J. Mol. Eiol. (1990) 212, 15-16
Crystallization
of a Chymotrypsin Inhibitor Erythrina caffra Seeds
fro
Huey-Sheng Shieh, Nancy K. Leimgruber, Mark 0. Palmier, Tze-Chein Wun Richard M. Leimgruber and Sherin S. Abdel-Meguid Corporate Research and Development Monsanto, 700 Chesterfield Village Parkway Chesterfield Village, MO 63198, U.S.A. (Received 16 August
1989; accepted 16 November 1989)
Crystals of a chymotrypsin inhibitor from Erythrina ca@ra seeds have been grown out of lithium sulfate, by the hanging drop method of vapor diffusion. The crystals belong to the rhombohedral space group R32, with a = 6’7.2 A and a = 99.4”, and diffract to 3 A resolution.
tion at 26,000 g for one hour. Triton X-100 was added to the clarified solution to attain a final concentration of 1%. This solution was passed through a chymotrypsin-Sepharose 4B column at 4°C. The column was washed with five column volumes of phosphate-buffered saline containing 0.4 iv-NaCl and 1 O/O(v/v) Triton X-100, then with phosphate-buffered saline until the effluent lbecame free of detergent. The bound inhibitors were eluted from the column with 0.2 M-glycine. HCI (pH 2.2), neutralized by adding Tris-OH and extensively dialyzed against 05 M-NaCl. Two molecular weight species of proteins were observed when the dialyzed eluent was analyzed by SDS/polyacrylamide gel electrophoresis. The molecular weights of these two species were estimated from the SDS/polyaerylamide gel to be 23,000 and 20,000. The two species were further separated by pooling the eluent from the chymotrypsin-Sepharose 4B column into two portions. One contained mostly the high molecular weight species and the other contained mostly the low molecular weight species. Eaeb of these two portions was then further purified by an additional pass through a chymotrypsin-Sepharose column, using the same procedure described above, and were tested for their inhibitory activities. The 20,000 M, species exhibited strong inhibitory activity against t-PA, while the 23,000 M, species exlnbited inhibitory activity against chymotrypsin but showed no t-PA inhibitory activity. Attempts to crystallize the species that showed inhibitory activity against t-PA have so far been unsuccessful. ere, however, we report the crystallization and space group determination of the species that exhibit cbymotrypsin inhibitory activity. Until now structural information on the Kunitz-type inhibitors is still scarce.
Seeds of the family Leguminosae contain a large number of proteinase inhibitors. These proteinase inhibitors can be classified into two groups based on their size and cystine content. One is the Kunitztype, which have molecular weights around 20,000 and low cystine content (usually 2 disulfide bridges) and the other is the Bowman-Birk-type, which have molecular weights around 8000 and high cystine content (usually 7 disulfide bridges). Joubert et al. (1987) further divided the Kunitztype proteinase inhibitors into three groups (a, b and e) based on their relative abilities to inhibit chymotrypsin, trypsin and tissue plasminogen activator (t-PAT). Group a inhibitors are relatively specific for chymotrypsin, but are poor inhibitors of trypsin and do not inhibit t-PA. Group b inhibitors are more specific towards trypsin than chymotrypsin, they also do not inhibit t-PA. Group c inhibitors inhibit trypsin, chymotrypsin and t-PA. Erythrina caffra plants belong to the Leguminosae family. Pour Kunitz-type inhibitors (DE-l to DE-4) were isolated from these plants (Joubert, 1982). In an attempt to devise a purification method that is simpler and easier to scale up, we tested the use of trypsin-Sepharose 4B and chymotrypsin-Sepharose 4 to purify these inhibitors. In this method, B. cafra seeds were pulverized in a blender by homogenization in liquid nitrogen. The resulting powder was extracted with cold ( -20°C) acetone to remove lipids, then it was vacuum dried. The dried powder was extracted overnight with 0.5 M-NaCl at 4°C. The liquid extract was clarified by centrifuga-
t Abbreviation activator.
used: t-PA, tissue plasminogen
002%2838/90/020015-02
$03.00/0
15
0 1990 Academii: Press Limited
16
H.-S.
Shieh et al.
