J. Mol. Biol. (1991) 221, 375-377
Crystals of Intact Elongation Factor Tu from Thermus thermophilus Diffracting to High Resolution L . S . Reshetnikova’+,
C. 0. A. Reiserl, N. K. Schirmer’, H. Berchtold2, R. Storm2 R. Hilgenfeld2 and M. Sprinzll fiir Biochemie. Universikit Bayreuth P.O. box 101251, D-8580 Bayreuth, Germany
‘Laboratorium
‘Protein
Crystallography, Central Research G 864, Hoechst AG P.O. box 800320, D-6230 Frankfurt 80, Germany (Received
9 April
1991: accepted 15 May
1991)
The intact elongation factor Tu from the extreme thermophile Thermus thermophilus has been crystallized as a complex with the GTP analogue guanosine-5’@,y-imido)triphosphate. The crystals are very stable in the X-ray beam and diffract to 1.9 A resolution. They exhibit space group (72; with a= 150.3(6) A, b=99.6(3) A, c=40.1(1) A, p=954(2)“, and contain one elongation factor Tu molecule per asymmet’ric unit. Keywords:
elongation
factor Tu; Thermus thermophilus; guanine crystallization: X-ray crystallography
Elongation factor Tu (EF-Tuf) is a guanine nucleotide-binding protein essential for bacterial protein biosynthesis. Ever since its detailed bio-chemical characterization (Miller & Weissbach, 1977), t’his protein (M, ~45,000) has attracted the attention of macromolecular crystallographers, but lack of suita,ble crystals has prevented a high-resolution study. To date, two structural studies using crystals of the Escherichia coli EF-Tu in a proteolytically modified form have been carried out at 2.9 and 2.7 A (1 ,&=O.l nm) resolution, respectively (Jurnak, 1985; la Cour et al., 1985). The resulting models comprised most of the G domain (domain I) of EF-Tu, but provided only preliminary information (Clark et al., 1990) on the chain fold of domains II and III, both of which are involved in the interact)ion of EF-Tu with aminoacyl-tRNA and with nucleotide-exchange factor EF-Ts. Also, the oligopeptide fragment between residues 44 and 59 is missing in these crystals, due to limited proteolysis. It has recently been shown that this segment, in particular residue Lys52, is located near the nucleotide binding site in the EF-Tu from Thermus thrrmophilus (Peter et al., 1988, 1990). In addition, t On leave of absence from Institute of Molecular Biology, Academy of Sciences of the U.S.S.R.. Moscow, U.S.S.R. 1 Abbreviations used: EF, elongation factor; GPPNP, guanosine-5’-(P,y-imido)triphosphate; PAGE, polyacrylamide gel electrophoresis. 002"-2836/91/18037.5~)3 $OXOO/tJ
nucleotide-hinding
protein:
Jurnak et al. (1990) have proposed an important role for Arg58 (E. coZi EF-Tu numbering) in recognition and binding of the nucleotide. Because of the shortcomings of the present structural models, recent efforts have concentrated on obtaining well-ordered crystals of intact, noncleaved EF-Tu. Small crystals of the unmodified E. coli factor have been mentioned in a recent review (Clark et al., 1990), and cryst’als of a GDP complex of intact T. aquaticus EF--Tu, which diffracted to a maximum resolution of 2.6 A when synchrotron radiation have been was used, described (Lippmann et al., 1988). We report on crystals of the intact EF-Tu from T. thermophilus in complex with the slowly hydrolyzable GTP analogue, GPPNP, which exhibit strong diffraction to 1.9 A on a conventional rotating anode source equipped with an area detect’or. Presumably, binding of GPPNP (which is frequently also called GMPPNP) will lock the EF-Tu in its activated, GTP-binding conformation, as it does in the case of the homologous p21 Ras protein (Pai et al., 1989; Schlichting et al., 1990). Therefore, our crystals offer the opportunity to look at this activated form of the EF-Tu G domain, while all structural information available to date concerns the inactive GDP form. Purification of EF-Tu from the extremely thermophilic bacterium, T. thermophilus HB8 was carried out according to the method of .Peter et al. (1990). Nucleotide-free EF-Tu was obtained according to the method of Seidler d al. (1987). (6 1991 Academic
I’ress Limited
376
Reshetnikova
1
2
3
4
5 u
PI
n Figure 1. SDS/PAGE of T. thermophilus EF-Tu. GDP (lane l), EF-Tu . GPPNP obtained from crystals dissolved after extensive rinsing (lane 2), protein from mother liquor of crystallization set-ups (lane 3). and partly trypsinized EF-Tu . GDP (lane 4). Lane 5, protein standard (Pharmacia): phosphorylase b (94,000 M,), bovine serum albumin (67,000 M,), ovalbumin (43,000 M,), carbonic anhydrase (30,000 M,), soybean trypsin inhibitor (20,100 M,), a-lactalbumin (14!400 M,).
