From the Institute of Molecular Biology, Academia
Sinica, Taipei, Taiwan 115, Republic of China, the
§ Department of Life Sciences, National Tsing-Hua
University, Hsinchu, Taiwan 300, Republic of China, the
¶ Institute of Microbiology & Immunology and ** Institute of
Neuroscience, School of Life Science, National Yang-Ming University,
Taipei, Taiwan 112, Republic of China, and the
Graduate Institute of Life Sciences,
National Defense Medical Center,
Taipei, Taiwan 114, Republic of China
Received for publication, November 16, 2000, and in revised form, February 14, 2001
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ABSTRACT |
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Ym1, a secretory protein synthesized by
activated murine peritoneal macrophages, is a novel mammalian lectin
with a binding specificity to GlcN. Lectins are responsible for
carbohydrate recognition and for mediating cell-cell and
cell-extracellular matrix interactions in microbes, plants, and
animals. Glycosaminoglycan heparin/heparan sulfate binding ability was
also detected in Ym1. We report here the three-dimensional structure of
Ym1 at 2.5-Å resolution by x-ray crystallography. The crystal
structure of Ym1 consists of two globular domains, a Macrophage displays marked heterogeneity during its
differentiation, activation, and distribution in various tissues. The list of secretory mediators produced by macrophage has grown to over
100; the mediators endow macrophages, through their abundance, distribution, and versatility, with the ability to influence
almost every aspect of the immune and inflammatory responses from the initial breach of epithelium to the ultimate repair of the inflamed tissue (1, 2).
Murine activated peritoneal macrophages elicited by oral
infection of nematodes (e.g. Trichinella
spiralis) were found to synthesize and secrete a novel protein Ym1
(63). Ym1 has been purified and characterized as a single chain
polypeptide with an estimated molecular mass of 45 kDa and a pI
of 5.7. Under low salt conditions, Ym1 has a tendency to crystallize
at its pI. Protein microsequence data derived from the NH2
terminus and CNBr cleavage fragments have facilitated the cloning of
Ym1 from a cDNA library of activated peritoneal macrophages. The
nucleotide sequence of a full-length cDNA clone was determined,
from which a single open reading frame of 398 amino acids with a
21-amino acid signal peptide typical of secretory protein was deduced. The induced expression of Ym1 by activated macrophages and the profound
cellular changes paralleling its appearance suggest that Ym1 may bear
functional significance to the development of either host defense
against or tolerance to nematode.
The existence of a multigene family was substantiated by data derived
from genomic organization and chromosomal mapping studies of Ym1 (3,
63) and the observation that a significant sequence homology (42-57%)
exists between Ym1 and several other "chitinase-like" proteins,
without known functions, such as HC-gp39 (4, 5), chitotriosidase (6),
chitinase A1 (7), gp38k (8), oviductin (9), and DS-47 (10).
Sequence analysis revealed significant homology between Ym1 and
microbial chitinases (~30%) (11, 63). However, no measurable chitinase activity was found associated with Ym1. Instead, Ym1 has been
identified as a novel animal lectin with a binding specificity toward
carbohydrates containing GlcN. In addition, its ability to bind
heparin/heparan sulfate was subsequently demonstrated (63).
By selective binding of oligosaccharides, lectins can modulate
cell-cell interactions pivotal during developmental processes and
immune defense mechanisms against infections and tumor metastasis (12,
13). Some crystal structures of animal lectins have been determined
that can be classified to several major groups (14-16), such as
galectins (17, 18), C-type animal lectins (19, 20), P-type animal
lectins (21-23), I-type animal lectins (24, 25), etc.
Selectins (26, 27) are calcium-dependent C-type lectins
that mediate adhesive interactions between leukocytes and the endothelium. Three known selectins, L-selectin (28), P-selectin (29),
and E-selectin (30), share structural features composed of carbohydrate
recognition domains, an epidermal growth factor domain, and several
complement-binding protein domains. The structures of E-selectin and
epidermal growth factor domain of P-selectin have been reported by the
studies of crystallography and NMR, respectively (29, 30). The
three-dimensional structure information of L-selectin is unavailable to
date; however, L-selectin has been shown to bind to
N-unsubstituted GlcN of heparin/heparan sulfate
proteoglycans (28, 31).
