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A high resolution diffracting crystal form of the complex between yeast tRNAAsp and aspartyl-tRNA synthetase
J. Mol. Biol. (1988)201, 235-236
A High Resolution Diffracting Crystal Form of the Complex Between Yeast tRNAAsp and Aspartyl-tRNA Synthetase Three new crystal forms of the complex between yeast tRNA*“p and aspartyl-tRNA synthetase have been produced. The best crystals, obtained after modifying both purification and crystallization conditions, belong to space group P212, 2, and diffract to 2.7 A. Unit cell parameters are a = 210.4 A, b = 145.3 A and c = 86.0 A (1 A = 0.1 nm), with one dimeric enzyme and two tRNA molecules in the asymmetric unit.
The aminoacylation of tRNAs, a key reaction in protein biosynthesis, is mainly responsible for the fidelity of translation of genetic information. The structure of a complex formed between aspartyltRNA synthetase from yeast and two molecules of its cognate yeast tRNA has recently been solved to a resolution of8 A(1 A = 0.1 nm) from crystals belonging to a cubic space group (see Table 1 and Podjarny et al., 1987). It provides the first information on global geometry of the interacting tRNAs with the synthetase molecule. However, it also clearly shows the necessity of obtaining data to atomic resolution in order to understand, at the molecular level, the basic mechanisms of the aminoacylation reaction including RNA and protein recognition. So far, five structures of distinct tRNAs are known, two of them to atomic resolution (Quigley et al., 1975; Jack et al., 1976; Sussman et al., 1978; Stout et al., 1978; Moras et al., 1980; Westhof et al., 1985 for the high resolution forms; and Schevitz et al., 1979; Wright et at., 1979; Woo et al., 1980 for the other forms), as well as that of one synthetase and one of its deletion mutant (Bhat et al., 1982; Bhat & Blow, 1983; Brick BEBlow, 1987) and of one tryptic fragment. (Risler et al., 1981; Brunie et al., 1987), which X-ray structures are known to a resolution better than 3 A. For a long time, cocrystallization experiments of tRNAs and synthetases have led to crystals diffracting only to low or medium resolution (GiegB et al., 1980, 1986; Lorber et aZ., 1983). We report here the first results on co-crystals
formed between yeast tRNAASp and its cognate aminoacyl-tRNA synthetase, diffracting to high resolution: X-ray pictures containing information to 2.7 A can currently be collected using a synchrotron radiation source. tRNAAsp (1M,= 24,160) was prepared from bulk brewer’s yeast tRNA as described for the crystallization of tRNAASp (GiegB et al., 1977). Stock solutions for crystallization experiments were prepared in 2 m&%-sodiumcacodylate buffer (pH 6.0) with 2 mM-MgCl,. Modifying the purification protocol of a protein is one of the classical approach in order to obtain new crystal forms diffracting to higher resolution. A new purification strategy has therefore been especially developed for crystallization purposes. Aspartylsynthetase (iM,= 125,000) from tRNA Saccharomycescerevisiae (baker’s yeast) was purified to homogeneity in three steps: (1) a polyethylene glycol 6000 partition; (2) a nucleic acid precipitation using protamine sulphate; and (3) an hydroxyapatite ULTROGEL column chromatography (a detailed description will be published elsewhere). The purification is carried out in three days, starting from 650 g of wild-type yeast cells, and reduces the important N-terminal proteolysis observed in the previous purification method (Lorber et al., 1987). About 15 to 20 mg of pure enzyme (with a yield of over 40%) can be produced. This enzyme exhibits an activity of 800 units/mg (1 unit, yields 1 nmol of aspartyl-tRNAASP/min at 37 “C under standard conditions).
Table 1 Crystallization Crystal system
Space group
Cubic Orthorhombic Orthorhombic Orthorhombie 2 is the number S, (oh) the solvent of 0.7 crt?/g. OOZZ-2836/88/090235-Z
I432 (I) (II) (III)
p2,2,21 P2,2,% p21212,
a(A)
Unit cell dimensions b(A) cm
354-o 225.7 222.4 210.4
354.0 211.7 133.2 145.3
of complex molecules (1 dimeric aspartyl-tRNA content; and V, the average crystal volume
$03.00/O
data
354.0 140.4 97.2 86.0
V(A3) 4.44 6.71 2.88 2.63
x x x x
10’ lo6 lo6 lo6
Resolution (A)
2
S,
V,
7 4 3.5 2.7
1 2 1 1
78 76 71 69
5.33 4.83 4.15 3.79
synthetaae molecule and 2 tRNAA’P molecules per asymmetric per unit molecular weight (&dalton), assuming a partial specific
