Proc. Nati. Acad. Sci. USA Vol. 74, No. 8, pp. 3345-3349, August 1977 Biochemistry

Crystallographic studies of bovinef2-microglobulin (x-ray diffraction/immunoglobulin domains/lactollin)

JOSEPH W. BECKER, JACK A. ZIFFER, GERALD M. EDELMAN, AND BRUCE A. CUNNINGHAM The Rockefeller University, New York, New York 10021

Contributed by Gerald M. Edelman, June 9, 1977

ABSTRACT 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.

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 6 M 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.

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Proc. Nati. Acad. Sci. USA 74 (1977)

BSA

c

0.90

0

Co

Lysc

c

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0.60

0 .0

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Tube number

FIG. 1. Isolation of lactollin by gel filtration of the appropriate fractions after chromatography on DEAE-cellulose (22) on Sephadex G-75 in phosphate-buffered saline; the column was calibrated with blue dextran (BD), ovalbumin (OA), cytochrome c (CC), human f2-microglobulin (HA2), and phenol red (PR).

using NaCl. Sephadex columns were calibrated with blue dextran, ovalbumin (Mr = 43,000), cytochrome c (Mr = 12,400), and phenol red. NaDodSO4/polyacrylamide gels were calibrated with bovine serum albumin (Mr = 68,000), ovalbumin, concanavalin A (Mr = 25,500), lysozyme (Mr = 13,500), and insulin (Mr = 6,000). X-ray crystallographic data were obtained from analysis of screened precession photographs taken on a rotating anode x-ray generator operated at 50 kV, 50 mA with a 2.0 X 0.2 mm focal spot.

RESULTS To optimize the production of purified lactollin, we modified the published procedure (22) to include gel filtration on Sephadex G-75 (Fig. 1) of the lactollin-containing fractions obtained from the DEAE-cellulose column. Polyacrylamide gel electrophoresis in NaDodSO4 of the material from this column gave the patterns shown in Fig. 2. Only material from fraction B gave significant amounts of material with a molecular weight corresponding to the known size of the lactollin subunit, and this material was found at the same position as human .02microglobulin in coelectrophoresis experiments. Material from this fraction was also the only material to give lactollin crystals. In preparations from colostrum, a small amount of material was eluted from gel filtration columns in the region of the ovalbumin marker (Fig. 1). This material also contained polypeptide chains with a molecular weight identical to that of the lactollin subunit. Material in fraction B was not homogeneous in every preparation, but frequently contained two kinds of contaminants: proteins of molecular weight higher than that of lactollin, and low-molecular-weight material that did not stain with Coomassie blue. The proteins, which may include a-lactalbumin (Mr = 14,000) (32), were removed by repeating the gel filtration or by crystallizing the lactollin. The low-molecular-weight material was detected by sequence analysis and by gel filtration of the reduced and alkylated protein on Sephadex G-75 in 2% (vol/vol).acetic acid. Amino acid analysis of this material in-

FIG. 2. NaDodSO41polyacrylamide gel electrophoresis of fractions isolated by gel filtration chromatography. Fractions are labeled as Fig. 1. Only fraction B contained significant amounts of material of molecular weight comparable to that of lactollin; the arrows indicate the positions where the lysozyme (Lyso, Mr = 13,500) and bovine

serum albumin (BSA, Mr = 68,000) standards migrated. dicated that it might be a fragment or component of casein (33),

and paper electrophoresis at pH 4.7 (28) showed the presence of at least four ninhydrin-positive components. These components were removed by crystallization of lactollin. Crystalline lactollin was homogeneous by amino acid sequence analysis, and the same sequence was obtained from crystals prepared from the material in fraction B (Fig. 1) from either milk or colostrum. The sequence we obtained was Ile-

Gln-Arg-Pro-(Pro)-Lys-Ile-Gln-Val-Tyr-(Ser)-Arg-His-Pro(Pro)-Glu-(Asx)-Gly, in excellent agreement with the results of Groves (20, 21) and strikingly similar to those of 032-micro-