Crystallization was carried out at 4°C using the hanging-drop variant of the vapor diffusion technique. The 10 ~1 droplets contained 5 to 7 mg protein/ml solution, 15 to 20% saturated ammonium sulfate in 20 mM-Tris. HCl or sodium cacodylate buffer, (pH 7 to 7.5). The ratio of GPPNP to EF-Tu against was 5 to 7 : 1. Droplets were equilibrated 1 ml of 40 to 45% saturated ammonium sulfate in the same buffer. Prismatic crystals appeared within three to five days and grew to a maximum size of extensive After O-3 mm x @35 mm X 1.2 mm. washing with 50% saturated ammonium sulfate, the crystals were analyzed by SDS/PAGE. Only one band, corresponding to the intact EF-Tu, was detected (Fig. 1). Diffraction qualities of crystals were inspected and three-dimensional data sets collected using a FAST television area detector system with a CAD4 four-circle goniostat mounted on an FR 571 rotating copper anode X-ray generator, operated at 4.5 kW, with @3 mm focus (all X-ray equipment was obtained from Enraf Nonius/Delft Instruments, Delft, The Netherlands). Data acquisition and processing were carried out under control of the MADNES software (Messerschmidt & Pflugrath, 1987). Unit cell constants and Bravais lattice type were determined by an autoindexing procedure (Kabsch, 1988) written by Dr P. Tucker, and confirmed by checking the systematic absences and the equivalence of symmetry-related reflections as well as by zero and first-layer precession photographs.
et al.
The crystals diffracted to a resolution limit of 1.9 Ii and displayed space group (‘2, with a=150.3(6) 8, h=996(3) 8, c=4@1(1) a and /?=,954(2)” (S.D va 1ues from a total of 4 crystals).
Assuming one EF-Tu complex per asymmetric unit. the packing density of these crystals was I’,,,= 3.24 Aj/dalton, which was well within the range normally observed (Matthews, 1978). and the solvent content was 62%. Native data sets were collected from four different crystals, all of which proved to be exceptionally stable in the X-ray beam. The reliability index, Rsymt. was between 3.3% and 62% for individual data sets, and the combined data had a Rmerge (on intensities, I) of 8.8% for all reflections with I>a(l), with each merged reflection measured three times on average. These merged data were 82 96 complete to 2.2 A. with measurements present for 61.1 y/, and 21.8?+, of the unique reflections in the 2.20 A to 2.04 A and 2.04 A to 1.91 a resolution ranges, respectively. This high-resolution data set forms a solid basis for the solution of the T. thermophilus EF-Tu structure by a combination of molecular and isomorphous replacement. References Clark, B. F. C., Kjeldgaard, M., la Cour, T. F. M.. Thirup. S. & Nyborg, tJ. (1990). Structural determination of the functional sites of E. coli elongation factor Tu. Biochim. Biophys. Acta, 1050, 203-208. Jurnak, F. (1985). Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins. Science, 230, 32-36. Jurnak, F., Heffron, S., Schick, B. & Delaria, K. (1990). Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu. Riochim. Biophys. Acta, 1050, 209-214. Kabsch, W. (1988). Automatic indexing of’ rotation diffraction patterns. J. Appl. Crystallogr. 21, 67-71. la Cour, T. F. M., Nyborg, J., Thirup. S. & Clark, B. F. (‘. (1985). Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. co& as studied by X-ray crystallography. EMBO J. 4, 191-219. Lippmann, C., Betzel, C., Dauter, Z.. Wilson, I(. Xr. V. A. (1988). C’rystallization and Erdmann, preliminary X-ray diffraction studies of intact EF-Tu from Thermus aquaticus YT-1 FEBB Letters, 240, 139-142. Matthews, B. W. (1978). X-ray structure of proteins. In The Proteins (Neurath, H. & Hill, R. I,., eds), 3rd edit. pp. 403-573, Academic Press, New York. Messerschmidt, A. & Pflugrath, J. W. (1987). Crystal orientation and X-ray pattern prediction routines for area-detector diffractometer systems J. A,:11 macromolecular crystallography. Crystallogr. 20, 306-315. t The reliability index is defined as R = [CiJli - l/&I,) x 100, where is the average of Ii over all its symmetry equivalents. RSymmeasures the agreement between symmetry-related reflections from the same crystal, and Rmergeg ives the overall agreement between intensities measured from different crystals.