Only the partial structure of soybean trypsin inhibitor (residues 1 to 93) has been determined from the crystal structure of its complex with porcine trypsin (Sweet et al., 1974). Before crystallization, samples of the purified chymotrypsin inhibitor from E. ca#ra seeds were dialyzed against 20 mM-Tris (pH 7.0), 100 mMNaCl. Numerous crystallization conditions were screened by the hanging drop method of vapor diffusion (McPherson, 1976); 5 ~1 of the protein solution were mixed with 5 ~1 of various concentrations of aqueous solutions of precipitants, and vapor equilibrated against the same solution added to the protein. Prismatic crystals of the chymotrypsin inhibitor from E. caffra seeds were grown out of 16 to 25% (w/v) saturated lithium sulfate in the presence of 100 m&r-sodium phosphate (pH 45 to 5.5) and 5 to 10 mg protein/ml. Isolated crystals achieved dimensions of 99 mm x 0.25 mm x 618 mm within four weeks at room temperature. The space group, R32, is unambiguously determined by examining precession photographs and a small hemisphere of diffractometer data. The lattice constants are a = 67.2 A (1 A = 0.1 nm) and a = 99.4” (the hexagonal lattice constants are a = 102.5 A and Edited
c = 95.5 A). The V, value (Matthews, 196%) is 2.1 A3/dalton, assuming one molecule (23,000 M,) per asymmetric unit. These crystals show diffraction to 3.0 A resolution on still photographs and are stable in the X-ray beam for several days. These chymotrypsin inhibitor crystals are suitable for single-crystal X-ray diffraction studies. Our strategy in solving the structure is to search for heavy-atom derivatives at 6 A resolution. As soon as phases have been determined and the structure has been solved at that resolution, high-resolution data collection on the native and heavy-atom derivatives will begin. We are grateful to Roderick A. Stegeman, ,%larkE. Gustafson and Kurt D. Junger for valuable assistance. References Joubert, F. J. (1982). Int. J. Biochem. 14, 187-193. Joubert, F. J., Merrifield, E. H. & Dowdle, E. B. D. (1987). Int. 2. Biochem. 19, 601-606. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. McPherson, A., Jr (1976). Methods Biochem. And. 23, 249-345. Sweet, R. M., Wright, H. T., Janin, J,, Chothia, C. H. & Blow, D. M. (1974). Biochemistry, 13, 42124228.
by R. Huber
Crystallization
of a Chymotrypsin Inhibitor Erythrina caffra Seeds
fro
Huey-Sheng Shieh, Nancy K. Leimgruber, Mark 0. Palmier, Tze-Chein Wun Richard M. Leimgruber and Sherin S. Abdel-Meguid Corporate Research and Development Monsanto, 700 Chesterfield Village Parkway Chesterfield Village, MO 63198, U.S.A. (Received 16 August
1989; accepted 16 November 1989)
Crystals of a chymotrypsin inhibitor from Erythrina ca@ra seeds have been grown out of lithium sulfate, by the hanging drop method of vapor diffusion. The crystals belong to the rhombohedral space group R32, with a = 6’7.2 A and a = 99.4”, and diffract to 3 A resolution.
tion at 26,000 g for one hour. Triton X-100 was added to the clarified solution to attain a final concentration of 1%. This solution was passed through a chymotrypsin-Sepharose 4B column at 4°C. The column was washed with five column volumes of phosphate-buffered saline containing 0.4 iv-NaCl and 1 O/O(v/v) Triton X-100, then with phosphate-buffered saline until the effluent lbecame free of detergent. The bound inhibitors were eluted from the column with 0.2 M-glycine. HCI (pH 2.2), neutralized by adding Tris-OH and extensively dialyzed against 05 M-NaCl. Two molecular weight species of proteins were observed when the dialyzed eluent was analyzed by SDS/polyacrylamide gel electrophoresis. The molecular weights of these two species were estimated from the SDS/polyaerylamide gel to be 23,000 and 20,000. The two species were further separated by pooling the eluent from the chymotrypsin-Sepharose 4B column into two portions. One contained mostly the high molecular weight species and the other contained mostly the low molecular weight species. Eaeb of these two portions was then further purified by an additional pass through a chymotrypsin-Sepharose column, using the same procedure described above, and were tested for their inhibitory activities. The 20,000 M, species exhibited strong inhibitory activity against t-PA, while the 23,000 M, species exlnbited inhibitory activity against chymotrypsin but showed no t-PA inhibitory activity. Attempts to crystallize the species that showed inhibitory activity against t-PA have so far been unsuccessful. ere, however, we report the crystallization and space group determination of the species that exhibit cbymotrypsin inhibitory activity. Until now structural information on the Kunitz-type inhibitors is still scarce.
