Báo cáo khoa học: Crystal structure of a staphylokinase variant A model for reduced antigenicity pptx

Crystal structure of a staphylokinase variant A model for reduced antigenicity Yuhang Chen 1 , I Gang Song 2 , Fan Jiang 1 , Liang Feng 1 , Xiaoxuan Zhang 1 , Yi Ding 1 , Mark Bartlam 1 , Ao Yang 1 , Xiang Ma 1 , Sheng Ye 1 , Yiwei Liu 1 , Hong Tang 1 , Houyan Song 2 and Zihe Rao 1 1 Laboratory of Structural Biology, MOE Laboratory of Protein Science, Tsinghua University, Beijing, China; 2 Department of Molecular Genetics, Shanghai Medical University, China Staphylokinase (SAK) is a 15.5-kDa protein from Staphy- lococcus aureus that activates plasminogen by forming a 1 : 1 complex with p lasmin. R ecombinant S AK has been shown in clinical trials to induce ®brin-speci®c clot lysis in patients with acute myocardial infarction. However, SAK elicits high titers of neutralizing antibodies. Biochemical and protein engineering studies h ave demonstrate d the f easibility of generating SAK variants with reduced antigenicity yet intact thrombolytic potency. Here, we present X-ray crystallo- graphic evidence that the SAK(S41G) mutant may assume a dimeric s tructure. T his dimer model, at 2.3-A Ê resolution, could explain a major antigenic epitope (residues A72±F76 and residues K135-K136) located in the vicinity of the dimer interface as identi®ed by phage-display. These results suggest that SAK antigenicity may be reduced by eliminating dimer formation. We propose several potential mutation sites at the dimer interface that may further reduce t he antigenicity of SAK. Keywords: staphylokinase; dimer; crystal structure; antige- nicity; p rotein enginee ring. Staphylokinase (SAK) is a 136-amino-acid protein pro- duced by the lysogenic phase of Staphylococcus a ureus and has been found to be a thrombolytic agent [1±3] w ith potency similar to streptokinase (SK). Unlike the endoge- nous urokinase (uPA) and tissue-type plasminogen activa- tor ( tPA), SAK has no proteolytic activity. S imilar to SK, SAK acts as a cofactor to form a 1 : 1 complex with human plasmin(ogen). The SAK±plasmin (cofactor±enzyme) com- plex, which has proteolytic activity, can form an enzyme± substrate c omplex with another plasminogen molecule, a nd ef®ciently convert the substrate p lasminogen to an active plasmin [4]. It has been demonstrated that recombinant SAK induces ®brinolysis speci®cally without ®brinogen depletion and has higher ®brinolytic activity compared with other plasminogen activators such as S K, urokinase and tPA [5±10]. In addition, SAK has been shown to be more ef®cient than SK for the dissolution of platelet-enriched and retracted blood clots [11,12]. Therefore in recent years, SAK has become a promising drug and stimulated much structural and protein engineering research. Unfortunately, S AK, like SK, elicits high t iters of antibodies from the second week after the fusion of SAK in patients [13,14]. Three nonoverlapping immunodominant epitopes of SAK were mapped b y a competitive antibody binding study. These included positions K35, E38, E80, and D82 in epitope 1, and K74, E75 and R77 in epitope 3 [15,16]. Recent studies on epitope mapping using negative selection of a phage-displayed antigen library [17,18] con®rmed these ®ndings and also identi®ed new antigenic areas. Combined with three-dimensional a tomic structural information, two major antigenic areas were deduced from these studies. Antigenic area I comprises residues A72±E75 while antigenic area II is located at r esidues N95±E99 [18]. Other minor areas are centered on positions W66, K135, and positions E19, N95, K1 02, and K121 [18]. Attempts have been made by comprehensive site-directed mutagenesis to reduce the immunogenicity of SAK [19±21]. Such SAKSTAR variants, e.g. SAKSTAR (K35A, E65Q, K74Q, D82A, S84A, T90A, E 99D, T101S, E108A, K109A, K130T, K135R, K136A, and insertion K137) have much reduced polyclonal human antibody binding capacity