From the Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110 029, India
Received for publication, September 3, 2002, and in revised form, January 9, 2003
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ABSTRACT |
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We have determined the crystal structure
of a novel regulatory protein (MGP-40) from the mammary gland. This
protein is implicated as a protective signaling factor that determines
which cells are to survive the drastic tissue remodeling that occurs
during involution. It has been indicated that certain cancers could
surreptitiously utilize the proposed normal protective signaling by
proteins of this family to extend their own survival and thereby allow
them to invade the organ and metastasize. In view of this, MGP-40 could form an important target for rational structure-based drug design against breast cancer. It is a single chain, glycosylated protein with a molecular mass of 40 kDa. It was isolated
from goat dry secretions and has been cloned and sequenced. It was
crystallized by microdialysis from 20 mg ml Mammary glands secrete a class of very important proteins during
involution. We have isolated a glycoprotein from goat dry secretions
which has a molecular mass of 40 kDa. This mammary gland protein has
been named MGP-40.1 It shows
a high sequence homology to the proteins of the chitinase-like family
(1, 2) but has no chitinase activity. Very similar proteins have been
reported earlier as prominent in the whey secretions of nonlactating
cows (3) and found in large amounts in the culture supernatants of the
MG-63 human osteosarcoma cell line (4), cultures of human synovial
cells (5), and human cartilage cells (1, 2). However, little is known
about the functions of these inactive chitinase-related proteins. Based
on their sequence homology it is also unclear whether one could assume
that they might bind to chitin-like polysaccharide or glycoproteins.
This molecular event would then help regulate various kinds of tissue remodeling and/or differentiation processes. For example, one of these
proteins appears only during wound repair in cartilage, and a second
works during the earliest event of pregnancy when a newly fertilized
ovum is implanted in the oviduct. A third member of these
chitinase-related proteins was identified in specific types of cancer
cells from the mammary gland of mice (6). It was also found later that
it was expressed by the normal gland once the young mouse pups were
weaned. During this period of involution, the structure and function of
the gland must revert back to the nonpregnant state. It appears that
MGP-40 acts normally as a protective signaling factor that determines
which cells are to survive the drastic tissue remodeling that must
occur during involution. Thus, many breast epithelial cells, which have
increased in number during pregnancy, must now be destroyed. These
cells die by a precise programmed cell death pathway called apoptosis,
but most of the breast tissues remain viable, and it is assumed that
MGP-40 contributes to regulating which cells in the gland are to
survive. It has been reported that certain types of breast cancer cells
also produce an MGP-40-like protein called BRP39 (7). To understand the role of MGP-40 and related proteins in the breast cancer growth, we
have analyzed the three-dimensional structure of MGP-40 by x-ray
diffraction method. The structure has revealed features indicating
unusual aspects of conformational variations leading to alter the scope
of functions.
Purification--
Fresh samples of dry secretions from goats
were obtained from National Dairy Research Institute, Karnal, India.
All purification steps were carried out at 277 K. The pooled sample of
dry secretions was diluted twice with distilled water. It was further
diluted twice with 50 mM Tris-HCl, pH 8.0. Cation exchanger
CM-Sephadex was added (7 g liter Sequence Determination--
The mammary gland tissue was
obtained from a nonlactating goat during the period of involution, and
the complete cDNA sequence was determined. The isolation of
poly(A)+ mRNA and cDNA synthesis were performed
following the manufacturer's protocol (Stratagene). The N-terminal
sequence of MGP-40 was compared with those of chitinase-like proteins,
and the sequences of conserved regions were used for the synthesis of
primers. PCR was performed with Taq polymerase (Promega)
using an MJ Research thermal cycler, model PTC-100. The nucleotide
sequencing was performed on the cloned double stranded DNA (pGEM-T)
using an automatic sequencer (ABI-377). The complete nucleotide and
derived amino acid sequences are given in Fig.
1.