235
0
1988 Academic
unit); volume
Press Limited
M. Ruff et al.
236
A systematic search for new crystallization conditions was undertaken using enzyme produced by the new purification method. Crystals were obtained using vapour diffusion techniques. Hanging drops, including 40 mbr-Trismaleate/NaOH (pH 7*5), 5 mw-MgCl,, 25% (w/v) ammonium sulphate, aspartyl-tRNA synthetase at 10 mg/ml (80 mnr), and tRNAA’r at 4.8 mglml (190 mM, corresponding to a stoichiometry of 2.4 molecules of tRNA for 1 molecule of enzyme), are set to crystallize in the cold (4%) against 60% (w/v) ammonium sulphate. The best crystals were obtained at pH 7.5, while urchins or spherolites tend to appear when lowering the pH to 6.0. Large plates (0.2 mm x O-4 mm x 1.0 mm) are formed within a few weeks, or in about one week using seeding techniques. Crystals grown at pH 7.5 from enzyme obtained using the previous preparation method and crystallization conditions similar to the one used for the cubic crystal form, have different cell parameters and diffract to 3.5 A (orthorhombic forms I and II; Table 1). The new crystals (form III; Table 1) belong to space group P2,2,2, and contain one synthetase and two tRNA molecules per asymmetric unit leading to 3.79 A3/dalton, a value slightly outside the standard range for proteins (1.68 to 3.6 Aldalton), but under that observed for the previously obtained crystal forms of the complex. This is well in agreement with the ability of the new crystals, not only to diffract to higher resolution but also to be less sensitive to X-ray damage, as a consequence of a lower content of solvent. Crystallographic data have been collected using synchrotron radiation at CHESS and LURE. A search for heavy-atom derivatives is under way. At present, crystals diffracting to high resolution (at least 3 A or higher) have been produced for both tRNA (Giege et al., 1977) and synthetase (Dietrich et al., 1980), molecules in the specific system of the aspartic acid from yeast and now for their complex, building a unique situation from which structures from molecules complexed and in their native state will be solved providing the first details at an atomic
level
on such a system
of the biosynthetic
pathway. During completion of this work another high resolution crystal form was obtained for the glutaminyl system from Escherichia coli; in that case crystals diffracting to 2.8 A resolution were grown with the monomeric synthetase interacting with one molecule of tRNA2’” (Perona et al., 1987). We are most indebted to C. Gaget, A. Garcia, V. Perret and A. Theobald for purification of tRNAAsP from counter current fractions provided by G. Dirheimer and M. Schlegel. We thank J. P. Samama for fruitful discussions about crystallization and Professor J. P. Ebel for his constant interest and support of this work.
M. Ruff J. Cavarelli V. Mikol B. Lorber
A. Mitschler R. Giege J. C. Thierry D. Moras Edited
Institut de Biologie Moleculaire et Cellulaire du CNRS 15 rue R. Descartes, 67084 Strasbourg-Cedex, Received 10 September
France
1987
References Bhat, T. N., & Blow, D. M. (1983). Actu Crystallogr. sect. A, 39, 166-170. Bhat, T. N., Blow, D. M., Brick, P. & Nyborg, J. (1982). J. Mol. Biol. 158, 699709. Brick, P. & Blow, D. M. (1987). J. Mol. Biol. 194, 287297. Brunie, S., Mellot, P., Zelwer, C., Risler, J. L., Blanquet, S. & Fayat, G. (1987). J. MOE. Graph. 5 1821. Dietrich, A., Giege, R., Comarmond, M. B., Thierry, J. C. & Moras, D. (1980). J. Mot. BioE. 138, 124-135. Giege, R., Moras, D. & Thierry, J. C. (1977). J. MOE. Biol. 115, 91-96. Giege, R., Lorber, B., Ebel, J. P., Thierry, J. C. & Moms, D. (1980). C.R. S&wee Acad. Sci (Parie), eer. D,
291, 393-396. Giege, R., Dock, A. C., Kern, D., Lo&r, B., Thierry, J. C. & Moras, D. (1986). J. Crystal Growth, 76, 554561. Jack, A., Ladner, J. E. & Klug, A. (1976). J. Mol. Biol. 108, 619649. Lorber, B., Giege, R., Ebel, J. P., Berthet, C., Thierry, J. C. & Moras, D. (1983). J. BioZ. Chem. 258, 84298435. Lorber, B., Kern, D., Mejdoub, H., Boulanger, Y., Reinbold, J. & Giege, R. (1987). Eur. J. Biochem.