Heparin/heparan sulfate is a sulfated, negatively charged
glycosaminoglycan that is abundant on cell surface and
extracellular matrix. The physiological roles of heparin/heparan
sulfate are highly diversified, including cell adhesion, motility,
proliferation, differentiation, and tissue morphogenesis. The ability
of Ym1 to bind heparin/heparan sulfate in vitro further
suggests that heparin/heparan sulfate proteoglycans may be the
functional substrate of Ym1 in vivo (32, 33, 63).
We took advantage of the fact that Ym1 has a tendency to
crystallize under low salt conditions to study its biophysical
characteristics by x-ray crystallography. The information derived
from three-dimensional structure of Ym1 and its carbohydrate binding
domain would not only unravel the structure-function relationship of
this protein but also further our understanding of other members in the
Ym1 gene family.
Data Collection and Structure Determination--
Ym1 was
purified and crystallized as described in the accompanying paper by
Chang et al. (63). After dialysis, Ym1 crystals were
retrieved from the bag and laid onto a 10-ml step gradient of 50, 40, 30, 20, and 10% glycerol in a sterile 15-ml tube. After the tube was
allowed to stand at room temperature for 6 h, large crystals that had settled at the bottom of the tube were collected, washed glycerol with deionized and distilled water, and laid again onto
a step gradient (1 ml; 50, 30, and 10%) in an Eppendorf tube. After
settling at room temperature for 4 h, the top glycerol layers were
removed. The crystals left in 50% glycerol were devoid of any
particulate. For the x-ray diffraction analysis, a crystal was mounted
in a thin walled glass capillary containing a small amount of mother
liquid, in this case 50% glycerol to prevent dehydration, and sealed
with diffusion pump oil. The systematic absences and Laue symmetry
indicated that the Ym1 crystal belongs to space group P21
with cell dimensions a = 51.34 Å, b = 60.66 Å, c = 60.76 Å, with
The heavy atom positions were located by difference Patterson maps and
refined using the CCP4 (36) implementation of MLPHARE (37). The
ISIR-ISAS program (38) was used to generate the initial MIRAS (multiple
isomorphous replacement including anomalous scattering) phases at
3.5-Å resolution followed by phase extension to 3.0 Å. The resulting
electron density map showed a molecular boundary indicative of a
two-domain structure and evidence of several
The initial model from the electron density map interpretation
contained four fragments, Tyr22-Thr67,
Arg75-Leu214,
Gly223-Lys293, and
Gly299-Leu387. Preliminary refinement was
calculated using bulk solvent correction. Torsion angle dynamic
restraint and isotropic B factor refinement were applied,
and most of the missing side chain and discontinuous backbone were
rebuilt. The final model contained 3158 nonhydrogen atoms including 372 residues and 214 oxygen atoms of water molecules. The side chains of
Glu71 and Gln72 were invisible from the
electron density map. Due to the structure disorder, the COOH-terminal
residues, 394-398, were excluded in the final model. The final model
was refined against data between 20.0 and 2.5 Å with 2 The Overall Structure--
Ym1 contains a single polypeptide chain
of 372 residues, excluding the first 21 leading peptides and the last
five COOH-terminal residues. Ribbon drawings of the overall structure
folding of Ym1 are shown in Fig. 1. The
structure is clearly divided into two globular domains, a large Sequence and Structure Similarity with "Family 18"
Glycosyl Hydrolases--
A DALI (43) search for structure similarity
to Ym1 showed good agreement with chitinase A of Serratia
marcescens (11), a glycosyl hydrolase. Chitinase A comprises three
domains, an all
In addition to chitinase A, concanavalin B (45) and narbonin (46) also
share structure similarity with Ym1 according to DALI (43) structure
alignment results. Both proteins also belong to the "family 18"
glycosyl hydrolases, which contain a similar TIM
domain motif and share sequence homology
as shown in Fig. 4 and Table
III. However, all of these proteins show
different biological functions. Chitinase A exhibits glycolytic
activity, Ym1 demonstrates glycosaminoglycan binding ability but no
glycosyl hydrolase activity (63), and concanavalin B and narbonin both lack chitinase activity.
