J. Mol. Biol. (1990) 214, 625-626
Crystals of a-Momorcharin A New Ribosome-inactivating
Protein
Zhang Feng, Wai Wan Li, Hin Wing Yeung Chinese
Chinese Medical Research Centre University of Hong Kong, Shatin, N.T.,
Hong Kong
Shi-zhi Chen, Yao-ping Wang, X¥ Lin, Yi-cheng Dong and Jia-huai Wang-f Institute
Academia
Sinica,
of Biophysics Beijing, 100080, China
(Received 27 October 1989; accepted 6 March
1990)
Crystals of a-momorcharin were obtained by vapour phase diffusion. The crystal belongs to the space group R,, with unit cell constants a = b = 131.3 8, c = 39.5 8. There is one cr-momorcharin molecule in the asymmetric unit; a data set at 3 .& has been collected.
It has been noticed that many, probably even all, plants contain at least one kind of the so-called ribosome-inactivating proteins (RIPsf: Barbieri & Stirpe, 1982; Stirpe & Barbieri, 1986; Roberts & Claude, 1986). The RIPS inactivate eukaryotic ribosomes by cleaving a single adenine base from a highly specific site on the 28 S RNA of the 60 S ribosomal subunit (Endo & Tsurugi, 1987). Although their biological significance in plants, where they are often present in quite high concentrations, is still puzzling plant physiologists, their potential use, conjugated with monoclonal antibodies, as immunotoxins to treat cancer has attracted an increasing number of molecular biologists. There are two types of RIP (Stirpe & Barbieri, 1986). Type I proteins are single-chained, whereas type II proteins are double-chained. The A chain possesses the RIP property and the B chain is responsible for binding the whole protein molecule to the target cell surface and helping the A chain in crossing the cell membrane. Type II RIPS are hence among the most toxic cytotoxins. We have shown by sequence comparison that a type I RIP, trichosanthin, is homologous to the A chain of the type II RIP, ricin (Zhang & Wang, 1986). The conformational similarities of the ricin A-chain and trichosanthin were demonstrated by circular dichroism studies (Kubota et al., 1987). The crystal structures
of both trichosanthin (Pan et al., 1986; Ma et al., 1987) and ricin (Montfort et al., 1987) have been elucidated. The overall fold of trichosanthin and the ricin A chain are almost identical. Since trichosanthin and ricin are from taxonomically distant species, Trichosanthes kirilowii of the Cucurbitaceae family and Ricinus communis of the Euphorbiaceae family, it is convincing that the widely distributed RIPS of both types must originate from the same ancestor and assume the same “RIP fold”. What interests us are the following questions regarding the structural basis of the RIP mechanism. (1) What is the three-dimensional entity of their N-glycosidase activity; (2) what conformakional feature determines the specificities of RIPS from different species; (3) what is the structural element that leads to membrane translocation of the proteins. More structural work is apparently needed. Other than trichosanthin and ricin, there are reports of RIP crystals such as abrin (Wei & Einstein, 1974) and pokeweed antiviral protein (Robertus et aZ., 1977), whose structures are not known. Here, we report our newly obtained crystal of a-momorcharin. The protein was extracted from the seeds of Momordica eharantia (Yeung et al., 1986), which belongs to the Cucurbitaceae family. Despite the fact that a-momorcharin is a glycoprotein, whereas trichosanthin does not contain carbohydrates, they share many common features. Like almost all type I RIPS (Stirpe & Barbieri, 1986), they have a similar molecular mass (26,000 to 31,000 Da) and a strongly basic p1 ( -pH 9). They also have similar secondary structure (Kubota et al.,
t Author to whom all correspondence should be addressed. $ Abbreviation used: RIP, ribosome-inactivating protein.
