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Crystallization of and preliminary X-ray diffraction data for TET-repressor and the TET-repressor-tetracycline complex
J. Mol. Biol. (1984) 180, 1189-1191
Crystallization of and Preliminary X-ray Diffraction Data for TET-repressor and the TET-repressor-Tetracycline Complex The TET-repressor encoded by the transposon TnlO has been crystallized along with the repressor-tetracycline complex. Both crystals belong to the space group P4,2,2 (or P4,2,2) with cell dimensions a = b = 74.3(l) A. c = 94.2(2) A and a = b = 73,3(l) A, c = 94.6(2) A for the free and complexed repressor, respectively. There is one molecule of molecular weight 23,666 per asymmetric unit. and the biologically active dimer therefore consists of two identically formed subunits which are related by a crystallographic 2-fold axis. This isomorphism of TET-repressor and its tetracycline complex suggests that only minor. subtle changes in structure trigger binding to or release of the operator. The crystals of the native protein permit X-ray data collection to 3.2 A and those of the complexed repressor to 2.8 A. The central role of protein-nucleic acid interactions in molecular biology is witnessed by the amount of research focused on structural studies on proteins that recognize specific sequences in double-stranded DNA and their complexes with segments of DNA. The structures of three such proteins which are involved in gene expression have been determined: the cro repressor, the N-terminal DNA binding domain of lambda repressor, and the catabolite gene activator protein (CAP) (Anderson et aE., 1981; Pabo & Lewis, 1982; McKay & Steitz, 1981). Furthermore, preliminary data have appeared on protein-DNA complexes (Anderson et al., 1983,1984) and recently the first structure of DNA in intimate association with the sequence-specific endonuclease EcoRI has been published (Frederick et al., 1984). The TET-repressor, encoded by the transposon TnlO regulates the expression of the tetracycline resistance determinant of this genetic element. It functions as a dimer in solution with molecular weight 2 x 23,000 and binds to the operator with an apparent association constant of 10” to 1013 M- ‘. An association constant for the inducer tetracycline cannot be measured but its binding is stronger than that of the operator (Hillen et al., 1984). The TET-repressor binds operator only in the absence of tetracycline. Upon addition of tetracycline the drug is bound to the repressor and causes the release of the operator. Tn order to investigate the structural features associated with the binding of TET-repressor to operator and to tetracycline, we have crystallized the free TETrepressor and its complex with tetracycline. Crystallization of the free repressor was effected at room temperature by vapour diffusion of 1% (w/v) protein/buffer A (buffer A: 50 mM-Tris . HCl. 50 mMNa(!l, 7 mM-B-mercaptoethanol, 1 mM-EDTA, 0.02% (w/v) NaN,, pH 8.0) against, 1.3 to 1.5 M-diammonium hydrogen phosphate in buffer A (pH 7.0). Crystals of the TET-repressor-tetracycline complex were obtained by co-crystallizing the protein with freshly prepared tetracycline solutions (5 to 10 mM) in the 1189 0022-2836/84/361189-03
$03.00/O
0
1984
Academic
Press
Inc.
(London)
Lt.d.
