J. Mol. Biol. (1990) 214, 25-26
Crystallization and Preliminary Diffraction Studies of Recombinant Human Granulocyte-stimulating Factor (KW2228) Yoshitomo
Nagahara, Noboru Konishi, Yoshihara Yokoo and Noriaki Hirayamat Tokyo Research Laboratories Kyowa Hakko Kogyo Co. Ltd 3-6-6 Asahimachi, Machida Tokyo 194, Japan (Received 19 March 1990; accepted 23 March 1990)
Human granulocyte colony-stimulating factor (hG-CSF) specifically stimulates proliferation of neutrophils. Two crystal forms of a mutant of hG-CSF expressed in Escherichia coli have been obtained using the hanging drop vapour diffusion method. One form is triclinic, space group Pl, with cell dimensions a= 3’7.3 A, b =464 A, c=47*7 A, a= 1055”, fl=980° and y= 1094”. The other is monoclinic, space group C2, with cell dimensions a=8200 A, b=492 A, c=494 A and fl= 1139”. Both crystal forms diffract beyond 2.0 A and are suitable for X-ray analysis.
Colony-stimulating factor is a family of glycoproteins that stimulate the proliferation and differentiation of hematopoietic cells in vitro. Granulocyte colony-stimulating factor, which is a member of this family, stimulates neutrophilic granulocyte colony formation from bone marrow stem cells and induces the terminal differentiation of leukemic cells (Nicola et al., 1983). hG-CSFS was isolated from tumour cell lines and the cDNA cloned (Nagata et al., 1986; splicing produces Souza et al., 1986). Alternative two different cDNAs that encode polypeptides of 177 and 174 amino acid residues (Nagata et al., 1986). The cDNA that encodes the latter was isolated from a cDNA library of lipopolysaccharidestimulating human peripheral blood macrophage (Komatsu et al., 1987). The recombinant hG-CSF was found to be useful in the treatment of 5-fluorouracil-induced hematopoietic injury in mice (Shimamura et al., 1987). A number of mutants have been made to investigate structure-activity relationships (Kuga et al., 1989). A mutant designated KW2228, in which Thrl, Leu3, Gly4, Pro5 and Cysl7 were replaced by Ala, Thr, Tyr, Arg and Ser, respectively, showed more potent granulopoietic activity than that of intact hG-CSF, both in vitro and in vivo (Kuga et al., 1989). Crystallization and preliminary X-ray studies of human granulo-
cyte-macrophage colony-stimulating factor, which has a similar biological function to hG-CSF, has been reported (La Londe et al., 1989) but the structure has not been described. The amino acid sequences of the two growth factors are not at all homologous. We undertook an X-ray analysis of KW2228 to better understand the function of this clinically important protein. The three-dimensional structure is undoubtedly useful to disclose the molecular mechanisms of proliferation and differentiation of hematopoietic progenies. KW2228 was purified by methods described (Konishi & Yokoo, 1990), dialyzed against 50 mbr-potassium/sodium phosphate with 2 mMNaN, (pH 60 or 7.0) or 50 m&i-Tris. HCl with 2 mM-NaN, (pH &O), and concentrated to 40 mg protein/ml by ultrafiltration with Centricon(Amicon) for crystallization. The first trials of crystallization were performed by the microdialysis method. A solution of protein in 50 mlrr-Tris . HCl (pH 8.0) was equilibrated against 10 mMpotassium/sodium phosphate buffer (pH 6.5) at 15 “C. Tabular crystals appeared reproducibly within a week, but various attempts to increase their size were unsuccessful. Large crystals suitable for X-ray diffraction experiments were obtained using the hanging drop vapour diffusion method with polyethylene glycol 6000 as precipitant. The reservoirs contained 50 m&r-KH,PO,, 2 mM-NaN, (pH 52) and 9 to 11 y0 (w/w) polyethylene glycol 6000. Protein solution (4 ~1) buffered with
t Author to whom correspondence should be addressed. $ Abbreviation used: hG-CSF, human granulocyte colony-stimulating factor.