165, 4@%417. Moras, D., Comarmond, M. B., Fischer, J., Weiss, R., Thierry, J. C., Ebel, J. P. & Giege, R. (1980). Nature (London), 288, 66%674. Perona, J., Steitz, T. A. $ SolI, D. (1987). Abstract of the Second In.!ernationul Cry&al Growth Meeting, B&her&erg, Strasbourg, France (abstract no. P2-17). Podjarny, A., Rees, B., Thierry, J. C., Cavarelli, J., Jesior, J. C., Roth, M., Lewitt-Bentley, A., Kahn, R., Lorber, B., Ebel, J. P., Giege, R. & Moras, D. (1987). J. Biumol. Struct. Dynam. 5, 187-198. Quigley, G. J., Wang, A., Seeman, N. C., Suddath, F. L.. Rich, A., Sussman, J. L. & Kim, S. H. (1975). Proc. Nat. Acad. Sci., U.S. A. 72, 486&4870. Risler, J. L., Zelwer, C. & Brunie, S. (1981). Nature (London), 292, 384-386. Schevitz, R. W., Podjarny, A. D., Krishnamachar, N., Hugues, J. J., Siegler, P. & Sussman, J. L. (1979). Nature (London), 278, 188190. Sussman, J. L., Holbrook, S. R., Warrant, R. W., Church, G. M. BEKim, S. H. (1978). J. MOE. BioZ. 123, 607-630. Stout, C. D., Mizuno, M., Rao, S. T., Swaminathan, P., Rubin, J., Brennan, T. & Sundaralingam, M. (1978). Actu Crystallogr. sect. B, 34, 15291540. Westhof, E., Dumas, P. & Moras, D. (1985). J. Mol. Biol. 184, 119145. Woo, N., Roe, B. & Rich, A. (1980). Nature (London), 286, 346-351. Wright, A. T., Manor, T. C., Beurling, K., Karpel, R. L. & Fresco, J. L. (1979). In Transfer RNA, Structure, Function and Recognition Cold Spring Harbor Monograph Series 9A, pp. 145-160, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
by R. Huber
A High Resolution Diffracting Crystal Form of the Complex Between Yeast tRNAAsp and Aspartyl-tRNA Synthetase Three new crystal forms of the complex between yeast tRNA*“p and aspartyl-tRNA synthetase have been produced. The best crystals, obtained after modifying both purification and crystallization conditions, belong to space group P212, 2, and diffract to 2.7 A. Unit cell parameters are a = 210.4 A, b = 145.3 A and c = 86.0 A (1 A = 0.1 nm), with one dimeric enzyme and two tRNA molecules in the asymmetric unit.
The aminoacylation of tRNAs, a key reaction in protein biosynthesis, is mainly responsible for the fidelity of translation of genetic information. The structure of a complex formed between aspartyltRNA synthetase from yeast and two molecules of its cognate yeast tRNA has recently been solved to a resolution of8 A(1 A = 0.1 nm) from crystals belonging to a cubic space group (see Table 1 and Podjarny et al., 1987). It provides the first information on global geometry of the interacting tRNAs with the synthetase molecule. However, it also clearly shows the necessity of obtaining data to atomic resolution in order to understand, at the molecular level, the basic mechanisms of the aminoacylation reaction including RNA and protein recognition. So far, five structures of distinct tRNAs are known, two of them to atomic resolution (Quigley et al., 1975; Jack et al., 1976; Sussman et al., 1978; Stout et al., 1978; Moras et al., 1980; Westhof et al., 1985 for the high resolution forms; and Schevitz et al., 1979; Wright et at., 1979; Woo et al., 1980 for the other forms), as well as that of one synthetase and one of its deletion mutant (Bhat et al., 1982; Bhat & Blow, 1983; Brick BEBlow, 1987) and of one tryptic fragment. (Risler et al., 1981; Brunie et al., 1987), which X-ray structures are known to a resolution better than 3 A. For a long time, cocrystallization experiments of tRNAs and synthetases have led to crystals diffracting only to low or medium resolution (GiegB et al., 1980, 1986; Lorber et aZ., 1983). We report here the first results on co-crystals
formed between yeast tRNAASp and its cognate aminoacyl-tRNA synthetase, diffracting to high resolution: X-ray pictures containing information to 2.7 A can currently be collected using a synchrotron radiation source. tRNAAsp (1M,= 24,160) was prepared from bulk brewer’s yeast tRNA as described for the crystallization of tRNAASp (GiegB et al., 1977). Stock solutions for crystallization experiments were prepared in 2 m&%-sodiumcacodylate buffer (pH 6.0) with 2 mM-MgCl,. Modifying the purification protocol of a protein is one of the classical approach in order to obtain new crystal forms diffracting to higher resolution. A new purification strategy has therefore been especially