Fluorescence Analysis of Protein-Carbohydrate Binding--
To
determine the binding of carbohydrates to MGP-40, the following
compounds were used: glucose,
N-acetyl-D-glucosamine (GlcNAc), glucosamine, galactose,
N-acetyl-D-galactosamine, mannose, lactose, and
trehalose (Sigma). For the experiment as positive control with a
chitinase from Penicillium chrysogenum (GlcNAc)4
was used. Solute quenching experiments were also performed using KI
with chitinase and MGP-40 in the presence and absence of sugars. The binding was monitored by measuring the tryptophan fluorescence. All
fluorescence experiments were performed on a Hitachi F-4500 fluorescence spectrophotometer. The excitation wavelength was fixed at
295 nm. Emission intensities were collected over a wavelength range of
300-400 nm. The excitation and emission slit widths were 5 nm.
Fluorescence emission scans were performed at room temperature by
titration of 50, 100, 150, 200, and 250 µM ligands,
respectively, with 1 µM of MGP-40 in 25 mM
Tris-HCl, pH 7.5. All data were corrected for blank titration without
MGP-40 by using the corresponding ligand in 25 mM Tris-HCl,
pH 7.5. As a control, ligands were also treated with 8 µM
tryptophan under the same conditions.
Protein Crystallization--
The purified samples of MGP-40 from
goat dry secretions were used for crystallization. The crystals were
obtained by microdialysis with a protein concentration of 20 mg
ml Diffraction Data--
The crystals of MGP-40 were stable in the
x-ray beam. One crystal with the dimensions 0.4 mm × 0.4 mm × 0.2 mm was used for data collection. The x-ray intensity data were
collected at 278 K using a MAR Research 300-mm diameter imaging plate
scanner mounted on a RU-200 rotating anode x-ray generator equipped
with a graphite monochromator. The data were integrated using DENZO and
SCALEPACK program packages (8). The crystals belong to space group
P212121 with cell parameters
a = 63.0 Å, b = 65.9 Å, and
c = 107.0 Å containing four molecules in the unit
cell. The data have an Rsym of 11.4% and an
overall completeness of 99.3% to 2.9 Å resolution. The statistics of
crystallographic data are shown in Table
I.
Structure Determination--
Because MGP-40 showed a sequence
identity up to 46% with the proteins of chitinase-like family (9, 10),
the structure determination was attempted with molecular replacement.
The structure was finally determined using AMORE (11) with a poly(Ala)
model of a novel mammalian protein YM1 (9; PDB 1E9L). After rigid body
refinement, it yielded an R factor of 49.4% and a
correlation coefficient of 38.1%. The solution was then transformed
from Eulerian coordinates to orthogonal coordinates and applied to
the model coordinates. These resulting coordinates were used for refinement.
Refinement--
Restrained least squares refinement was carried
out with the CNS package version 1.0 (12) using atomic
coordinates as obtained from molecular replacement with an starting
R value of 0.494. Omit and difference electron density maps
(2Fo Sequence Analysis--
As seen from Fig. 1, the mature protein
consists of 361 amino acids. The complete nucleotide and amino acid
sequences have been deposited with the protein sequence data bank with
a GenBank Accession Number AY081150. The protein contains five
cysteines, four are involved in disulfide bridges,
Cys5-Cys30 and
Cys279-Cys343, and one, Cys20, is
free. The sequence also indicated two possible glycosylation sites at
Asn39-Ile40-Ser41 and
Asn346-Leu347-Thr348. MGP-40 showed
a sequence identity of 24-46% with chitinases (14-16) and other
chitinase-like proteins (9, 10). The amino acids essential for
catalytic activity in chitinases are three acidic residues Asp, Glu,
and Asp. The corresponding residues in MGP-40 are Asp115,
Leu119, and Asp186. The mutation of Glu to Leu
in MGP-40 shows that it lacks chitinase activity. There is another
chitinase-like family of proteins that lack chitinase activity with
varying mutations of three essential residues as His/Asn from Asp at
the first position, Gln for Glu at the second position and Asn for the
third position. These proteins also lacked chitinase-like catalytic
activity but were reported to bind saccharides/polysaccharides in a
manner similar to that observed in chitinases (14-16). A search using
BLAST (17) for further sequence analysis of MGP-40 revealed striking
sequence identities with five proteins: CLP-1 (GenBank AF011373), gp38k (18; GenBank U19900), HCgp39 (2; GenBank M80927), Ratgp (GenBank
AF062038), and BRP39 (6; GenBank X93035) as high as 95, 90, 83, 76, and
69%, respectively (Table III). Some
regions such as 1-16, 34-41, 110-121, 151-156, 232-249, 273-282,
298-305, and 325-332 are highly conserved in these proteins. These
proteins contain identical locations of cysteine residues and form two identical disulfide bridges. However, there is an exception in BRP39,
which has four cysteine residues, and the free Cys20
present in other similar proteins is replaced by Phe20 in
BRP39 (Table III). The most striking observation in these proteins pertains to the presence of an identical sequence of three residues Asp115, Leu119, and Asp186
corresponding to the three essential residues Asp, Glu, and Asp in
chitinases. These proteins were either expressed in mammary gland
during involution/mammary tumor/breast cancer (3, 4, 6) or in articular
chondrocytes of patients with rheumatoid arthritis (1, 2). The exact
physiological functions of these proteins are not yet fully understood.
