PROTEINS: Structure, Function, and Genetics 27:160–161 (1997) Crystallization and Preliminary X-Ray Analysis of Recombinant Staphylokinase Anja Rabijns,1 Katrien Baeyens,1 Hendrik L. De Bondt,1 and Camiel De Ranter1* 1Laboratorium voor Analytische Chemie en Medicinale Fysicochemie, Faculteit Farmaceutische Wetenschappen, Katholieke Universiteit Leuven, Leuven, Belgium ABSTRACT Diffraction quality crystals of recombinant staphylokinase (STAR) have been grown by the hanging drop vapor diffusion technique from a solution containing MgCl2, Tris buffer (pH 8.5), and polyethylene glycol 4000. The crystals belong to the monoclinic space group C2 with unit cell dimensions a 5 60.6 Å, b 5 43.7 Å, c 5 54.3 Å, and b 5 115.6°. A complete native data set to 1.8 Å resolution has been collected using synchrotron radiation. Proteins 27:160–161 r 1997 Wiley-Liss, Inc. Key words: protein crystallization; x-ray crystallography; thrombolytic agents; staphylokinase; STAR INTRODUCTION Staphylokinase (STA), a protein produced by certain strains of Staphylococcus aureus, has been shown to have profibrinolytic properties.1 Recently, its mechanism of action2,3 and its potential for thrombolytic therapy4–7 have been the subject of intensive studies. Like streptokinase (SK), which is a routinely used thrombolytic agent, STA is not an enzyme, but it activates plasminogen indirectly by forming a 1:1 complex with plasmin, which in turn activates other plasminogen molecules. In contrast to SK, however, STA is able to induce clot lysis in a plasma milieu without causing systemic plasminogen activation and marked fibrinogen breakdown, thereby decreasing the risk of severe bleeding.8,9 Furthermore, in experimental animal models, STA is relatively more efficient than SK for the dissolution of plateletenriched and/or retracted clots, and STA appears to be less immunogenic than SK.10,11 All these observations prove that STA may be promising as an alternative to streptokinase for the treatment of acute myocardial infarction. Mature STA consists of 136 amino acids (calculated Mr 5 15.5 kDa), but also lower molecular weight derivatives of mature STA, lacking the 6 or the 10 NH2-terminal amino acids, were obtained.12,13 Recent studies have shown that the amino acid at position 26 in STA appears to be important for the activation process of plasminogen by STA. Indeed, substitution of the Met residue at position 26 with Arg or Val results in total loss of the functional r 1997 WILEY-LISS, INC. activity of STA, while substitution with Leu or Cys has little or no influence.14,15 In order to get a better understanding of the mechanism of the nonenzymatic plasminogen activation, we report here the crystallization and some preliminary x-ray data obtained from crystals of recombinant staphylokinase (STAR). MATERIALS AND METHODS Production and Purification of STAR STAR was produced and purified as described previously.5 The final material was obtained at a concentration of 2 to 3 mg/ml. NH2-terminal amino acid sequence analysis revealed a main sequence of Ser-Ser-Ser-Phe, and a minor sequence (approximately 8%) of Gly-Lys-Tyr-Lys.5 Finally, the material was dialyzed and concentrated against 50 mM Hepes (pH 7.5) to a final concentration of 35 mg/ml. Crystallization and Data Collection Crystallization was carried out by the hanging drop vapor diffusion technique at 4°C using Linbro multiwell tissue plates. Each well was filled with 700 µl of reservoir solution that contained 0.2 M MgCl2, 0.1 M Tris buffer (pH 8.5) and 35% polyethylene glycol 4000. Drops consisting of 1.5 µl protein solution (35 mg/ml protein in 50 mM Hepes buffer, pH 7.5) and 1.5 µl of reservoir solution, were placed on coverslips and set to equilibrate against the reservoir solution in the wells. Chuncky x-ray quality crystals of STAR generally appeared after 2 or 3 months. These spontaneous crystals could be utilized for microseeding. To produce a seeding solution, a crystal was placed in 10 µl of the original reservoir solution and crushed with a glass rod. Next, tenfold serial dilutions of the crushed crystal were made in the reservoir solution, and hanging drops consisting of 1.5 µl protein solution and 1.5 µl seeding solution (104-fold dilution) were assembled. Diffraction data were collected at DESY/EMBL *Correspondence to: Camiel De Ranter, Laboratorium voor Analytische Chemie en Medicinale Fysicochemie, Faculteit Farmaceutische Wetenschappen, Katholieke Universiteit Leuven, Van Evenstraat 4, B-3000 Leuven, Belgium. Received 25 April 1996; accepted 17 May 1996. ARTOCARPUS INTEGRIFOLIA REFERENCES 1. Santos-Oliveira, R., Dias-Baruffi, M., Thomaz, S.M.O., Beltramini, L.M., Roque-Barreira, M.C. A Neutrophil Migration Inducing Lectin from Artocarpus integrifolia. J. Immunology 153:1798–1807, 1994. 2. Rot, A. Neutrophil Attractant/Ativation protein-1 (Interleukine-8) Induces in vitro Neutrophil Migration by Haptotatic Mechanism. Eur. J. Immunology 23:303, 1993. 3. Sharon, N., Lis, H. Carbohydrates in Cell Recognition. Scientific American, January, 82–89, 1993. 4. Travis, J. Biotech Gets a Grip on Cell Adhesion. Science 260:906–908, 1993. 5. Mathews, B.W. Solvent content of protein crystals. J. Mol. Biol. 33:491–497, 1968. 161 6. Higashi, T. The processing of diffraction data taken on a screenless Wissenberg camera for macromolecular crystallography. J. App. Cryst. 22:9–18, 1989. 7. Sakabe, N. X-ray diffraction data collection system for modern protein crystallography in a Weissenberg camera and an imaging plate using synchrotron radiation. Nucl Instrum. Methods, A303, 448–463, 1991. 8. CCP4—Collaborative Computer Project, Number 4. The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50:760–763, 1994. 9. Crowther, T. The Fast Rotation Function. In: Rossmann, MG, ed. Molecular Replacement Method. N. York, Gordon and Breach, 173–178, 1972.