while retaining f ull ®brinolytic potency and ®brin-selectivity in a human plasma milieu [20]. Nevertheless, the r esidual prevalence of speci®c immunocompetence against SAK remains too high for mu ltiple c linical uses. The antibodies induced by treatment with the SAK variants were com- pletely absorbed by the SAK, indicating that immunization was not due to neoepitopes generated by the amino-acid substitutions but to a residual epitope in the variants [19]. The present work was initiated in light of our observa- tions that the dimer of SAK was formed when the lyophilized p owder was stored for 1 month a t 4 °C. Taylor et al. have suggested that dimerization may be the cause of increased antigenicity of acetyl cholinesterase [22], thus deleterious for clinical use. In this report, we present a dimer model from t he crystal structure of SAK(S41G) and extend the epitope search to the quaternary s tructure level. The results indicate that both o f the two well-de®ned epitope areas a re in the vicinity of the dimer interface. This model Correspondence to Z. Rao, Laboratory of Structural Biology, School of Life Science and Engineering, Tsinghua University, Beijing, 100084, China. Fax: + 8 6 10 6277 3145, Tel.: + 86 10 6277 1493, E-mail: raozh@xtal.tsinghua.edu.cn Abbreviations: SAK, staphylokinase; SK, streptokinase; uPA, urokinase; tPA, tissue-type plasminogen activator; a-cyano- 4-hydroxycinnamic, acid; PEG, poly(ethylene glycol). Note: the atomic coordinates f or the SAK a±a dimer has been deposited in the RCSB Protein Data Bank with ac cession no. 1C78. (Received 2 8 August 2001, revised 26 November 2001, accepted 28 November 2001) Eur. J. Biochem. 269, 705±711 (2002) Ó FEBS 2002 may provide detailed information and new insight into the origin of SAK antigenicity, especially in relation to dimer formation. New approaches may also be developed to eliminate the residual antigenicity of SAK by the use of site- directed mutagenesis to disrupt or prevent S AK dimer formation. METHODS AND MATERIAL Protein expression and puri®cation The SAK(S41G) g ene was cloned into the plasmid pSTE- SAK, and then transformed to the Escherichia coli JF1125 strain [23]. The SAK(S41G) protein was overexpressed in soluble form by temperature inductio n and puri®ed by two ion-exchange and one gel ® ltration chromatography steps. The ®nal SAK(S41G) p rotein was o ver 95% pure by SDS/ PAGE and fully active in animal thrombolytic tests [24]. Identi®cation of dimerization of SAK Lyophilized SAK, which had been stored as a powder at 4 °C for a month, was dissolved in Mini-Q water. The SAK dimer w as detected by SDS/PAGE (15%), gel-®ltration chromatography and MALDI-TOF mass spectroscopy. Gel-®ltration analysis was carried out using a Superdex75 column (HR, 10/30, Amersham Pharmacia Biotech), eluting with 25 m M Tris/HCl, pH 8.0, 1 m M phenylmethanesulfo- nyl ¯uoride, 150 m M NaCl. The peak fractions were collected and analyzed by SDS/PAGE. The MALDI-TOF spectrum was obtained in positive ion mode with a Bruker BIFLEX III MALDI-TOF mass spectrometer using a-cyano-4-hydroxycinnamic acid (CCA) as the matrix. Protein crystallization Crystallization trials were carried out using the hanging- drop vapor-diffusion method at 293 K. Crystals were obtained a few days after mixing 2 lL of SAK protein solution (5 mgámL )1 in 10 m M Tris/HCl, pH 8.0) with 2 lL of the reservoir solution [45±50%, w/v, poly(ethylene glycol) (PEG)1000, 100 m M Tris/HCl, pH 7.5±8.5]. Data collection X-ray d iffraction data were collected using an i n-house Rigaku rotating anode X-ray generator with a MAR Research MAR345 image plat e detector. The radiation wavelength was 1.5418 A Ê . The crystal d iffracted to beyond 2.3 A Ê , a nd a data set was collected at 2.3 A Ê resolution with 90.5% completeness. All t he raw data were processed w ith DENZO andscaledwith SCALEPACK [25]. Structure determination The c rystal structure has been determined at 