Crystal Structure of a Novel Regulatory 40-kDa Mammary Gland Protein (MGP-40) Secreted during Involution*

Ashok K. Mohanty, Garima SinghDagger, Murugan ParamasivamDagger, Kolandaivelu Saravanan, Talat Jabeen, Sujata Sharma, Savita Yadav, Punit Kaur, Pravindra KumarDagger, Alagiri Srinivasan, and Tej P. Singh§

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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES

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-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 beta /alpha domain structure with a triose-phosphate isomerase barrel conformation in the core and a small alpha +beta 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 beta  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

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES

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-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.

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.


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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 ***.

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-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.

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.


                              
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Table I
Crystallographic data

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 - 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 2sigma .


                              
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Table II
Summary of crystallographic refinement


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES

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.


                              
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Table III
Sequence alignment of proteins having identical Asp-115, Leu-119, Asp-186 residues as a characteristic feature
These residues are shaded in green. The locations of glycosylation sites and cysteine residues are shaded in blue and yellow, respectively. The critical residues involved in the binding of saccharides/polysaccharides are indicated in red. Sources are as follows: MGP-40, goat mammary gland (GenBank AY081150); CLP-1, bovine articular chondrocyte (GenBank AF011373); Gp38k, porcine differentiating vascular smooth muscle (GenBank U19900); HCgp39, human articular chondrocyte (GenBank M80927); Ratgp, rat articular chondrocyte (GenBank AF062038); BRP39, mouse mammary gland (during involution)/mouse mammary tumor (GenBank X93035).


                              
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Table IV
Sequence comparisons of MGP-40 with a selected chitinase-like protein (YM1) and a chitinase (Chit1)
The active site residues are indicated in green, glycosylation site in blue, and critical residues involved in the binding of saccharides/polysaccharides are indicated in red. The cysteine residues are represented in yellow. Sources are as follows: YM1; mouse macrophage (GenBank M94584); Chit1; Coccidiodes (GenBank L41663).

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 beta /alpha TIM barrel (21) domain and a small alpha +beta 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 alpha +beta 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 beta 1 of the TIM barrel domain and folds into beta 1, alpha 1-1, alpha 1-2, alpha 1-3, beta 2-1, beta 2-2, alpha 2, beta 3, alpha 3-1, alpha 3-2, beta 4 alpha 4, beta 5, alpha 5-1, alpha 5-2, beta 6, alpha 6-1, alpha 6-2, beta 7 (Fig. 4a), then switches over to the small domain at residue 240 to form beta 1', beta 2', alpha 1', beta 3', beta 4', and beta 5' (Fig. 4b). At residue 310, it returns to the TIM barrel domain to generate alpha 7, beta 8, alpha 8-1, and alpha 8-2 to complete the folding of the chain. The eight stranded parallel beta  sheet structure forms the core of the protein structure, and eight pieces of alpha  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 beta  (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 1.2sigma level. The superimposed model is part of beta  barrel strands 4 and 5.


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Fig. 4.   Ribbon diagrams (29) of MGP-40. A, top view orientation. The eight parallel beta  strands that form the core are labeled beta 1-beta 8. alpha -Helices of the TIM barrel domain are indicated by alpha 1-alpha 8. B, side view orientation. The beta  strands and alpha  helices of the alpha +beta small domain are represented as beta 1'-beta 5' and alpha 1', respectively. The disulfide bridges are also indicated.


                              
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Table V
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 delta 2 of the Asn39. Arg84 NH2 forms a hydrogen bond network with O5 of the linked GlcNAc, as Odelta 2 of Asn39 and O of Ile40.

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 Calpha 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 beta  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 beta  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 beta  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 beta  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 Calpha positions of the beta  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 beta  barrel. Similar differences were also observed between MGP-40 and Chit1 (Fig. 8b). It indeed clearly shows that the free space in the beta  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 Cbeta -Cxi 2 Trp360) in YM1 and 8.4 Å (Trp131 Cbeta -Cepsilon 2 Trp378) in Chit1, whereas it is only 3.0 Å (Trp78 Czeta 3-Czeta 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 beta  barrel unlike those in YM1 (Fig. 8a) and Chit1 (Fig. 8b) where the corresponding residues Trp99 and Trp131, respectively, are located away from the beta  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 beta  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 beta  barrel of the TIM domain. The space in the beta  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 beta  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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Fig. 9.   Comparison of the carbohydrate binding sites in the beta  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).

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 phi , psi  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 beta  barrel of the TIM domain. The corresponding loops in YM1 and Chit1 adopt a well defined conformation with two overlapping type III beta  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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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).

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 beta  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 beta  barrel which subsequently triggers the rearrangement of several peripheral residues to move closure to the interior of the beta  barrel to pack it tightly.

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.


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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

MGP-40 contains a beta  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).

    FOOTNOTES

* 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/).

Dagger 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

    ABBREVIATIONS

The abbreviations used are: MGP-40, 40-kDa mammary gland protein; TIM, triose-phosphate isomerase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
REFERENCES

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