1 190
H. E. PARGE
ET
AL
precipitating agent at pH values between 7-O and 7.4. X-ray precession photography shows both crystals to be tetragonal and the systematic absences identify the space group as P432,2 (or P4,2,2) with cell dimensions a = 74.3( 1) A, c = 94.2(2) A and a = 73.3(l) A, c = 94.6(2) A for the free and complexed repressor, respectively. The density of 1.27(2) g cme3 (measured in a hexane/ chloroform gradient) yields the solvent content in the repressor crystal as 54% assuming one protein monomer per asymmetric unit. This is in agreement with a l’, value of 2.82 A3/dalton (Matthews, 1968). In accord with the results on cro, lambda, CAP and the tryptophan trp-repressor (Joachimiak et al, 1983) the biologically active form of the TET-repressor is a dimer to match, on a structural basis, the 2-fold symmetry inherent in the DNA double helix. Thus, the arrangement of the protein monomers in the unit cell is presumably about a crystallographic 2-fold axis. The binding of tetracycline to the repressor does not destroy this symmetry as evidenced by the isomorphism between the two crystal forms. Moreover, we can infer from the isomorphism and from the small changes in diffraction pattern that occur upon inducer binding, that the repressor does not undergo major conformational changes nor does the protein surface change enough to influence the packing in the crystal. Attempts to determine the precise stoichiometry of tetracycline-TET-repressor in the complex crystals by spectrophotometric methods yielded a ratio of 20 : 1, suggesting that binding of inducer also occurs non-specifically at the protein surface. These observations are in stark contrast when compared with, for instance, the conformational change detected in the CAP protein upon binding of the effector, cyclic-AMP, in solution (Eilen & Krakow, 1977; Wu et al., 1976; Wu & Wu, 1974), and the recent observation that a conformational change in the EcoRI endonuclease is necessary to allow the DNA to approach the active site of the enzyme (Frederick et al., 1984). Significant differences are also apparent if we compare the TET and trp repressors. First, TET-repressor binds to DNA in its uncomplexed form and releases DNA upon binding of tetracycline, whereas the trp-repressor binds DNA only if complexed with tryptophan. Second, both the free and tetracycline complexed species could be crystallized in an isomorphous form suitable for X-ray diffraction studies. As for the trp-repressor, only the tryptophan complex has been crystallized thus far in a manageable size, whereas the native aporepressor gives only very small, probably non-isomorphous crystals unsuitable for X-ray characterization. This suggests, that in the trp-repressor, the aporepressor and the tryptophan complexed form differ to a much greater extent than do the free and tetracycline complexed TET-repressor.. A complete X-ray crystallographic analysis of the native, tetracycline and operator complexed repressor is necessary to elucidate these subtle protein. Heavy-atom “conformational changes” in the TET-repressor derivatization and co-crystallization experiments with a 13 base-pair oligonucleotide fragment of the operator are in progress. One of us (H.E.P.) would like to acknowledge financial support from the European Molecular Biology Organization. L.A. is supported by a predoctoral training grant from the Fonds der Chemischen Industrie. This work profited from a DFG grant in the frame of
LETTERS
Schwerpunktprogramm Dynamik der zellularen
“Biophysik Bausteine”
Institut fur Kristallographie Freie UniversitBt Berlin Takustr. 6. D-1000 Berlin
TO
THE
der Organisation der Zelle-Wechselwirkungen (to W.S. and W.H.).
und
H. E. PARGE M. SCHNEIDER 33, F.D.R.
C. HAHN W. SAENGER
Institut fur Organische Chemie und Biochemie der Technischen Hochschule Petersenstr. 22, D-6100 Darmstadt, F.D.R. Received 3 September
1191
EDITOR
L. ALTSCHMIED IV.
HILLEN
1984
REFERENCES J., Ptashne, M. & Harrison, S. C. (1984). Proc. Nat. Acad. Sci., U.S.A. 81. 1307-
Anderson, 1311. Anderson, W. F., Ohlendorf, D. H., Takeda, Y. & Matthews. B. W. (1981). X&m (London), 290. 754-758. Anderson, W. F., Cygler, M., Vandonselaar, M., Ohlendorf, D. H.. Matthews, B. W.. Kim, J. & Takeda, Y. (1983). J. MOE. Viol. 168, 9033906. Eilen, E. & Krakow, J. S., (1977). J. Mol. Biol. 114, 47-60. Frederick. C. A., Grable, J., Melia, M., Samudzi, C., Jen-Jacobson, L., Wang, B.C., Green, P., Boyer, H. W. & Rosenberg, J. M. (1984). Nature (London), 309> 327-331. Hillen, W., Schollmeier, K. C Gatz, C. (1984). J. Mol. Biol. 172, 185-201.