2.3-A Ê resolu- tion using molecular replacement with a model from a previously determined SAKSTAR structure (RCSB PDB accession no. 2SAK) as a search probe. There is a single amino-acid mutation, S41G, between the m odel and the target molecule. There are two molecules in the asymmetric unit. Molecular replacement was performed using AMORE [26] and subsequent re®nement was carried out using X - PLOR [27]. The re¯ection data used in the model re®ne- ment were in the resolution range 20±2.3 A Ê . The initial R work and R free after rigid body re®nement were 36.5 and 40.1%, respectively. A fter several cycles of simulated annealing together with model rebuilding in O [28], R work and R free were reduced to 19.8 and 26.7%, r espectively. The ®nal model statistics for the structure were 0.020 A Ê for bond length and 2.07° for bond angles, with 89.2% of residues in the most-favored regions as determined by PROCHECK [29] (Table 1 2 ) 2 . RESULTS AND DISCUSSION Evidence for SAK dimer in solution The dimerization of SAK in solution was de tected by S DS/ PAGE, gel ®ltration chromatography and MALDI-TOF mass spectroscopy. In a ddition to the band for the S AK monomer (15 k Da), one other band with molecular mass 31 k Da corresponding to the SAK dimer was observed (Fig. 1A) by SDS/PAGE. The MALDI-TOF mass s pec- trum of the power (Fig. 1B) showed that both monomer (m  15 456 Da) and dimer (m  30 910 D a) were present. The gel ®ltration chromatography elution pro®le (Fig. 1 C) of the s tored lyophilized SAK showed two p eaks that were assigned to the dimer (shorter retention time) and the monomer (lo nger r etention time) b y S DS/PAGE analysis (Fig. 1D). These results demonstrated that highly puri®ed SAK could form dimers in solution. SAK dimer model as suggested by the crystal structure Because there is strong evidence of dimer formation in solution from various studies, extensive crystallization trials were performed on the SAK(S41G) variant to explore the dimer model in crystals. We obtained a new crystal form that belonged to the s pace group (P2 1 2 1 2 1 ) and has two molecules in the asymmetric unit. This crystal structure is Table 1. Data collection and re®nement statistics. Numbe rs i n p aren - theses are the co rresponding numbers for the highest resolution shell (2.4±2.3 A Ê ). Data statistics Space group P2 1 2 1 2 1 Unit cell (A Ê ) Resolution (A Ê ) a  43.87, b  59.26, c  102.42, 30±2.3 R merge (%) 5.8 (20.4) No. of unique re¯ections 48 705 (12 387) Completeness 99.8% (99.9%) Re®nement statistics R working (%) 19.7 for 11 959 re¯ections R free (%) 26.0 for 11 959 re¯ections No. of nonhydrogen atoms Protein 1084 ´ 2 Solvent (%) 56 Rmsd from ideal values Bond length (A Ê 2 ) 0.020 Bond angle (deg) 2.07 Average B-factor (A Ê 2 ) 33.7% 706 Y. Chen et al. (Eur. J. Biochem. 269) Ó FEBS 2002 the ®rst to contain more than one SAK molecule in an asymmetric unit. The SAK(S41G) structure presented here is similar to the monomer structure, SAKSTAR, previously reported by Rabijns and coworkers [30]. Comparing the two structures, t here are 16 residues (residue S1±S16) missing from SAKSTAR, while the N-terminus in the SAK(S41G) structure was more structurally de®ned. Molecule A could be traced to residue G7 and molecule B to residue Y9. The structures could be superimposed (residue S16±residue K136) with a rmsd for C a of 0.62 A Ê for the molecule A±molecule B pair; 0.65 A Ê for the SAKSTAR±mole- cule A pair; and 1.49 A Ê for the SAKSTAR±molecule B pair. Inspection of the graphics showed that the largest differences between molecule A and molecule B were localized at the N-terminal ÔarmÕ, which had different conformations. After examining the packing geometries between the two molecules of an asymmetric unit in t he SAK(S41G) crystal, we identi®ed three possible dimer geometries, designated as a±a, h ead±tail, and b±b.Thea±a dimer h as a diad 3 and is characterized as helix-helix packing between