J. Mol. Biol. (1992) 228, 1271-1273
Crystallization and Preliminary X-ray Diffraction Analysis of a Plant Ribonuclease from the Seeds of the Bitter Gourd Momordica charantia Amitabha Protein Xtructure 44 Lincoln’s
Laboratory, Inn Fields,
Imperial
London
and Gunki Faculty
De-f Research Fund WCZA 3PX, U.K. Cancer
Funatsu
Laboratory of Protein Chemistry and Engineering of Agriculture, Kyushu University, Fukuoka 812, Japan
(Received 9 September 1992; accepted 15 September
1992)
Single crystals of ribonuclease MC, a new class of plant ribonuclease from the seeds of the bitter gourd, were obtained from solutions of polyethylene glycol 8000 by the hanging-drop vapour diffusion method. The crystals belong to the orthorhombic space group P2,2,2, with cell dimensions a = 67.28 A, b = 7521 A, c = 38.54 A. The assumption of one monomer per asymmetric unit gives rise to a V, value of 2.29 As/Da. The crystals diffract beyond 2.0 A resolution and are suitable for high resolution X-ray structure analysis.
Keywords: crystallization;
ribonuclease;
Recognition of nucleotide bases in nucleic acids by proteins is of central importance in molecular biology. Many structural studies of complexes between proteins and their target nucleic acids have been carried out by X-ray diffraction and other physical methods. A large number of RNases with different base specificities have been isolated from a variety of organisms and investigated extensively from both structural and functional viewpoints and are well understood in terms of a model system of nucleic acid/protein interaction (Heinemann & Saenger, 1982; Wlodawer et al.; 1982; Arni et al., 1988). These enzymes are relatively small proteins with molecular masses of 11 to 14 kDa. A vast amount of information has been accumulated on these microbial and mammalian RNases and it has turned out that they share a common structural folding (for structural comparison of this class of RNases, see Hill et al., 1983). Little information is available on the structure-function relationship of RNase with the higher molecular mass of 20 to 30 kDa, and although a vast amount of information has been accumulated on microbial and mammalian
T Author addressed.
to whom
all correspondence
0022%2836/92/241271-03$08.00/O
should be
X-ray
crystallography
RNases, very few studies of the plant RNases have been pursued so far. RNase Mc$ was isolated from the seeds of bitter gourd Momordica charantia by Hiroyuki et al. (1991). The enzyme consists of a single polypeptide chain of 191 residues and has a calculated molecular mass of 21,259 Da. RNase MC has a remarkably high specificity toward CPU, ApU and UpU (Hiroyuki et al., 1991). Comparison of the amino acid sequence of RNase MC with those of RNase T2 (Kawata et al., 1988) and RNase Rh (Horiuchii et al., 1988), which are base non-specific RNases from fungi, shows some homology (approx. 28%). Although the substrate specificity of RNase MC is different from those of fungal RNases and thle level of sequence identity is relatively low, they share two segments of highly conserved residues. The t’wo longest regions of homology at positions 32 to 38 and 81 to 92 contain His34, His89 and lGlu85. Similar residues have been identified as active site residues in RNase T2 (Kawata et al., 1990) and RNase Rh (Sanda et al., 1985) and in the recently solved crystal structure of RNase Rh (Hiroyuki et
$ Abbreviation used: RNase Momordica charantia.