globulins from other species. Lactollin samples from milk and colostrum also appear identical when their tryptic peptides are compared, and these peptides closely resemble those from human (#2-microglobulin (Fig. 3). At least seven major tryptic peptides of the bovine protein were absent in the human protein, but at least nine components of the proteins from the two species appear identical and the overall patterns are strikingly similar. All of our data indicate that the molecular weight in solution is 12,000. Material in fraction B (Fig. 1) was eluted from the Sephadex G-75 column at a position corresponding to a molecular weight of 12,000. When the purified material was chromatographed on the same column or on Sephadex G-75 in 0.05 M sodium acetate, pH 5.0 (F/2 = 0.1) it also was eluted in a position corresponding to a molecular weight of 12,000. Similar results were obtained by sedimentation equilibrium experiments. Studies in the ultracentrifuge did indicate that the protein redissolved from crystals may have a greater tendency to aggregate. Gel filtration of redissolved crystals on Sephadex G-75 in phosphate-buffered saline, however, gave the same pattern as the protein prior to crystallization, i.e., a single component of molecular weight 12,000. The lactollin crystals are parallelepipeds, approximately 0.5 X 0.1 X 0.1 mm in size. The crystals produce x-ray diffraction patterns extending to Bragg spacings of at least 2.8 A. The diffraction symmetry and systematic extinctions observed in precession photographs are consistent only with the orthorhombic space group P212121 (Fig. 4). The molecular weight, space group, and unit cell dimensions, a = 77.4, b = 47.9, and c = 34.3 A, combined with the average volume to mass ratio (Vm) of protein crystals (34), suggest that the asymmetric unit contains one polypeptide chain. An occupancy of one poly-

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Proc. Natl. Acad. Sci. USA 74 (1977)

00Z

I

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. 00

.V

4

I

A

B

C

FIG. 3. Comparison of tryptic peptides from human ,62-microglobulin (A), lactollin from milk (B), and lactollin from colostrum (C) by electrophoresis at pH 4.7 (left, right) followed by chromatography in n-butanol/acetic acid/water/pyridine (15:3:12:10) (top to bottom). Peptides were detected with 0.5% ninhydrin in acetone; I denotes origin.

peptide chain per asymmetric unit gives a Vm of 2.69 A3/dalton and a solvent volume fraction of 55%, both well within the range normally observed in protein crystals. An occupancy of two chains per asymmetric unit, the only other value allowed by the unit cell volume, yields a Vm of 1.35 A3/dalton and a solvent volume fraction of 7%. Both of these values are well outside the normally observed range and are highly unlike-

ly.

DISCUSSION Examination of the lactollin crystals indicates that their diffraction patterns are of sufficient quality to support the determination of the structure of this protein at high resolution. The structure of lactollin is of particular importance because this molecule apparently is bovine fl2-microglobulin. The results reported previously (20, 21) and the present data strongly support this hypothesis. The most convincing evidence is the amino acid sequence data (20, 21), which clearly indicate the similarity between lactollin and all known ,32-microglobulins, and the comparison of the tryptic peptides (Fig. 3), which shows a number of similarities with human f32-microglobulin. The

FIG. 4. Precession photograph of the hOl zone of crystalline lactollin. Reflections at the outer edge of the pattern correspond to a Bragg spacing of 4.0 A.

molecular weight of the lactollin subunit (12,000) is comparable to that of other ,#2-microglobulins.

The unusual feature of lactollin is its ability to form wellordered crystals readily. We have crystallized human and rabbit (B2-microglobuhn, but the crystals are quite small and produce only weak, low-resolution diffraction patterns. Lactollin has solubility properties that are different from those of human 02-microglobulin (8, 22), and these differences may be accounted for when the complete amino acid sequence of lactollin is known and can be compared to that of human &2-microglobulin (2). The extensive sequence homology between ,B2microglobulin and the constant regions of immunoglobulins suggests the possibility of solving the three-dimensional structure of lactollin using molecular replacement methods (35) based on one or more of the already known immunoglobulin structures (17, 36-39), as well as by the method of multiple isomorphous replacement (40). Previous studies have suggested that lactollin forms species of molecular weight 43,000 at pH 5.0 (22). The crystal data indicate that the dominant molecular form of lactollin is the 12,000-dalton monomer. The unit cell size suggests that the crystallographic asymmetric unit can contain only a single polypeptide chain, and the crystal packing required by space group P212121 precludes the possibility that the molecular species is any larger than one asymmetric unit, except in the case of a polymer. That is, the crystalline molecular species may be one asymmetric unit or an infinite polymer. Thus, the molecular species in the crystal cannot be an oligomeric assembly. In lactollin prepared from milk, we find no evidence for a 43,000-dalton species, either at pH 5.0 or at pH 7.4 (in phosphate-buffered saline). However, we have seen such components on gel filtration of crude lactollin from colostrum. Previous studies (22) suggesting that lactollin appeared as a 43,000-dalton species also made use of material from colostrum. Lactollin prepared from the two sources, however, does not appear to differ by amino-terminal sequence analysis and by comparison of tryptic peptides (Fig. 3). The presence of higher aggregates on gel filtration of lactollin-containing material from colostrum might result from the increased concentration of the protein in colostrum (22). Alternatively, differences between the two proteins might be due to differences in other components of the two fluids that copurify with lactollin and bind to it. Knowledge of any molecule that can bind or interact with 2-rMicroglobulin subunits would be important because it might bear upon some aspects of Ormicroglobulin function. Purified lactollin from both sources appears to aggregate at

