of the side chains of Asp-531and Trp-532 in this loop allow interactions with the substrate.Overall there is an excellent agreement between the cocrystalstructure and the binding studies involving altered residues (com-pare Figs. 2C and 3B).

The C-Terminal Domain Enhances the Catalytic Activity of MousePNGase. To investigate whether the C-terminal domain contributesto the deglycosylation activity of mouse PNGase (Fig. 3C and Fig.6B), we compared the enzymatic activity of full-length mousePNGase, mouse PNGase �C (residues 1–450), and the mousePNGase K527A variant, a mutant in which the binding of the

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 theN-linked oligosaccharide. Orange squares, N-acetylglucosamine; blue circles, mannose residues with numbers. Mannoses 1 and 2 and mannoses 2 and 3 are linkedby �-1,6-glycosidic linkages, whereas mannoses 2 and 4 and mannoses 1 and 5 are connected by �-1,3-glycosidic bonds. (C) Close-up view into the putativeoligomannose binding pocket. Residues altered by site-directed mutagenesis are shown in stick representation. Carbon atoms of residues that retain no bindingaffinity to mannopentaose after mutation to Ala are colored in yellow, whereas those of residues that retain partial binding affinity are colored in blue. Carbonatoms of residues that have no effect after substitution with Ala are shown in cyan.

Zhou et al. PNAS � November 14, 2006 � vol. 103 � no. 46 � 17217

BIO

CHEM

ISTR

Y

Dow

nloa

ded

by g

uest

on

Dec

embe

r 19

, 202

1

C-terminal domain to mannopentaose is abolished according to ourITC studies. RNase B, a high-mannose type N-glycosylated proteinand well characterized PNGase substrate, was used as a substrate(12). The assay showed that the half life (t1�2

) of glycosylated RNaseB in the presence of full-length mouse PNGase is �2.5 min, whereasit is �80 min in the presence of mouse PNGase �C. This dramaticdifference between the full-length protein and the mouse PNGase�C truncation confirmed that the C-terminal domain is veryimportant for mouse PNGase activity. The mouse PNGase K527Amutant displayed an intermediate activity in this assay with a t1�2

of�5 min. This finding indicates that although no binding to man-nopentaose of the corresponding mouse PNGase C-terminal do-main variant could be detected in our ITC experiments, a residualaffinity presumably remains, which increases the catalytic activity of

the core domain. In conclusion, although the catalytic core domainof mouse PNGase exhibits de-N-glycosylation activity in the ab-sence of the C-terminal domain, the presence of this domain greatlyaccelerates substrate turnover.

Relative Arrangement of the C-Terminal and Catalytic Domains ofPNGase. Recently, the structure of the core domain of mousePNGase in complex with the XPC binding domain of HR23B hasbeen determined (10). This structure, especially the complex withthe inhibitor Z-VAD-fmk, provides additional information on howPNGase recognizes its substrate. As demonstrated here, substratebinding by mouse PNGase involves not only the core domain, butalso the C-terminal domain. Because the relative arrangement ofthe two domains is important in understanding the substratespecificity of PNGase from higher eukaryotes, possible domainorientations were investigated by manually positioning the struc-tures of the mouse PNGase core and C-terminal domains while atthe same time fulfilling two conditions: (i) The C terminus of thecore domain and the N terminus of the C-terminal domain have tobe in proximity because there are only four residues in between.Nevertheless, certain movements within this region appear possiblebecause both ends are flexible. (ii) Because the active site cysteine(Cys-306) in the core domain has to be in close proximity to theN-glycosidic bond of the substrate, the distance between its sidechain and the center of the mannose-binding motif in the C-terminal domain was required to be within 25 Å, the maximumlength of four pyranoses.

In the resulting model (Fig. 4), a continuous cleft is visible thaton one end accommodates the peptide represented by the Z-VAD-

Fig. 3. Mannopentaose binding activity of the C-terminal domain. (A)Stereoview of a 2Fo � Fc omit map contoured at 1 times the rms deviation, withthe mannose residues omitted from molecule B. The carbon atoms of themannotetraose 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 atomsof interacting residues are shown in yellow, potential hydrogen bonds areindicated as dashed lines in magenta, and water molecules are shown as redspheres. (C) RNase B digestion by PNGase. Time course of the reactionsinvolving full-length mouse PNGase, mouse PNGase �C (residues 1–450), andthe K527A variant of full-length mouse PNGase, with RNase B as the substrate.The curves were obtained by densitometric analysis of SDS�PAGE gels andrepresent the average of two experiments. The error bars indicate the result-ing standard deviations; however, in some instances, the standard deviationsare smaller than the geometric symbols that represent the data points.