1271
MC, ribonuclease
of
0 1992 Academic Press Limited
1272
A. De & G. Punatsu
al., 1992), these residues are found to be clustered forming the active site. This suggests that in RNase MC, these homologous residues may also be involved in the catalytic mechanism or in the formation of the active sit’e. Recently, it has been found that S-allelic glyeoproteins from style extracts of Nicotiana alata involved in self-incompatibility in flowering plants are RNase (McClure et al.: 1989), which exhibit this activity in arresting pollen tubule growth (McClure et al., 1990). The primary amino acid sequences of three S-glycoproteins S2, S3 and S6, show some regions that are perfectly conserved and other regions that are less homologous (Anderson et al.; 1986). Sequence analysis of RNase MC shows a high identical residues) to these homology (4P y. S-glycoproteins S2, 53 and S6 (Anderson et al., 1986, 1989), much higher in comparison with those of fungal RNases, RNase T2 and RNase Rh (approx. 10%). In addition to the two highly conserved segments described above, there are also other individual conserved positions in the sequence of all S2, S3 and S6 glycoproteins. All eight cysteine residues in RNase MC are totally conserved, whereas only five cyst,eine residues are conserved in the sequence of RNase T2 and RNase Rh. Since the local homology around the essential residues is fairly high in this new class of RNases, it is possible that they may share a common backbone fold as in the much smaller class of microbia,l RNases. In order to understand the structure-function relationships of RNases from plan% origin and to obtain knowledge of its evolutionary relationships to other RNases and S-glycoproteins, we have crystallized RNase Me to determine its three-dimensional structure. Lyophilized protein was dissolved in 20 marITris ’ HCl buffer at pH 7.0 to a final concentration of 10 mg/ml. Crystals were obtained by the hangingdrop vapour diffusion method (McPherson, 1982) using polyethylene glycol (PEG) 8000 as precipitant at 20°C. Drops containing 5 ~1 of RNase MC and 5 ~1
Figure I. An orthorhombic crystal form of RNase MC grown by vapour diffusion using the hanging-drop method using PEG 8000: 200 rnM-sodium acetate and 100 mivr-sodium cacodylate buffer at pH 6%.
Figure 2. 4 screened 15’ precession photograph oi’ tine Ok1zone. The exposure time was 22 h at, 45 kV and 88 mA, at a crystal-to-film distance of 60 mm. This photograph reveals Laue symmetry mm and the absence of reflections with odd indices of the classes Ok0 and 001. An additional precession photograph of the hQZ zone reveals Laue symmetry mm and the additional absence of reflections with odd indices of the class hO0, indicating the space group P2,2,2,.
of well solution, were equilibrated against 1 ml of well solution comprising 30 to 31 ye (w/v) PEG 8000, 200 rnx-sodium acetate and 100 mysodium cacodylat’e buffer (pH 6.6 to 6.8). Rhombic prism-like crystals appeared aft,er three t,o four days and reached their maximal dimensions (0.25 mm x 0.3 mm x 0.7 mm) after two weeks (Fig. 1). Preliminary X-ray diffraction experiments were performed using a precession Camera with Nifiltered CuKcl radiation generated by a Rigaku RU-200 X-ray generator (45 kV: 80 mA, focal size 0.3 mm). Screened 15” precession photographs of two principal zones Ok1 and h01 (Fig. 2) loca,ted 90” apart were taken. These photographs reveal Laue symmetry mm and the absence of reflections with odd indices of the classes hO0, Ok0 or 001; indicating the space group P2,2,2,. The unit cell dimensions are a = 67.28 A, b = 75.21 & c = 38.54 8; giving the volume unit 1.95 x IO5 A3 of the cell as (1 A = 0-I nm). Assuming one RNase MC molecule per asymmetric unit, V, has a value of 2.29 A3/Da, which corresponds to a solvent content of 46 ?A. These values are well within the range for protein crystals (Matt,hews, 1968). The V, value is very similar to the value of 2.60 A3/Da report’ed for the homologous RNase Rh (Hiroyuki et al.; 1989). The crystals diffract beyond 2.0 A resolution and only a slight deterioration was observed after 80 hours of exposure to X-rays at room temperature. Attempt’s are now underway to find a suitable heavy-atom derivat’ive.
Crystallization
This work has been carried out in the Paul S. Freemont at ICRF, London. We Freemont, Michael A. Gorman, John M. Vrielink for their helpful discussions Freemont for the use of X-ray facilities. to thank all his colleagues at Khalisani support.
