Proc. Nati. Acad. Sci. USA
Vol. 73, No. 10, pp. 3529-3533, October 1976 Biochemistry
The functional repressor parts of a tetrameric lac repressor-f3galactosidase chimaera are organized as dimers (protein chimaera/electron microscopy/subunit crosslinking)
JURGEN KANIA AND DENNIS T. BROWN Institut fur Genetik der Universitit zu K6ln, Weyertal 121, 5000 Cologne, Federal Republic of Germany
Communicated by Thomas F. Anderson, August 9,1976
ABSTRACT The chimaeric protein repressor-galactosidase, in which fully active Jac repressor is covalently linked to the active enzyme #-galactosidase, was used as a system for probing the quaternary structure of Jac repressor. Electron micrographs revealed repressor-galactosidase to be a tetrameric aggregate. When Jac repressor, alone, was crosslinked with dimethyl suberimidate, dimers, trimers, tetramers, and oligomers of the protein subunit were produced, whereas crosslinking of the tetrameric repressor-galactosidase resulted in the production of only dimers of the chimaera. Treatment of Jac repressor with iodine resulted in the formation of protein dimers; the same result was obtained with repressor galactosidase. After limited proteolysis of Jac repressor, no crosslinking was obtained after treatment with dimethyl suberimidate, whereas iodine still produced a covalent linkage. These results are interpreted as evidence that the Jac repressor parts of the tetrameric repressor-galactosidase-chimaera are organized as dimers on the tetrameric-ftgalactosidase core. Because this chimaera has been previously shown to have normal repressor activity [B. MullerHill and J. Kania (1974) Nature, 249,561-63,l we conclude that Jac repressor still is biologically active as a dimeric aggregate. lac repressor, a tetramer of identical subunits, specifically interacts with its DNA target, the lac operator, thus preventing the expression of the structural genes of the lac operon. Genetic and biochemical analysis point to the NH2-terminus of lac repressor as the region which interacts specifically with DNA [see review by Muller-Hill (1)]. The primary structure of the lac repressor protein (2) and the lac operator (3) have been determined. Little, however, is known about the tertiary and quaternary structure of wac repressor since crystals large enough for single crystal x-ray diffraction analysis have not yet been obtained. Electron micrographs of the protein in solution exist (4, 5). The most detailed structural analysis was carried out by Steitz et al. (6), who proposed a model of quaternary structure from electron micrographs and powder x-ray diffraction analysis of microcrystals. For this model, lac repressor subunits are arranged in an exact or quasi D2 symmetry. As a conclusion to their investigations Steitz et al. proposed a model for repressor-operator interaction, where it was suggested that two operator binding sites exist per tetramer, in the case of D2 symmetry. This model, if correct, might imply that lac repressor dimers are capable of recognizing the lac operator sequence, if somehow the repressor tetramer could be dissociated into the appropriate stable dimers. Repressor-galactosidase* is a fusion-protein, in which fully active lac repressor is covalently attached by its COOH-terminus to the NH2-terminus of active Abbreviation: NaDodSO4, sodium dodecyl sulfate. * We will use the term "repressor-galactosidase" for the protein chimaera purified from Escherichia coil strain iql (71-56-14), as described (7). The terms "repressor-part" and "galactosidase-part" are used for those amino acid sequences in repressor-galactosidase, which are coded by the lac repressor gene and by the fl-galactosidase gene, respectively.
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fl-galactosidase (7), and is an appropriate system for the further examination of models of the quaternary structure of lac repressor. Because the covalent linkage of lac repressor to fl-ga-
lactosidase limits the theoretically possible arrangements of (ac repressor subunits, investigation on the geometry of this hybrid molecule enables us to test the model of Steitz et al. As a result of the application of electron microscopy and crosslinking agents on repressor-galactosidase, we propose that lac repressor is able to recognize lac operator as a dimer, fixed via its COOH-terminus to a soluble matrix, i.e., fl-galactosidase. MATERIALS AND METHODS Purification of Repressor-Galactosidase. Repressor-galactosidase protein was purified as described previously (7), with the modification that chromatography on DEAE-cellulose (Biorad) was performed after the phosphocellulose step. DEAE-cellulose was equilibrated with buffer D [0.01 M Tris at pH 7.27 (at 22°), 0.01 M MgAc, 0.01 M 2-mercaptoethanol, and 0.1 M NaCI]; the pure protein eluted at 0.21 M NaCl, when a linear gradient of 0.1 M NaCI to 0.5 M NaCl in buffer applied to the column. According to sodium dodecyl sulfate (NaDodSO4) gels, the protein was about 90% pure. Electron Microscopy. A concentrated solution (15 mg/ml) of repressor-galactosidase was diluted 1000-fold into 1% NH4Ac at pH 7.2 with or without 0.35% glutaraldehyde. After 5 min at room temperature, the protein was mounted on copper grids (400 mesh) having a thin carbon film. The carbon film had been made hydrophilic by glow discharging prior to use. The mounted specimens were washed with distilledtwater, and negatively stained with a solution of 1.5%-uranyl-acetate in distilled water. Micrographs were made in a Siemens 101 electron microscope, the magnification of which was calibrated with a carbon grating replica, having 2160 lines/mm (Ernest Fullam Co. no. 1002). Crosslinking with Dimethyl Suberimidate and Dimethyl Adipinimidate. Diimidates were obtained from Pierce. Crosslinking was performed essentially as described by Davies and Stark (8). Repressor-galactosidase was crosslinked at 3-4 mg/ml of protein in the reaction mixture, lac repressor at an approximate concentration of 6 mg/ml. The pH was 8.5 and the diimidate concentration was 0.7 mg/ml. The concentrations of protein and crosslinker were not critical within a factor of two. More concentrated repressor-galactosidase solutions only resulted in the appearance of numerous multimeres apparently due to intermolecular crosslinking. The reaction was stopped after 30 min at room temperature by making the solution- 1% of NaDodSO4 and 2-mercaptoethanol and incubating for 20 min at 650. The presence of either 10 mM isopropyl-thio-flD-galactoside or 10 mM o-nitrophenyl-,6-rfucoside had no effect on the yield of crosslinked material. Crosslinking with 12/KI. lodination was performed by slightly modifying the conditions described by Fanning (9).