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3348

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Froc. Natl. Acad. Sci. USA 74 (1977) ASP

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dominantly as the monomer, while interactions between complementary Cy3 domains are required to stabilize the F, dimer (42), and free COy3 domains are known to form-*ioncovalent dimers (44). Knowledge of the three-dimensional structure of (32-microglobulin will be of great interest. Because of this molecule's close structural relationship to the constant regions of immunoglobulins, its structure should yield further insight into the three-dimensional structures of immunoglobulins as well as into the variations in those structures induced by the known sequence variations. The fact that flrmicroglobulin occurs closely associated with histocompatibility and'thymus leukemia antigens also suggests that parts of its structure may be complementary to the structures of those antigens. Knowledge of the structure of lactollin may therefore provide some insight into the structures of these cell surface molecules as well.

LYS GLLJ

FIG. 5. Structural relationship between human j12-microglobulin and the human immunoglobulin G domain Cy3. The observed polypeptide fold of human Cyl (37) is depicted with the amino acid sequence of human 132-microglobulin. Residues that are identical in the two proteins are enclosed in rectangles. The cysteine residues that participate in the intradomain disulfide bond are shaded, and ,structure type hydrogen bonds observed in Cyl are indicated by dashed lines.

high concentration. This tendency may result from structural features that are responsible for the fact that it crystallizes readily. Other (32-microglobulins do aggregate. Rabbit (32microglobulin tends to aggregate more readily than the human protein, which does, however, form aggregates on long standing in solution. These aggregates appear to be nonspecific, random aggregates, and may result from denaturation of the protein. In the native form, all (32-microglobulins studied so far, including lactollin, appear to exist as monomers in association with other polypeptides, such as the heavy chains of HLA antigens, H-2 antigens, or thymus leukemia antigens. Clues to the interaction of #2-microglobulin with these glycoproteins may come from comparison of the structure of (32-microglobulin and the homology regions of immunoglobulin. Of the immunoglobulin domains whose amino acid sequences are known, the carboxyl-terminal domain, e.g., C-y3 of IgG, displays the greatest homology with 02-microglobulin (2). Comparison of the amino acid sequence of human (32microglobulin (2) with known immunoglobulin structures (17, 36-39, 41, 42) indicates that half of the residues that are common to (32-microglobulin and the Cy3 homology region are confined to a single region of the CSy3 structure, the three strands (S1, S2, and S7) of the four-stranded ,3-sheet that forms the interdomain Cy3-Cy3 interface in Fc (42). In fact, 50% of the residues are identical in this region of the structure, while only 19% of the residues in the remainder of the structure are the same in the two molecules (Fig. 5). The fact that 59% of the conserved residues in this (-sheet are hydrophobic, as opposed to 28% of those that are not conserved, suggests that the conserved residues are likely to be in the interior of the molecule and therefore involved with maintaining its three-dimensional structure, rather than on the surface, participating in intermolecular interactions. This conservation of sequence in what may be considered "framework" residues (43) strongly supports the hypothesis (1) that the three-dimensional structure of (32microglobulin resembles that of the constant regions of immunoglobulin. On the other hand, the absence of sequence identity between 02-microglobulin and Cy3 in residues expected to lie on the molecular surface is consistent with the observations that (32-microglobulin occurs in solution pre-

We thank Dr. W. Einar Gall for help in the molecular weight determination. Dr. Merton L. Groves of the Eastern Regional Research Center, Philadelphia, PA kindly made his data available to us prior to publication. We are grateful to Mrs. Kerri Jones Mulry and Miss Rosanne Apfeldorf for excellent technical assistance. This work was supported by National Institutes qf Health Grants A111378 and A109273 and by a career scientist award (B.A.C.) from the Irma T. Hirschl Charitable Trust. 1. Peterson, P. A., Cunningham, B. A., BerggArd, I. & Edelman, G. M. (1972) "32-Microglobulin: A free immunoglobulin domain," Proc. Nati. Acad. Sci. USA 69, 1697-1701. 2. Cunningham, B. A., Wang, J. L., BerggArd, I. & Peterson, P. A. (1973) "The complete amino acid sequence of (.2-microglobulin," Biochemistry 12, 4811-4821. 3. Smithies, 0. & Poulik, M. D. (1972) "Initiation of protein synthesis at an unusual position in an immunoglobulin gene?" Science 175, 187-189. 4. Grey, H. M., Kubo, R. T., Colon, S. M., Poulik, M. D., Cresswell, P., Springer, T., Turner, M. & Strominger, J. L. (1973) "The small subunit of HL-A antigens is 02-microglobulin," J. Exp. Med. 138, 1608-1612. 5. Nakamuro, K., Tanigaki, N. & Pressman, D. (1973) "Multiple common properties of human (32-microglobulin and the common portion fragment derived from HL-A antigen molecules," Proc. Natl. Acad. Sci. USA 70, 2863-2867. 6. Silver, J. & Hood, L. (1974) "Detergent-solubilized H-2 alloantigen is associated with a small molecular weight peptide," Nature 249,764-765. 7. Peterson, P. A., Rask, L. & Lindblom, J. B. (1974) "Highly purified papain-solubilized HL-A antigens contain 02-microglobulin," Proc. Natl. Acad. Sd. USA 71,3-39. 8. Berggard, I. & Beam, A.- G. (1968) "Isolation and properties of a low molecular weight 02-globulin occurring in human biological fluids," J. Biol. Chem. 243, 4095-4103. 9. Bernier, G. M. & Fanger, M.. W.. (1972) "Synthesis of (B2-mi-

