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Crystallization and preliminary X-ray analysis of the human c-H-ras-oncogene product p21 complexed with GTP analogues
J. Mol. Biol. (1989) 206, 257-259
Crystallization and Preliminary X-ray Analysis of the Human c-H-ras-Oncogene Product ~21 Complexed with GTP Analogues The catalytic domain (amino acid residues 1 to 166) of the human ras-oncogene product ~21 complexed with the GTP analogues b.lt-imido-GTP (GMPPNP), j?,y-methylene-QTP (GMPPCP), and guanosine-5’-(y-thiotriphosphate) (GTPyS) h ave been crystallized. Crystals of the GMPPNP and GMPPCP complexes are well suited for high resolution X-ray crystallography. They belong to space group P3,21 (or it,s enantiomorph P3,21) with unit cell axes a = b = 40.3 A and c = 162.2 A. -
~21 proteins are the products of the N-, K- and H-ras genes, which have been identified as oncogenes in many human tumours. The molecular basis of activation of these genes has always been found to be a single point mutation, which changes the amino acid sequence at residues 12/13 or 59/61 (for reviews, see Gibbs et al., 1985; Barbacid, 1987). The native proteins are of molecular weight 21,000. They bind GDP and GTP and show slow intrinsic GTPase activity (Gibbs et aE., 1984; McGrath et al., 1984; Sweet et al., 1984). They are homologous to the G proteins of the signaltransducing pathway (Dever et al., 1987; Gilman, 1980) and their oncogenic variants have growthpromoting and transforming properties. Therefore, it has been postulated that they are involved in the signal-transduction pathway growth-promoting (Barbacid, 1987). Together with G proteins and elongation factors, 1~21 proteins share a common property: they cycle between an “inactive” GDPand an “active” GTP-binding form (Kaziro, 1978). The C-t,erminal part of native p21 proteins, which is highly variable among the different p21 proteins, is responsible for anchoring the protein to cell membranes (Willumsen et al., 1984; Willingham et al., 1980). Furthermore, it has been postulated to be flexible in structure (Willingham et al., 1980; Taparowsky et al.. 1983; Willumsen et al.. 1984), which might cause problems in the crystallization of this protein. Therefore, in c:-H-ras cDNA we have mutated the Lys167 codon, AAA, to a TAA stop codon (John et al., unpublished results) and cloned the mutated DNA in the ptac-ras expression vector in Escherichia co& (John et al., 1988). Expression was induced with 0.2 mM-isopropyl-/-n-thiogalactoside and produced high concentrations of the gene product, truncated p21 (~21,‘). The mutated protein was easily purified following a procedure described elsewhere (Tucker et al., 1986). In a separate report we show that this domain of ~21, called the catalytic domain, has biochemical properties very similar to those of the intact protein (John et al., unpublished results). Recent’ly. deVos et al. (1988) reported on the
three-dimensional structure of the GDP complex of the guanosine nucleotide-binding domain of p21 (residues 1 to 171). We were also able to obtain crystals of the GDP complex of p21,‘, as were others using the same construction as did deVos et al. (Morikawa et al., 1988). In contrast to these reports, our crystals of the GDP complex proved to be very fragile and highly susceptible to radiation damage. In order to understand the structural basis of the conformational transition between the diphosphateand the triphosphate-binding forms of t,he protein, we have investigated the possibility of crystallizing complexes of p21,’ with guanosine triphosphates. Since p21 proteins are slow GTPases. the GTP complex having a half-life of hours at room temperature, and, as it usually takes at least days to grow protein crystals suitable for X-ray crystallographic structure determination. we tested whether slowly hydrolysable analogues of GTP would bind to ~21. The binding affinities were tested by incubating the complex of p21 and labelled GDP with increasing concentrations of analogue and filtering the incubation mixture through nitrocellulose filters (Tucker et al., 1986). Table 1 shows the affinities of the different t’riphosphates relative to GDP. Binding to cellular p21 increases in the order GMPPCP, GMPPNP, GTPyS. Since the binding constant for GTP is 6 x 