25 OOZZ-2836/99/13002cW2
$03.00/O
0 1990 Academic Press Limited
26
Y. Nagahara et al.
50 miw-potassium/sodium phosphate (pH 6.0) was mixed with an equal volume of each reservoir solution and left to equilibrate in the reservoir at 15°C. Within a few hours a liquid-liquid phase separation occurred yielding what appeared to be coacervate droplets. A similar phenomenon was reported with recombinant human granulocyte-macrophage colony-stimulation factor (La Londe et al., 1989). Prismatic microcrystals appeared on the surface of the coacervate droplets after about one month. The coacervate droplets were gently dissolved and the crystal gradually grew over a period of two to three months to reach maximum dimensions of 065 mm x 955 mm x 0.35 mm. When growth reached the upper limit usually no coacervate droplets were observed at all. Preliminary X-ray data were collected with an Enraf-Nonius precession camera and CuKcl radiation (40 kV, 200 mA) from a rotating anode X-ray generator (Enraf-Nonius FR571) at, 15°C. The cell constants were measured on an Enraf-Nonius CAD-4 diffractometer. In the course of the preliminary diffraction experiments it was found that there were two different crystal forms in the hanging drops. The first, form (form I) is triclinic. space group PI ! with cell dimensions a= 37.3 A. h=46.4 A, c=47.7 A (1 A=@1 nm), cr=1055”, /I= 9%0” and y = 109.4”, and a unit cell volume of 7.26 x IO4 A3. Using these data and a molecular weight of 18,910, t,he volume occupied per dalton (l’,,,) is calculated to be 1.92 for the presence of two molecules per asymmetric unit. The second form (form TT) is monoclinic, space group C2. with cell dimensions a = 82.0 A, b = 49.2 A, c = 494 A and fl= 113.9”. and a unit cell volume of I.82 x IO5 A3. On the assumption that the asymmetric unit is a monomer, one obtains 1’,,,=2.40 A3/dalton. The V,,, values of the two forms are reasonable for protein crystals (Matthew, 1968). Roth crystal forms diffract beyond 2.3 A resolution on still photographs and are sufficiently stable in the X-ray beam. Therefore, both forms appear suitable for at least medium-resolution structure determination. Since form II is more useful for solving phase problems we it. st,arted the collection of dat’a A three-dimensional native dataset, to a resolution
of 1.9 A. has been measured using synchrotron radiation at the Photon Factory (Tsukuba, Japan). The intensity data were collecbed on a screenless Weissenberg camera for macromolecules (Sakabe. 1983). A Fuji imaging plate (Fuji Film) was used as a two-dimensional detector in place of an ordinary X-ray film pack. Collection of the first derivat,ive data is underway. Details of t.his work will he presented in a future publication. in which vve expect to be able to describe t,he overall conformation of this clinically prospective growth factor. We thank Professor N. Sakabe and A. Nakagawa their help with data collection.
for
References Komatsu, Y.. Matsumoto. ‘I’.. Kuga. ‘I’.. Nishi. T.. Sekine, S , Saito, A., Okabe, M.. Morimoto; M.. Itoh, S., Okabe, T. & Takaku. F. (1987). Jpn. .I. Cancer Res. 78, 11791181. Konishi, N. & Yokoo, Y. (1990). J. Biochenl. Jn the press. Kuga. T.. Komatsu, Y.. Yamasaki, M., Sekine. S.. Miyaji. H., Nishi, T.. Sat,o. M., Yokoo, Y Asano, M.. Okabe, M.. Morimot’o, M. 8: Tt’oh, K. (1989). Hiochern. Hiophys. Res. (‘ommun. 159. 103~lll. La Londe. .J. M.. Hanna. I,. S.. Rottoballi. R~.. Berman, H. M. si Vort,. I). (1989). ,I. ;lilol. Hiol. 205. 783-785. Matthews, B. L2’. (1968). J. ~vol. Biol. 33. 491-497. Nagata, S., Tsuchiya. M.. Asano. S.. Kaziro. Y.. Yamazaki. T.. Yamamotao 0 Hirata. Y.. Kubota. N.. Oheda. M., Nomura. H. Di Ono. M. (1986). L17ature(Londcm), 319. 435-418. Nicola, N. A.. Metcalf, I>.. Matsumoto, Al. bi .Johnsotr. G. R. (1983). ,/. Biol. (‘hem. 258, 9017-9023. Sakabe, N. (1983). J. Appl. C’rystallogr. 16. !i42--547. Shimamura, M., Kobayashi, Y.. Yuo. A.. lirabe. A.. Okabe. T.. Komatsu. I’.. Itoh. S. & Takaku. F. (1987). Blood. 69. 353%3X5. Souza, 1,. M., Boone. T. C’., Gabrilove. J., Lai. I’. H.. Zsebo. K. M.. Murdock. I). c’., (‘hazin. V. R.. Bruszewski. tJ.. Lu, H.. Chrn. K. K.. Barendt. ,I.. Platzer, E.. Moore, M. A. 8.. Mertelsmann. R. b Welte. K. (1986). Science. 232. 61L6.5.
