Structural and biochemical studies of the C-terminal
domain of mouse peptide-N-glycanase identify
it as a mannose-binding module
Xiaoke 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 Virchow
Center for Experimental Biomedicine and Institute of Structural Biology, University of Wu ¨rzburg, Versbacher Strasse 9, 97078 Wu ¨rzburg, 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 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 glyco-
proteins. 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
domain of PNGase is involved in protein–protein interactions, the
function 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 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.
endoplasmic reticulum ? N-linked glycoproteins ? proteasome ?
protein degradation ? deglycosylation
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 oligosaccha-
ride chains are attached via N-glycosidic bonds to the side-chain
amide 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 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, deglycosy-
lation, 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 struc-
tures of yeast and mouse PNGase in complex with Rad23?
HR23B and an inhibitor (8), carbobenzyloxy-Val-Ala-Asp-?-
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
roteins need to acquire their native conformation after
protein synthesis to carry out their biological functions.
on the ?-N-glycosidic bond linking the Asn side chain and the
first GlcNAc residue, thereby cleaving the glycan chain from the
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 oligosac-
charide-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 (resi-
dues 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 isomor-
phous replacement and anomalous scattering with the aid of a
Hg derivative (Table 2, which is published as supporting infor-
mation on the PNAS web site) and was refined at 1.9-Å
resolution (Table 1) to an R factor of 0.167 (Rfree? 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
(Rfree? 0.207). The two structures are very similar as reflected
in a rms deviation in the C?positions of 0.24 Å. However, in the
His-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 the
Protein 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.
© 2006 by The National Academy of Sciences of the USA
November 14, 2006 ?
vol. 103 ?
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
of yeast PNGase. The presence of the oligomannose binding site in
of the enzyme. Moreover, because both the SCFFbs1E3 ligase and
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,
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 Li2SO4and 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
Phase refinement was carried out with SHARP (28) to 3.3 Å,
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
between ?16 and ?17. 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
ligands were dissolved in the same buffer to minimize the heat of
dilution. Proteins at concentrations ranging from 15 to 30 ?M were
titrated with 0.6–1.2 mM ?3,?6-mannopentaose, N,N-diacetyl-
chitobiose, or ?3,?6-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 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 the
to substrate was 1:40 in each reaction. The RNase B substrate
(Sigma, St. Louis, MO), at a concentration of 5 ?g??l, 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
We thank Jae-Hyun Cho for help with CD spectroscopy. This work was
supported by National Institutes of Health Grants GM33814 (to W.J.L.)
and DK54835 (to H.S.).
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