10” (Feuerstein et al., 1987), all t,hese analogues are still very tightly bound to ~21. nucleotide-binding p21, like most guanine proteins, contains one mole of bound nucleotide, usually GDP, per mole of protein (Poe rt al., 1985; Tucker et al., 1986). In fact, the protein is unstable in the absence of nucleotides. We have developed a method to replace GDP quantitatively by several GTP analogues including GMPPCP, GMPPNP and GTPyS (John et al., unpublished results). Crystals of the GMPPCP and GMPPNP complexes of ~21,’ could be grown from a solution of 3 to 4 mg 64 m&r-Tris . HCI protein/ml in (pH 7.5), 1 mM-dithioerithritol, 10 mM-MgCl,, 1 mw-sodium azide using a broad range of ethylene glycols as
-
r(min) -
t?! -t(min)
-
t(min) -
-
Figure 1. Analysis of ~21,’ crystals for nucleotide content. One crystal each of p21,.GMPPCP and ~21,‘. GMPPNP were washed with mother liquor and dissolved in 20 ~1 of water. A 10 ~1 portion was analysed on a C-18 reversed phase high-pressure liquid chromatography column (Shandon Hypersil 5 pm). The column was run at ambient temperature with phosphate buffer (50 mM (pH 6.5)) containing 0.2 mM-tertiary butylammonium bromide, 1.8% (v/v) acetonitrile, and 0.2 mM-sodium azide (Tucker et al., 1986). (a) Standard mixture containing guanosine (elution time: 2.072 min), GMP (2.343 min), GDP (3.155 min), GMPPCP (3.832 min) and GTP (4.463 min); (b) crystal of GMPPCP complex, GMPPCP (3.885 min); (c) standard mixture containing guanosine (2.032 min), GMP (2.297 min), GDP (3.113 min), GMPPNP (3-730 min) and GTP (4-438 min); (d) crystal of GMPPNP complex, GMPPNP (3-737 min).
259
Letters to the Editor
Table 1 Relative
afinities
of binding of GDP, GTP, and “non-hydrolysable” to p21 as measured by inhibition
Nudrotide
Rel. affinities
GTP-analogues
binding
GDP
GTP
GTPyS
GMPPNP
GMPPCP
I.0
1.9
0.7
0.09
0.01
precipitants. The largest crystals were obtained with 20% (w/v) polyethylene glycol (PEG?) 1450 (Sigma Co., St Louis, MO). In general, crystals grew better at. room temperature than at 4°C. Figure I shows the data of a high-pressure liquid chromatography analysis of a redissolved crystal that had been used for X-ray data collection. It can be seen that GDP has been exchanged completely and that the triphosphate analogue is still intact after collection of a complete data set. Both the GMPPCP and the GMPPNP complex crystallized in the trigonal space group P3,21 (or its enantiomorph P3,21) with unit cell axes a = b = 40.3 A and c = 162.2 A (1 d=@l
nm).
The
crystals are mechanically very stable, diffract to about 1.5 b resolution, and show little radiation damage in the X-ray beam. Determination of crystal density was done by setting up, in a graduated measuring cylinder, a gradient of xylene and carbon tetrachloride and calibrating it by adding small droplets of CsCl solutions of known densities. This method gave an average pCR= 1.288 gl cm3. The unit cell volume (in A3/dalton: Matthews, 1968) is 2.1, with one molecule in the asymmetric unit, equivalent to 60% of the crystal volume being occupied by protein. Complete native data sets were collected on a Nicolet/Xentronics area detector for GMPPCP crystals grown from solutions containing PEG 400, PEG 1450, or PEG 4000 as precipitant, as well as the GMPPNP crystals grown from PEG 1450 solutions. Whereas all crystals of the GMPPCP complex showed the same diffraction pattern, regardless of the precipitant used, there was a distinct change in intensities when GMPPCP and GMPPNP data were compared, most, probably indicating that, the conformation of the p21 molecule is sensitive to differences in the geometry of the b-7 phosphate bond, reflecting the improved binding of GMPPNP. This agrees well with the fact that crystals of the GTPyS complex have a different crystal shape. So far their size is still insufficient for crystallographic analysis. Jn our crystallization conditions, even at 4”C, we also find crystals of a shape characteristic for the GDP complex. Most probably hydrolysis of this GTP analogue even at low temperature is still too fast, producing a sufficiently high concentration of GDP to enable formation of the GDP complex and hence its crystallization. T Abbreviation
of 13HIC;lDP
used: PEG, polyethylene
glycol. Edited
We thank W. Kabsch for helpful discussions.