by A. Klug
Crystallization of and Preliminary X-ray Diffraction Data for TET-repressor and the TET-repressor-Tetracycline Complex The TET-repressor encoded by the transposon TnlO has been crystallized along with the repressor-tetracycline complex. Both crystals belong to the space group P4,2,2 (or P4,2,2) with cell dimensions a = b = 74.3(l) A. c = 94.2(2) A and a = b = 73,3(l) A, c = 94.6(2) A for the free and complexed repressor, respectively. There is one molecule of molecular weight 23,666 per asymmetric unit. and the biologically active dimer therefore consists of two identically formed subunits which are related by a crystallographic 2-fold axis. This isomorphism of TET-repressor and its tetracycline complex suggests that only minor. subtle changes in structure trigger binding to or release of the operator. The crystals of the native protein permit X-ray data collection to 3.2 A and those of the complexed repressor to 2.8 A. The central role of protein-nucleic acid interactions in molecular biology is witnessed by the amount of research focused on structural studies on proteins that recognize specific sequences in double-stranded DNA and their complexes with segments of DNA. The structures of three such proteins which are involved in gene expression have been determined: the cro repressor, the N-terminal DNA binding domain of lambda repressor, and the catabolite gene activator protein (CAP) (Anderson et aE., 1981; Pabo & Lewis, 1982; McKay & Steitz, 1981). Furthermore, preliminary data have appeared on protein-DNA complexes (Anderson et al., 1983,1984) and recently the first structure of DNA in intimate association with the sequence-specific endonuclease EcoRI has been published (Frederick et al., 1984). The TET-repressor, encoded by the transposon TnlO regulates the expression of the tetracycline resistance determinant of this genetic element. It functions as a dimer in solution with molecular weight 2 x 23,000 and binds to the operator with an apparent association constant of 10” to 1013 M- ‘. An association constant for the inducer tetracycline cannot be measured but its binding is stronger than that of the operator (Hillen et al., 1984). The TET-repressor binds operator only in the absence of tetracycline. Upon addition of tetracycline the drug is bound to the repressor and causes the release of the operator. Tn order to investigate the structural features associated with the binding of TET-repressor to operator and to tetracycline, we have crystallized the free TETrepressor and its complex with tetracycline. Crystallization of the free repressor was effected at room temperature by vapour diffusion of 1% (w/v) protein/buffer A (buffer A: 50 mM-Tris . HCl. 50 mMNa(!l, 7 mM-B-mercaptoethanol, 1 mM-EDTA, 0.02% (w/v) NaN,, pH 8.0) against, 1.3 to 1.5 M-diammonium hydrogen phosphate in buffer A (pH 7.0). Crystals of the TET-repressor-tetracycline complex were obtained by co-crystallizing the protein with freshly prepared tetracycline solutions (5 to 10 mM) in the 1189 0022-2836/84/361189-03
$03.00/O
0
1984
Academic
Press
Inc.
(London)
Lt.d.