3530
Biochemistry: Kania and Brown
Proc. Natl. Acad. Sci. USA 73 (1976)
W>z..-,.'9K>7x~~~~ -r.
FIG. 1. Electron micrographs of negatively stained repressor-galactosidase. (A) Low magnification field showing a number of tetrameric complexes. A number of the tetramers have a prominent centrally located hole (1). In other tetramers the hole is less easily seen (2). (B) Six similarly oriented tetramers superimposed and printed as a single image. The four subunits of the tetramer are easily seen. Magnification (A) X161,000 and (B) X737,000.
Proteins were dialyzed against TMS buffer [0.01 M Tris at pH 7.5 (at 220), 0.01 M MgAc, 0.2 M KCl, and 0.1 mM EDTA] without 2-mercaptoethanol at 40. To 100 ,l of both lac repressor (8 mg/ml) and repressor-galactosidase (10 mg/ml), we added 10 Al from a stock iodine solution (0.01 M 12, 0.04 M KI) made up in distilled water at 40. After 1 min, the reaction was stopped by adding 10 ,l of 2-mercaptoethanol. Doubling the reaction time or the amount of iodine did not result in an increase in crosslinked material, neithr was the concentration of the protein critical. Isopropyl-thio-f3-D-galactoside or o-nitrophenyl-f3D-fucoside at 10-2 M had no effect. Some protein precipitated during the reaction, but could be dissolved by adding NaDodSO4 to a final concentration of 1%. After incubation for 20 min at 650, each gel received 50-100 ,ug of protein. NaDodSO4/Acrylamide Gel Electrophoresis. NaDodSO4 gels were made by standard procedures (10). In the case of repressor-galactosidase, 4% acrylamide gels were used and run for 8.5 hr at 8 mA per tube. Crosslinked lac repressor was run on 5% acrylamide for 4.5 hr (if treated with proteases, then the time was reduced to 3-4 hr) at 8 mA per tube.
RESULTS Electron Microscopy of Repressor-Galactosidase. We have previously shown that repressor-galactosidase is a stable and homogeneous oligomer which is similar in its sedimentation behavior to fl-galactosidase itself (16S) (7). As the knowledge of the number of subunits is essential for further investigations on the geometry of this protein, we attempted to establish the
state of aggregation of repressor-galactosidase subunits directly by electron microscopy. By electron microscopy, the repressor-galactosidase molecule was found to be square in shape. Frequently, the complex could be clearly seen to be composed of four subunits (Fig. 1). The tetramers were of constant size and possessed a centrally located hole which was somewhat variable in diameter. The complexes shown in Fig. 1 were obtained from glutaraldehyde treated preparations. Similar results were obtained with unfixed material. The tetrameric configuration of the complex was enhanced by superimposing several similarly oriented molecules and printing them photographically as a single image (Fig. 1). For this purpose the tetramers having the centrally located hole were chosen. The width of the tetramers along the edge was 217 A and measured on the diagonal (corner to corner) 251 A. Each of the four subunits comprising the tetramer were 82 A in cross section. We have also examined the structure of purified j3-galactosidase by electron microscopy (data not shown). We found fl-galactosidase was not as well preserved in preparations for electron microscopy as repressor-galactosidase. f3-Galactosidase was found to be tetrameric in structure as has been previously reported (11). The measured length of the edge of the (3-galactosidase tetramer was found to be slightly larger than that of repressor-galactosidase at 220 A. The larger size was probably due to measuring structures in less well-preserved specimens. Lac repressor alone, although tetrameric in structure (12), is less than one half the size of repressor-galactosidase (4, 5, 13). Thus, the size and shape of the repressor-galactosidase complex,
Biochemistry: a
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Proc. Nati. Acad. Sci. USA 73 (1976)
Kania and Brown d e
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FIG. 2. NaDodSO4/gel electrophoresis of crosslinked lac repressor and crosslinked repressor-galactosidase. The positions of monomers (mono), dimers (di), trimers (tri), and tetramers (tetra) of the protein subunit are indicated by arrows. (A) lac repressor crosslinked with dimethyl suberimidate and with I2/K1 (a) untreated; (b) treated with chymotrypsin before incubation with dimethyl suberimidate; (c) crosslinked with dimethyl suberimidate; (d) same as in (c) except that repressor-galactosidase was added as marker; (e) untreated lac repressor, bovine serum albumin, and repressor-galactosidase added as marker proteins; (f) crosslinked with iodine. (B) Repressor-galactosidase crosslinked with dimethyl suberimidate (a) untreated; (b) crosslinked with dimethyl suberimidate; (c) same as in (b) except that myosin was added as marker; (d) untreated repressor-galactosidase and myosin. (C) Repressor-galactosidase crosslinked with I2/K1 (a) untreated; (b) crosslinked with iodine; (c) same as in (b) except that myosin was added. (D) lac repressor crosslinked with 12/KI after limited proteolysis (a) untreated; (b) untreated core after incubation with chymotrypsin; (c) same as in (b), treated with iodine; (d) untreated after incubation with trypsin and chymotrypsin; (e) same as in (d), treated with iodine; (f) uncleaved lac repressor after treatment with iodine; (g) untreated lac repressor, bovine serum albumin, and repressor-galactosidase added as marker proteins. as revealed in the electron microscope, suggests that the galactosidase part of the repressor-galactosidase has the same structural organization as galactosidase alone and further implies that the glactosidase moiety of the repressor-galactosidase complex forms the structural core of the tetramer (see Fig. 3). If the smaller repressor part of the repressor-galactosidase formed the structural core of the aggregate, then one would have expected to see "dumbbell" shaped structures consisting of large dimers of the high-molecular-weight galactosidase part connected by the smaller repressor portions of the complex. Such structures were never found. Crosslinking with Diimidoesters. If lac repressor is treated with dimethyl suberimidate or dimethyl adipinimidate, several bands are found on NaDodSO4 gels which can be interpreted as monomer, dimer, two trimers, one or more tetramers, and oligomers (Fig. 2A). Repressor-galactosidase, however, if treated with the same diimidoesters in a parallel experiment, can be crosslinked only to dimers (Fig. 2B). 3-Galactosidase alone cannot be crosslinked with these diimidoesters under the conditions described (see also ref. 14). This suggests that only two lac repressor-parts, in the repressor-galactosidase aggregate, are in the configuration of operator-binding lac repressor which allows them to be crosslinked. This result would be expected if the repressor parts of the complex were peripherally located as dimers on a fl-galactosidase core which has the tetrameric organization described in the preceding section (see Fig. 3). Crosslinking in the Presence of Iodine. Iodine proved to be an excellent agent for crosslinking lac repressor, by applying the procedure described in Materials and Methods. After iodination of lac repressor, two main bands can be resolved on NaDodSO4 gels at the positions of dimers and also some minor