developed for crystallization purposes. Aspartylsynthetase (iM,= 125,000) from tRNA Saccharomycescerevisiae (baker’s yeast) was purified to homogeneity in three steps: (1) a polyethylene glycol 6000 partition; (2) a nucleic acid precipitation using protamine sulphate; and (3) an hydroxyapatite ULTROGEL column chromatography (a detailed description will be published elsewhere). The purification is carried out in three days, starting from 650 g of wild-type yeast cells, and reduces the important N-terminal proteolysis observed in the previous purification method (Lorber et al., 1987). About 15 to 20 mg of pure enzyme (with a yield of over 40%) can be produced. This enzyme exhibits an activity of 800 units/mg (1 unit, yields 1 nmol of aspartyl-tRNAASP/min at 37 “C under standard conditions).
Table 1 Crystallization Crystal system
Space group
Cubic Orthorhombic Orthorhombic Orthorhombie 2 is the number S, (oh) the solvent of 0.7 crt?/g. OOZZ-2836/88/090235-Z
I432 (I) (II) (III)
p2,2,21 P2,2,% p21212,
a(A)
Unit cell dimensions b(A) cm
354-o 225.7 222.4 210.4
354.0 211.7 133.2 145.3
of complex molecules (1 dimeric aspartyl-tRNA content; and V, the average crystal volume
$03.00/O
data
354.0 140.4 97.2 86.0
V(A3) 4.44 6.71 2.88 2.63
x x x x
10’ lo6 lo6 lo6
Resolution (A)
2
S,
V,
7 4 3.5 2.7
1 2 1 1
78 76 71 69
5.33 4.83 4.15 3.79
synthetaae molecule and 2 tRNAA’P molecules per asymmetric per unit molecular weight (&dalton), assuming a partial specific
235
0
1988 Academic
unit); volume
Press Limited
M. Ruff et al.
236
A systematic search for new crystallization conditions was undertaken using enzyme produced by the new purification method. Crystals were obtained using vapour diffusion techniques. Hanging drops, including 40 mbr-Trismaleate/NaOH (pH 7*5), 5 mw-MgCl,, 25% (w/v) ammonium sulphate, aspartyl-tRNA synthetase at 10 mg/ml (80 mnr), and tRNAA’r at 4.8 mglml (190 mM, corresponding to a stoichiometry of 2.4 molecules of tRNA for 1 molecule of enzyme), are set to crystallize in the cold (4%) against 60% (w/v) ammonium sulphate. The best crystals were obtained at pH 7.5, while urchins or spherolites tend to appear when lowering the pH to 6.0. Large plates (0.2 mm x O-4 mm x 1.0 mm) are formed within a few weeks, or in about one week using seeding techniques. Crystals grown at pH 7.5 from enzyme obtained using the previous preparation method and crystallization conditions similar to the one used for the cubic crystal form, have different cell parameters and diffract to 3.5 A (orthorhombic forms I and II; Table 1). The new crystals (form III; Table 1) belong to space group P2,2,2, and contain one synthetase and two tRNA molecules per asymmetric unit leading to 3.79 A3/dalton, a value slightly outside the standard range for proteins (1.68 to 3.6 Aldalton), but under that observed for the previously obtained crystal forms of the complex. This is well in agreement with the ability of the new crystals, not only to diffract to higher resolution but also to be less sensitive to X-ray damage, as a consequence of a lower content of solvent. Crystallographic data have been collected using synchrotron radiation at CHESS and LURE. A search for heavy-atom derivatives is under way. At present, crystals diffracting to high resolution (at least 3 A or higher) have been produced for both tRNA (Giege et al., 1977) and synthetase (Dietrich et al., 1980), molecules in the specific system of the aspartic acid from yeast and now for their complex, building a unique situation from which structures from molecules complexed and in their native state will be solved providing the first details at an atomic
level
on such a system
of the biosynthetic
pathway. During completion of this work another high resolution crystal form was obtained for the glutaminyl system from Escherichia coli; in that case crystals diffracting to 2.8 A resolution were grown with the monomeric synthetase interacting with one molecule of tRNA2’” (Perona et al., 1987). We are most indebted to C. Gaget, A. Garcia, V. Perret and A. Theobald for purification of tRNAAsP from counter current fractions provided by G. Dirheimer and M. Schlegel. We thank J. P. Samama for fruitful discussions about crystallization and Professor J. P. Ebel for his constant interest and support of this work.