625 002%2836/90/150625-02
$03.00/O
0 1990 Academic Press Limited
626
2. Feng et a!.
1986). They both induced mid-term abortion in pregnant mice and inhibit cell-free protein synthesis with similar potency (Yeung et al., 1988). We
believe that a-momorcharin should have a conformation even closer to that of trichosanthin than the ricin A chain does. Carefully examining the similarities and differences of three-dimensional structures and a a-momorcharin should help of trichosanthin to answer the questions raised above. a-Momorcharin was purified as described (Robertus et al., 1977). The conventional hanging droplet method was used to screen crystallization conditions. Various precipitants, buffers of different pH, organic solvents and ionic metals were tested. Xormally, 4 ~1 of buffered protein solution at a concentration of 5% (w/v) was placed on a silinized cover-glass. The cover-glass was then placed on a well, which contained about 1 ml of crystallization liquor with higher-concentration salts or organic solvents. In some trials, crystals appeared after a few days of vapour diffusion. However, in most cases crystals grow as clusters of needles or interdigitated twins. Occasionally, single rhombohedral crystals emerged. Under the microscope, they looked very neat, though usually tiny. The best condition obtained so far is 10% (w/v) (MH,),SO,, 0.05 M-Tris. HCl (pH 7.65), 4% (w/v) protein in the droplet
and 23%
(w/v)
buffered
(NH,),SO,
in the
well. The largest crystal size obtained was 0.4 mm x 0.4 mm x 0.1 mm. It seems that single crystals are less stable than twinned crystals. If single crystals are left in t,he droplet too long, their size will gradually decrease as the twins grow. This makes it difficult to harvest large, single crystals. One such relatively large crystal was mounted in a quartz capillary and set on a PWllOO four-circle The crystallographic parameters diffractometer. were measured as a = b = 131.3 8, c = 39.5 a (1 ,& = 0.1 nm) in space group R, (hexagonal setting). Assuming one a-momorcharin molecule of 30,000 Da in the asymmetric unit,, the Mattews, coefficient will then be 2.15 A3/Da, i.e. the crystal contains 43% solvent, the most probable value for protein crystals is given by Matthews (1968). The crystal diffracted at least to 3 L%and stood under a 40 kV, 25 mA X-ray source without significant Edited
decay after one 3 x data set had been collected for two weeks. Single-chained RIPS are thought to be better candidates than double-chain RIPS like ricin in constructing immunotoxins, because they do not have their own cell recognition and binding ability. We hope that our efforts of carrying out structural studies on trichosant,hin, momorcharin and their analogues will contribute in this promising field. This work was supported Foundation.
by grant from the Croueher
References Barbieri, L. & St,irpe, F. (1982). Cancer Surveys, IO. 489-520. Endo, Y. & Tsurugi, K. (1987). .J. Biol. Chem. 262, 8128-8130. Kubota, S.; Yeung, H. W. & Yang; J. T. (1986). &ochim. Biophys. Acta, 871, 101-106. Kubota, S.. Yeung, H. W. & Yang, 9. T. (1987). ink. j. Pept. Protein Res.. 30, 646-651. Ma, X.-Q., Wang, Y.-P. & Wang, J.-H. (1987). Scientia Sinica, B30, 692-697. Matthews, B. W. (1968). J. &i’ol. Biol. 33, 491-497. Montfort, W., Villafranca, J. E., Monzingo, A. F.. Ernst: S. R., Katzin, B., Rutenber; E.? Xuong, K. I-I.; Hamlin, R,. 8: Robertus J. D. (1987). J. BioZ. Chem. 262, 5398-5403. Pan, K.-Z., Zang: Y.-M., Lin. Y.-J., Wu; Z.-W.; Zhang, A., Chen, Y.-Z., Dong, Y.-C.; Ma; X.-Q.. Wang, Y.-P., Wu, S.? Zhang, M.-A.. Chen, S.-Z.. Xia; Z.-X.. Tian, G.-Y., Nix C.-Z., Fan, Z.-C., Ma, Y.-L. & Sun, X.-X. (1986). Scientia Xinica, B29, 26-32. Roberts? W. K. & Claude, P. S. E. (1986). Biosci. Rep. 6, 19-29. Robertus; J. D., Monzongo. A. F. & Irvin, J. D. (1977). Biochem. Biophys. Res. Commun. 74, 775-779. Stirpe, F. & Barbieri; L. (1986). FE&S Letters, 195. l-8. Wei, C. H. & Einstein: R. J. (1974). J. Biol. Chem. 249, 2985-2986. Yueng, H. W., Li, W. W.. Law, L. K.; Chan, W. Y. & Sg, T. B. (1986). Int. J. Pept. Protein Res. 28, 518-524. Yeung, H. W.; Li, W. W., Feng, Z., Barbieri. L. & Stirpe, F. (1988). Int. J. Pept. Protein Res. 31, 265-268. Zhang, X.-J. 8: Wang, J.-H. (1986). Nature {London): 321, 477-478.