bands at trimer and tetramer positions (Fig. 2A and D). If flgalactosidase is treated with iodine in the same way, faint bands at dimer, trimer, and tetramer positions were visible on NaDodSO4 gels, only if the gel was heavily overloaded. However, treatment of repressor-galactosidase with iodine resulted in a strong band at the dimer position on NaDodSO4 gels (Fig. 2C). Thus, iodine as well as diimidoesters crosslink repressor-galactosidase only to dimers, whereas lac repressor is crosslinked primarily into dimers by iodine and to dimers, trimers, and tetramers by diimidoesters. Crosslinking of lac Repressor after Limited Proteolysis. Platt et al. have shown that if lac repressor is incubated with trypsin and chymotrypsin under native conditions, peptides are released only from the termini of the molecule thus leaving an unnicked, aggregated core protein (15). We applied this technique to distinguish between the crosslinking sites of dimethyl suberimidate and I2/KI. After treatment of lac repressor with chymotrypsin (3% wt/wt, 1 hr at 370), no crosslinking could be achieved by dimethyl suberimidate within the chymotryptic core (Fig. 2A). This implies that the crosslinking sites (lysine residues) are located at the termini of the polypeptide chain of lac repressor. Excessive treatment of lac repressor with chymotrypsin and a mixture of trypsin and chymotrypsin (each 2% wt/wt, 30 min at 370, and then by incubation at 40 for 12 hr) did not result in an abolition of crosslinking by iodine (Fig. 2D). Gel (c) of Fig. 2 reveals that after digestion the iodinated sample consisted of a mixture of polypeptide chains of different lengths, due to the prolonged incubation with the protease. The upper band presumably consists of dimers which lack the NH2-termini; the crosslinked material from the lower band should be free of both the NH2- and COOH-termini. Since trypsin si-
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Biochemistry: Kania and Brown
multaneously acts at both termini of the lac repressor, a mixture of trypsin and chymotrypsin leads to the double-band pattern found on gels (d) and (e). Precise molecular weight determinations of the iodinated material are difficult from gels (c) and (e) since iodinated, but not crosslinked polypeptides, seem to migrate faster than iodinated, while the uncleaved, uncrosslinked but iodinated subunit shows the same mobility as noniodinated lac repressor [gel (f)]. From these results we conclude that iodine catalyzes the crosslinking of the protease resistant core fragments of lac repressor at or near their aggregation sites.
DISCUSSION Protein chemical investigations have revealed that no more than five amino acids are missing at the COOH-terminus of lac repressor in repressor-galactosidase, because the second to last tryptic peptide can be identified after limited tryptic digestion of the protein (16). Because the subunit is calculated to be about 155,000 daltons (7) (with RNA polymerase as a standard on NaDodSO4 gels), about 70 amino acids are missing from the f3-galactosidase portion. Because the increase in the molecular mass which results from the attachment of lac repressor does not result in a faster sedimentation relative to f3-galactosidase, we propose that the structure of repressor-galactosidase is ellipsoidal rather than spherical. By electron microscopy, we were able to establish the active repressor-galactosidase to be a tetramer with morphology and size similar to 03-galactosidase alone. #-Galactosidase and lac repressor are also both stable tetramers (12, 17). Langley et al. have shown that defective galactosidase protein from the deletion mutant strain M15 of E. coli, which lacks residues 11-41 of intact ,B-galactosidase, is a dimer under native conditions (18). This observation excludes C4 symmetry for the ,B-galactosidase tetramer, since in the case of such a symmetrical arrangement, dissociation by mutation would lead exclusively to monomers [see review by Matthews and Bernhard (19)]. This must also be true for lac repressor, because substitutions of amino acids in the presumed aggregation sites of lac repressor lead to dissociation of the repressor tetramer into a mixture of dimers and V monomers, as found recently by Schmitz et al. (20). Our results, obtained by iodine-catalysed crosslinking of lac repressor, support this assumption. Actually, this is not surprising since all other tetrameric proteins of known structure are of D2 symmetry (19). Therefore, in the repressor-galactosidase tetramer, lac repressor parts cannot be aggregated into a tetramer if f,-galactosidase parts are tetrameric with D2 symmetry and vice versa. Consequently, one of the two protein-parts must retain its biological activity in the repressor-galactosidase tetramer in the state of a dimer. The crosslinking patterns of lac repressor and repressor-galactosidase produced by diimidoesters and iodine in this study support the hypothesis that it is the lac repressor portion of the repressor-galactosidase complex which is organized into dimers. As mentioned above, Schmitz et al. have found mutations in the repressor gene which result in the dissociation of lac repressor into dimers and monomers which are no longer capable of operator binding (20). Such mutations map in two regions, one around tryptophan 209 and phenylalanine 215, the other one around tyrosine 260 and tyrosine 269 in the repressor sequence. It is at these positions that Schmitz et al. propose subunit-aggregation sites. It is attractive to propose that these particular aromatic amino acids are involved in the iodinecatalyzed crosslinking reaction. Crosslinking of repressor-galactosidase catalyzed by iodine revealed that the aggregation sites necessary for operator-binding (as proposed by Schmitz
Proc. Natl. Acad. Sci. USA 73 (1976) lac Repressor
Repressor-Ga I actosidase FIG. 3. Schematic drawing of the proposed model of repressorgalactosidase organization. The fl-galactosidase-parts are aggregated as tetramers in a D2 symmetry as in the wild type molecule. Each of those two fl-galactosidase parts, which have point symmetry to the x-axis, carry the repressor-parts which, as shown by the crosslinking data, must be aggregated as dimers. The native lac repressor tetramer is also aggregated in a D2 symmetry; this is schematically indicated in the above drawing. By comparing the corresponding dimers in these two models, it becomes evident that the DNA binding site in the repressor tetramere is made by those two subunits, which have point symmetry to the x-axis. Consequently, the NH2-terminus of the lac repressor polypeptide chain, which contains the DNA-binding site, must be located at the free narrow-end of each subunit in the above model.