However, the expression of MGP-40 in mammary gland during involution
supports its possible role in apoptosis. The strikingly similar
characteristics suggest that these proteins might have similar
three-dimensional structures and closely related functions. Therefore,
this group of proteins can be considered proteins of one family.
Because the crystal structure of MGP-40 is the first report from this
class, they can be called proteins of MGP-40 family. The crystal
structures of some of the distantly related chitinases (14-16) and
other chitinase-like proteins (9, 10) are available. To understand the
relationship among the proteins of these three families,
i.e. MGP-40, chitinase-like, and chitinases, the sequences
of representative proteins from the three classes have been compared.
The sequences of MGP-40 from the MGP-40 family, YM1 from the family of
chitinase-like proteins, and Chit1 from chitinases are listed in Table
IV. YM1 and Chit1 show sequence
identities with MGP-40 at the level of 46 and 25%, respectively. It is
noteworthy that the chain lengths in these three classes of proteins
are not similar. Compared with MGP-40, YM1 contains two extra stretches
of residues 146-150 and residues 391-398, whereas Chit1 possesses two
large segments of residues 76-91 and residues 387-401, which are
absent in both MGP-40 and YM1. Furthermore, YM1 and Chit1 are not
glycosylated. The number and locations of disulfide bridges in YM1 are
identical to those observed in MGP-40, but the locations of free
cysteines in the two sequences are different. On the other hand, Chit1
has only one disulfide bridge, which is widely different from both MGP-40 and YM1. These variations among the proteins of three families are expected to provide interesting structural and functional comparisons.
Overall Structure--
MGP-40 contains a single polypeptide chain
of 361 residues. Structural evaluations of the final model of the
protein using PROCHECK (19) indicated that 87.3% of the residues are
in the most allowed regions of the Ramachandran plot (20). Fig.
3 shows a section of the final electron
density map superimposed upon the final model. The refined model
included all 361 residues, 48 water molecules, and 2 molecules of
GlcNAc, yielding an R factor of 18.0% and a free
R factor of 23.5%. The root mean square deviations in bond
lengths and angles were 0.009 Å and 1.7 °, respectively. The
overall folding of the protein is shown in Fig.
4, a and b. The
structure is broadly divided into two globular domains, a Saccharide/Polysaccharide Binding Site--
The key
residues involved in the catalysis of chitinases are three acidic amino
acids Asp167, Glu171, and Asp240
(15). The corresponding residues in MGP-40 are Asp115,
Leu119, and Asp186. The presence of Leu at
position 119 in the sequence of MGP-40 completely rules out its role as
a glycolytic enzyme. Similar substitutions were also observed in other
chitinase-like proteins, although these were not as drastic but
significant enough to cause the loss of chitinase activity. These
residues were Asn136, Gln140, and
Asp213 in YM1 (9), His127, Gln131,
and Asn190 in concanavalin B (22), and Asp128,
Glu132, and Asn194 in narbonin (23).