Laboratory of Dr thank Dr Paul S. Lally & Dr Alice and Dr Paul S. A.D. would like College for their
References Anderson, M. A.; Cornish, E. C., Mau, S.-L., Williams, E. G., Hoggart, R., Atkinson, A., Bonig, I.; Grego, B., Simpson, R.; Roche, P. J., Haley, J. D., Penschow, J. D., Niall, H. D., Tregear, G. W., Coghlan, J. P., Crawford, R. J. & Clarke, A. E. (1986). Cloning of cDNA for a stylar glycoprotein associated with expression of self-incompatibility in AVicotiana alata. Nature (London), 321, 38-44. Anderson M. A., McFadden, G. I., Bernatzky, R., Atkinson, A.; Orpin, T., Dedman, H., Tregear, G., Fernley, R. & Clarke, A. E. (1989). Sequence variability of three alleles of the self-incompatibility gene of Nicotiana alata. Plant Cell, 1, 483-492. Arni, R., Heinemann, U., Tokuoka, R. & Saenger, W. (1988). Three-dimensional structure of the ribonuclease T, *2’-GMP complex at 1.9 A resolution. J. Biol. Chem. 263, 15358-15368. Heinemann, U. 8: Saenger, W. (1982). Specific proteinnucleic acid recognition in ribonuclease Ti-2’.guanylic acid complex: an X-ray study. Nature (London), 299, 27-31. Hill; C., Dodson, G., Heinemann, U., Saenger, W.; Mitsui, Y., Nakamura; K.; Borisov, S., Tischenko, G.; Polyakov, K. & Pavlovsky, S. (1983). The structural and sequence homology of a family of microbial ribonucleases. Trends Biochem. Sci. 8, 364-369. Y. i Nakamura, K. T., Hiroyuki, K., Mitsui, Wakabayashi, E., Ohgi, K. & Irie, M. (1989). Crystallization of a new class of microbial ribonuclease from Rhizopus niveus. J. Mol. Biol. 206, 791-792.
Edited
Notes
1273
Hiroyuki, I., Kimura, M., Arai, M. 8: Fanatsu, G. (1991). The complete amino acid sequence of ribonuclease from the seeds of bitter gourd (Momordica &am&a). FEBS Letters, 284, 161-164. Hiroyuki, K.; Mitsui, Y. E.; Ohgi, K., Irie, M., Mizumo, H. & Nakamura, K. T. (1992). Crystal and molecular structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus. FEBS Letters, 306, 189-192. Horiuchi, H.; Yanai, K., Takagi, M., Yano, K., Wakabayashi, E., Sanda, A., Mine, S., Ohgi, K. & Irie; M. (1988). Primary structure of a base nonribonuclease specific from Rhizopus niveus. J. Biochem. (Tokyo), 103, 408418. Kawata, Y., Sakiyama, F. 8: Tamaoki, H. (1988). A.minoacid sequence of ribonuclease T2 from Aspergillus oryzae. Eur. J. Biochem. 176, 683-697. Kawata, Y., Sakiyama, F., Hayashi, F. & Kyogoku, Y. (1990). Identification of two essential histidine residues of ribonuclease T2 from Aspergillus oryzae. Eur. J. Bioehem. 187: 255-262. Matthews, B. W. (1968). Solvent content of protein crystals. J. Mol. Biol. 33, 491-497. McClure, B. A., Haring, V., Ebert, P. R., Anderson, M. A., Simpson, R. J., Sakiyama, F’. & Clarke, A. E. (1989). Style self-incompatibility gene products of alata are ribonucleases. Nature (London), Nicotiana 342, 955-957. McClure, B. A., Gray, J. E., Anderson M. A. & Clarke, A. E. (1990). Self-incompat’ibility in Nicotiana alata involves degradation of pollen rRNA. Nature (London), 347; 757-760. McPherson, A. (1982). Preparation and Analysis of Protein Crystals; pp. 96-97, J. Wiley & Sons Inc., New York. Sanda, A., Takizawa, Y., Iwama, M. & Irie, M. (1985). Modification of a ribonuclease from Rhizopus Sp. with 1-cyclohexyl-3-(2.morpholinyl-(4)-ethyl) carbodiimide p-toluenesulfonate. J. Biochem. (Tokyo), 98, 125-132. Wlodawer, A., Bott, R. & Sjolin, L. (1982). The refined crystal structure of ribonuclease A at 2.0 A resolution. J. Biol. Chem. 257, 1325-1332.