by R. Huber
Crystals of a-Momorcharin A New Ribosome-inactivating
Protein
Zhang Feng, Wai Wan Li, Hin Wing Yeung Chinese
Chinese Medical Research Centre University of Hong Kong, Shatin, N.T.,
Hong Kong
Shi-zhi Chen, Yao-ping Wang, X¥ Lin, Yi-cheng Dong and Jia-huai Wang-f Institute
Academia
Sinica,
of Biophysics Beijing, 100080, China
(Received 27 October 1989; accepted 6 March
1990)
Crystals of a-momorcharin were obtained by vapour phase diffusion. The crystal belongs to the space group R,, with unit cell constants a = b = 131.3 8, c = 39.5 8. There is one cr-momorcharin molecule in the asymmetric unit; a data set at 3 .& has been collected.
It has been noticed that many, probably even all, plants contain at least one kind of the so-called ribosome-inactivating proteins (RIPsf: Barbieri & Stirpe, 1982; Stirpe & Barbieri, 1986; Roberts & Claude, 1986). The RIPS inactivate eukaryotic ribosomes by cleaving a single adenine base from a highly specific site on the 28 S RNA of the 60 S ribosomal subunit (Endo & Tsurugi, 1987). Although their biological significance in plants, where they are often present in quite high concentrations, is still puzzling plant physiologists, their potential use, conjugated with monoclonal antibodies, as immunotoxins to treat cancer has attracted an increasing number of molecular biologists. There are two types of RIP (Stirpe & Barbieri, 1986). Type I proteins are single-chained, whereas type II proteins are double-chained. The A chain possesses the RIP property and the B chain is responsible for binding the whole protein molecule to the target cell surface and helping the A chain in crossing the cell membrane. Type II RIPS are hence among the most toxic cytotoxins. We have shown by sequence comparison that a type I RIP, trichosanthin, is homologous to the A chain of the type II RIP, ricin (Zhang & Wang, 1986). The conformational similarities of the ricin A-chain and trichosanthin were demonstrated by circular dichroism studies (Kubota et al., 1987). The crystal structures
of both trichosanthin (Pan et al., 1986; Ma et al., 1987) and ricin (Montfort et al., 1987) have been elucidated. The overall fold of trichosanthin and the ricin A chain are almost identical. Since trichosanthin and ricin are from taxonomically distant species, Trichosanthes kirilowii of the Cucurbitaceae family and Ricinus communis of the Euphorbiaceae family, it is convincing that the widely distributed RIPS of both types must originate from the same ancestor and assume the same “RIP fold”. What interests us are the following questions regarding the structural basis of the RIP mechanism. (1) What is the three-dimensional entity of their N-glycosidase activity; (2) what conformakional feature determines the specificities of RIPS from different species; (3) what is the structural element that leads to membrane translocation of the proteins. More structural work is apparently needed. Other than trichosanthin and ricin, there are reports of RIP crystals such as abrin (Wei & Einstein, 1974) and pokeweed antiviral protein (Robertus et aZ., 1977), whose structures are not known. Here, we report our newly obtained crystal of a-momorcharin. The protein was extracted from the seeds of Momordica eharantia (Yeung et al., 1986), which belongs to the Cucurbitaceae family. Despite the fact that a-momorcharin is a glycoprotein, whereas trichosanthin does not contain carbohydrates, they share many common features. Like almost all type I RIPS (Stirpe & Barbieri, 1986), they have a similar molecular mass (26,000 to 31,000 Da) and a strongly basic p1 ( -pH 9). They also have similar secondary structure (Kubota et al.,
t Author to whom all correspondence should be addressed. $ Abbreviation used: RIP, ribosome-inactivating protein.