Structurally, the three key residues in MGP-40, YM1, and Chit1 are
located at similar sites, having similar distances of C
The observed conformational differences involving Trp78 in
MGP-40 and the corresponding Trp99 in YM1 and
Trp131 in Chit1 can be traced through sequence variations
in these proteins. The most important feature of the MGP-40 structure
is presented by the loop Val75-Phe85 (Fig.
10a) compared with the
corresponding segments in YM1 (Ile96-Phe106)
(Fig. 10b) and Chit1 (Ile128-Phe137)
(Fig. 10c). In MGP-40, the residue at 84 is Arg whereas in
YM1, it is Pro, and in Chit1, it is absent. In the structure of MGP-40, Arg84 stretches to interact with a distant region
containing Asn39, Ile40, and GlcNAc (Fig.
10a). All of these interactions are absent in YM1 and Chit1.
It is particularly noteworthy that the glycosylation is a unique
feature in proteins of MGP-40 family (Table III) and is absent in the
chitinases and chitinase-like proteins (Table IV). It may be reiterated
that this is the first protein structure from the MGP-40 family. The
interactions involving Arg84 observed in MGP-40 apparently
influence the conformation of the loop
Val75-Phe85 considerably. Hence corresponding
loops in YM1 and Chit1 adopt a different conformation with several
intraloop attractive interactions. The central segment
Trp78-Gly81 of this loop in MGP-40 has
In yet another remarkable feature leading to conformational differences
regarding MGP-40, there is an extra stretch of
Pro76-Lys91 in Chit1 which facilitates the
formation of an essential conformation of Trp131.
Similarly, in YM1, Arg145 interacts with Trp99
O through two strong hydrogen bonds (Fig. 10b). These
interactions seem to determine the orientation of Trp99
with respect to the
Both MGP-40 and YM1 have five cysteines with two disulfide bridges and
the remaining Cys20 in MGP-40 and Cys49 in YM1
are free. Cys20 in MGP-40 is buried in a highly hydrophobic
environment, whereas Cys49 in YM1 is found on the surface
of the protein in close proximity of several polar groups including its
C-terminal segment. In fact, the protein chain in the C-terminal
regions of MGP-40 and YM1 turn to opposite directions. Perhaps the
location of Cys49 and an extra length of protein chain at
the C terminus facilitate the observed conformation in YM1. Although a
free cysteine is not present in Chit1, its C-terminal chain folds into
a separate domain.
There is yet another striking gross difference in the form of overall
charge distribution on the surfaces of MGP-40, YM1, and Chit1. The
surface of MGP-40 shows a basic charge, whereas the surfaces of YM1 and
Chit1 are negatively charged (Fig. 11). The pI values for MGP-40, YM1, and Chit1 are 9.0, 5.3, and 5.8, respectively.
MGP-40 contains a 1
solution in 0.1 M Tris-HCl, pH 8.0, and equilibrated
against the same solution containing 19% ethanol. Its x-ray structure has been determined by molecular replacement and refined to a 2.9 Å resolution. The protein adopts a
/
domain structure with a
triose-phosphate isomerase barrel conformation in the core and a small
+
folding domain. A single glycosylation site containing two
N-acetylglucosamine units has been observed in the
structure. Compared with chitinases and chitinase-like proteins the
most important mutation in this protein pertains to a change from Glu to Leu at position 119, which is part of the so-called active site
sequence in the form of Asp115, Leu119, and
Asp186 and in this case resulting in the loss of chitinase
activity. The orientations of two Trp residues Trp78
and Trp331 in the
barrel reduces the free space,
drastically impairing the binding of saccharides/polysaccharides.
