Crystallographic studies of bovinef 2 - microglobulin

Crystals of the bovine milk protein lactollin yield x-ray diffraction data extending to a resolution of 2.8 A. Lactollin is a bovine analogue of #2-microglobulin, a protein that is homologous in amino acid sequence to the constant domains of immunoglobulins and is the light chain of the human and murine major histocompatability antigens. The protein crystallizes in the orthorhombic space group P212121 with a = 77.4, b = 47.9, and c = 34.3 A. The unit cell parameters and physical chemical solution studies indicate that the molecule exists in the crystal and in solution as a single polypeptide chain of 12,000 daltons. #2-Microglobulin is a protein distinguished by its similarity to certain portions of immunoglobulins (1-3) and by its association on cell surfaces with the major histocompatability antigens of man (HLA) and mouse (H-2) (4-7). Since its discovery in human urine in 1968, the overall properties, synthesis, and amino acid sequence of this protein have been characterized (2, 8-11). Similar proteins have been detected in a variety of species, and all appear as a single polypeptide [molecular weight (Mr) = 11,600], with few differences in amino acid sequence among the proteins from different species (2, 3, 12-14). Comparison of the amino acid sequence and other features of human #32-microglobulin with immunoglobulins (1, 15) has indicated that it closely resembles the constant homology regions (16) of immunoglobulins G, M, and E. In intact immunoglobulin molecules, each homology region is closely associated with the corresponding homology region of another immunoglobulin chain in pairs of domains (16, 17), whereas ,62microglobulin is found associated with the heavy chain of HLA or H-2 antigens or the closely related thymus leukemia antigen (4-7, 18, 19), suggesting that such paired domains may be structural features of these cell surface molecules. In solution, purified Or-microglobulin, however, is generally unpaired; i.e., it appears as a free monomer (8) The elucidation of the three-dimensional structure of ,32-microglobulin would be of particular interest because it would provide an opportunity to compare this molecule in detail with immunoglobulin domains, and thus contribute to an understanding of its interaction with the heavy chains of H-2 and HLA antigens. It has recently been found that lactollin, a crystalline protein isolated from bovine milk and colostrum, has an amino-terminal amino acid sequence sufficiently similar to those of 6B2-microglobulins from several species to indicate that it is probably a bovine homologue of the protein (M. L. Groves, private communication; refs. 20 and 21). Although the reported molecular weight of lactollin is 43,000 (22), it is composed of subunits of 12,000 daltons. We have confirmed the molecular weight of the subunit and the similarity in amino acid sequence to 62microglobulin. We report here x-ray diffraction studies of The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3345 crystals of lactollin preliminary to a complete structural analysis. Our results indicate that the protein crystallizes in the orthorhombic space group P212121 with a = 77.4, b = 47.9, and c = 34.3 A and one polypeptide chain per asymmetric unit. MATERIALS AND METHODS Fresh bovine milk and colostrum were obtained from local dairy farmers. Lactollin was prepared as described by Groves et al. (22), except that the protein isolated by chromatography on DEAE-cellulose was further purified by gel filtration prior to crystallization. Fractions containing lactollin from the ionexchange column were dialyzed against water, lyophilized, and dissolved in phosphate-buffered saline (pH 7.4; 8.00 g of NaCl, 0.20 g of KCI, 0.20 g of KH2 P04, and 0.15 g of Na2 HPO4 per liter) containing 0.001% thymol. Gel filtration on columns (1.7 X 100 cm) of Sephadex G-75 was carried out at room temperature. Fractions from this column were dialyzed against water, lyophilized, and prepared for crystallization. Single crystals were prepared by dialysis in microdiffusion cells (23) of the purified lactollin against 0.05-0.005 M sodium phosphate at pH values between 7.0 and 8.5. Final purification was monitored by amino acid sequence analysis. Amino acid sequence analysis of the fully reduced and alkylated protein (24) was carried out in the automatic sequencer (Beckman 890C) using the Quadrol double cleavage program. Residues released were identified by thin-layer chromatography (25), by back hydrolysis in 6M HCI under reduced pressure for 24 hr at 1500 followed by amino acid analysis, and by colorimetric assay (His and Arg) (26, 27). Three separate determinations made on separately prepared samples, two from milk and one from colostrum, gave equivalent results. To compare tryptic peptides, 0.5 mg of fully reduced and alkylated (24) lactollin and human 132-microglobulin were digested with trypsin (treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone) at 370 for a total of 4 hr. The protein was suspended in 1% NH4HCO3 and tryspin was added at 0 and 2 hr. A protein:enzyme ratio of 50:1 was used for each addition. After electrophoresis in the first dimension at pH 4.7 for 45 min at 4500 V on Whatman 3MM paper (28), second dimension chromatography was carried out in n-butanol/acetic acid/ water/pyridine (15:3:12:10). Peptides were detected by briefly immersing the maps in a solution of 0.5% ninhydrin in acetone that was 0.07% (vol/vol) in pyridine. Molecular weights were estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (NaDodSO4) (29, 30), sedimentation equilibrium (31), and gel filtration on columns of Sephadex G-75 in phosphate-buffered saline and in 0.05 M sodium acetate, pH 5.0, with the ionic strength adjusted to 0.1 Abbreviations: phosphate-buffered saline, (per liter; pH 7.4) 8.00 g of NaCl, 0.20 g of KCl, 0.20 g of KH2PO4, 0.15 g of Na2HPO4; NaDodSO4, sodium dodecyl sulfate; HLA and H-2, major histocompatibility antigens of man and mouse, respectively; Mr, molecular weight. Proc. Nati. Acad. Sci. USA 74 (1977)