by A. Klug
Crystallization and Preliminary X-ray Diffraction Analysis of a Plant Ribonuclease from the Seeds of the Bitter Gourd Momordica charantia Amitabha Protein Xtructure 44 Lincoln’s
Laboratory, Inn Fields,
Imperial
London
and Gunki Faculty
De-f Research Fund WCZA 3PX, U.K. Cancer
Funatsu
Laboratory of Protein Chemistry and Engineering of Agriculture, Kyushu University, Fukuoka 812, Japan
(Received 9 September 1992; accepted 15 September
1992)
Single crystals of ribonuclease MC, a new class of plant ribonuclease from the seeds of the bitter gourd, were obtained from solutions of polyethylene glycol 8000 by the hanging-drop vapour diffusion method. The crystals belong to the orthorhombic space group P2,2,2, with cell dimensions a = 67.28 A, b = 7521 A, c = 38.54 A. The assumption of one monomer per asymmetric unit gives rise to a V, value of 2.29 As/Da. The crystals diffract beyond 2.0 A resolution and are suitable for high resolution X-ray structure analysis.
Keywords: crystallization;
ribonuclease;
Recognition of nucleotide bases in nucleic acids by proteins is of central importance in molecular biology. Many structural studies of complexes between proteins and their target nucleic acids have been carried out by X-ray diffraction and other physical methods. A large number of RNases with different base specificities have been isolated from a variety of organisms and investigated extensively from both structural and functional viewpoints and are well understood in terms of a model system of nucleic acid/protein interaction (Heinemann & Saenger, 1982; Wlodawer et al.; 1982; Arni et al., 1988). These enzymes are relatively small proteins with molecular masses of 11 to 14 kDa. A vast amount of information has been accumulated on these microbial and mammalian RNases and it has turned out that they share a common structural folding (for structural comparison of this class of RNases, see Hill et al., 1983). Little information is available on the structure-function relationship of RNase with the higher molecular mass of 20 to 30 kDa, and although a vast amount of information has been accumulated on microbial and mammalian
T Author addressed.
to whom
all correspondence
0022%2836/92/241271-03$08.00/O
should be
X-ray
crystallography
RNases, very few studies of the plant RNases have been pursued so far. RNase Mc$ was isolated from the seeds of bitter gourd Momordica charantia by Hiroyuki et al. (1991). The enzyme consists of a single polypeptide chain of 191 residues and has a calculated molecular mass of 21,259 Da. RNase MC has a remarkably high specificity toward CPU, ApU and UpU (Hiroyuki et al., 1991). Comparison of the amino acid sequence of RNase MC with those of RNase T2 (Kawata et al., 1988) and RNase Rh (Horiuchii et al., 1988), which are base non-specific RNases from fungi, shows some homology (approx. 28%). Although the substrate specificity of RNase MC is different from those of fungal RNases and thle level of sequence identity is relatively low, they share two segments of highly conserved residues. The t’wo longest regions of homology at positions 32 to 38 and 81 to 92 contain His34, His89 and lGlu85. Similar residues have been identified as active site residues in RNase T2 (Kawata et al., 1990) and RNase Rh (Sanda et al., 1985) and in the recently solved crystal structure of RNase Rh (Hiroyuki et
$ Abbreviation used: RNase Momordica charantia.