Edited by A. Klug
Crystallization and Preliminary Diffraction Studies of Recombinant Human Granulocyte-stimulating Factor (KW2228) Yoshitomo
Nagahara, Noboru Konishi, Yoshihara Yokoo and Noriaki Hirayamat Tokyo Research Laboratories Kyowa Hakko Kogyo Co. Ltd 3-6-6 Asahimachi, Machida Tokyo 194, Japan (Received 19 March 1990; accepted 23 March 1990)
Human granulocyte colony-stimulating factor (hG-CSF) specifically stimulates proliferation of neutrophils. Two crystal forms of a mutant of hG-CSF expressed in Escherichia coli have been obtained using the hanging drop vapour diffusion method. One form is triclinic, space group Pl, with cell dimensions a= 3’7.3 A, b =464 A, c=47*7 A, a= 1055”, fl=980° and y= 1094”. The other is monoclinic, space group C2, with cell dimensions a=8200 A, b=492 A, c=494 A and fl= 1139”. Both crystal forms diffract beyond 2.0 A and are suitable for X-ray analysis.
Colony-stimulating factor is a family of glycoproteins that stimulate the proliferation and differentiation of hematopoietic cells in vitro. Granulocyte colony-stimulating factor, which is a member of this family, stimulates neutrophilic granulocyte colony formation from bone marrow stem cells and induces the terminal differentiation of leukemic cells (Nicola et al., 1983). hG-CSFS was isolated from tumour cell lines and the cDNA cloned (Nagata et al., 1986; splicing produces Souza et al., 1986). Alternative two different cDNAs that encode polypeptides of 177 and 174 amino acid residues (Nagata et al., 1986). The cDNA that encodes the latter was isolated from a cDNA library of lipopolysaccharidestimulating human peripheral blood macrophage (Komatsu et al., 1987). The recombinant hG-CSF was found to be useful in the treatment of 5-fluorouracil-induced hematopoietic injury in mice (Shimamura et al., 1987). A number of mutants have been made to investigate structure-activity relationships (Kuga et al., 1989). A mutant designated KW2228, in which Thrl, Leu3, Gly4, Pro5 and Cysl7 were replaced by Ala, Thr, Tyr, Arg and Ser, respectively, showed more potent granulopoietic activity than that of intact hG-CSF, both in vitro and in vivo (Kuga et al., 1989). Crystallization and preliminary X-ray studies of human granulo-
cyte-macrophage colony-stimulating factor, which has a similar biological function to hG-CSF, has been reported (La Londe et al., 1989) but the structure has not been described. The amino acid sequences of the two growth factors are not at all homologous. We undertook an X-ray analysis of KW2228 to better understand the function of this clinically important protein. The three-dimensional structure is undoubtedly useful to disclose the molecular mechanisms of proliferation and differentiation of hematopoietic progenies. KW2228 was purified by methods described (Konishi & Yokoo, 1990), dialyzed against 50 mbr-potassium/sodium phosphate with 2 mMNaN, (pH 60 or 7.0) or 50 m&i-Tris. HCl with 2 mM-NaN, (pH &O), and concentrated to 40 mg protein/ml by ultrafiltration with Centricon(Amicon) for crystallization. The first trials of crystallization were performed by the microdialysis method. A solution of protein in 50 mlrr-Tris . HCl (pH 8.0) was equilibrated against 10 mMpotassium/sodium phosphate buffer (pH 6.5) at 15 “C. Tabular crystals appeared reproducibly within a week, but various attempts to increase their size were unsuccessful. Large crystals suitable for X-ray diffraction experiments were obtained using the hanging drop vapour diffusion method with polyethylene glycol 6000 as precipitant. The reservoirs contained 50 m&r-KH,PO,, 2 mM-NaN, (pH 52) and 9 to 11 y0 (w/w) polyethylene glycol 6000. Protein solution (4 ~1) buffered with
t Author to whom correspondence should be addressed. $ Abbreviation used: hG-CSF, human granulocyte colony-stimulating factor.