the two monomers, as shown in Fig. 2A. The head±tail dimer is formed by a crystallographic translation alo ng one crystal axis of t he SAK monomer, as shown in Fig. 2B. The b±b dimer is formed through contacts between two b turns from the two monomers, which are related to each other by a diad perpendicular to the b sheet (Fig. 2C ). We then examined the crystal packing of the SAKSTAR structure [30]. In t his structure with one molecule in the asymmetric unit, only two dimer geometries were observed. These were similar to the h ead±tail an d b±b geometries, while the a±a packing geometry was not present (see Table 2). The signi®cance of the dimer geometries observed in the current crystal structure can be partially deduced from the buried surface area and the interactions in the dimer interface. The total buried surface areas of the a±a or head± tail geometries were more than 1000 A Ê 2 , m uch larger than the a verage buried surface area of random crystal packing [31]. The interaction between the monomers in the b±b dimer packing geometry was the weakest, and thus this dimer should have the lowest probability o f persisting in solution among these three dimer models. The residues involved in the head±tail dimer interface were not relevant to the known e pitopes. We therefore f ocused on the a±a dimer model, which w as most likely t o b e biologically relevant. Characteristics of the a±a dimer interface The a±a SAK dimer interface is complementary and extensive, burying 1009 A Ê 2 surface area from each mono- mer. In this model, the single a helix in each monomer is juxtaposed with each other in a n antiparallel manner. Most of the residues involved in dimer formation are located within the a helix. In the central region of the a±a dimer interface, the exposed polar side chains of the helices participate in an extended network of salt bridges a nd hydrogen bonds, which are almost completely shielded from the bulk solvent by the hydrophobic side-chains nearby (Fig. 3 A). The A65E±B77R salt bridge is stabilized by intramolecular hydrogen bonding with residues A 78V and B65E nearby, and the network is further strengthened by two additional hydrogen bonds (A65E±B62Y, B65E± A62Y). In the central position of the a±a dimer interface and close to B77R , B 136K forms an i ntermolecular hydrogen bond with A61E. The exposed hydrophobic side-chains A62Y, A66W, B62Y an d B66W of the helix wheels face each other and a re in close van der Waals contact (Fig. 3B). One SAKSTAR variant, which has the s ubstitution of K136A a nd the addition of Lys i n position 137 (ad137K), has reduced antigenicity [19,20]. According to the a±a dimer geometry presented here, a long and bulky lysine residue inserted at position K137 will interfere with the interactions at the dimer interface, and most like ly will disrupt dimer formation. Therefore, we can reasonably argue that the a±a dimer pattern of SAK i n the crystal lattice may b e the one that exists in the solution. Antigenicity and activity of the dimer The a±a dimer model was used to investigate the charac- teristics of the dimer interface. A detailed list of all the important residues located at the antigenic sites, the substrate binding site and t he dimer interface is given in Table 3. Fig. 1. Evidence for SAK dimer in solution. 6 (A) SDS/PAGE analysis of the lyophilized highly puri®ed SAK (A). Lane 1 n ewly puri®ed SAK; lane 2 lyo philized puri®e d SAK after storage at 4 °C for 1 month; Lane M, molecular mass stand ards. (B ) Th e M ALDI-TOF mass spectrum of the lyophilized puri®ed SAK; the peak a t 15456 Da indicates th e S AK mo nomer, the peak at 30 910 Da indica tes t he SAK dimer. (C) The elution pro® le of lyophiliz ed SAK on a S uperdex75 column (HR 10/30). (D). SD S/PAGE analysis of the eluted peaks from the gel-®ltration column; lane 1 the peak corresponding to SAK dimer at sho rter retention tim e; lane 2 the p eak corresponding to SAK monomer at longer retention time; Lane M, m olecular mass standards. Ó FEBS 2002 A SAK dimer model: implications in SAK antigenicity (Eur. J. Biochem. 