Anna Scherer Jacob John Rosita Linke Roger S. Goody Alfred Wittinghofer Emil F. Pai Kenneth C. Holmes Abt. Biophysik, Max-Planck-Institut fiii Medizinische Forschung, Jahnstrasse 29 D-6900 Heidelberg. F.R.G. Received 25 August 1988, and in revised form 31 October 1988
References Barbacid, M. (1987). Annu. Rev. Biochem. 56, 779-827. Dever, T. E., Glynias, M. J. & Merrick, W. C. (1987). I’roc. Nat. Acad. Sci., U.S.A. 84, 1814-1818. deVos, A. M., Tong, L., Milburn, M. V., Matias, P. M., Jancarik, J., Noguchi, S., Nshimura, S., Miura, K., Ohtsuka, E. & Kim, S.-H. (1988). Science, 239, 888893. Feuerstein, J., Goody, R. S. & Wittinghofer, A. (1987). J. Biol. Chem. 262, 8455-8458. Gibbs, J., Sigal, 1. S., Poe, M. & Scolnick, E. M. (1984). Proc. Nat. Acad. Sci., U.S.A. 81, 5704-5708. Gibbs, J., Sigal, I. S. & Scolnick, E. M. (1985). Trends Biochem. Sci. 10, 350-353. Gilman, A. G. (1980). Annu. Rev. Biochem. 56, 615649. John, J., Frech, M. & Wittinghofer, A. (1988). J. Riol. Chew 263, 11792-11799. Kaziro, Y. (1978). Biochim. Biophys. Acta, 505, 95-127. Matthews, B. W. (1968). J. Mol. Biol. 33, 491497. McGrath, J. P.. Capon, D. J., Goeddel, D. V. & Levinson, A. D. (1984). Nature (London), 310. 64M44. Morikawa, K., Tsujimoto, M., Higashi, T.. Matsumoto, 0.. Miura, K. & Ohtsuka, E. (1988). .I. Mol. Biol.
201, 237-238. Poe, M., Scolnik, E. M. & Stein. R. B. (1985). J. Biol. Chem.
260, 39063909.
Sweet. R. W., Yokoyama, S.: Kamata, T.. Frramisco, ,J. R., Rosenberg, M. & Gross, M. (1984). ,Wure (London), 311, 273-275. Taparowsky, E., Shimizu, K., Goldfarb, M. & Wigler, M. (1983). Cell, 34, 581-586. Tucker, J., Sczakiel. C., Feuerstein, J., John, J ,, Goody. R. S. & Wittinghofer, A. (1986). EMBOJ. 5, 1351 1358. Willingham, M. C., Pastan, I., Shih, T. Y. & Scolnick, E. M. (1980). CPU, 19, 10051014. Willumsen, B. M., Christensen, A.. Hubbert. S. I,.. Papageorge, A. G. & Lowy. D. R. (1984). Natuw (London). 3 10, 583-586. by R. Huber
Crystallization and Preliminary X-ray Analysis of the Human c-H-ras-Oncogene Product ~21 Complexed with GTP Analogues The catalytic domain (amino acid residues 1 to 166) of the human ras-oncogene product ~21 complexed with the GTP analogues b.lt-imido-GTP (GMPPNP), j?,y-methylene-QTP (GMPPCP), and guanosine-5’-(y-thiotriphosphate) (GTPyS) h ave been crystallized. Crystals of the GMPPNP and GMPPCP complexes are well suited for high resolution X-ray crystallography. They belong to space group P3,21 (or it,s enantiomorph P3,21) with unit cell axes a = b = 40.3 A and c = 162.2 A. -
~21 proteins are the products of the N-, K- and H-ras genes, which have been identified as oncogenes in many human tumours. The molecular basis of activation of these genes has always been found to be a single point mutation, which changes the amino acid sequence at residues 12/13 or 59/61 (for reviews, see Gibbs et al., 1985; Barbacid, 1987). The native proteins are of molecular weight 21,000. They bind GDP and GTP and show slow intrinsic GTPase activity (Gibbs et aE., 1984; McGrath et al., 1984; Sweet et al., 1984). They are homologous to the G proteins of the signaltransducing pathway (Dever et al., 1987; Gilman, 1980) and their oncogenic variants have growthpromoting and transforming properties. Therefore, it has been postulated that they are involved in the signal-transduction pathway growth-promoting (Barbacid, 1987). Together with G proteins and elongation factors, 1~21 proteins share a common property: they cycle between an “inactive” GDPand an “active” GTP-binding form (Kaziro, 1978). The C-t,erminal part of native p21 proteins, which is highly variable among the different p21 proteins, is responsible for anchoring the protein to cell membranes (Willumsen et al., 1984; Willingham et al., 1980). Furthermore, it has been postulated to be flexible in structure (Willingham et al., 1980; Taparowsky et al.. 1983; Willumsen et al.. 1984), which might cause problems in the crystallization of this protein. Therefore, in c:-H-ras cDNA we have mutated the Lys167 codon, AAA, to a TAA stop codon (John et al., unpublished results) and cloned the mutated DNA in the ptac-ras expression vector in Escherichia co& (John et al., 1988). Expression was induced with 0.2 mM-isopropyl-/-n-thiogalactoside and produced high concentrations of the gene product, truncated p21 (~21,‘). The mutated protein was easily purified following a procedure described elsewhere (Tucker et al., 1986). In a separate report we show that this domain of ~21, called the catalytic domain, has biochemical properties very similar to those of the intact protein (John et al., unpublished results). Recent’ly. deVos et al. (1988) reported on the
