Three-dimensional structure of j32-microglobulin

The three-dimensional structure of 132microglobulin, the light chain of the major histocompatibility complex class I antigens, has been determined by x-ray crystallography. An electron density map of the bovine protein was calculated at a nominal resolution of 2.9 A by using the methods of multiple isomorphous replacement and electron density modification refinement. The molecule is approximately 45 x 25 x 20 A in size. Almost half of the amino acid residues participate in two large .3 structures, one of four strands and the other of three, linked by a central disulfide bond. The molecule thus strongly resembles Ig constant domains in polypeptide chain folding and overall tertiary structure. Amino acid residues that are the same in the sequences of 132-microglobulin and Ig constant domains are predominantly in the interior of the molecule, whereas residues conserved among 182-microglobulins from different species are both in the interior and on the molecular surface. In the crystals studied, the molecule is clearly monomeric, consistent with the observation that 132-microglobulin, unlike Ig constant domains, apparently does not form dimers in vivo but associates with the heavy chains of major histocompatibility complex antigens. Our results demonstrate that, at the level of detailed threedimensional structure, the light chain of the major histocompatibility class I antigens belongs to a superfamily of structures related to the Ig constant domains. 032-microglobulin (,82m) was discovered in the urine of patients with chronic kidney dysfunction (1). It has since been found in a variety of physiological fluids as well as on the surfaces of nearly all cells as the light chain of the major histocompatibility complex (MHC) class I antigens of man (HLA) and other vertebrates (2-5). These antigens play central roles in two widely studied activities involving immune recognition by T cells: the rejection of foreign tissue grafts through direct recognition of foreign MHC antigens and the recognition of viral and other antigens in conjunction with self-MHC antigens (6-8). The MHC class I antigens display an extraordinary polymorphism that is the apparent basis for the diversity and specificity of these recognition events. The heavy chains of these antigens are integral membrane proteins, and their polymorphism is confined to their NH2-terminal 180 amino acid residues, those farthest from the cell surface. In contrast, 832m and the 90 extracellular residues closest to the membrane are highly conserved. Although the function of P32m in these antigens is unknown, there is evidence that its presence is necessary for posttranslational processing and insertion of HLA heavy chains into the membrane (9, 10). It also appears to stabilize the structure of the heavy chain in that its removal causes loss of alloantigenic sites on HLA (11, 12). 832ms from different species have similar chemical structures. Approximately 50% of the residues are identical in the five sequences that are known completely (13-17). 832ms from different species apparently can replace one another in the quaternary structure of the MHC class I antigens (18-20), suggesting that the conservation of sequence reflects strong evolutionary pressure to conserve a functionally important conformation. By amino acid sequence homology, f32m belongs to a "superfamily" of proteins related to the Ig constant domains and believed to have evolved from a common ancestor (21). The observation that the chains of Ig contain regions homologous to one another in amino acid sequence led Edelman to suggest that these regions would be folded into distinct compact domains with similar three-dimensional structures (22). Such domains have subsequently been observed in all Igs whose three-dimensional structures have been determined (23). Recently, several other proteins have been shown to have all or part of their amino acid sequences homologous to those of Ig constant domains. These molecules include the T-cell differentiation antigen Thy-1 (24), the a and f3 chains of the T-cell antigen receptor (25-29), the MHC class I and II antigens (7, 30), and 32m (13-17). The similarities in primary structure have led to the suggestion that the homologous portions of these molecules may resemble Ig domains in detailed three-dimensional structure as well. Chemical studies indicate that in the MHC class I antigen complex, f32m associates noncovalently with the region of the heavy chain that is homologous to the Ig constant domains, the 90 extracellular residues adjacent to the membrane (31). In this case, the mode ofassociation as well as the three-dimensional structure may resemble that of Ig constant domains. Here we report the determination, at 2.9 A nominal resolution, of the three-dimensional structure of (32m. We show that the molecule closely resembles the constant domains of Ig with significant differences only in the polypeptide loops connecting the p structures. Most of the amino acid residues that are identical in the aligned sequences of f32m and Ig constant domains have their side chains in the interior of the molecule, consistent with the common peptide folding. There is much more variation in the residues on the molecular surface, consistent with the different functional roles of these molecules. MATERIALS AND METHODS The preparation and crystallization of 832m from bovine milk and colostrum have been reported previously (32, 33). The molecule crystallizes in the orthorhombic space group P212121 with a = 77.27, b = 47.99, and c = 34.42 A. Heavy-atom derivatives were prepared by soaking crystals in crystallization buffer (0.05 M phosphate/0.02% NaN3, pH 7.80) in which heavy-atom reagents had been dissolved. If the reagent was insoluble in this buffer, crystals were transferred to a solution of 0.02 M Tris N03/0.02% NaN3, pH 7.80, for at least 2 hours and then treated with heavy-atom reagents Abbreviations: f32m, 