269) 707 Table 2. Buried surface areas and hydrogen bonds of the SAK dimer models. Accessible surface areas are calculated with a probe radius 1.4 A Ê added to the van der Waals rad ius. Dimer model Buried surface area (A Ê 2 ) Hydrogen bonds Salt bridges Residues involved in hydrophobic interaction P2 1 2 1 2 1 1009 A 65Glu OE1±B 62Tyr OH, A 77Arg NH 1 ±B 65Glu OE 2 A 62Tyr, A 66Trp a±a A 77Arg NH 2 ±B 78 Val O A 65Glu OE 1 ±B 77Arg NH 2 , B 62Tyr, B 65Trp A 62Tyr OH±B 65Glu OE 1 , A 65Glu OE 2 ±B 77Arg NH 1 , A 61Glu OE 1 ±B136Arg N Z A 58Glu OE 1 ±B 74Lys N Z P2 1 2 1 2 1 1139 A 39Leu O±B 115Asp N, None A8Lys, A39Leu, A72Ala Head±tail A 42Pro O±B 118 OE2 A 73Tyr OH±B 113Val O A 41Ser OG±B 115Asp O A73Tyr, A76Phe, B51Pro, B52Gly, B112Val, B121Lys, P2 1 2 1 2 1 492 A 99GluOE 1 ±B 130LysN Z , None None b±b A 130LysN Z ±B 99GluOE 1 SAKSTAR Head±tail 872 73 Tyr OH±113 Val¢N, 39 Leu O±B 115 Asp¢N, 72 Ala O±50 Lys¢N Z None 39Leu, 72Ala, 73Tyr, 76Phe, 51¢Pro, 52¢Gly, 112¢Val, 135 Arg N Z ±54 Thr¢OG 1 SAKSTAR 553 99Glu OE 2 )130Lys¢N Z None None b±b 130Lys N Z ±99Glu¢OE 2 Fig. 2. The packing of SAK molecules in the P2 1 2 1 2 1 crystal and the dimer model based on the crystal structure. (A) The a±a dimer has a diad and is characterized as h elix±helix packing between the two monomers. (B) The head±tail d imer is formed by a crystallographic t ranslation along one crystal axis of the SAK monomer. (C) The b±b dimer is formed through contacts betwe en two b turns from the two monomers, which are related to each other by a diad perpendicular to the b sheet. The ®gures a re drawn with MOLSCRIPT [34] and r endered by RASTER 3 D [35]. 708 Y. Chen et al. (Eur. J. Biochem. 269) Ó FEBS 2002 First, we c ompared the locations of the buried surface areas at the dimer interface with the antigenic sites identi®ed in previous studies. We found that one of the major epitopes, comprising residues A72±F76 and K135±K136, lies in t he vicinity and overlaps the dimer surface at the interface (Fig. 4A,B). Therefore, dimerization could bury some of the major epitopes (for instance the one containing residue K136), making it inaccessible to an antibody (Fig. 4 A), or dimerization could pull some of th e major epitopes (for instance those containing residues E75, etc.) together, making a new speci®c B cell e pitope (Fig. 4B). Another possibility is that t he dimer will be more likely t o activate B cells because of t he presence of more epitopes in the dimer than in the monomer. Second, residues E65 and D69 buried in the dimer interface are involved in cofactor±substrate binding in the ternary enzyme±cofactor±substrate complex [32,33], there- fore a±a dimer formation will block cofactor±substrate binding (Fig. 4B). Model rebuilding stud ies of the ternary enzyme±cofactor±substrate c omplex based on th e a±a dimer model and the t ernary microplasmin±SAK±micro- plasmin crystal complex [32] also showed that the a±a dimer cannot form the ternary complex due to steric hindrance, suggesting that a±a dimer formation may destroy SAK thrombolytic activity. Implications in the design of SAK variants The discovery of a previously unknown a±a dimer geometry and the consequent mapping (Fig. 4A,B) of the antigenic sites in r elation to the dimer i nterface can explain many of the previous studies (summarized in Table 3). Dimer formation p rovides a convincing interpretation of some previous mutation studies, particularly those of the mutant Table 3. Antigenic sites and binding sites identi®ed by s tructural and protein engineering studies. 