1271
MC, ribonuclease
of
0 1992 Academic Press Limited
1272
A. De & G. Punatsu
al., 1992), these residues are found to be clustered forming the active site. This suggests that in RNase MC, these homologous residues may also be involved in the catalytic mechanism or in the formation of the active sit’e. Recently, it has been found that S-allelic glyeoproteins from style extracts of Nicotiana alata involved in self-incompatibility in flowering plants are RNase (McClure et al.: 1989), which exhibit this activity in arresting pollen tubule growth (McClure et al., 1990). The primary amino acid sequences of three S-glycoproteins S2, S3 and S6, show some regions that are perfectly conserved and other regions that are less homologous (Anderson et al.; 1986). Sequence analysis of RNase MC shows a high identical residues) to these homology (4P y. S-glycoproteins S2, 53 and S6 (Anderson et al., 1986, 1989), much higher in comparison with those of fungal RNases, RNase T2 and RNase Rh (approx. 10%). In addition to the two highly conserved segments described above, there are also other individual conserved positions in the sequence of all S2, S3 and S6 glycoproteins. All eight cysteine residues in RNase MC are totally conserved, whereas only five cyst,eine residues are conserved in the sequence of RNase T2 and RNase Rh. Since the local homology around the essential residues is fairly high in this new class of RNases, it is possible that they may share a common backbone fold as in the much smaller class of microbia,l RNases. In order to understand the structure-function relationships of RNases from plan% origin and to obtain knowledge of its evolutionary relationships to other RNases and S-glycoproteins, we have crystallized RNase Me to determine its three-dimensional structure. Lyophilized protein was dissolved in 20 marITris ’ HCl buffer at pH 7.0 to a final concentration of 10 mg/ml. Crystals were obtained by the hangingdrop vapour diffusion method (McPherson, 1982) using polyethylene glycol (PEG) 8000 as precipitant at 20°C. Drops containing 5 ~1 of RNase MC and 5 ~1
Figure I. An orthorhombic crystal form of RNase MC grown by vapour diffusion using the hanging-drop method using PEG 8000: 200 rnM-sodium acetate and 100 mivr-sodium cacodylate buffer at pH 6%.
Figure 2. 4 screened 15’ precession photograph oi’ tine Ok1zone. The exposure time was 22 h at, 45 kV and 88 mA, at a crystal-to-film distance of 60 mm. This photograph reveals Laue symmetry mm and the absence of reflections with odd indices of the classes Ok0 and 001. An additional precession photograph of the hQZ zone reveals Laue symmetry mm and the additional absence of reflections with odd indices of the class hO0, indicating the space group P2,2,2,.
of well solution, were equilibrated against 1 ml of well solution comprising 30 to 31 ye (w/v) PEG 8000, 200 rnx-sodium acetate and 100 mysodium cacodylat’e buffer (pH 6.6 to 6.8). Rhombic prism-like crystals appeared aft,er three t,o four days and reached their maximal dimensions (0.25 mm x 0.3 mm x 0.7 mm) after two weeks (Fig. 1). Preliminary X-ray diffraction experiments were performed using a precession Camera with Nifiltered CuKcl radiation generated by a Rigaku RU-200 X-ray generator (45 kV: 80 mA, focal size 0.3 mm). Screened 15” precession photographs of two principal zones Ok1 and h01 (Fig. 2) loca,ted 90” apart were taken. These photographs reveal Laue symmetry mm and the absence of reflections with odd indices of the classes hO0, Ok0 or 001; indicating the space group P2,2,2,. The unit cell dimensions are a = 67.28 A, b = 75.21 & c = 38.54 8; giving the volume unit 1.95 x IO5 A3 of the cell as (1 A = 0-I nm). Assuming one RNase MC molecule per asymmetric unit, V, has a value of 2.29 A3/Da, which corresponds to a solvent content of 46 ?A. These values are well within the range for protein crystals (Matt,hews, 1968). The V, value is very similar to the value of 2.60 A3/Da report’ed for the homologous RNase Rh (Hiroyuki et al.; 1989). The crystals diffract beyond 2.0 A resolution and only a slight deterioration was observed after 80 hours of exposure to X-rays at room temperature. Attempt’s are now underway to find a suitable heavy-atom derivat’ive.
Crystallization
This work has been carried out in the Paul S. Freemont at ICRF, London. We Freemont, Michael A. Gorman, John M. Vrielink for their helpful discussions Freemont for the use of X-ray facilities. to thank all his colleagues at Khalisani support.