et al.) are present in repressor-galactosidase. This implies that the two repressor parts are properly aggregated for operator
binding. Our conclusions, from this study, are presented in model form in Fig. 3 and may be summarized as follows: the fl-galactosidase parts in repressor-galactosidase are aggregated in a D2 symmetry. Each of those f3-galactosidase parts, which are similarly oriented, carry at their NH2-termini lac repressor-parts which, aggregated as dimers, are able to bind to lac operator DNA (7). Similar conclusions have been made based on other types of evidence by Geisler and Weber (21) and by J. Miwa and J. R. Sadler (personal communication). This model supports the implication of the model of Steitzet al. that lac repressor dimers are capable of recognizing the lac operator and is also consistent with some aspects of the model suggested by Adler et al. (22), who proposed that the DNA binding site is situated on a protrusion at the NH2-terminus of the polypeptide chain. The model presented here predicts as yet undiscovered point mutations in the i gene of lac repressor, in which the substitution of an amino acid in an aggregation site different from those aggregation sites described by Schmitz et al. would lead to a dissociation of the lac repressor tetramer into stable dimers exhibiting all biologicl activities of native lac repressor. Since repressor-galactosidase does not aggregate through its repressor-parts into long chains, one might conclude the COOH-terminus of lac repressor is involved in the aggregation of repressor dimers to tetramers. This is supported by the fact that Ll-lac repressor, in which the COOH-terminus of lac repressor is missing, is still able to repress at low levels in vow and forms exclusively dimers [Miller et al. (23)]. This third predicted aggregation site could be the hydrophobic cluster existing around proline 307.
Biochemistry:
Kania, and Brown
We thank B. Muller-Hill for his stimulating interest and helpful discussion throughout this work. Thanks are also due to T. G. Fanning and K. Beyreuther for advice and suggestions and for providing purified lac repressor; and to K. Weber for providing myosin. This investigation was supported by Deutsche Forschungsgemeinschaft through SFB 74 by grants to B.M.-H. and D.T.B. 1. Muller-Hill, B. (1975) Prog. Biophys. Mol. Biol. 30,227-252. 2. Beyreuther, K., Adler, K., Geisler, N. & Klemm. A. (1973) Proc. Nati. Acad. Sci. USA 70,3576-580. 3. Gilbert, W. & Maxam, A. (1973) Proc. Nati. Acad. Sci. USA 70, 3581-584. 4. Oshima, Y., Horiuchi, T. & Yanagida, M. (1975) J. Mol. Biol. 91, 515-519. 5. Abermann, R., Bahl, C. P., Marians, K. J., Salpeter, M. M. & Wu, R. (1976) J. Mol. Biol. 100, 109-114. 6. Steitz, T. A., Richmond, T. J., Wise, D. & Engelman, D. (1974) Proc. Natl. Acad. Sci. USA 71,593-597. 7. Muller-Hill, B. & Kania, J. (1974) Nature 249,561-563. 8. Davies, G. E. & Stark, G. R. (1970) Proc. Natl. Acad. Sci. USA 66,651-656. 9. Fanning, T. G. (1975) Biochemistry 14,2512-2520. 10. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 44064412. 11. Zabin, I. (1963) Cold Spring Harbor Symp. Quant. Biol. 28, 431-435. 12. Muller-Hill, B., Beyreuther, K. & Gilbert, W. (1971) in Methods in Enzymology, eds. Grossman, L. & Moldave, K. (Academic
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Press, New York), Vol. 21, pp. 483-487. 13. Bourgeois, S. & Pfahl, M. (1976) Advances in Protein Chemistry eds. Anfinsen, C. B., Edsall, J. T. & Richards, F. M. (Academic Press, New York), Vol. 30, pp. 1-99. 14. Mfillner, H., Hucho, F. & Sund, H. (1975) Hoppe-Seylers Z. Physiol. Chem. 356, 256. 15. Platt, T., Files, J. G. & Weber, K. (1973) J. Blol. Chem. 248, 110-121. 16. Kania, J., Ruth, C. & Mfiller-Hill, B. (1975) Hoppe-Seylers Z. Physlol. Chem. 356,243. 17. Craven, G., Steers, E. & Anfinsen, C. B. (1965) J. Blol. Chem. 240, 2468-2477. 18. Langley, K. E., Villarejo, M. R., Fowler, A. V., Zamenhof, P. J. & Zabin, I. (1975) Proc. Natl. Acad. Scd. USA 72, 1254-1257. 19. Matthews, B. W. & Bernhard, S. A. (1973) "Structure and symmetry of oligomeric enzymes," in Annual Review of Biophysics and Bioengineering, eds. Mullins, L. J., Hagins, W. A. & Stryer, L. (Annual Reviews Inc., Palo Alto, Calif.), Vol. 2. 20. Schmitz, A., Schmeissner, U., Miller, J. H. & Lu, P. (1976) J. Biol. Chem. 251,3359-3366. 21. Geisler, N. & Weber, K. (1976) Proc. Nati. Acad. Sci. USA 73, 3103-3106. 22. Adler, K., Beyreuther, K., Fanning; E., Geisler, N., Gronenborn, B., Klemm, A., Mfiller-Hill, B., Pfahl, M. & Schmitz, A. (1972) Nature 237,322-327. 23. Miller, J. H., Platt, T. & Weber, K. (1970) in The Lactose Operon, eds. Beckwith, J. R. & Zipser, D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), pp. 343-351.