Fig. 4. Hypothetical model of the relative arrangements of the core andC-terminal domains of mouse PNGase. (A) Ribbon diagram of the core domainin green and the C-terminal domain in cyan. In the core domain, residues ofthe catalytic triad are colored in red, and other conserved residues in thebinding pocket are colored in light orange. The mannose-binding residues inthe C-terminal domain are displayed in yellow. The C terminus of the coredomain 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 surfacerepresentation. The carbon atoms of the chitobiose are colored in yellow, andthose of the mannotetraose are colored in green. The carbon atoms of thePNGase inhibitor Z-VAD-fmk are shown in magenta.

17218 � www.pnas.org�cgi�doi�10.1073�pnas.0602954103 Zhou et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 19

, 202

1

fmk inhibitor, followed by the chitobiose moiety in the center, andfinally, the mannotetraose in the C-terminal domain. The peptideinhibitor and the chitobiose are adjacent to the catalytic triad in thecore domain, whereas additional conserved residues in the coredomain may be involved in chitobiose and peptide binding becauseof their close spatial proximity. The chitobiose orientation is basedon a docking calculation with the core domain (24). Although wedid not detect binding of chitobiose to the core domain in ITCexperiments, Suzuki et al. (25) recently used mass spectrometry todetect binding of affinity-labeled chitobiose to the catalytic cysteineof yeast PNGase. The presence of the oligomannose binding site inthe C-terminal domain extends the substrate-binding cleft of mousePNGase and therefore presumably enhances the catalytic efficiencyof the enzyme. Moreover, because both the SCFFbs1 E3 ligase andmouse PNGase bind to p97, the degradation efficiency of misfoldedprotein may be increased as p97 could function as a commonplatform for the ubiquitination of misfolded glycoproteins andremoval of N-glycans.

Materials and MethodsCrystallization and Structure Determination. Protein expression andpurification 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 anintein-fusion protein were grown by using the hanging-drop vapordiffusion method against a reservoir solution containing 14–16%PEG 8000, 0.1 M Tris�HCl (pH 8.5), 0.12 M MgCl2, and 10 mMDTT. Larger and better diffracting crystals were obtained by usingseeding techniques. The heavy atom derivative was prepared bysoaking with 10 mM sodium ethylmercurithiosalicylate for 10 min.Crystals of the His6-tagged C-terminal domain were obtained witha reservoir solution containing 1.6 M Li2SO4 and 0.1 M Hepes (pH7.0). Crystals of the complex were grown in a solution containing19% PEG 4000, 11.5% 2-propanol, 0.1 M Tris�HCl (pH 7.5), and0.2 M calcium acetate. Diffraction data of the apo structures werecollected on beam line X26C and that of the complex structure onbeam line X25 of the National Synchrotron Light Source atBrookhaven National Laboratory at 100 K. Diffraction data wereindexed, integrated and scaled with HKL2000 (26).

The structure of the mouse PNGase C-terminal domain wasdetermined by single isomorphous replacement and anomalousscattering (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 preliminarymodel consisting of �80% of the residues without side chains wasbuilt with the aid of the program O (30), and starting from thismodel ARP�wARP (31) was able to build 160 of 201 residues. Theprotein model was completed manually and was refined withREFMAC5 (32). Water molecules were added automatically withARP�wARP. Structures of the His6-tagged protein and complexwere solved by molecular replacement by using MOLREP (33). Inthe complex, there are two molecules (A and B) in the asymmetricunit, which are very similar to each other, except in the flexible loopbetween �16 and �17. Overall, molecule B is slightly better definedin the electron density maps and is shown in Fig. 3 A and B.

ITC Experiments. ITC measurements were carried out by using aVP-ITC microcalorimeter (MicroCal, Northampton, MA). Beforethe experiment, the proteins were dialyzed overnight at 4°C againsta buffer containing 10 mM Tris�HCl (pH 8.5) and 0.15 M NaCl. Theligands were dissolved in the same buffer to minimize the heat ofdilution. Proteins at concentrations ranging from 15 to 30 �M weretitrated with 0.6–1.2 mM �3,�6-mannopentaose, N,N-diacetyl-chitobiose, or �3,�6-mannotriose at 18°C. The binding parameterswere 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 tem-perature 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 thesame time, following the same protocol. The molar ratio of enzymeto substrate was 1:40 in each reaction. The RNase B substrate(Sigma, St. Louis, MO), at a concentration of 5 �g��l, wasdenatured by incubation at 95°C for 15 min before the start of theassay. The reaction was stopped by the addition of SDS samplebuffer and heating at 95°C for 10 min. The resulting Coomassie-stained gels were quantitated by densitometry with the ImageJprogram (34).

We thank Jae-Hyun Cho for help with CD spectroscopy. This work wassupported by National Institutes of Health Grants GM33814 (to W.J.L.)and DK54835 (to H.S.).