Saccharide Binding Environment in "Family 18" Glycosyl
Hydrolases--
The remarkably symmetrical eight-stranded
The key residues of the catalytic domain of chitinases in "family
18" glycosyl hydrolases have been identified. Site-directed mutagenesis studies conducted in chitinase A1 of Bacillus
circulans clearly demonstrated that both Asp200 and
Glu204 are essential for chitinolytic activity of the
enzyme (7). The mutation of glutamic acid to glutamine completely
abolished the enzymatic activity. Conversion of Asp to Asn also
significantly reduced the activity (7). It was intriguing to find that
the corresponding residue Glu315 of Serratia
chitinase A is not only key to substrate binding but also essential for
its catalytic activity (7, 11). The three essential residues
Asp311, Glu315, and Asp391 are
highly conserved in majority of chitinases known to date.
The corresponding residues in Ym1 are Asn136,
Gln140, and Asp213, located at Identification of a Putative Saccharide Binding Site in
Ym1--
Although no oligosaccharide was added during the
crystallization process, from the Fo Comparison of the Substrate Binding Pocket between Ym1 and
Chitosanase--
Although Ym1 exhibits a binding activity toward
glucosamine, the sequence and three-dimensional structure of Ym1 do not
share any significant similarity with chitosanase. Chitosanase
hydrolyzes chitosan, a polymer of GlcN produced by partial or full
deacetylation of chitin. To our knowledge, chitosanases from
Streptomyces N174 (6) and B. circulans (51) are
the only ones in "family 46" glycosyl hydrolases with known crystal
structures. The chitosanase molecule is dumbbell-shaped,
containing two globular domains linked by a bent helix. The
chitosan-binding pocket of chitosanase is located in the two helices
and the three-stranded Carbohydrate-binding Proteins and Ym1--
Since Ym1 has the
glucosamine binding activity and the heparin/heparan sulfate
proteoglycans might be the functional substrate of Ym1 in
vivo (32, 33, 63), we examined the available structure information
of several carbohydrate-binding proteins. For example, mannose-binding
protein (19, 20) and E-selectin (30) both belong to the
Three-dimensional structures of some heparin/heparan-binding
proteins have been determined (56-59), such as human heparin-binding protein (57) and fibroblast growth factor (56). Heparin-binding protein
(57) consists of a close
Structurally, Ym1 does not exhibit any three-dimensional homology with
the aforementioned carbohydrate-binding proteins. Ym1 contains a large
Ym1 and Chitinase-like Proteins--
Chang et al.
(63) discussed the Ym1 sequence related to secretory proteins,
including HC-gp39, chitotriosidase, gp38k, and DS-47. gp38k has been
identified as a heparin-binding glycoprotein (8). From our molecular
modeling studies (data not shown), the three-dimensional structures of
these chitinase-like proteins are quite similar to Ym1, except for
several loop insertions on DS-47 and a longer COOH-terminal region on
chitotriosidase. Under "Identification of a Putative
Saccharide-binding Site in Ym1," we proposed that residues
Gln140 and Asp213 of Ym1 are the essential
residues that participate in sugar binding. From the sequence
comparison, Asp213 of Ym1 is completely conserved within
this superfamily. However, residue Gln140 varies greatly
and might perform different functions. Hydrophobic residues
Leu140 and Ile140 in HC-gp39 and gp38k
substitute the corresponding residue, Gln140 of Ym1,
respectively. In DS-47, Gln165 is the same as that of Ym1.
In chitotriosidase, Gln140 is replaced by
Glu140, which is conserved in "family 18" glycosyl
hydrolases. This may explain why only chitotriosidase has the chitinase
activity. Furthermore, several hydrophobic residues, such as
Tyr27, Phe58, Trp99,
Tyr212, and Trp360 of Ym1, are highly conserved
within this superfamily. These hydrophobic residues may play
significant functional roles, since they are seated in the
Another noteworthy similarity among this superfamily is the disulfide
bond linkage. Two Cys pairs, Cys26-Cys51
and Cys307-Cys372, were determined in
the Ym1 crystal structure from a total of six cysteine residues.
Surprisingly, both disulfide bonds are strictly conserved in all of
these chitinase-like proteins. It should be noted that
Cys49 may form the third disulfide bond with
Cys394. However, due to the quality of the electron density
map, the third possible disulfide bond could not be determined in the
structure. Meanwhile, residues Cys49 and Cys394
are not conserved in these secretory proteins. Based on the Ym1 crystal
structure and the sequence comparison of these chitinase-like proteins,
we suggest that they may share structure similarity and saccharide
binding activity like Ym1.