However, the site and mode of binding of this protein to cell surface
receptors are not yet known.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
1) and stirred slowly for
1 h using a mechanical stirrer. The gel was allowed to settle, the
milk was decanted, and the gel was washed with an excess of 50 mM Tris-HCl, pH 8.0, packed in a column (25 cm × 2.5 cm) and washed with the same buffer containing 0.1 M NaCl,
which facilitated the removal of impurities. MGP-40 was then eluted
with the same buffer containing 0.3 M NaCl. The protein solution was dialyzed against triple distilled water and again passed
through a CM-Sephadex C50 column (10 cm × 2.5 cm) preequilibrated with 50 mM Tris-HCl, pH 8.0, and eluted with a linear
gradient of 0.05-0.35 M NaCl in the same buffer. The
protein was concentrated using an Amicon ultrafiltration cell. The
concentrated samples were passed through a Sephadex G-150 column (100 cm × 2 cm) using 50 mM Tris-HCl, pH 8.0. The second
peak in this final chromatographic step corresponded to a molecular
mass of 40 kDa. The N-terminal sequence was determined and showed a
high identity with a protein isolated earlier from the secretions of a
nonlactating cow (3). Because the function of this protein was not yet
known and because it was secreted from the mammary gland and had
molecular mass of 40 kDa, we named it MGP-40.
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Fig. 1.
Nucleotide and deduced amino acid
sequences of MGP-40. The amino acids are shown in three-letter
code. The triangle indicates the N-terminal amino acid of
the mature protein. The stop codon is indicated by ***.
1 in 10 mM Tris-HCl, dialyzing against the
same buffer containing 19% (v/v) ethanol at pH 8.0 and 277 K. The
colorless irregularly shaped crystals with the dimensions 0.5 mm × 0.4 mm × 0.2 mm grew after a period of 3-4 months.
Crystallographic data
Fc) and
(Fo
Fc) were
calculated with the same program. Model building was performed using a
Silicon graphics O2 work station using graphics program O
(13). The complete sequence of the protein was built into the electron
density. The backbone of helix comprising
His188-Thr194 and a loop region comprising
residues Arg203-Arg212 did not follow the path
indicated by the original model. These regions were deleted from the
calculations, and their directions were followed as indicated by the
electron density in the omit maps. An extra density was also observed
in the vicinity of
Asn39-Ile40-Ser41 into which two
units of GlcNAc were interpreted (Fig.
2), and they were included in the
subsequent refinement cycles. 48 water molecules with good hydrogen
bonding geometry were included in the model (Ow - - - O/N
distances in the range of 2.5-3.5 Å). The final crystallographic
R value is 18.0%, and the free R is 23.5%.
Relevant numerical data for the refinement are given in Table
II.
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Fig. 2.
Difference electron density map
(Fo Fc) for two units of
GlcNAc (NAG) linked to Asn39. The contour
level begins at 2
.
Summary of crystallographic refinement
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
Sequence alignment of proteins having identical Asp-115, Leu-119,
Asp-186 residues as a characteristic feature
Sequence comparisons of MGP-40 with a selected chitinase-like protein
(YM1) and a chitinase (Chit1)
/
TIM
barrel (21) domain and a small
+
domain. The TIM barrel domain
contains both the N and C termini of MGP-40. This domain is made up of
two polypeptide segments that are from 2-237 and 310-360 (Fig.
4a). The TIM barrel domain includes about 79% of the
residues. The small
+
domain contains the remaining 21% of the
residues of MGP-40. There are two disulfide bonds formed by the
residues Cys5-Cys30 and
Cys279-Cys343 (Fig. 4b). The latter
disulfide bond is formed between the two domains and apparently holds
them together. There is a free Cys20 in MGP-40, which is
located in a tightly packed hydrophobic pocket containing residues
Tyr7, Ala24, Ile25,
Phe338, and Phe349. It is practically
inaccessible to solvent. The secondary structure elements of the TIM
barrel and that of the small domain are listed in Table
V. The polypeptide chain of MGP-40 starts
with
1 of the TIM barrel domain and folds into
1,
1-1,
1-2,
1-3,
2-1,
2-2,
2,
3,
3-1,
3-2,
4
4,
5,
5-1,
5-2,
6,
6-1,
6-2,
7 (Fig. 4a), then
switches over to the small domain at residue 240 to form
1',
2',
1',
3',
4', and
5' (Fig. 4b). At residue 310, it
returns to the TIM barrel domain to generate
7,
8,
8-1, and
8-2 to complete the folding of the chain. The eight stranded parallel
sheet structure forms the core of the protein structure, and eight pieces of
helices surround it covering at least
three-fourths of the barrel from outside. The interior of the barrel is
filled predominantly with hydrophobic residues. Although there are two possible sites of glycosylation, only one of them,
Asn39-Ile40-Ser41, is actually
glycosylated with two units of GlcNAc through
(1-4) linkage (Fig.