1. Yoshida Y (2003) J Biochem (Tokyo) 134:183–190.2. Suzuki T, Park H, Hollingsworth NM, Sternglanz R, Lennarz WJ (2000) J Cell

Biol 149:1039–1052.3. Suzuki T, Kwofie MA, Lennarz WJ (2003) Biochem Biophys Res Commun

304:326–332.4. Hirsch C, Blom D, Ploegh HL (2003) EMBO J 22:1036–1046.5. Suzuki T, Park H, Kwofie MA, Lennarz WJ (2001) J Biol Chem 276:21601–21607.6. Prakash S, Prakash L (2000) Mutat Res 451:13–24.7. Park H, Suzuki T, Lennarz WJ (2001) Proc Natl Acad Sci USA 98:11163–11168.8. Misaghi S, Pacold ME, Blom D, Ploegh HL, Korbel GA (2004) Chem Biol

11:1677–1687.9. Lee JH, Choi JM, Lee C, Yi KJ, Cho Y (2005) Proc Natl Acad Sci USA

102:9144–9149.10. Zhao G, Zhou X, Wang L, Li G, Kisker C, Lennarz WJ, Schindelin H (2006)

J Biol Chem 281:13751–13761.11. Suzuki T, Kitajima K, Inoue S, Inoue Y (1994) Glycoconjugate J 11:469–476.12. Hirsch C, Misaghi S, Blom D, Pacold ME, Ploegh HL (2004) EMBO Rep

5:201–206.13. Joshi S, Katiyar S, Lennarz WJ (2005) FEBS Lett 579:823–826.14. Holm L, Sander C (1994) Proteins 19:165–173.15. Mizushima T, Hirao T, Yoshida Y, Lee SJ, Chiba T, Iwai K, Yamaguchi Y, Kato

K, Tsukihara T, Tanaka K (2004) Nat Struct Mol Biol 11:365–370.16. Yoshida Y, Tokunaga F, Chiba T, Iwai K, Tanaka K, Tai T (2003) J Biol Chem

278:43877–43884.17. Yoshida Y, Chiba T, Tokunaga F, Kawasaki H, Iwai K, Suzuki T, Ito Y,

Matsuoka K, Yoshida M, Tanaka K, Tai T (2002) Nature 418:438–442.18. Garcia-Hernandez E, Zubillaga RA, Rodriguez-Romero A, Hernandez-Arana

A (2000) Glycobiology 10:993–1000.

19. Taroni C, Jones S, Thornton JM (2000) Protein Eng 13:89–98.20. Yoshida Y, Adachi E, Fukiya K, Iwai K, Tanaka K (2005) EMBO Rep 6:239–244.21. McNeill H, Knebel A, Arthur JS, Cuenda A, Cohen P (2004) Biochem J

384:391–400.22. Rabinovich E, Kerem A, Frohlich KU, Diamant N, Bar-Nun S (2002) Mol Cell

Biol 22:626–634.23. Jarosch E, Taxis C, Volkwein C, Bordallo J, Finley D, Wolf DH, Sommer T

(2002) Nat Cell Biol 4:134–139.24. Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, Olson

AJ (1998) J Comput Chem 19:1639–1662.25. Suzuki T, Hara I, Nakano M, Zhao G, Lennarz WJ, Schindelin H, Taniguchi

N, Totani K, Matsuo I, Ito Y (2006) J Biol Chem 281:22152–22160.26. Otwinowski Z, Minor W (1997) Methods Enzymol 276:307–326.27. Sheldrick G, Schneider T (1997) Methods Enzymol 277:319–343.28. Bricogne G, Vonrhein C, Flensburg C, Schiltz M, Paciorek W (2003) Acta

Crystallogr D 59:2023–2030.29. Abrahams JP, Leslie AG (1996) Acta Crystallogr D 52:30–42.30. Jones TA, Zou JY, Cowan SW, Kjeldgaard M (1991) Acta Crystallogr A 47:110–119.31. Perrakis A, Morris R, Lamzin VS (1999) Nat Struct Biol 6:458–463.32. Murshudov GN, Vagin AA, Dodson EJ (1997) Acta Crystallogr D 53:

240–255.33. Vagin A, Teplyakov A (1997) J Appl Crystallogr 30:1022–1025.34. Abramoff MD, Magelhaes PJ, Ram SJ (2004) Biophotonics Int 11:36–42.35. Collaborative Computational Project, Number 4 (1994) Acta Crystallogr D

50:760–763.36. DeLano WL (2002) The PyMOL User’s Manual (DeLano Scientific, San

Carlos, CA).37. Gouet P, Courcelle E, Stuart DI, Metoz F (1999) Bioinformatics 15:305–308.

Zhou et al. PNAS � November 14, 2006 � vol. 103 � no. 46 � 17219

BIO

CHEM

ISTR

Y

Dow

nloa

ded

by g

uest

on

Dec

embe

r 19

, 202

1