three-dimensional structure of the GDP complex of the guanosine nucleotide-binding domain of p21 (residues 1 to 171). We were also able to obtain crystals of the GDP complex of p21,‘, as were others using the same construction as did deVos et al. (Morikawa et al., 1988). In contrast to these reports, our crystals of the GDP complex proved to be very fragile and highly susceptible to radiation damage. In order to understand the structural basis of the conformational transition between the diphosphateand the triphosphate-binding forms of t,he protein, we have investigated the possibility of crystallizing complexes of p21,’ with guanosine triphosphates. Since p21 proteins are slow GTPases. the GTP complex having a half-life of hours at room temperature, and, as it usually takes at least days to grow protein crystals suitable for X-ray crystallographic structure determination. we tested whether slowly hydrolysable analogues of GTP would bind to ~21. The binding affinities were tested by incubating the complex of p21 and labelled GDP with increasing concentrations of analogue and filtering the incubation mixture through nitrocellulose filters (Tucker et al., 1986). Table 1 shows the affinities of the different t’riphosphates relative to GDP. Binding to cellular p21 increases in the order GMPPCP, GMPPNP, GTPyS. Since the binding constant for GTP is 6 x 10” (Feuerstein et al., 1987), all t,hese analogues are still very tightly bound to ~21. nucleotide-binding p21, like most guanine proteins, contains one mole of bound nucleotide, usually GDP, per mole of protein (Poe rt al., 1985; Tucker et al., 1986). In fact, the protein is unstable in the absence of nucleotides. We have developed a method to replace GDP quantitatively by several GTP analogues including GMPPCP, GMPPNP and GTPyS (John et al., unpublished results). Crystals of the GMPPCP and GMPPNP complexes of ~21,’ could be grown from a solution of 3 to 4 mg 64 m&r-Tris . HCI protein/ml in (pH 7.5), 1 mM-dithioerithritol, 10 mM-MgCl,, 1 mw-sodium azide using a broad range of ethylene glycols as
-
r(min) -
t?! -t(min)
-
t(min) -
-
Figure 1. Analysis of ~21,’ crystals for nucleotide content. One crystal each of p21,.GMPPCP and ~21,‘. GMPPNP were washed with mother liquor and dissolved in 20 ~1 of water. A 10 ~1 portion was analysed on a C-18 reversed phase high-pressure liquid chromatography column (Shandon Hypersil 5 pm). The column was run at ambient temperature with phosphate buffer (50 mM (pH 6.5)) containing 0.2 mM-tertiary butylammonium bromide, 1.8% (v/v) acetonitrile, and 0.2 mM-sodium azide (Tucker et al., 1986). (a) Standard mixture containing guanosine (elution time: 2.072 min), GMP (2.343 min), GDP (3.155 min), GMPPCP (3.832 min) and GTP (4.463 min); (b) crystal of GMPPCP complex, GMPPCP (3.885 min); (c) standard mixture containing guanosine (2.032 min), GMP (2.297 min), GDP (3.113 min), GMPPNP (3-730 min) and GTP (4-438 min); (d) crystal of GMPPNP complex, GMPPNP (3-737 min).
259
Letters to the Editor
Table 1 Relative
afinities
of binding of GDP, GTP, and “non-hydrolysable” to p21 as measured by inhibition
Nudrotide
Rel. affinities
GTP-analogues
binding
GDP
GTP
GTPyS
GMPPNP
GMPPCP
I.0
1.9
0.7
0.09
0.01
precipitants. The largest crystals were obtained with 20% (w/v) polyethylene glycol (PEG?) 1450 (Sigma Co., St Louis, MO). In general, crystals grew better at. room temperature than at 4°C. Figure I shows the data of a high-pressure liquid chromatography analysis of a redissolved crystal that had been used for X-ray data collection. It can be seen that GDP has been exchanged completely and that the triphosphate analogue is still intact after collection of a complete data set. Both the GMPPCP and the GMPPNP complex crystallized in the trigonal space group P3,21 (or its enantiomorph P3,21) with unit cell axes a = b = 40.3 A and c = 162.2 A (1 d=@l
nm).