1 190
H. E. PARGE
ET
AL
precipitating agent at pH values between 7-O and 7.4. X-ray precession photography shows both crystals to be tetragonal and the systematic absences identify the space group as P432,2 (or P4,2,2) with cell dimensions a = 74.3( 1) A, c = 94.2(2) A and a = 73.3(l) A, c = 94.6(2) A for the free and complexed repressor, respectively. The density of 1.27(2) g cme3 (measured in a hexane/ chloroform gradient) yields the solvent content in the repressor crystal as 54% assuming one protein monomer per asymmetric unit. This is in agreement with a l’, value of 2.82 A3/dalton (Matthews, 1968). In accord with the results on cro, lambda, CAP and the tryptophan trp-repressor (Joachimiak et al, 1983) the biologically active form of the TET-repressor is a dimer to match, on a structural basis, the 2-fold symmetry inherent in the DNA double helix. Thus, the arrangement of the protein monomers in the unit cell is presumably about a crystallographic 2-fold axis. The binding of tetracycline to the repressor does not destroy this symmetry as evidenced by the isomorphism between the two crystal forms. Moreover, we can infer from the isomorphism and from the small changes in diffraction pattern that occur upon inducer binding, that the repressor does not undergo major conformational changes nor does the protein surface change enough to influence the packing in the crystal. Attempts to determine the precise stoichiometry of tetracycline-TET-repressor in the complex crystals by spectrophotometric methods yielded a ratio of 20 : 1, suggesting that binding of inducer also occurs non-specifically at the protein surface. These observations are in stark contrast when compared with, for instance, the conformational change detected in the CAP protein upon binding of the effector, cyclic-AMP, in solution (Eilen & Krakow, 1977; Wu et al., 1976; Wu & Wu, 1974), and the recent observation that a conformational change in the EcoRI endonuclease is necessary to allow the DNA to approach the active site of the enzyme (Frederick et al., 1984). Significant differences are also apparent if we compare the TET and trp repressors. First, TET-repressor binds to DNA in its uncomplexed form and releases DNA upon binding of tetracycline, whereas the trp-repressor binds DNA only if complexed with tryptophan. Second, both the free and tetracycline complexed species could be crystallized in an isomorphous form suitable for X-ray diffraction studies. As for the trp-repressor, only the tryptophan complex has been crystallized thus far in a manageable size, whereas the native aporepressor gives only very small, probably non-isomorphous crystals unsuitable for X-ray characterization. This suggests, that in the trp-repressor, the aporepressor and the tryptophan complexed form differ to a much greater extent than do the free and tetracycline complexed TET-repressor.. A complete X-ray crystallographic analysis of the native, tetracycline and operator complexed repressor is necessary to elucidate these subtle protein. Heavy-atom “conformational changes” in the TET-repressor derivatization and co-crystallization experiments with a 13 base-pair oligonucleotide fragment of the operator are in progress. One of us (H.E.P.) would like to acknowledge financial support from the European Molecular Biology Organization. L.A. is supported by a predoctoral training grant from the Fonds der Chemischen Industrie. This work profited from a DFG grant in the frame of
LETTERS
Schwerpunktprogramm Dynamik der zellularen
“Biophysik Bausteine”
Institut fur Kristallographie Freie UniversitBt Berlin Takustr. 6. D-1000 Berlin
TO
THE
der Organisation der Zelle-Wechselwirkungen (to W.S. and W.H.).
und
H. E. PARGE M. SCHNEIDER 33, F.D.R.
C. HAHN W. SAENGER
Institut fur Organische Chemie und Biochemie der Technischen Hochschule Petersenstr. 22, D-6100 Darmstadt, F.D.R. Received 3 September
1191
EDITOR
L. ALTSCHMIED IV.
HILLEN
1984
REFERENCES J., Ptashne, M. & Harrison, S. C. (1984). Proc. Nat. Acad. Sci., U.S.A. 81. 1307-
Anderson, 1311. Anderson, W. F., Ohlendorf, D. H., Takeda, Y. & Matthews. B. W. (1981). X&m (London), 290. 754-758. Anderson, W. F., Cygler, M., Vandonselaar, M., Ohlendorf, D. H.. Matthews, B. W.. Kim, J. & Takeda, Y. (1983). J. MOE. Viol. 168, 9033906. Eilen, E. & Krakow, J. S., (1977). J. Mol. Biol. 114, 47-60. Frederick. C. A., Grable, J., Melia, M., Samudzi, C., Jen-Jacobson, L., Wang, B.C., Green, P., Boyer, H. W. & Rosenberg, J. M. (1984). Nature (London), 309> 327-331. Hillen, W., Schollmeier, K. C Gatz, C. (1984). J. Mol. Biol. 172, 185-201.
by A. Klug