Abstract
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 A 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 Asp(115), Leu(119), and Asp(186) and in this case resulting in the loss of chitinase activity. The orientations of two Trp residues Trp(78) and Trp(331) 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.
Highlights
Mammary glands secrete a class of very important proteins during involution
It was 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
The mutation of Glu to Leu in MGP-40 shows that it lacks chitinase activity
Summary
Purification—Fresh samples of dry secretions from goats were obtained from National Dairy Research Institute, Karnal, India. MGP-40 was 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 ϫ 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. Fluorescence emission scans were performed at room temperature by titration of 50, 100, 150, 200, and 250 M ligands, respectively, with 1 M of MGP-40 in 25 mM Tris-HCl, pH 7.5. 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 mlϪ1 in 10 mM Tris-HCl, dialyzing against the same buffer containing 19% (v/v) ethanol at pH 8.0 and 277 K.
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