Communications
Miller, D. L. & Weissbach, H. (1977). Factors involved in the transfer of aminoacyl-tRNA to the ribosome. In Molecular Mechanisms of Protein Biosynthesis (Weissbach, H. & Pestka, S., eds), pp. 323-373, Academic Press, New York. Pai, E. F., Kabsch, W., Krengel, U., Holmes, K. C., John, J. & Wittinghofer, A. (1989). Structure of the guanine-nucleotide-binding domain of the Ha-ras product p21 in the triphosphate oncogene conformation. Nature (London), 341, 209214. Peter, M. E., Wittmann-Liebold, B. & Sprinzl, M. (1988). Affinity labeling of the GDP/GTP binding site in elongation factor Tu. thermophilus Thermus Biochemistry, 27, 9132-9139.
377
Peter, M. E., Schirmer, N. K., Reiser, C. 0. A. & Sprinzl, M. (1990). Mapping the effector region in Thermus thermophilus elongation factor Tu. Bioch,emistry, 29, 287tV-2884. Schlichting, I., Almo, S. C., Rapp, G., Wilson, K., Petratos, K., Lentfer, A., Wittinghofer, A., Kabsch, W., Pai, E. F., Petsko, G. A. & Goody, R. S. (1990). Time-resolved X-ray crystallographic study of the conformational change in Ha-ras p21 protein on GTP hydrolysis. Nature (London), 345, 309-315. Seidler, L., Peter, M., Meissner, F. & Sprinzl, M. (1987). Sequence and identification of the nucleotide binding site for the elongation factor Tu from Thermus thermophilus HB8. Nucl. Acids Res. 15, !I263-9277.
Edited by A. R. Fersht
Crystals of Intact Elongation Factor Tu from Thermus thermophilus Diffracting to High Resolution L . S . Reshetnikova’+,
C. 0. A. Reiserl, N. K. Schirmer’, H. Berchtold2, R. Storm2 R. Hilgenfeld2 and M. Sprinzll fiir Biochemie. Universikit Bayreuth P.O. box 101251, D-8580 Bayreuth, Germany
‘Laboratorium
‘Protein
Crystallography, Central Research G 864, Hoechst AG P.O. box 800320, D-6230 Frankfurt 80, Germany (Received
9 April
1991: accepted 15 May
1991)
The intact elongation factor Tu from the extreme thermophile Thermus thermophilus has been crystallized as a complex with the GTP analogue guanosine-5’@,y-imido)triphosphate. The crystals are very stable in the X-ray beam and diffract to 1.9 A resolution. They exhibit space group (72; with a= 150.3(6) A, b=99.6(3) A, c=40.1(1) A, p=954(2)“, and contain one elongation factor Tu molecule per asymmet’ric unit. Keywords:
elongation
factor Tu; Thermus thermophilus; guanine crystallization: X-ray crystallography
Elongation factor Tu (EF-Tuf) is a guanine nucleotide-binding protein essential for bacterial protein biosynthesis. Ever since its detailed bio-chemical characterization (Miller & Weissbach, 1977), t’his protein (M, ~45,000) has attracted the attention of macromolecular crystallographers, but lack of suita,ble crystals has prevented a high-resolution study. To date, two structural studies using crystals of the Escherichia coli EF-Tu in a proteolytically modified form have been carried out at 2.9 and 2.7 A (1 ,&=O.l nm) resolution, respectively (Jurnak, 1985; la Cour et al., 1985). The resulting models comprised most of the G domain (domain I) of EF-Tu, but provided only preliminary information (Clark et al., 1990) on the chain fold of domains II and III, both of which are involved in the interact)ion of EF-Tu with aminoacyl-tRNA and with nucleotide-exchange factor EF-Ts. Also, the oligopeptide fragment between residues 44 and 59 is missing in these crystals, due to limited proteolysis. It has recently been shown that this segment, in particular residue Lys52, is located near the nucleotide binding site in the EF-Tu from Thermus thrrmophilus (Peter et al., 1988, 1990). In addition, t On leave of absence from Institute of Molecular Biology, Academy of Sciences of the U.S.S.R.. Moscow, U.S.S.R. 1 Abbreviations used: EF, elongation factor; GPPNP, guanosine-5’-(P,y-imido)triphosphate; PAGE, polyacrylamide gel electrophoresis. 002"-2836/91/18037.5~)3 $OXOO/tJ