Seeds of the family Leguminosae contain a large number of proteinase inhibitors. These proteinase inhibitors can be classified into two groups based on their size and cystine content. One is the Kunitztype, which have molecular weights around 20,000 and low cystine content (usually 2 disulfide bridges) and the other is the Bowman-Birk-type, which have molecular weights around 8000 and high cystine content (usually 7 disulfide bridges). Joubert et al. (1987) further divided the Kunitztype proteinase inhibitors into three groups (a, b and e) based on their relative abilities to inhibit chymotrypsin, trypsin and tissue plasminogen activator (t-PAT). Group a inhibitors are relatively specific for chymotrypsin, but are poor inhibitors of trypsin and do not inhibit t-PA. Group b inhibitors are more specific towards trypsin than chymotrypsin, they also do not inhibit t-PA. Group c inhibitors inhibit trypsin, chymotrypsin and t-PA. Erythrina caffra plants belong to the Leguminosae family. Pour Kunitz-type inhibitors (DE-l to DE-4) were isolated from these plants (Joubert, 1982). In an attempt to devise a purification method that is simpler and easier to scale up, we tested the use of trypsin-Sepharose 4B and chymotrypsin-Sepharose 4 to purify these inhibitors. In this method, B. cafra seeds were pulverized in a blender by homogenization in liquid nitrogen. The resulting powder was extracted with cold ( -20°C) acetone to remove lipids, then it was vacuum dried. The dried powder was extracted overnight with 0.5 M-NaCl at 4°C. The liquid extract was clarified by centrifuga-
t Abbreviation activator.
used: t-PA, tissue plasminogen
002%2838/90/020015-02
$03.00/0
15
0 1990 Academii: Press Limited
16
H.-S.
Shieh et al.
Only the partial structure of soybean trypsin inhibitor (residues 1 to 93) has been determined from the crystal structure of its complex with porcine trypsin (Sweet et al., 1974). Before crystallization, samples of the purified chymotrypsin inhibitor from E. ca#ra seeds were dialyzed against 20 mM-Tris (pH 7.0), 100 mMNaCl. Numerous crystallization conditions were screened by the hanging drop method of vapor diffusion (McPherson, 1976); 5 ~1 of the protein solution were mixed with 5 ~1 of various concentrations of aqueous solutions of precipitants, and vapor equilibrated against the same solution added to the protein. Prismatic crystals of the chymotrypsin inhibitor from E. caffra seeds were grown out of 16 to 25% (w/v) saturated lithium sulfate in the presence of 100 m&r-sodium phosphate (pH 45 to 5.5) and 5 to 10 mg protein/ml. Isolated crystals achieved dimensions of 99 mm x 0.25 mm x 618 mm within four weeks at room temperature. The space group, R32, is unambiguously determined by examining precession photographs and a small hemisphere of diffractometer data. The lattice constants are a = 67.2 A (1 A = 0.1 nm) and a = 99.4” (the hexagonal lattice constants are a = 102.5 A and Edited
c = 95.5 A). The V, value (Matthews, 196%) is 2.1 A3/dalton, assuming one molecule (23,000 M,) per asymmetric unit. These crystals show diffraction to 3.0 A resolution on still photographs and are stable in the X-ray beam for several days. These chymotrypsin inhibitor crystals are suitable for single-crystal X-ray diffraction studies. Our strategy in solving the structure is to search for heavy-atom derivatives at 6 A resolution. As soon as phases have been determined and the structure has been solved at that resolution, high-resolution data collection on the native and heavy-atom derivatives will begin. We are grateful to Roderick A. Stegeman, ,%larkE. Gustafson and Kurt D. Junger for valuable assistance. References Joubert, F. J. (1982). Int. J. Biochem. 14, 187-193. Joubert, F. J., Merrifield, E. H. & Dowdle, E. B. D. (1987). Int. 2. Biochem. 19, 601-606. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. McPherson, A., Jr (1976). Methods Biochem. And. 23, 249-345. Sweet, R. M., Wright, H. T., Janin, J,, Chothia, C. H. & Blow, D. M. (1974). Biochemistry, 13, 42124228.
by R. Huber