Conclusion--
In the present study, we determined the crystal
structure of Ym1 by x-ray diffraction method. Ym1 is composed of two
domains, a large
We have observed a saccharide binding site in the Ym1 crystal structure
and elucidated the saccharide binding environment. The substrate
monosaccharide is located inside the TIM domain at the COOH terminal
end of the
The glucosamine binding ability of Ym1 suggests that it may belong to
the chitinase-like animal lectins, and it may have other important
biological functions yet to be discovered. There is no structure
information available for other chitinase-like proteins (e.g. HC-gp39, chitotriosidase, gp38k, and DS-47). Results
from the present study should provide a framework for understanding the
possible structure conformation and related biological functions of
these Ym1 superfamily proteins. We suggest that these Ym1 superfamily proteins may also have saccharide binding activity similar to Ym1 and
may share a structure conformation similar to that of Ym1.
/
triose-phosphate isomerase barrel domain and a small
+
folding domain. A notable electron density of sugar is detected in the
Ym1 crystal structure. The saccharide is located inside the
triose-phosphate isomerase domain at the COOH terminal end of the
-strands. Both hydrophilic and hydrophobic interactions are noted in
the sugar-binding site in Ym1. Despite the fact that Ym1 is not a
chitinase, structurally, Ym1 shares significant homology with chitinase
A of Serratia marcescens. Ym1 and chitinase A have a
similar carbohydrate binding cleft. This study provides new structure
information, which will lead to better understanding of the biological
significance of Ym1 and its putative gene members.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
= 94.61°. The
crystal diffracts to 2.5 Å. The value of Vm (34) was
calculated to be 2.08 Å3/dalton, and the solvent content
was estimated to be 41%, presuming one molecule per asymmetric unit.
After an extensive heavy atom search, two useful derivatives,
UO2(AC)2 and EtHgCl, were obtained by soaking
native crystals in the corresponding heavy atom solution. Heavy atom
soak conditions were 1 mM UO2(AC)2
for 1 day and 50% EtHgCl-saturated solution for 1 day. All data used
for Ym1 structure determination were collected on a Rigaku R-Axis II
imaging plate system using double mirror-focused CuK
x-ray radiation
generated from a RigakuRU-300 rotating anode operating at 50 kV and 80 mA. The data were indexed, integrated, and scaled using DENZO and SCALEPACK (35) (Table I).
X-ray crystallography data statistics
-helices and
-sheet
strands in both domains. The connectivities between some of the
secondary structure elements were difficult to determine. The initial
MIRAS phases were therefore further improved by the program DM (39),
using a combination of solvent flattening, histogram mapping, and
Sayer's equation, in which the skeletonization was excluded in this
calculation. This map showed continuous electron density with well
defined side chains for almost the entire molecule.
(F) and an R factor of 19.8. Using a 10% reflection test set (1154 reflections), the
Rfree value (40) was 27.2%. The model has a
reasonable stereochemistry with root mean square deviations in bond
lengths and angles of 0.008 Å and 1.4°, respectively. Analysis of
the Ramachandran plot (41) showed no violation of acceptable backbone
torsion angles. Atomic coordinates have been deposited with the Protein
Data Bank (42), accession code 1E9L.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
/
barrel (triose-phosphate isomerase (TIM)1 barrel) domain and a
small
+
domain. The TIM barrel domain contains both the
NH2 and COOH termini of Ym1. This domain is built from two
separate peptide segments from residues 22-266 and 338-393. The TIM
barrel domain includes about 80% of the Ym1 residues. The small
+
domain consists of residues 268-336, accounting for another 20%
of the total residues of Ym1. There are two disulfide bonds formed by
residues Cys26-Cys51 and residues
Cys307-Cys372. The second disulfide bond is
within the interdomain region to stabilize the two domains. A topology
diagram of the secondary structure of the
polypeptide backbone of Ym1 is shown in Fig. 2 and Table
II. Briefly, the polypeptide chain of Ym1
starts in the
1 of the TIM barrel domain folding as a
secondary topology of seven
-strands and six
-helices and forms the major part of the TIM
barrel motif. The chain then traverses from strand
7 to the small
domain and forms the entire structure of the small domain, which
consists of one helix and six-stranded antiparallel
sheets, making
an
+
folding motif. From the residue Asn338, the
polypeptide chain returns back to the large domain and completes the
entire (
)8 TIM barrel motif. The eight-stranded
parallel
-sheet is located inside, and eight parallel
helices
are surrounded outside. The TIM barrel is elliptical in a donut shape
with axes about 13 and 10 Å inside and about 49 and 42 Å outside.