Vol. 73, No. 10, pp. 3529-3533, October 1976 Biochemistry
The functional repressor parts of a tetrameric lac repressor-f3galactosidase chimaera are organized as dimers (protein chimaera/electron microscopy/subunit crosslinking)
JURGEN KANIA AND DENNIS T. BROWN Institut fur Genetik der Universitit zu K6ln, Weyertal 121, 5000 Cologne, Federal Republic of Germany
Communicated by Thomas F. Anderson, August 9,1976
ABSTRACT The chimaeric protein repressor-galactosidase, in which fully active Jac repressor is covalently linked to the active enzyme #-galactosidase, was used as a system for probing the quaternary structure of Jac repressor. Electron micrographs revealed repressor-galactosidase to be a tetrameric aggregate. When Jac repressor, alone, was crosslinked with dimethyl suberimidate, dimers, trimers, tetramers, and oligomers of the protein subunit were produced, whereas crosslinking of the tetrameric repressor-galactosidase resulted in the production of only dimers of the chimaera. Treatment of Jac repressor with iodine resulted in the formation of protein dimers; the same result was obtained with repressor galactosidase. After limited proteolysis of Jac repressor, no crosslinking was obtained after treatment with dimethyl suberimidate, whereas iodine still produced a covalent linkage. These results are interpreted as evidence that the Jac repressor parts of the tetrameric repressor-galactosidase-chimaera are organized as dimers on the tetrameric-ftgalactosidase core. Because this chimaera has been previously shown to have normal repressor activity [B. MullerHill and J. Kania (1974) Nature, 249,561-63,l we conclude that Jac repressor still is biologically active as a dimeric aggregate. lac repressor, a tetramer of identical subunits, specifically interacts with its DNA target, the lac operator, thus preventing the expression of the structural genes of the lac operon. Genetic and biochemical analysis point to the NH2-terminus of lac repressor as the region which interacts specifically with DNA [see review by Muller-Hill (1)]. The primary structure of the lac repressor protein (2) and the lac operator (3) have been determined. Little, however, is known about the tertiary and quaternary structure of wac repressor since crystals large enough for single crystal x-ray diffraction analysis have not yet been obtained. Electron micrographs of the protein in solution exist (4, 5). The most detailed structural analysis was carried out by Steitz et al. (6), who proposed a model of quaternary structure from electron micrographs and powder x-ray diffraction analysis of microcrystals. For this model, lac repressor subunits are arranged in an exact or quasi D2 symmetry. As a conclusion to their investigations Steitz et al. proposed a model for repressor-operator interaction, where it was suggested that two operator binding sites exist per tetramer, in the case of D2 symmetry. This model, if correct, might imply that lac repressor dimers are capable of recognizing the lac operator sequence, if somehow the repressor tetramer could be dissociated into the appropriate stable dimers. Repressor-galactosidase* is a fusion-protein, in which fully active lac repressor is covalently attached by its COOH-terminus to the NH2-terminus of active Abbreviation: NaDodSO4, sodium dodecyl sulfate. * We will use the term "repressor-galactosidase" for the protein chimaera purified from Escherichia coil strain iql (71-56-14), as described (7). The terms "repressor-part" and "galactosidase-part" are used for those amino acid sequences in repressor-galactosidase, which are coded by the lac repressor gene and by the fl-galactosidase gene, respectively.
3529
fl-galactosidase (7), and is an appropriate system for the further examination of models of the quaternary structure of lac repressor. Because the covalent linkage of lac repressor to fl-ga-
lactosidase limits the theoretically possible arrangements of (ac repressor subunits, investigation on the geometry of this hybrid molecule enables us to test the model of Steitz et al. As a result of the application of electron microscopy and crosslinking agents on repressor-galactosidase, we propose that lac repressor is able to recognize lac operator as a dimer, fixed via its COOH-terminus to a soluble matrix, i.e., fl-galactosidase. MATERIALS AND METHODS Purification of Repressor-Galactosidase. Repressor-galactosidase protein was purified as described previously (7), with the modification that chromatography on DEAE-cellulose (Biorad) was performed after the phosphocellulose step. DEAE-cellulose was equilibrated with buffer D [0.01 M Tris at pH 7.27 (at 22°), 0.01 M MgAc, 0.01 M 2-mercaptoethanol, and 0.1 M NaCI]; the pure protein eluted at 0.21 M NaCl, when a linear gradient of 0.1 M NaCI to 0.5 M NaCl in buffer applied to the column. According to sodium dodecyl sulfate (NaDodSO4) gels, the protein was about 90% pure. Electron Microscopy. A concentrated solution (15 mg/ml) of repressor-galactosidase was diluted 1000-fold into 1% NH4Ac at pH 7.2 with or without 0.35% glutaraldehyde. After 5 min at room temperature, the protein was mounted on copper grids (400 mesh) having a thin carbon film. The carbon film had been made hydrophilic by glow discharging prior to use. The mounted specimens were washed with distilledtwater, and negatively stained with a solution of 1.5%-uranyl-acetate in distilled water. Micrographs were made in a Siemens 101 electron microscope, the magnification of which was calibrated with a carbon grating replica, having 2160 lines/mm (Ernest Fullam Co. no. 1002). Crosslinking with Dimethyl Suberimidate and Dimethyl Adipinimidate. Diimidates were obtained from Pierce. Crosslinking was performed essentially as described by Davies and Stark (8). Repressor-galactosidase was crosslinked at 3-4 mg/ml of protein in the reaction mixture, lac repressor at an approximate concentration of 6 mg/ml. The pH was 8.5 and the diimidate concentration was 0.7 mg/ml. The concentrations of protein and crosslinker were not critical within a factor of two. More concentrated repressor-galactosidase solutions only resulted in the appearance of numerous multimeres apparently due to intermolecular crosslinking. The reaction was stopped after 30 min at room temperature by making the solution- 1% of NaDodSO4 and 2-mercaptoethanol and incubating for 20 min at 650. The presence of either 10 mM isopropyl-thio-flD-galactoside or 10 mM o-nitrophenyl-,6-rfucoside had no effect on the yield of crosslinked material. Crosslinking with 12/KI. lodination was performed by slightly modifying the conditions described by Fanning (9).