M. Ruff J. Cavarelli V. Mikol B. Lorber
A. Mitschler R. Giege J. C. Thierry D. Moras Edited
Institut de Biologie Moleculaire et Cellulaire du CNRS 15 rue R. Descartes, 67084 Strasbourg-Cedex, Received 10 September
France
1987
References Bhat, T. N., & Blow, D. M. (1983). Actu Crystallogr. sect. A, 39, 166-170. Bhat, T. N., Blow, D. M., Brick, P. & Nyborg, J. (1982). J. Mol. Biol. 158, 699709. Brick, P. & Blow, D. M. (1987). J. Mol. Biol. 194, 287297. Brunie, S., Mellot, P., Zelwer, C., Risler, J. L., Blanquet, S. & Fayat, G. (1987). J. MOE. Graph. 5 1821. Dietrich, A., Giege, R., Comarmond, M. B., Thierry, J. C. & Moras, D. (1980). J. Mot. BioE. 138, 124-135. Giege, R., Moras, D. & Thierry, J. C. (1977). J. MOE. Biol. 115, 91-96. Giege, R., Lorber, B., Ebel, J. P., Thierry, J. C. & Moms, D. (1980). C.R. S&wee Acad. Sci (Parie), eer. D,
291, 393-396. Giege, R., Dock, A. C., Kern, D., Lo&r, B., Thierry, J. C. & Moras, D. (1986). J. Crystal Growth, 76, 554561. Jack, A., Ladner, J. E. & Klug, A. (1976). J. Mol. Biol. 108, 619649. Lorber, B., Giege, R., Ebel, J. P., Berthet, C., Thierry, J. C. & Moras, D. (1983). J. BioZ. Chem. 258, 84298435. Lorber, B., Kern, D., Mejdoub, H., Boulanger, Y., Reinbold, J. & Giege, R. (1987). Eur. J. Biochem.
165, 4@%417. Moras, D., Comarmond, M. B., Fischer, J., Weiss, R., Thierry, J. C., Ebel, J. P. & Giege, R. (1980). Nature (London), 288, 66%674. Perona, J., Steitz, T. A. $ SolI, D. (1987). Abstract of the Second In.!ernationul Cry&al Growth Meeting, B&her&erg, Strasbourg, France (abstract no. P2-17). Podjarny, A., Rees, B., Thierry, J. C., Cavarelli, J., Jesior, J. C., Roth, M., Lewitt-Bentley, A., Kahn, R., Lorber, B., Ebel, J. P., Giege, R. & Moras, D. (1987). J. Biumol. Struct. Dynam. 5, 187-198. Quigley, G. J., Wang, A., Seeman, N. C., Suddath, F. L.. Rich, A., Sussman, J. L. & Kim, S. H. (1975). Proc. Nat. Acad. Sci., U.S. A. 72, 486&4870. Risler, J. L., Zelwer, C. & Brunie, S. (1981). Nature (London), 292, 384-386. Schevitz, R. W., Podjarny, A. D., Krishnamachar, N., Hugues, J. J., Siegler, P. & Sussman, J. L. (1979). Nature (London), 278, 188190. Sussman, J. L., Holbrook, S. R., Warrant, R. W., Church, G. M. BEKim, S. H. (1978). J. MOE. BioZ. 123, 607-630. Stout, C. D., Mizuno, M., Rao, S. T., Swaminathan, P., Rubin, J., Brennan, T. & Sundaralingam, M. (1978). Actu Crystallogr. sect. B, 34, 15291540. Westhof, E., Dumas, P. & Moras, D. (1985). J. Mol. Biol. 184, 119145. Woo, N., Roe, B. & Rich, A. (1980). Nature (London), 286, 346-351. Wright, A. T., Manor, T. C., Beurling, K., Karpel, R. L. & Fresco, J. L. (1979). In Transfer RNA, Structure, Function and Recognition Cold Spring Harbor Monograph Series 9A, pp. 145-160, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
by R. Huber