25 OOZZ-2836/99/13002cW2
$03.00/O
0 1990 Academic Press Limited
26
Y. Nagahara et al.
50 miw-potassium/sodium phosphate (pH 6.0) was mixed with an equal volume of each reservoir solution and left to equilibrate in the reservoir at 15°C. Within a few hours a liquid-liquid phase separation occurred yielding what appeared to be coacervate droplets. A similar phenomenon was reported with recombinant human granulocyte-macrophage colony-stimulation factor (La Londe et al., 1989). Prismatic microcrystals appeared on the surface of the coacervate droplets after about one month. The coacervate droplets were gently dissolved and the crystal gradually grew over a period of two to three months to reach maximum dimensions of 065 mm x 955 mm x 0.35 mm. When growth reached the upper limit usually no coacervate droplets were observed at all. Preliminary X-ray data were collected with an Enraf-Nonius precession camera and CuKcl radiation (40 kV, 200 mA) from a rotating anode X-ray generator (Enraf-Nonius FR571) at, 15°C. The cell constants were measured on an Enraf-Nonius CAD-4 diffractometer. In the course of the preliminary diffraction experiments it was found that there were two different crystal forms in the hanging drops. The first, form (form I) is triclinic. space group PI ! with cell dimensions a= 37.3 A. h=46.4 A, c=47.7 A (1 A=@1 nm), cr=1055”, /I= 9%0” and y = 109.4”, and a unit cell volume of 7.26 x IO4 A3. Using these data and a molecular weight of 18,910, t,he volume occupied per dalton (l’,,,) is calculated to be 1.92 for the presence of two molecules per asymmetric unit. The second form (form TT) is monoclinic, space group C2. with cell dimensions a = 82.0 A, b = 49.2 A, c = 494 A and fl= 113.9”. and a unit cell volume of I.82 x IO5 A3. On the assumption that the asymmetric unit is a monomer, one obtains 1’,,,=2.40 A3/dalton. The V,,, values of the two forms are reasonable for protein crystals (Matthew, 1968). Roth crystal forms diffract beyond 2.3 A resolution on still photographs and are sufficiently stable in the X-ray beam. Therefore, both forms appear suitable for at least medium-resolution structure determination. Since form II is more useful for solving phase problems we it. st,arted the collection of dat’a A three-dimensional native dataset, to a resolution
of 1.9 A. has been measured using synchrotron radiation at the Photon Factory (Tsukuba, Japan). The intensity data were collecbed on a screenless Weissenberg camera for macromolecules (Sakabe. 1983). A Fuji imaging plate (Fuji Film) was used as a two-dimensional detector in place of an ordinary X-ray film pack. Collection of the first derivat,ive data is underway. Details of t.his work will he presented in a future publication. in which vve expect to be able to describe t,he overall conformation of this clinically prospective growth factor. We thank Professor N. Sakabe and A. Nakagawa their help with data collection.
for
References Komatsu, Y.. Matsumoto. ‘I’.. Kuga. ‘I’.. Nishi. T.. Sekine, S , Saito, A., Okabe, M.. Morimoto; M.. Itoh, S., Okabe, T. & Takaku. F. (1987). Jpn. .I. Cancer Res. 78, 11791181. Konishi, N. & Yokoo, Y. (1990). J. Biochenl. Jn the press. Kuga. T.. Komatsu, Y.. Yamasaki, M., Sekine. S.. Miyaji. H., Nishi, T.. Sat,o. M., Yokoo, Y Asano, M.. Okabe, M.. Morimot’o, M. 8: Tt’oh, K. (1989). Hiochern. Hiophys. Res. (‘ommun. 159. 103~lll. La Londe. .J. M.. Hanna. I,. S.. Rottoballi. R~.. Berman, H. M. si Vort,. I). (1989). ,I. ;lilol. Hiol. 205. 783-785. Matthews, B. L2’. (1968). J. ~vol. Biol. 33. 491-497. Nagata, S., Tsuchiya. M.. Asano. S.. Kaziro. Y.. Yamazaki. T.. Yamamotao 0 Hirata. Y.. Kubota. N.. Oheda. M., Nomura. H. Di Ono. M. (1986). L17ature(Londcm), 319. 435-418. Nicola, N. A.. Metcalf, I>.. Matsumoto, Al. bi .Johnsotr. G. R. (1983). ,/. Biol. (‘hem. 258, 9017-9023. Sakabe, N. (1983). J. Appl. C’rystallogr. 16. !i42--547. Shimamura, M., Kobayashi, Y.. Yuo. A.. lirabe. A.. Okabe. T.. Komatsu. I’.. Itoh. S. & Takaku. F. (1987). Blood. 69. 353%3X5. Souza, 1,. M., Boone. T. C’., Gabrilove. J., Lai. I’. H.. Zsebo. K. M.. Murdock. I). c’., (‘hazin. V. R.. Bruszewski. tJ.. Lu, H.. Chrn. K. K.. Barendt. ,I.. Platzer, E.. Moore, M. A. 8.. Mertelsmann. R. b Welte. K. (1986). Science. 232. 61L6.5.
Edited by A. Klug