5 Study method Area Amino acid residues on SAK variants Competitive antibody binding [15] Epitope I K74, E75, R77 Epitope II ? Unknown (? could be dimer speci®c) Epitope III K35, E65, E80, D82, K130, K135 Negative selection of phage display library [12] Major Minor 72±76, 95±99 6,19,66,102,121,135 Cofactor-enzyme binding [32] Ternary complex K10, K11, E19, Y24, M26, N28, E38, S41, R43, Y44, E46 Cofactor-substrate binding [32] Ternary complex E46, P48, Y62, W66, A70, Y73 Variants with reduced antigenicity [19±21] SAKSTAR variant I E65D, K79R, E80A, D82A, K130T, K135R SAKSTAR variant II K35A, E65Q, K74Q, D82A, S84A, T90A, E99D, T101S, E108A, K109A, K130T, K135R, K136A, and insertion K137 Dimer a±a interface a Molecule A E58, E61, Y62, E65, W66, D69, R77, V78, V79, E134, K136 Molecule B E58, Y62, E65, W66, D69, K74, R77, V78, K136 a The residues involved in the dimer interface are calculated using a cut-o distance of 4.5 A Ê . Fig. 3. Views of the salt bridge and hydrogen bonding networks (A) and the hydrophobic residues in the dimer interface (B). (A) Close up view of the salt bridge and hydr ogen bonding networks in the dimer interface; more details are shown in Table 2. (B) The hydrophobic residues in the dimer interface. The ®gures are drawn with MOLSCRIPT [34] and ren- dered by RASTER 3 D [35]. Ó FEBS 2002 A SAK dimer model: implications in SAK antigenicity (Eur. J. Biochem. 269) 709 SAKSTAR (E65Q, K 74Q, D82A, S84A, T90A, E99D, T101S, E 108A, K 109A, K 130T, K135R), and the peculiar binding that the substitution of K136A and addition of Lys in position 137 (ad137K) 4 reduced antibody binding from 50% to around 30% [19,20]. The insertion of residue K137 may b e suf®cient to block dimer formation d irectly. The model also suggested that some of the antigenic sites identi®ed by previous studies are conformationally dimer speci®c, particular epitope II [15]. Based on the a±a dimer model, we propose a promising strategy f or designing SAK variants with reduced antigenicity, namely, mutations aimed at disrupting the complementarities of the dimer interface or blocking the dimer interactions directly. We can either target the residues that are directly involved in dimer formation or introduce new b arrier residues that could disrupt the dimer interface. All these potential sites are listed in Table 3. CONCLUSION In this study, we have presented evidence for SAK dimer formation i n solution and a dimer model based on its structure in a new crystal form. The dimerization of SAK may be deleterious for clinical use. The a±a dimer model provides a novel basis for designing mutations aimed at further reducing the antigenicity by disruption o f S AK dimer formation. ACKNOWLEDGEMENTS We thank Dr Robert S im and Dr L. L. Wong f or helpful discussions. This research was supported by the following grants: NSFC no. 39870174 and no. 39 970155; Project Ô863Õ no. 103130306; Project Ô973Õ no. G1999075602, no. G1999011902 and no. G19 9805110 5. REFERENCES 1. Collen, D., Schlott, B., Engelborghs, Y., Van Hoef, B., Hartmann, M., Lijnen, H.R. & Behnke, D. (1993) O n the mechanism of the activation of human plasmin ogen by recom binant staphylok inase. J. Biol. Chem. 268, 8284±8289. 2. Collen,D.,VanHoef,B.,Schlott,B.,Hartmann,M.,Guhrs,K.H. & Lijnen, H.R. (1993) Mechanisms of activation of mamma- lian plasma ® brinolytic systems with streptokinase a nd with recombinant staphylokinase. Eur. J. Biochem. 216 , 307±314. 3. Silence, K., Collen, D. & Lijnen, H.R. (1993) Interaction between staphylokinase, plasmin(ogen), and alpha 2 -antiplasmin. Recycling of staphylokinase after neutralization o f the plasm in-staphylo- kinase c omplex by a lpha 2 -antiplasmin. J. Biol. C hem. 268, 9811± 9816. 4. Lijnen, H.R. 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Ó FEBS 2002 A SAK dimer model: implications in SAK antigenicity (Eur. J. Biochem. 269) 711 . Crystal structure of a staphylokinase variant A model for reduced antigenicity Yuhang Chen 1 , I Gang Song 2 , Fan Jiang 1 , Liang Feng 1 , Xiaoxuan. hydrophobic side-chains A6 2Y, A6 6W, B62Y an d B66W of the helix wheels face each other and a re in close van der Waals contact (Fig. 3B). One SAKSTAR variant, which has