nucleotide-hinding
protein:
Jurnak et al. (1990) have proposed an important role for Arg58 (E. coZi EF-Tu numbering) in recognition and binding of the nucleotide. Because of the shortcomings of the present structural models, recent efforts have concentrated on obtaining well-ordered crystals of intact, noncleaved EF-Tu. Small crystals of the unmodified E. coli factor have been mentioned in a recent review (Clark et al., 1990), and cryst’als of a GDP complex of intact T. aquaticus EF--Tu, which diffracted to a maximum resolution of 2.6 A when synchrotron radiation have been was used, described (Lippmann et al., 1988). We report on crystals of the intact EF-Tu from T. thermophilus in complex with the slowly hydrolyzable GTP analogue, GPPNP, which exhibit strong diffraction to 1.9 A on a conventional rotating anode source equipped with an area detect’or. Presumably, binding of GPPNP (which is frequently also called GMPPNP) will lock the EF-Tu in its activated, GTP-binding conformation, as it does in the case of the homologous p21 Ras protein (Pai et al., 1989; Schlichting et al., 1990). Therefore, our crystals offer the opportunity to look at this activated form of the EF-Tu G domain, while all structural information available to date concerns the inactive GDP form. Purification of EF-Tu from the extremely thermophilic bacterium, T. thermophilus HB8 was carried out according to the method of .Peter et al. (1990). Nucleotide-free EF-Tu was obtained according to the method of Seidler d al. (1987). (6 1991 Academic
I’ress Limited
376
Reshetnikova
1
2
3
4
5 u
PI
n Figure 1. SDS/PAGE of T. thermophilus EF-Tu. GDP (lane l), EF-Tu . GPPNP obtained from crystals dissolved after extensive rinsing (lane 2), protein from mother liquor of crystallization set-ups (lane 3). and partly trypsinized EF-Tu . GDP (lane 4). Lane 5, protein standard (Pharmacia): phosphorylase b (94,000 M,), bovine serum albumin (67,000 M,), ovalbumin (43,000 M,), carbonic anhydrase (30,000 M,), soybean trypsin inhibitor (20,100 M,), a-lactalbumin (14!400 M,).
Crystallization was carried out at 4°C using the hanging-drop variant of the vapor diffusion technique. The 10 ~1 droplets contained 5 to 7 mg protein/ml solution, 15 to 20% saturated ammonium sulfate in 20 mM-Tris. HCl or sodium cacodylate buffer, (pH 7 to 7.5). The ratio of GPPNP to EF-Tu against was 5 to 7 : 1. Droplets were equilibrated 1 ml of 40 to 45% saturated ammonium sulfate in the same buffer. Prismatic crystals appeared within three to five days and grew to a maximum size of extensive After O-3 mm x @35 mm X 1.2 mm. washing with 50% saturated ammonium sulfate, the crystals were analyzed by SDS/PAGE. Only one band, corresponding to the intact EF-Tu, was detected (Fig. 1). Diffraction qualities of crystals were inspected and three-dimensional data sets collected using a FAST television area detector system with a CAD4 four-circle goniostat mounted on an FR 571 rotating copper anode X-ray generator, operated at 4.5 kW, with @3 mm focus (all X-ray equipment was obtained from Enraf Nonius/Delft Instruments, Delft, The Netherlands). Data acquisition and processing were carried out under control of the MADNES software (Messerschmidt & Pflugrath, 1987). Unit cell constants and Bravais lattice type were determined by an autoindexing procedure (Kabsch, 1988) written by Dr P. Tucker, and confirmed by checking the systematic absences and the equivalence of symmetry-related reflections as well as by zero and first-layer precession photographs.
et al.
The crystals diffracted to a resolution limit of 1.9 Ii and displayed space group (‘2, with a=150.3(6) 8, h=996(3) 8, c=4@1(1) a and /?=,954(2)” (S.D va 1ues from a total of 4 crystals).