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Fig. 1.
Ribbon diagrams (60,
61) of Ym1 in side view orientation (a) and top view
orientation (b). The proposed saccharide
substrate-binding site is shown as a ball and
stick. The -helices are shown as cylinders in
violet, and the
-strands are shown as arrows
in cyan. The amino and carboxyl termini are labeled.
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Fig. 2.
A topology diagram of Ym1 is consisting of
two domains, the TIM domain and a small +
domain. The
-helices are
shown as cylinders in lavender and labeled
1-
8 for the TIM domain and
C for the small domain. The
-strands are
shown as arrows in cyan and labeled
1-
8 for the TIM domain and
A,
B,
D,
E, and
F for the small domain. The amino and carboxyl
termini are labeled.
Secondary structure elements of Ym1 protein
-stranded NH2-terminal domain, a
catalysis
/
barrel (TIM barrel) domain, and a small
+
folding domain. The superimposition between Ym1 and chitinase A in C
is shown in Fig. 3a. When the NH2-terminal domain (residues 1-159) was excluded from
chitinase A, the superimposition (Fig. 3b) showed a strong
similarity in the supersecondary structure. The C
positions of the
TIM and
+
domains from two proteins could be superimposed with
a root mean square deviation of 0.74 Å. The orientation of Fig.
3b is rotated along the z axis (perpendicular to
the plane of the paper) of Fig. 3a by
90°. The sequence
alignment of Ym1 and chitinase A using BLAST (44) showed that the two
proteins have 26% sequence identity and 46% sequence similarity.
Although Ym1 shares high structure conservation with the catalytic and
small domains of chitinase A, there are three noteworthy structure
deviations. First, there is an extra 29-residue insertion (residues
195-224) that forms as two helices in chitinase A, whereas the
corresponding region in Ym1 is located between
2-1 and
2-2. This
is the most divergent region between Ym1 and chitinase A. The
differences in conformation were found in this region, two
strands
in Ym1 and two
helices in chitinase A. Second, there is an extra
18-residue loop insertion (residues 234-252) in chitinase A. The
corresponding region of Ym1 is around residue Asp73,
located in the middle of
2. Third, there is a 10-residue insertion (residues 365-375) in Ym1 to form a long loop. The corresponding region of chitinase A is located between
5 and
5-1.
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Fig. 3.
The superimposition of Ym1 and chitinase A in
C . Shown are the superimposition of the
overall structures of Ym1 and chitinase A (a) and the
superimposition of Ym1 and two domains of chitinase A (residues
159-561) (b). Ym1 is colored in blue, and
chitinase A is colored in cyan. The NH2 and COOH
termini are labeled.
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Fig. 4.
Ribbon plots (60, 61) of the TIM domain motif
of four family 18 chitinase-like proteins: Ym1 (residues 22-266 and
338-393), chitinase A (residues 159-442 and 518-561)
(1ctn), concanavalin B (residues 1-283)
(1cnv), and narbonin (residues 1-289)
(1nar). The three corresponding residues
(Asn136, Gln140, and Asp213 in Ym1;
Asp311, Glu315, and Asp391 in
chitinase A; His127, Gln131, and
Asn190 in concanavalin B; and Asp128,
Glu132, and Asn194 in narbonin) are represented
as balls and sticks.
Alignment of eight -strands of TIM barrel domain of four
chitinase-like proteins: Ym1, chitinase A (Protein Data Bank 1ctn),
concanavalin B (Protein Data Bank 1cnv), and narbonin (Protein Data
Bank 1nar)
/
-barrel structure was first found in the glycolytic enzyme,
TIM (47). The typical arrangement of structural elements in the
/
TIM barrel domain consists of eight alternating
-helices and
-strands coiled into a barrel. Structurally, the presence of a
highly symmetrical, eight-stranded
/
-barrel structure in many
unrelated enzymes seems to have significance for similar substrate
binding sites (48). The saccharide substrate of Ym1 might be bound in a
fashion similar to that of chitinase A, since Ym1 shares significant
sequence and structure conservation with chitinase A. From Perrakis's
study (11), the substrate-binding site of chitinase A is formed by a
long groove, located at the carboxyl-terminal end of the
-strands of
the TIM barrel. Many studies on glycosyl hydrolases have delineated that two residues (e.g. in chitinase A of S. marcescens, glutamic acid (Glu315) and aspartic acid
(Asp391)) participate in sugar binding when glycosidic
bonds are hydrolyzed (11). Despite the possibility that glycosyl
hydrolases may mediate their activities through different protein
folding patterns or mechanisms of catalysis, these two residues are key
to substrate binding (49).