croglobulin by stimulated lymphocytes," J. Immunol. 109,

407-409. 10. Htitteroth, T. H., Cleve, H., Litwin, S. 0. & Poulik, M. D. (1973)

"The relationship between 132-microglobulin and immunoglobulin in cultured human lymphoid cell lines," J. Exp. Med. 137, 838-843. 11. Evrin, P. E. & Nilsson, K. (1974) "j32-Microglobulin production in vitro by human hematopoietic, mesenchymal, and epithelial cells," J. Immunol. 112, 137-144. 12. Cunningham, B. A. & Berggird, I. (1975) "Partial amino acid sequence of rabbit #2-microglobulin," Science 187, 1079-

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30. Weber, K. & Osborn, M. (1969) "The reliability of molecular weight determinations by dodecyl sulfate-polycrylamide gel electrophoresis," J. Biol. Chem. 244, 4406-4412. 31. Yphantis, D. A. (1964) "Equilibrium ultracentrifugation of dilute solutions," Biochemistry 3, 297-317. 32. Gordon, W. G. (1971) "a-Lactalbumin," in The Milk Proteins, ed. McKenzie, H. A. (Academic Press, New York), pp. 331366. 33. Dayhoff, M. O., ed. (1972) Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, Washington, DC). 34. Matthews, B. W. (1968) "Solvent content of protein crystals," J. Mol. Biol. 33,491-497. 35. Rossmann, M. G., ed. (1972) The Molecular Replacement Method (Gordon and Breach, New York). 36. Segal, D. M., Padlan, E. A., Cohen, G. H., Rudikoff, S., Potter, M. & Davies, D. R. (1974) "The three-dimensional structure of a phosphorylcholine-binding mouse immunoglobulin Fab and the nature of the antigen binding site," Proc. Natl. Acad. Sci. USA 71,4298-4302. 37. Poljak, R. J., Amzel, L. M., Chen, B. L., Phizackerley, R. P. & Saul, F. (1974) "The three-dimensional structure of the Fab' fragment of a human myeloma immunoglobulin at 2.0-A resolution," Proc. Natl. Acad. Sci. USA 71,3440-3444. 38. Epp, O., Lattman, E. E., Schiffer, M., Huber, R. & Palm, W. (1975) "The molecular structure of a dimer composed of the variable portions of the Bence-Jones protein REI refined at 2.0-A resolution," Biochemistry 14, 4943-4952. 39. Schiffer, M., Girling, R. L., Ely, K. R. & Edmundson, A. B. (1973) "Structure of a X-type Bence-Jones protein at 3.5-A resolution," Biochemistry 12, 4620-4631. 40. Blow, D. M. & Crick, F. H. C. (1959) "The treatment of errors in the isomorphous replacement method," Acta Crystallogr. 12, 794-802. 41. Edelman, G. M., Cunningham, B. A., Gall, W. E., Gottlieb, P. D., Rutishauser, U. & Waxdal, M. J. (1969) "The covalent structure of an entire -yGimmunoglobulin molecule," Proc. Natl. Acad. Sci. USA 63,78-5. 42. Deisenhofer, J., Colman, P. M., Huber, R., Haupt, H. & Schwick, G. (1976) "Crystallographic structural studies of a human F.fragment," Hoppe-Seyler's Z. Physiol. Chem. 357, 435-445. 43. Padlan, E. A. & Davies, D. R. (1975) "Variability of three-dimensional structure in immunoglobulins," Proc. Natl. Acad. Sci. USA 72, 819-823. 44. Turner, M. W. & Bennich, H. (1968) "Subfragments from the Fc fragment of human immunoglobulin G," Biochem. J. 107, 171-178.