Assuming one EF-Tu complex per asymmetric unit. the packing density of these crystals was I’,,,= 3.24 Aj/dalton, which was well within the range normally observed (Matthews, 1978). and the solvent content was 62%. Native data sets were collected from four different crystals, all of which proved to be exceptionally stable in the X-ray beam. The reliability index, Rsymt. was between 3.3% and 62% for individual data sets, and the combined data had a Rmerge (on intensities, I) of 8.8% for all reflections with I>a(l), with each merged reflection measured three times on average. These merged data were 82 96 complete to 2.2 A. with measurements present for 61.1 y/, and 21.8?+, of the unique reflections in the 2.20 A to 2.04 A and 2.04 A to 1.91 a resolution ranges, respectively. This high-resolution data set forms a solid basis for the solution of the T. thermophilus EF-Tu structure by a combination of molecular and isomorphous replacement. References Clark, B. F. C., Kjeldgaard, M., la Cour, T. F. M.. Thirup. S. & Nyborg, tJ. (1990). Structural determination of the functional sites of E. coli elongation factor Tu. Biochim. Biophys. Acta, 1050, 203-208. Jurnak, F. (1985). Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins. Science, 230, 32-36. Jurnak, F., Heffron, S., Schick, B. & Delaria, K. (1990). Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu. Riochim. Biophys. Acta, 1050, 209-214. Kabsch, W. (1988). Automatic indexing of’ rotation diffraction patterns. J. Appl. Crystallogr. 21, 67-71. la Cour, T. F. M., Nyborg, J., Thirup. S. & Clark, B. F. (‘. (1985). Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. co& as studied by X-ray crystallography. EMBO J. 4, 191-219. Lippmann, C., Betzel, C., Dauter, Z.. Wilson, I(. Xr. V. A. (1988). C’rystallization and Erdmann, preliminary X-ray diffraction studies of intact EF-Tu from Thermus aquaticus YT-1 FEBB Letters, 240, 139-142. Matthews, B. W. (1978). X-ray structure of proteins. In The Proteins (Neurath, H. & Hill, R. I,., eds), 3rd edit. pp. 403-573, Academic Press, New York. Messerschmidt, A. & Pflugrath, J. W. (1987). Crystal orientation and X-ray pattern prediction routines for area-detector diffractometer systems J. A,:11 macromolecular crystallography. Crystallogr. 20, 306-315. t The reliability index is defined as R = [CiJli - l/&I,) x 100, where is the average of Ii over all its symmetry equivalents. RSymmeasures the agreement between symmetry-related reflections from the same crystal, and Rmergeg ives the overall agreement between intensities measured from different crystals.
Communications
Miller, D. L. & Weissbach, H. (1977). Factors involved in the transfer of aminoacyl-tRNA to the ribosome. In Molecular Mechanisms of Protein Biosynthesis (Weissbach, H. & Pestka, S., eds), pp. 323-373, Academic Press, New York. Pai, E. F., Kabsch, W., Krengel, U., Holmes, K. C., John, J. & Wittinghofer, A. (1989). Structure of the guanine-nucleotide-binding domain of the Ha-ras product p21 in the triphosphate oncogene conformation. Nature (London), 341, 209214. Peter, M. E., Wittmann-Liebold, B. & Sprinzl, M. (1988). Affinity labeling of the GDP/GTP binding site in elongation factor Tu. thermophilus Thermus Biochemistry, 27, 9132-9139.
377
Peter, M. E., Schirmer, N. K., Reiser, C. 0. A. & Sprinzl, M. (1990). Mapping the effector region in Thermus thermophilus elongation factor Tu. Bioch,emistry, 29, 287tV-2884. Schlichting, I., Almo, S. C., Rapp, G., Wilson, K., Petratos, K., Lentfer, A., Wittinghofer, A., Kabsch, W., Pai, E. F., Petsko, G. A. & Goody, R. S. (1990). Time-resolved X-ray crystallographic study of the conformational change in Ha-ras p21 protein on GTP hydrolysis. Nature (London), 345, 309-315. Seidler, L., Peter, M., Meissner, F. & Sprinzl, M. (1987). Sequence and identification of the nucleotide binding site for the elongation factor Tu from Thermus thermophilus HB8. Nucl. Acids Res. 15, !I263-9277.
Edited by A. R. Fersht