182-microglobulin; MHC, major histocompatibility complex; m.i.r., multiple isomorphous replacement; HLA, the major histocompatibility antigens of man. 4225 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 4226 Immunology: Becker and Reeke Table 1. Data collection and reduction Concentration, Soaking No. of Total Unique Bdert Pmaxt Name mM time, days crystals observations reflections R* A2 -2 Native 1 56,102 2892 0.0451 0.0288 Hg(OAc)2§ 0.1 8 3 32,478 3049 0.0637 -0.24 0.0256 Pt(NH3)2(NO2)2 1.0 7 3 31,494 2965 0.0463 -5.66 0.0240 Hg4¶ 12 3 40,078 2996 0.0487 1.31 0.0256 *R = I[I(H) T(H)]/ET(H) for the averaging of symmetry-equivalent reflections, where T is the average intensity of reflection H and I is any measurement of that reflection. tBder = Bdenvative Bnative, the difference between the isotropic temperature factors. tPmax = (sin2O/X2) for the highest resolution data used in any calculation. §The Hg(OAc)2 derivative was prepared in Tris NO3 buffer. IThe preparation of the HgIderivative is discussed in the text. dissolved in the latter buffer. Approximately 90 compounds were tested before three usable derivatives, Hg(OAc)2, Pt(NH3)2(NO2)2, and HgIJ2, were found. The HgI2derivative was discovered in soaking experiments using CH2(HgI)2. In subsequent control experiments, it was found that this derivative was not formed when the soaking solution was shielded from room light. A high-resolution difference electron density map of the site of substitution, using multiple isomorphous replacement (m.i.r.) phases based on the other two derivatives, revealed a clearly tetrahedral moiety. This result suggested that the derivatizing substance was HgI2-, formed by photodecomposition of CH2(HgI)2. This suggestion was confirmed by the observation that crystals treated with pure K2HgI4 have projection diffraction patterns and difference maps identical to those treated with CH2(HgI)2 and light. Three-dimensional diffraction data were collected to a maximum resolution of 2.9 A on 20 screenless oscillation photographs. Graphite monochromatized copper radiation from a rotating-anode generator operated at 40 kV, 60 mA was used. Except where noted, all computation was carried out by use of the ROCKS system of crystallographic computer programs (34). During film scanning, crystal slippage was assessed by comparing each photograph with a plot of the diffraction pattern predicted from the crystal parameters measured in alignment and early data photographs. When necessary, new orientation parameters were calculated from data obtained from the data photographs (35). Data reduction and scaling were performed as described (36). The native diffraction data were placed on an absolute scale and an isotropic temperature factor was estimated by using differential Wilson plot procedures, with the parameters of the refined structure of concanavalin A (37) as the reference. Heavy-atom derivative data were processed similarly, using the native protein as reference. Wilson plots of derivative intensity differences (Ider Inat) were used to assess the highest resolution at which derivative data could be considered isomorphous to native. Data collection and reduction are summarized in Table 1. Heavy atoms were located by inspection of difference Patterson and difference electron density maps. Anomalous difference Patterson maps indicated that the Hg(OAc)2 derivative provided useful anomalous dispersion data and these data were included in the m.i.r. phasing and used to establish the absolute hand of the protein. Heavy-atom parameters were refined by the method of Blow and Matthews (38) in which each derivative was refined separately, using m.i.r. phases calculated from the other two derivatives. After this refinement had converged, the parameters were refined in a final series of joint refinements. Anisotropic temperature parameters were included for the mercuric acetate and platinum derivatives because difference electron density maps showed nonspherical heavy-atom sites for these derivatives. The final assignment of heavy-atom sites was confirmed by calculating a difference map of each derivative using m.i.r. phases based on the other two derivatives. The overall figure-of-merit was 0.611 for the final heavy-atom parameters given in Table 2. The ratio of lack of closure to atomic scattering factor, E/f, was 0.78, 0.69, and 1.0 for the Hg(OAc)2, Pt(NH3)2(NO2)2, and HgI2derivatives, respectively. The boundary of the molecule was clearly visible in a map calculated with these phases and much of the polypeptide chain could be interpreted with the aid of the published amino acid sequence (17). After this preliminary interpretation, the protein phases were further refined by electron density modification methods. For these refinements, a molecular envelope was defined by interpretation of the m.i.r. map. This envelope Table 2. Refined heavy-atom parameters Fractional coordinates Atom Occupancy x y z Thermal parameters* Hg1t 0.550 0.0481 0.1336 0.1416 1.01, -1.32, 3.92, 2.40, -3.86, -2.23 Ptl 0.176 0.0334 0.1226 0.1948 1.77, -1.70, 15.22, 3.69, -3.45, -0.01 Pt2 0.261 0.1052 0.1292 0.1631 1.83, -1.71, 18.42, -0.49, -3.32, 6.30 Hg2t 0.242t 0.1124 0.1905 0.2967 11.971 I1 0.242 0.0877 0.1469 0.2404 24.923§ 12 0.242 0.1445 0.1557 0.3079 24.923 B3 0.242 0.1051 0.2387 0.3270 24.923 14 0.242 0.1016 0.1552 0.3164 24.923 *Single values are isotropic thermal parameters, B (A2). When six values are given, they are respectively the parameters (B11, B22, B33, B12, B13, B23) x 103 in the anisotropic temperature factor expression: exp[-(h2B1l + k2B22 + 12B33 + hkB12 + hlB13 + klB23)]. tHgl is the site of Hg(OAc)2 substitution; Hg2, of HgI4substitution. MThe Hg and I sites of the HgI4derivative were constrained to have identical occupancies. §The isotropic temperature factors of the I atoms were fixed at the values obtained in a Wilson plot of the isomorphous differences. Proc. Natl. Acad. Sci. USA 82 (1985) Proc. Natl. Acad. Sci. USA 82 (1985) 4227