5). In addition to covalent linkage with Asn39, it forms hydrogen bonds with Arg84
NH2 and Ile40 through O5. This is a unique
feature of MGP-40 structure because the residue corresponding to
Arg84 does not exist in chitinases, and it is mutated to
Pro in chitinase-like proteins. It should also be mentioned here that
Arg84 is conserved in the proteins of MGP-40 family,
reimposing their similarity. Furthermore, the interactions of
Arg84 with GlcNAc and Asn39 influence the
backbone conformation of loop Val75-Phe85,
which in turn alters the disposition of Trp78 leading to
the differences of the saccharide/polysaccharide binding to the
protein.
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Fig. 3.
Electron density (2 Fo Fc) map for a section of
the final MGP-40 model. The contours are drawn at the
1.2
level. The superimposed model is part of
barrel strands 4 and 5.
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Fig. 4.
Ribbon diagrams (29) of MGP-40. A,
top view orientation. The eight parallel strands that form the core
are labeled
1-
8.
-Helices of the TIM
barrel domain are indicated by
1-
8. B,
side view orientation. The
strands and
helices of the
+
small domain are represented as
1'-
5' and
1', respectively. The disulfide bridges are also
indicated.
Organization of secondary structure elements in MGP-40
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Fig. 5.
Linkage of the chain of the two units of
GlcNAc (NAG). It is linked to 2 of the
Asn39. Arg84 NH2 forms a hydrogen
bond network with O5 of the linked GlcNAc, as O
2 of
Asn39 and O of Ile40.
positions
between the corresponding residues, although their side chain
dispositions were not similar (Fig. 6,
a and b). The chitinases (14-16) and chitinase-like proteins (9) bind carbohydrate. The presence of saccharides/oligosaccharides has been observed in the
barrel of TIM domains of chitinase-like and chitinase proteins. The
locations of the saccharides/polysaccharides in the TIM domains of
these proteins are highly conserved. In the present case, both binding
studies as well as the absence of electron density in the
barrel of
the TIM domain indicated the absence of a saccharide/polysaccharide.
The studies of binding to various sugars have shown that the emission
spectrum of MGP-40 has a maximum at 328 nm with an excitation at 295 nm, which is characteristic of a tryptophan residue located in a
hydrophobic environment. The shift in emission maximum and changes in
the emission intensity on titration with ligands are indicative of the
binding/stacking of ligands against a tryptophan residue (24-26). None
of the several sugars used in the present binding assay caused any
observable shift in the emission maximum of 328 nm. There were also no
significant changes in the fluorescence intensities at 328 nm at
different concentrations of ligands. Furthermore, the addition of KI to MGP-40 showed a substantial quenching; however, the extent of tryptophan fluorescence quenching was the same in both the presence and
absence of sugars. The positive control experiments with P. chrysogenum chitinase using (GlcNAc)4 showed a shift
in the emission maximum from 331 nm to 328 nm together with an increase
in the emission intensity. In the case of chitinase there was less
reduction in the quenching of tryptophan fluorescence by KI in the
presence of sugar. This indicated a shielding of an exposed tryptophan residue(s) by KI. These changes could result from the stacking of a
sugar against a tryptophan residue, an interaction common to many
protein-carbohydrate interactions (25). These results clearly indicated
that the saccharides/oligosaccharides did not bind to MGP-40.
Consistent with this analysis, attempts to co-crystallize MGP-40 with
various carbohydrates were unsuccessful. Unlike other structures with
similar scaffolding, the top of the
barrel in the TIM domain of
MGP-40 is tightly packed, leaving no space for saccharide binding (Fig.
7). The key residues that pack the top of
the
barrel tightly are Tyr6, Phe37,
Trp78, Tyr185, and Trp331. The
corresponding residues in YM1 are Tyr27, Phe58,
Trp99, Tyr212, and Trp360. The
superimposition of the C
positions of the
barrels of MGP-40 and
YM1 together with the side chains of Phe37 (58),
Trp78 (99), Tyr185 (212), and
Trp331 (360) (Fig.