4 and the
COOH-terminal ends of
4 and
6 in the TIM barrel, respectively.
Amidated residues, Asn136 and Gln140, may
explain why Ym1 neither binds chitin nor exhibits chitinase activity.
Similar residue substitutions and the lack of chitinase activity were
noted in concanavalin B (His127, Gln131,
Asn190) (45) and narbonin (Asp128,
Glu132, Asn194) (46). Structurally, the three
key residues are located at a similar, if not identical, position in
the TIM barrel in all four chitinase-like proteins (shown in Fig. 4 and
Table III). In chitinase A, the C
distance between
Glu315 and Asp391 is 13.1 Å, whereas the
corresponding C
distance for Ym1 (Gln140,
Asp213), concanavalin B (Gln131,
Asn190), and narbonin (Glu132,
Asn194) is 13.6, 11.7, and 10.4 Å, respectively.
Fc difference Fourier map (Fig.
5), surprisingly, an extra saccharide
density was observed at the top of the
barrel of the TIM domain.
This location of the saccharide is highly conserved with the
oligosaccharide-binding site in many glycosyl hydrolases (50). Since
Ym1 has GlcN binding ability (63), we have tentatively modeled the
monoglucosamine into this electron density map. The orientation of the
sugar ring cannot be definitively determined at the current level of
resolution. However, based on its similarity to the location and shape
of the saccharide density observed in chitinase A complexed with GlcNAc
(11), the six carbon atoms were positioned into the tentative orientation as shown in Fig. 5. The sugar ring of the putative glucosamine appeared to fit snugly into the electron density map and
sat right between the two putative key residues, Gln140 and
Asp213. The orientations of Gln140 and
Asp213 of Ym1 were very similar to that of the two
corresponding residues, glutamic acid and aspartic acid, key to sugar
binding of many glycosyl hydrolases (50). The sugar ring was modeled
with its O1 and O5 atoms oriented toward Gln140 OE1 and
Gln140 NE2 with distances of 2.9 and 3.2 Å, respectively.
The O6 of GlcN is 3.6 Å apart from Asp213. The charged
residue Asp138 might also participate in sugar binding, for
its OD1 is modeled 2.7 Å away from the N2 atom of the putative GlcN
(Fig. 5). In addition, several aromatic residues, such as
Tyr27, Phe58, Trp99,
Tyr212, and Trp360, surrounding the binding
pocket might contribute to the hydrophobic interactions for sugar
binding. It should be pointed out that the face of the glucosamine
sugar ring (GlcN) is packed against the ring of Trp360 as
seen in other lectin-saccharide interactions (14, 15). All surrounding
hydrophobic residues are involved in van der Waals interactions about
4.0 Å from the sugar. As shown in Table III, these important
hydrophobic residues are highly conserved in chitinase-like proteins,
i.e. chitinase A, concanavalin B, and narbonin. The average
temperature factor for the putative sugar binding pocket is about 15 Å2 for those residues surrounding the Ym1 protein and 20 Å2 for atoms of the proposed glucosamine, respectively.
The locations of these residues are evenly distributed into
1,
2,
3,
6, and
8 secondary structures. This might provide the basis
for explaining why the (
)8 TIM barrel motif plays an
important role in sugar binding. However, the small domain of Ym1,
which is mainly
structure, is not involved in the saccharide
binding. Thus far, we have been unable to assign any definitive
function for this domain.
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Fig. 5.
Difference density map (Fo Fc with phase from the Ym1 model) of the possible bound sugar
substrate, glucosamine, has been tentatively modeled into the electron
density with its O1, O5, and O6 (in red) and N2 (in
deep blue) as marked. Two essential
residues, Gln140 and Asp213, and several
hydrophobic residues, Tyr27, Phe58,
Trp99, Tyr212, and Trp360, are
shown. Density is contoured at the 2
level and drawn by O/OPLOT
(62).