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Proc. Natl. Acad. Sci. USA 73 (1976)
W>z..-,.'9K>7x~~~~ -r.
FIG. 1. Electron micrographs of negatively stained repressor-galactosidase. (A) Low magnification field showing a number of tetrameric complexes. A number of the tetramers have a prominent centrally located hole (1). In other tetramers the hole is less easily seen (2). (B) Six similarly oriented tetramers superimposed and printed as a single image. The four subunits of the tetramer are easily seen. Magnification (A) X161,000 and (B) X737,000.
Proteins were dialyzed against TMS buffer [0.01 M Tris at pH 7.5 (at 220), 0.01 M MgAc, 0.2 M KCl, and 0.1 mM EDTA] without 2-mercaptoethanol at 40. To 100 ,l of both lac repressor (8 mg/ml) and repressor-galactosidase (10 mg/ml), we added 10 Al from a stock iodine solution (0.01 M 12, 0.04 M KI) made up in distilled water at 40. After 1 min, the reaction was stopped by adding 10 ,l of 2-mercaptoethanol. Doubling the reaction time or the amount of iodine did not result in an increase in crosslinked material, neithr was the concentration of the protein critical. Isopropyl-thio-f3-D-galactoside or o-nitrophenyl-f3D-fucoside at 10-2 M had no effect. Some protein precipitated during the reaction, but could be dissolved by adding NaDodSO4 to a final concentration of 1%. After incubation for 20 min at 650, each gel received 50-100 ,ug of protein. NaDodSO4/Acrylamide Gel Electrophoresis. NaDodSO4 gels were made by standard procedures (10). In the case of repressor-galactosidase, 4% acrylamide gels were used and run for 8.5 hr at 8 mA per tube. Crosslinked lac repressor was run on 5% acrylamide for 4.5 hr (if treated with proteases, then the time was reduced to 3-4 hr) at 8 mA per tube.
RESULTS Electron Microscopy of Repressor-Galactosidase. We have previously shown that repressor-galactosidase is a stable and homogeneous oligomer which is similar in its sedimentation behavior to fl-galactosidase itself (16S) (7). As the knowledge of the number of subunits is essential for further investigations on the geometry of this protein, we attempted to establish the
state of aggregation of repressor-galactosidase subunits directly by electron microscopy. By electron microscopy, the repressor-galactosidase molecule was found to be square in shape. Frequently, the complex could be clearly seen to be composed of four subunits (Fig. 1). The tetramers were of constant size and possessed a centrally located hole which was somewhat variable in diameter. The complexes shown in Fig. 1 were obtained from glutaraldehyde treated preparations. Similar results were obtained with unfixed material. The tetrameric configuration of the complex was enhanced by superimposing several similarly oriented molecules and printing them photographically as a single image (Fig. 1). For this purpose the tetramers having the centrally located hole were chosen. The width of the tetramers along the edge was 217 A and measured on the diagonal (corner to corner) 251 A. Each of the four subunits comprising the tetramer were 82 A in cross section. We have also examined the structure of purified j3-galactosidase by electron microscopy (data not shown). We found fl-galactosidase was not as well preserved in preparations for electron microscopy as repressor-galactosidase. f3-Galactosidase was found to be tetrameric in structure as has been previously reported (11). The measured length of the edge of the (3-galactosidase tetramer was found to be slightly larger than that of repressor-galactosidase at 220 A. The larger size was probably due to measuring structures in less well-preserved specimens. Lac repressor alone, although tetrameric in structure (12), is less than one half the size of repressor-galactosidase (4, 5, 13). Thus, the size and shape of the repressor-galactosidase complex,
Biochemistry: a
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Proc. Nati. Acad. Sci. USA 73 (1976)
Kania and Brown d e
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FIG. 2. NaDodSO4/gel electrophoresis of crosslinked lac repressor and crosslinked repressor-galactosidase. The positions of monomers (mono), dimers (di), trimers (tri), and tetramers (tetra) of the protein subunit are indicated by arrows. (A) lac repressor crosslinked with dimethyl suberimidate and with I2/K1 (a) untreated; (b) treated with chymotrypsin before incubation with dimethyl suberimidate; (c) crosslinked with dimethyl suberimidate; (d) same as in (c) except that repressor-galactosidase was added as marker; (e) untreated lac repressor, bovine serum albumin, and repressor-galactosidase added as marker proteins; (f) crosslinked with iodine. (B) Repressor-galactosidase crosslinked with dimethyl suberimidate (a) untreated; (b) crosslinked with dimethyl suberimidate; (c) same as in (b) except that myosin was added as marker; (d) untreated repressor-galactosidase and myosin. (C) Repressor-galactosidase crosslinked with I2/K1 (a) untreated; (b) crosslinked with iodine; (c) same as in (b) except that myosin was added. (D) lac repressor crosslinked with 12/KI after limited proteolysis (a) untreated; (b) untreated core after incubation with chymotrypsin; (c) same as in (b), treated with iodine; (d) untreated after incubation with trypsin and chymotrypsin; (e) same as in (d), treated with iodine; (f) uncleaved lac repressor after treatment with iodine; (g) untreated lac repressor, bovine serum albumin, and repressor-galactosidase added as marker proteins. as revealed in the electron microscope, suggests that the galactosidase part of the repressor-galactosidase has the same structural organization as galactosidase alone and further implies that the glactosidase moiety of the repressor-galactosidase complex forms the structural core of the tetramer (see Fig. 3). If the smaller repressor part of the repressor-galactosidase formed the structural core of the aggregate, then one would have expected to see "dumbbell" shaped structures consisting of large dimers of the high-molecular-weight galactosidase part connected by the smaller repressor portions of the complex. Such structures were never found. Crosslinking with Diimidoesters. If lac repressor is treated with dimethyl suberimidate or dimethyl adipinimidate, several bands are found on NaDodSO4 gels which can be interpreted as monomer, dimer, two trimers, one or more tetramers, and oligomers (Fig. 2A). Repressor-galactosidase, however, if treated with the same diimidoesters in a parallel experiment, can be crosslinked only to dimers (Fig. 2B). 3-Galactosidase alone cannot be crosslinked with these diimidoesters under the conditions described (see also ref. 14). This suggests that only two lac repressor-parts, in the repressor-galactosidase aggregate, are in the configuration of operator-binding lac repressor which allows them to be crosslinked. This result would be expected if the repressor parts of the complex were peripherally located as dimers on a fl-galactosidase core which has the tetrameric organization described in the preceding section (see Fig. 3). Crosslinking in the Presence of Iodine. Iodine proved to be an excellent agent for crosslinking lac repressor, by applying the procedure described in Materials and Methods. After iodination of lac repressor, two main bands can be resolved on NaDodSO4 gels at the positions of dimers and also some minor