8a) shows remarkable
conformational differences in the dispositions of several of these side
chains in the
barrel. Similar differences were also observed
between MGP-40 and Chit1 (Fig. 8b). It indeed clearly shows
that the free space in the
barrel of MGP-40 is rather small to
accommodate a saccharide/polysaccharide molecule. The distance between
the two nearest atoms from opposite sides of the barrel is 7.5 Å (Trp99 C
-C
2
Trp360) in YM1 and 8.4 Å (Trp131
C
-C
2 Trp378) in Chit1,
whereas it is only 3.0 Å (Trp78
C
3-C
2 Trp331) in MGP-40. In
fact the interior of MGP-40 is so tightly filled by residues
Tyr6, Phe37, Trp78,
Leu119, Tyr185, and Trp331 that the
strong hydrophobic interactions were observed between the side chains
of various residues inside the barrel. The most striking variation was
observed in the conformation of Trp78 of MGP-40, which has
moved toward the center of the
barrel unlike those in YM1 (Fig.
8a) and Chit1 (Fig. 8b) where the corresponding residues Trp99 and Trp131, respectively, are
located away from the
barrel (Fig. 8, a and
b). This conformational change is responsible for the
drastic functional difference observed in MGP-40 as compared with both YM1 and Chit1 where saccharides bind to both YM1 and Chit1 at the
barrel, whereas MGP-40 is unable to accommodate any ligand at the
corresponding site (Fig. 9, a
and b). Therefore, this rules out the possibility of
accommodating the saccharide/oligosaccharide ligands in the so-called
carbohydrate binding site in MGP-40. This suggests that other
possibilities such as protein-protein binding with little or no
involvement of carbohydrate might exist. However, a final answer must
await biochemical studies to define the ligand binding
specifically.
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Fig. 6.
Superimpositions of three essential residues
of MGP-40, YM1, and Chit1. A, MGP-40 (green) and
YM1 (red). B, MGP-40 (green) and Chit1
(red).
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Fig. 7.
Top view of the barrel of the TIM domain. The space in the
barrel of
MGP-40 is tightly packed leaving no space for saccharide binding unlike
other chitinases and chitinase-like proteins. The residues filling the
interior of the
barrel are represented as balls and
sticks.
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Fig. 8.
Superimpositions of key residues involved in
saccharide binding. A, MGP-40 (green) and
YM1 (red). B, MGP-40 (green) and Chit1
(red). The residues are represented as balls and
sticks.
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[in a new window]
Fig. 9.
Comparison of the carbohydrate binding sites
in the barrel of TIM domain. a,
glucosamine (GCS) shown in red, fits well in YM1
(yellow), whereas there is a clash with the corresponding
residues Phe37, Trp78, Tyr185, and
Trp331 in MGP-40 (green). b,
similarly allosamidin (AMI) shown in red fits
well in Chit1 (yellow), whereas AMI clashes with residues of
MGP-40 (green).
,
torsion angles
81°,
31°;
84°, 13°;
118°, 113°;
63°, 121°. The corresponding values in YM1 and chitinase are
72°,
46°;
62°,
40°;
52°,
45°; 80°, 172°, and
52°,
50°;
65°,
40°;
88°,
5°, and
53°,
36°. The conformation of loop Val75-Phe85
is also influenced by Trp48 in MGP-40. This residue is
oriented toward the loop Val75-Phe85 and pushes
Phe80 away unlike in YM1 and Chit1 where the corresponding
residues are Thr69 and Ser83. Thus the steric
effects caused by Trp48 stabilize the observed conformation
of the loop very differently than those in YM1 and Chit1 by pushing the
side chain of Trp78 into the center of the
barrel of
the TIM domain. The corresponding loops in YM1 and Chit1 adopt a well
defined conformation with two overlapping type III
turns that are
rather tightly held (Fig. 10, a-c). The distinctly
different conformation of the loop Val75-Phe85
in MGP-40 is supported further by the presence Trp48 whose
side chain is turned toward this loop thus preventing favorable disposition of Phe80 which otherwise would have clashed
with the side chain of Trp48. The corresponding residues in
YM1 and Chit1 are Thr69 and Ser83 and lack the
level of steric constraints generated by the corresponding Trp48 in MGP-40.