The
crystals are mechanically very stable, diffract to about 1.5 b resolution, and show little radiation damage in the X-ray beam. Determination of crystal density was done by setting up, in a graduated measuring cylinder, a gradient of xylene and carbon tetrachloride and calibrating it by adding small droplets of CsCl solutions of known densities. This method gave an average pCR= 1.288 gl cm3. The unit cell volume (in A3/dalton: Matthews, 1968) is 2.1, with one molecule in the asymmetric unit, equivalent to 60% of the crystal volume being occupied by protein. Complete native data sets were collected on a Nicolet/Xentronics area detector for GMPPCP crystals grown from solutions containing PEG 400, PEG 1450, or PEG 4000 as precipitant, as well as the GMPPNP crystals grown from PEG 1450 solutions. Whereas all crystals of the GMPPCP complex showed the same diffraction pattern, regardless of the precipitant used, there was a distinct change in intensities when GMPPCP and GMPPNP data were compared, most, probably indicating that, the conformation of the p21 molecule is sensitive to differences in the geometry of the b-7 phosphate bond, reflecting the improved binding of GMPPNP. This agrees well with the fact that crystals of the GTPyS complex have a different crystal shape. So far their size is still insufficient for crystallographic analysis. Jn our crystallization conditions, even at 4”C, we also find crystals of a shape characteristic for the GDP complex. Most probably hydrolysis of this GTP analogue even at low temperature is still too fast, producing a sufficiently high concentration of GDP to enable formation of the GDP complex and hence its crystallization. T Abbreviation
of 13HIC;lDP
used: PEG, polyethylene
glycol. Edited
We thank W. Kabsch for helpful discussions.
Anna Scherer Jacob John Rosita Linke Roger S. Goody Alfred Wittinghofer Emil F. Pai Kenneth C. Holmes Abt. Biophysik, Max-Planck-Institut fiii Medizinische Forschung, Jahnstrasse 29 D-6900 Heidelberg. F.R.G. Received 25 August 1988, and in revised form 31 October 1988
References Barbacid, M. (1987). Annu. Rev. Biochem. 56, 779-827. Dever, T. E., Glynias, M. J. & Merrick, W. C. (1987). I’roc. Nat. Acad. Sci., U.S.A. 84, 1814-1818. deVos, A. M., Tong, L., Milburn, M. V., Matias, P. M., Jancarik, J., Noguchi, S., Nshimura, S., Miura, K., Ohtsuka, E. & Kim, S.-H. (1988). Science, 239, 888893. Feuerstein, J., Goody, R. S. & Wittinghofer, A. (1987). J. Biol. Chem. 262, 8455-8458. Gibbs, J., Sigal, 1. S., Poe, M. & Scolnick, E. M. (1984). Proc. Nat. Acad. Sci., U.S.A. 81, 5704-5708. Gibbs, J., Sigal, I. S. & Scolnick, E. M. (1985). Trends Biochem. Sci. 10, 350-353. Gilman, A. G. (1980). Annu. Rev. Biochem. 56, 615649. John, J., Frech, M. & Wittinghofer, A. (1988). J. Riol. Chew 263, 11792-11799. Kaziro, Y. (1978). Biochim. Biophys. Acta, 505, 95-127. Matthews, B. W. (1968). J. Mol. Biol. 33, 491497. McGrath, J. P.. Capon, D. J., Goeddel, D. V. & Levinson, A. D. (1984). Nature (London), 310. 64M44. Morikawa, K., Tsujimoto, M., Higashi, T.. Matsumoto, 0.. Miura, K. & Ohtsuka, E. (1988). .I. Mol. Biol.
201, 237-238. Poe, M., Scolnik, E. M. & Stein. R. B. (1985). J. Biol. Chem.
260, 39063909.
Sweet. R. W., Yokoyama, S.: Kamata, T.. Frramisco, ,J. R., Rosenberg, M. & Gross, M. (1984). ,Wure (London), 311, 273-275. Taparowsky, E., Shimizu, K., Goldfarb, M. & Wigler, M. (1983). Cell, 34, 581-586. Tucker, J., Sczakiel. C., Feuerstein, J., John, J ,, Goody. R. S. & Wittinghofer, A. (1986). EMBOJ. 5, 1351 1358. Willingham, M. C., Pastan, I., Shih, T. Y. & Scolnick, E. M. (1980). CPU, 19, 10051014. Willumsen, B. M., Christensen, A.. Hubbert. S. I,.. Papageorge, A. G. & Lowy. D. R. (1984). Natuw (London). 3 10, 583-586. by R. Huber