bands at trimer and tetramer positions (Fig. 2A and D). If flgalactosidase is treated with iodine in the same way, faint bands at dimer, trimer, and tetramer positions were visible on NaDodSO4 gels, only if the gel was heavily overloaded. However, treatment of repressor-galactosidase with iodine resulted in a strong band at the dimer position on NaDodSO4 gels (Fig. 2C). Thus, iodine as well as diimidoesters crosslink repressor-galactosidase only to dimers, whereas lac repressor is crosslinked primarily into dimers by iodine and to dimers, trimers, and tetramers by diimidoesters. Crosslinking of lac Repressor after Limited Proteolysis. Platt et al. have shown that if lac repressor is incubated with trypsin and chymotrypsin under native conditions, peptides are released only from the termini of the molecule thus leaving an unnicked, aggregated core protein (15). We applied this technique to distinguish between the crosslinking sites of dimethyl suberimidate and I2/KI. After treatment of lac repressor with chymotrypsin (3% wt/wt, 1 hr at 370), no crosslinking could be achieved by dimethyl suberimidate within the chymotryptic core (Fig. 2A). This implies that the crosslinking sites (lysine residues) are located at the termini of the polypeptide chain of lac repressor. Excessive treatment of lac repressor with chymotrypsin and a mixture of trypsin and chymotrypsin (each 2% wt/wt, 30 min at 370, and then by incubation at 40 for 12 hr) did not result in an abolition of crosslinking by iodine (Fig. 2D). Gel (c) of Fig. 2 reveals that after digestion the iodinated sample consisted of a mixture of polypeptide chains of different lengths, due to the prolonged incubation with the protease. The upper band presumably consists of dimers which lack the NH2-termini; the crosslinked material from the lower band should be free of both the NH2- and COOH-termini. Since trypsin si-
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Biochemistry: Kania and Brown
multaneously acts at both termini of the lac repressor, a mixture of trypsin and chymotrypsin leads to the double-band pattern found on gels (d) and (e). Precise molecular weight determinations of the iodinated material are difficult from gels (c) and (e) since iodinated, but not crosslinked polypeptides, seem to migrate faster than iodinated, while the uncleaved, uncrosslinked but iodinated subunit shows the same mobility as noniodinated lac repressor [gel (f)]. From these results we conclude that iodine catalyzes the crosslinking of the protease resistant core fragments of lac repressor at or near their aggregation sites.
DISCUSSION Protein chemical investigations have revealed that no more than five amino acids are missing at the COOH-terminus of lac repressor in repressor-galactosidase, because the second to last tryptic peptide can be identified after limited tryptic digestion of the protein (16). Because the subunit is calculated to be about 155,000 daltons (7) (with RNA polymerase as a standard on NaDodSO4 gels), about 70 amino acids are missing from the f3-galactosidase portion. Because the increase in the molecular mass which results from the attachment of lac repressor does not result in a faster sedimentation relative to f3-galactosidase, we propose that the structure of repressor-galactosidase is ellipsoidal rather than spherical. By electron microscopy, we were able to establish the active repressor-galactosidase to be a tetramer with morphology and size similar to 03-galactosidase alone. #-Galactosidase and lac repressor are also both stable tetramers (12, 17). Langley et al. have shown that defective galactosidase protein from the deletion mutant strain M15 of E. coli, which lacks residues 11-41 of intact ,B-galactosidase, is a dimer under native conditions (18). This observation excludes C4 symmetry for the ,B-galactosidase tetramer, since in the case of such a symmetrical arrangement, dissociation by mutation would lead exclusively to monomers [see review by Matthews and Bernhard (19)]. This must also be true for lac repressor, because substitutions of amino acids in the presumed aggregation sites of lac repressor lead to dissociation of the repressor tetramer into a mixture of dimers and V monomers, as found recently by Schmitz et al. (20). Our results, obtained by iodine-catalysed crosslinking of lac repressor, support this assumption. Actually, this is not surprising since all other tetrameric proteins of known structure are of D2 symmetry (19). Therefore, in the repressor-galactosidase tetramer, lac repressor parts cannot be aggregated into a tetramer if f,-galactosidase parts are tetrameric with D2 symmetry and vice versa. Consequently, one of the two protein-parts must retain its biological activity in the repressor-galactosidase tetramer in the state of a dimer. The crosslinking patterns of lac repressor and repressor-galactosidase produced by diimidoesters and iodine in this study support the hypothesis that it is the lac repressor portion of the repressor-galactosidase complex which is organized into dimers. As mentioned above, Schmitz et al. have found mutations in the repressor gene which result in the dissociation of lac repressor into dimers and monomers which are no longer capable of operator binding (20). Such mutations map in two regions, one around tryptophan 209 and phenylalanine 215, the other one around tyrosine 260 and tyrosine 269 in the repressor sequence. It is at these positions that Schmitz et al. propose subunit-aggregation sites. It is attractive to propose that these particular aromatic amino acids are involved in the iodinecatalyzed crosslinking reaction. Crosslinking of repressor-galactosidase catalyzed by iodine revealed that the aggregation sites necessary for operator-binding (as proposed by Schmitz
Proc. Natl. Acad. Sci. USA 73 (1976) lac Repressor
Repressor-Ga I actosidase FIG. 3. Schematic drawing of the proposed model of repressorgalactosidase organization. The fl-galactosidase-parts are aggregated as tetramers in a D2 symmetry as in the wild type molecule. Each of those two fl-galactosidase parts, which have point symmetry to the x-axis, carry the repressor-parts which, as shown by the crosslinking data, must be aggregated as dimers. The native lac repressor tetramer is also aggregated in a D2 symmetry; this is schematically indicated in the above drawing. By comparing the corresponding dimers in these two models, it becomes evident that the DNA binding site in the repressor tetramere is made by those two subunits, which have point symmetry to the x-axis. Consequently, the NH2-terminus of the lac repressor polypeptide chain, which contains the DNA-binding site, must be located at the free narrow-end of each subunit in the above model.