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[in a new window]
Fig. 10.
Critical interactions those are responsible
for the observed orientations of Trp78 in MGP-40
(A), Trp99 in YM1 (B), and
Trp131 in Chit1 (C).
barrel of TIM domain in YM1. This particular interaction is absent in MGP-40. The appropriate placement of Arg145 in YM1 is facilitated by the conformation of the
loop Pro142-Phe155. As seen from Table IV, the
corresponding loop in MGP-40 is Pro121-Leu129
and is shorter by five residues. As a result of deletions, the loop in
MGP-40 folds very differently compared with the corresponding loop in
YM1 and does not provide the interactions with Trp78 in
MGP-40 as observed in YM1. In both YM1 and Chit1, these extra segments
of protein which help in producing the required orientations of
Trp99 and Trp131, respectively, are responsible
in generating the suitable spaces for the binding of carbohydrates in
these proteins. In contrast, not only these facilitating interactions
are absent in MGP-40, it has unique substitution of Arg84
and the presence of an important GlcNAc to introduce specific interactions that drag the crucial Trp78 into the center of
the
barrel which subsequently triggers the rearrangement of several
peripheral residues to move closure to the interior of the
barrel
to pack it tightly.
View larger version (31K):
[in a new window]
Fig. 11.
Electrostatic surface potential of MGP-40
(a), YM1 (b), and Chit1
(c). The negative potentials are shown in
deep red and positive potentials deep blue. The
neutral surface potential regions are depicted in white. The
orientations of molecules in a, b, and
c are the same.
CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES
barrel in its core with an overall similar
scaffolding to those observed in chitinases and chitinase-like proteins. The length of protein chain in MGP-40 is 361 residues, whereas those in YM1 and Chit1 are 377 and 392 residues respectively. MGP-40 shows sequence identities of 46 and 25% with YM1 and Chit1, respectively. The important features of their sequences are related to
deletions, alterations, and additions. The presence of extra stretches
of Gly146-Lys150 in YM1,
Pro76-Lys91 in Chit1, and Arg84 in
MGP-40 play crucial roles in the formations of saccharide binding
sites. Both YM1 and Chit1 bind saccharides, whereas MGP-40 is unable to
do so. Furthermore, the chitinases are capable of hydrolyzing chitin
molecules, whereas chitinase-like proteins and MGP-40 cannot because of
mutations of one or more essential residues of the catalytic triad. The
proteins of MGP-40 family are glycosylated, and the glycosylation sites
in all of them are conserved (Table III), whereas the proteins of
chitinase-like and chitinases are not glycosylated. The binding to
carbohydrates and hydrolyzing chitin polymers by chitinases, binding to
carbohydrates and not being able to hydrolyze chitins by chitinase-like
proteins, and finally neither binding to carbohydrates nor hydrolyzing
chitins by MGP-40 indicate that the proteins of MGP-40 family may have evolved from chitinases to acquire new properties. The possibility exists that MGP-40 may instead be a primarily protein-binding molecule.
There are reports that members of C-type lectins family recognize
proteins directly, with little or no involvement of carbohydrates (27,
28).
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FOOTNOTES |
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* This work was supported in part by the Department of Science and Technology, Government of India, New Delhi.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 nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY081150.
The atomic coordinates and the structure factors (code 1LJY) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Recipients of fellowships from the Council of Scientific and
Industrial Research, New Delhi.
§ To whom correspondence should be addressed. Tel.: 91-11-2659-3201 and 91-11-2658-8931; Fax: 91-11-2658-8663 and 91-11-2658-8641; E-mail: tps@aiims.aiims.ac.in.
Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M208967200
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ABBREVIATIONS |
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The abbreviations used are: MGP-40, 40-kDa mammary gland protein; TIM, triose-phosphate isomerase.
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REFERENCES |
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