625 002%2836/90/150625-02
$03.00/O
0 1990 Academic Press Limited
626
2. Feng et a!.
1986). They both induced mid-term abortion in pregnant mice and inhibit cell-free protein synthesis with similar potency (Yeung et al., 1988). We
believe that a-momorcharin should have a conformation even closer to that of trichosanthin than the ricin A chain does. Carefully examining the similarities and differences of three-dimensional structures and a a-momorcharin should help of trichosanthin to answer the questions raised above. a-Momorcharin was purified as described (Robertus et al., 1977). The conventional hanging droplet method was used to screen crystallization conditions. Various precipitants, buffers of different pH, organic solvents and ionic metals were tested. Xormally, 4 ~1 of buffered protein solution at a concentration of 5% (w/v) was placed on a silinized cover-glass. The cover-glass was then placed on a well, which contained about 1 ml of crystallization liquor with higher-concentration salts or organic solvents. In some trials, crystals appeared after a few days of vapour diffusion. However, in most cases crystals grow as clusters of needles or interdigitated twins. Occasionally, single rhombohedral crystals emerged. Under the microscope, they looked very neat, though usually tiny. The best condition obtained so far is 10% (w/v) (MH,),SO,, 0.05 M-Tris. HCl (pH 7.65), 4% (w/v) protein in the droplet
and 23%
(w/v)
buffered
(NH,),SO,
in the
well. The largest crystal size obtained was 0.4 mm x 0.4 mm x 0.1 mm. It seems that single crystals are less stable than twinned crystals. If single crystals are left in t,he droplet too long, their size will gradually decrease as the twins grow. This makes it difficult to harvest large, single crystals. One such relatively large crystal was mounted in a quartz capillary and set on a PWllOO four-circle The crystallographic parameters diffractometer. were measured as a = b = 131.3 8, c = 39.5 a (1 ,& = 0.1 nm) in space group R, (hexagonal setting). Assuming one a-momorcharin molecule of 30,000 Da in the asymmetric unit,, the Mattews, coefficient will then be 2.15 A3/Da, i.e. the crystal contains 43% solvent, the most probable value for protein crystals is given by Matthews (1968). The crystal diffracted at least to 3 L%and stood under a 40 kV, 25 mA X-ray source without significant Edited
decay after one 3 x data set had been collected for two weeks. Single-chained RIPS are thought to be better candidates than double-chain RIPS like ricin in constructing immunotoxins, because they do not have their own cell recognition and binding ability. We hope that our efforts of carrying out structural studies on trichosant,hin, momorcharin and their analogues will contribute in this promising field. This work was supported Foundation.
by grant from the Croueher
References Barbieri, L. & St,irpe, F. (1982). Cancer Surveys, IO. 489-520. Endo, Y. & Tsurugi, K. (1987). .J. Biol. Chem. 262, 8128-8130. Kubota, S.; Yeung, H. W. & Yang; J. T. (1986). &ochim. Biophys. Acta, 871, 101-106. Kubota, S.. Yeung, H. W. & Yang, 9. T. (1987). ink. j. Pept. Protein Res.. 30, 646-651. Ma, X.-Q., Wang, Y.-P. & Wang, J.-H. (1987). Scientia Sinica, B30, 692-697. Matthews, B. W. (1968). J. &i’ol. Biol. 33, 491-497. Montfort, W., Villafranca, J. E., Monzingo, A. F.. Ernst: S. R., Katzin, B., Rutenber; E.? Xuong, K. I-I.; Hamlin, R,. 8: Robertus J. D. (1987). J. BioZ. Chem. 262, 5398-5403. Pan, K.-Z., Zang: Y.-M., Lin. Y.-J., Wu; Z.-W.; Zhang, A., Chen, Y.-Z., Dong, Y.-C.; Ma; X.-Q.. Wang, Y.-P., Wu, S.? Zhang, M.-A.. Chen, S.-Z.. Xia; Z.-X.. Tian, G.-Y., Nix C.-Z., Fan, Z.-C., Ma, Y.-L. & Sun, X.-X. (1986). Scientia Xinica, B29, 26-32. Roberts? W. K. & Claude, P. S. E. (1986). Biosci. Rep. 6, 19-29. Robertus; J. D., Monzongo. A. F. & Irvin, J. D. (1977). Biochem. Biophys. Res. Commun. 74, 775-779. Stirpe, F. & Barbieri; L. (1986). FE&S Letters, 195. l-8. Wei, C. H. & Einstein: R. J. (1974). J. Biol. Chem. 249, 2985-2986. Yueng, H. W., Li, W. W.. Law, L. K.; Chan, W. Y. & Sg, T. B. (1986). Int. J. Pept. Protein Res. 28, 518-524. Yeung, H. W.; Li, W. W., Feng, Z., Barbieri. L. & Stirpe, F. (1988). Int. J. Pept. Protein Res. 31, 265-268. Zhang, X.-J. 8: Wang, J.-H. (1986). Nature {London): 321, 477-478.
by R. Huber