Laboratory of Dr thank Dr Paul S. Lally & Dr Alice and Dr Paul S. A.D. would like College for their
References Anderson, M. A.; Cornish, E. C., Mau, S.-L., Williams, E. G., Hoggart, R., Atkinson, A., Bonig, I.; Grego, B., Simpson, R.; Roche, P. J., Haley, J. D., Penschow, J. D., Niall, H. D., Tregear, G. W., Coghlan, J. P., Crawford, R. J. & Clarke, A. E. (1986). Cloning of cDNA for a stylar glycoprotein associated with expression of self-incompatibility in AVicotiana alata. Nature (London), 321, 38-44. Anderson M. A., McFadden, G. I., Bernatzky, R., Atkinson, A.; Orpin, T., Dedman, H., Tregear, G., Fernley, R. & Clarke, A. E. (1989). Sequence variability of three alleles of the self-incompatibility gene of Nicotiana alata. Plant Cell, 1, 483-492. Arni, R., Heinemann, U., Tokuoka, R. & Saenger, W. (1988). Three-dimensional structure of the ribonuclease T, *2’-GMP complex at 1.9 A resolution. J. Biol. Chem. 263, 15358-15368. Heinemann, U. 8: Saenger, W. (1982). Specific proteinnucleic acid recognition in ribonuclease Ti-2’.guanylic acid complex: an X-ray study. Nature (London), 299, 27-31. Hill; C., Dodson, G., Heinemann, U., Saenger, W.; Mitsui, Y., Nakamura; K.; Borisov, S., Tischenko, G.; Polyakov, K. & Pavlovsky, S. (1983). The structural and sequence homology of a family of microbial ribonucleases. Trends Biochem. Sci. 8, 364-369. Y. i Nakamura, K. T., Hiroyuki, K., Mitsui, Wakabayashi, E., Ohgi, K. & Irie, M. (1989). Crystallization of a new class of microbial ribonuclease from Rhizopus niveus. J. Mol. Biol. 206, 791-792.
Edited
Notes
1273
Hiroyuki, I., Kimura, M., Arai, M. 8: Fanatsu, G. (1991). The complete amino acid sequence of ribonuclease from the seeds of bitter gourd (Momordica &am&a). FEBS Letters, 284, 161-164. Hiroyuki, K.; Mitsui, Y. E.; Ohgi, K., Irie, M., Mizumo, H. & Nakamura, K. T. (1992). Crystal and molecular structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus. FEBS Letters, 306, 189-192. Horiuchi, H.; Yanai, K., Takagi, M., Yano, K., Wakabayashi, E., Sanda, A., Mine, S., Ohgi, K. & Irie; M. (1988). Primary structure of a base nonribonuclease specific from Rhizopus niveus. J. Biochem. (Tokyo), 103, 408418. Kawata, Y., Sakiyama, F. 8: Tamaoki, H. (1988). A.minoacid sequence of ribonuclease T2 from Aspergillus oryzae. Eur. J. Biochem. 176, 683-697. Kawata, Y., Sakiyama, F., Hayashi, F. & Kyogoku, Y. (1990). Identification of two essential histidine residues of ribonuclease T2 from Aspergillus oryzae. Eur. J. Bioehem. 187: 255-262. Matthews, B. W. (1968). Solvent content of protein crystals. J. Mol. Biol. 33, 491-497. McClure, B. A., Haring, V., Ebert, P. R., Anderson, M. A., Simpson, R. J., Sakiyama, F’. & Clarke, A. E. (1989). Style self-incompatibility gene products of alata are ribonucleases. Nature (London), Nicotiana 342, 955-957. McClure, B. A., Gray, J. E., Anderson M. A. & Clarke, A. E. (1990). Self-incompat’ibility in Nicotiana alata involves degradation of pollen rRNA. Nature (London), 347; 757-760. McPherson, A. (1982). Preparation and Analysis of Protein Crystals; pp. 96-97, J. Wiley & Sons Inc., New York. Sanda, A., Takizawa, Y., Iwama, M. & Irie, M. (1985). Modification of a ribonuclease from Rhizopus Sp. with 1-cyclohexyl-3-(2.morpholinyl-(4)-ethyl) carbodiimide p-toluenesulfonate. J. Biochem. (Tokyo), 98, 125-132. Wlodawer, A., Bott, R. & Sjolin, L. (1982). The refined crystal structure of ribonuclease A at 2.0 A resolution. J. Biol. Chem. 257, 1325-1332.
by A. Klug