et al.) are present in repressor-galactosidase. This implies that the two repressor parts are properly aggregated for operator
binding. Our conclusions, from this study, are presented in model form in Fig. 3 and may be summarized as follows: the fl-galactosidase parts in repressor-galactosidase are aggregated in a D2 symmetry. Each of those f3-galactosidase parts, which are similarly oriented, carry at their NH2-termini lac repressor-parts which, aggregated as dimers, are able to bind to lac operator DNA (7). Similar conclusions have been made based on other types of evidence by Geisler and Weber (21) and by J. Miwa and J. R. Sadler (personal communication). This model supports the implication of the model of Steitzet al. that lac repressor dimers are capable of recognizing the lac operator and is also consistent with some aspects of the model suggested by Adler et al. (22), who proposed that the DNA binding site is situated on a protrusion at the NH2-terminus of the polypeptide chain. The model presented here predicts as yet undiscovered point mutations in the i gene of lac repressor, in which the substitution of an amino acid in an aggregation site different from those aggregation sites described by Schmitz et al. would lead to a dissociation of the lac repressor tetramer into stable dimers exhibiting all biologicl activities of native lac repressor. Since repressor-galactosidase does not aggregate through its repressor-parts into long chains, one might conclude the COOH-terminus of lac repressor is involved in the aggregation of repressor dimers to tetramers. This is supported by the fact that Ll-lac repressor, in which the COOH-terminus of lac repressor is missing, is still able to repress at low levels in vow and forms exclusively dimers [Miller et al. (23)]. This third predicted aggregation site could be the hydrophobic cluster existing around proline 307.
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Kania, and Brown
We thank B. Muller-Hill for his stimulating interest and helpful discussion throughout this work. Thanks are also due to T. G. Fanning and K. Beyreuther for advice and suggestions and for providing purified lac repressor; and to K. Weber for providing myosin. This investigation was supported by Deutsche Forschungsgemeinschaft through SFB 74 by grants to B.M.-H. and D.T.B. 1. Muller-Hill, B. (1975) Prog. Biophys. Mol. Biol. 30,227-252. 2. Beyreuther, K., Adler, K., Geisler, N. & Klemm. A. (1973) Proc. Nati. Acad. Sci. USA 70,3576-580. 3. Gilbert, W. & Maxam, A. (1973) Proc. Nati. Acad. Sci. USA 70, 3581-584. 4. Oshima, Y., Horiuchi, T. & Yanagida, M. (1975) J. Mol. Biol. 91, 515-519. 5. Abermann, R., Bahl, C. P., Marians, K. J., Salpeter, M. M. & Wu, R. (1976) J. Mol. Biol. 100, 109-114. 6. Steitz, T. A., Richmond, T. J., Wise, D. & Engelman, D. (1974) Proc. Natl. Acad. Sci. USA 71,593-597. 7. Muller-Hill, B. & Kania, J. (1974) Nature 249,561-563. 8. Davies, G. E. & Stark, G. R. (1970) Proc. Natl. Acad. Sci. USA 66,651-656. 9. Fanning, T. G. (1975) Biochemistry 14,2512-2520. 10. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 44064412. 11. Zabin, I. (1963) Cold Spring Harbor Symp. Quant. Biol. 28, 431-435. 12. Muller-Hill, B., Beyreuther, K. & Gilbert, W. (1971) in Methods in Enzymology, eds. Grossman, L. & Moldave, K. (Academic
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Press, New York), Vol. 21, pp. 483-487. 13. Bourgeois, S. & Pfahl, M. (1976) Advances in Protein Chemistry eds. Anfinsen, C. B., Edsall, J. T. & Richards, F. M. (Academic Press, New York), Vol. 30, pp. 1-99. 14. Mfillner, H., Hucho, F. & Sund, H. (1975) Hoppe-Seylers Z. Physiol. Chem. 356, 256. 15. Platt, T., Files, J. G. & Weber, K. (1973) J. Blol. Chem. 248, 110-121. 16. Kania, J., Ruth, C. & Mfiller-Hill, B. (1975) Hoppe-Seylers Z. Physlol. Chem. 356,243. 17. Craven, G., Steers, E. & Anfinsen, C. B. (1965) J. Blol. Chem. 240, 2468-2477. 18. Langley, K. E., Villarejo, M. R., Fowler, A. V., Zamenhof, P. J. & Zabin, I. (1975) Proc. Natl. Acad. Scd. USA 72, 1254-1257. 19. Matthews, B. W. & Bernhard, S. A. (1973) "Structure and symmetry of oligomeric enzymes," in Annual Review of Biophysics and Bioengineering, eds. Mullins, L. J., Hagins, W. A. & Stryer, L. (Annual Reviews Inc., Palo Alto, Calif.), Vol. 2. 20. Schmitz, A., Schmeissner, U., Miller, J. H. & Lu, P. (1976) J. Biol. Chem. 251,3359-3366. 21. Geisler, N. & Weber, K. (1976) Proc. Nati. Acad. Sci. USA 73, 3103-3106. 22. Adler, K., Beyreuther, K., Fanning; E., Geisler, N., Gronenborn, B., Klemm, A., Mfiller-Hill, B., Pfahl, M. & Schmitz, A. (1972) Nature 237,322-327. 23. Miller, J. H., Platt, T. & Weber, K. (1970) in The Lactose Operon, eds. Beckwith, J. R. & Zipser, D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), pp. 343-351.