-sheet between the two domains (52). Two
essential residues, Glu22 and Asp40 in
Streptomyces N174, participate in the catalytic mechanism of
the chitosanase enzymatic reaction (52, 53). The putative glycosaminoglycan binding site of Ym1, however, is located inside the TIM barrel domain and at the carboxyl-terminal end of the
-strands. To understand the interaction of glycosaminoglycan and
proteins, we have examined the binding surfaces of Ym1 and performed
the electrostatic surface potential calculations on Ym1 and
chitosanase. Fig. 6 shows two color-coded
images produced according to the electrostatic surface potential of Ym1
and chitosanase generated by the program GRASP (54). The surfaces of
the possible substrate binding cleft of Ym1 and chitosanase exhibited
polarized negative potentials, as shown in red. These
results indicate that the substrate binding cleft is appropriate for a
positively charged substrate such as GlcN or chitosan. Consequently,
in vivo Ym1 may bind to N-substituted
glycosaminoglycan ligand, structurally similar to chitosan, which is
not present in mammals.
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Fig. 6.
Electrostatic surface potential of the
TIM domain of Ym1 and chitosanase (Protein Data Bank number 1CHK)
displayed by the program GRASP (54). The putative saccharide
binding site of both Ym1 and chitosanase shows a relatively strong
electronegative character. Negative potentials (<10 kiloteslas)
are colored in deep red, and positive potentials
(>10 kiloteslas) are colored in deep
blue. The neutral surface potential regions are depicted in
white. The orientation of Ym1 is the same as in Fig.
1b.
/
class
with C-type lectin-like folding. The carbohydrate ligands are bound
around the loop 3 and 4 regions, the relatively shallow depressions of
the molecular surface (55). The carbohydrate binding activity is
Ca2+- and pH-dependent (55).
-barrel of six antiparallel
-strands, a
typical serine protease fold; fibroblast growth factor (56) is composed
entirely of
-sheet structure, composed of a three-fold repeat of a
four-stranded antiparallel
-meander motif as a trefoil fold
structure. The heparin/heparan sulfate binding is mediated by the
electrostatic interaction between the positively charged residues of
protein and the negatively charged groups of heparin/heparan sulfate.
/
barrel domain, a typical TIM motif folding, and a small
+
fold domain. The saccharide binding site of Ym1 is found inside the
TIM domain at the COOH terminal end of the
-strands. The biological
relevance of heparin/heparan sulfate binding of Ym1 is not defined at
this stage.
strands
of the TIM barrel domain and located around the position of the
putative sugar-binding pocket in Ym1.
/
barrel (TIM barrel) domain and a small
+
fold domain. Surprisingly, the three-dimensional structure of Ym1, rather than being similar to the known animal lectins, is highly homologous to chitinase structure. Although no chitinase activity can
be detected in Ym1, Ym1 has a similar structure to that of the
"family 18" glycosyl hydrolases, which consist of a
(
)8 topology.
-strands. Meanwhile, the heparin/heparan sulfate
proteoglycan binding activity has been found associated with Ym1. The
heparin/heparan sulfate proteoglycans may be the functional substrate
of Ym1 in vivo. Ym1 may be a novel
heparin/heparan sulfate-binding protein with a common TIM barrel
folding. The three-dimensional structure of Ym1 presented here provides
important information toward understanding how Ym1 binds with its
biological targets.
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FOOTNOTES |
---|
* This work was supported by National Science Council Grants NSC-87-2316-B001-010 (to C. D. H.) and NSC-87-2316-B010-012 (to N. C. C.) and a grant from the Academia Sinica, Republic of China (to C. D. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1E9L) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence may be addressed. Tel.:
886-2-2826-7114; Fax: 886-2-2820-2593; E-mail: acchang@ym.edu.tw.
§§ To whom correspondence may be addressed. Tel.: 886-2-2788-2743; Fax: 886-2-2782-6085; E-mail: mbhsiao@ccvax.sinica.edu.tw.
Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M010416200
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ABBREVIATIONS |
---|
The abbreviation used is: TIM, triose-phosphate isomerase.
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