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

This dimer model, at 2.3-A resolution, could explain a major antigenic epitope residues A72—-F 76 and residues K 135-K 136 located in the vicinity of the dimer interface as identified by

Crystal structure of a staphylokinase variant

A model for reduced antigenicity

Yuhang Chen’, Gang Song’, Fan Jiang’, Liang Feng’, Xiaoxuan Zhang’, Yi Ding’, Mark Bartlam',

Ao Yang’, Xiang Ma’, Sheng Ye", Yiwei Liu’, Hong Tang', Houyan Song? and Zihe Rao’

"Laboratory of Structural Biology, MOE Laboratory of Protein Science, Tsinghua University, Beijing, China; 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

complex with plasmin Recombinant SAK has been shown

in clinical trials to induce fibrin-specific clot lysis in patients

with acute myocardial infarction However, SAK elicits high

titers of neutralizing antibodies Biochemical and protein

engineering studies have demonstrated the feasibility 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 structure This dimer model, at 2.3-A resolution,

could explain a major antigenic epitope (residues A72—-F 76 and residues K 135-K 136) located in the vicinity of the dimer interface as identified 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 the antigenicity

of SAK

Keywords: staphylokinase; dimer; crystal structure; antige- nicity; protein engineering

Staphylokinase (SAK) is a 136-amino-acid protein pro-

duced by the lysogenic phase of Staphylococcus aureus and

has been found to be a thrombolytic agent [1-3] with

potency similar to streptokinase (SK) Unlike the endoge-

nous urokinase (uPA) and tissue-type plasminogen activa-

tor (tPA), SAK has no proteolytic activity Similar to SK,

SAK acts as a cofactor to forma 1 : 1 complex with human

plasmin(ogen) The SAK—plasmin (cofactor-enzyme) com-

plex, which has proteolytic activity, can form an enzyme—

substrate complex with another plasminogen molecule, and

efficiently convert the substrate plasminogen to an active

plasmin [4] It has been demonstrated that recombinant

SAK induces fibrinolysis specifically without fibrinogen

depletion and has higher fibrinolytic activity compared with

other plasminogen activators such as SK, urokinase and

tPA [5-10] In addition, SAK has been shown to be more

efficient 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, SAK, like SK, elicits high titers of

antibodies from the second week after the fusion of SAK

in patients [13,14] Three nonoverlapping immunodominant

Correspondence to Z Rao, Laboratory of Structural Biology, School

of Life Science and Engineering, Tsinghua University, Beijing, 100084,

China Fax: + 86 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; «-cyano-

4-hydroxycinnamic, acid; PEG, poly(ethylene glycol)

Note: the atomic coordinates for the SAK o-o dimer has been

deposited in the RCSB Protein Data Bank with accession no 1C78

(Received 28 August 2001, revised 26 November 2001, accepted 28

November 2001)

epitopes of SAK were mapped by 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] confirmed these findings and also identified new antigenic areas Combined with three-dimensional atomic 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 residues N95—E99 [18] Other minor areas are centered on positions W66, K135, and positions E19, N95, K102, 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, E99D, T101S, E108A, K109A, K130T, K135R, K136A, and insertion K137) have much reduced polyclonal human antibody binding capacity while retaining full fibrinolytic potency and fibrin-selectivity in a human plasma milieu [20] Nevertheless, the residual prevalence of specific immunocompetence against SAK remains too high for multiple clinical 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 powder was stored for 1 month at 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 the crystal structure of SAK(S41G) and extend the epitope search to the quaternary structure level The results indicate that both of the two well-defined epitope areas are in the vicinity of the dimer interface This model

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 SAK dimer

formation

METHODS AND MATERIAL

Protein expression and purification

The SAK(S41G) gene 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 induction and purified by two

ion-exchange and one gel filtration chromatography steps

The final SAK(S41G) protein was over 95% pure by SDS/

PAGE and fully active in animal thrombolytic tests [24]

Identification 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 was detected by SDS/PAGE (15%), gel-filtration

chromatography and MALDI-TOF mass spectroscopy

Gel-filtration analysis was carried out using a Superdex75

column (HR, 10/30, Amersham Pharmacia Biotech), eluting

with 25 mm Tris/HCl, pH 8.0, 1 mm phenylmethanesulfo-

nyl fluoride, 150mm 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 nL of SAK protein

solution (5 mgmL"! in 10 mm Tris/HCl, pH 8.0) with 2 pL

of the reservoir solution [45—50%, w/v, poly(ethylene glycol)

(PEG)1000, 100 mm Tris/HCl, pH 7.5-8.5]

Data collection

X-ray diffraction data were collected using an in-house

Rigaku rotating anode X-ray generator with a MAR

Research MAR345 image plate detector The radiation

wavelength was 1.5418 A The crystal diffracted to beyond

2.3 A, and a data set was collected at 2.3 A resolution with

90.5% completeness All the raw data were processed with

DENZO and scaled with sCALEPACK [25]

Structure determination

The crystal structure has been determined at 2.3-A 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 model and the

target molecule There are two molecules in the asymmetric

unit Molecular replacement was performed using AMORE

[26] and subsequent refinement was carried out using

Table 1 Data collection and refinement statistics Numbers in paren- theses are the corresponding numbers for the highest resolution shell

(2.4-2.3 A)

Data statistics

Unit cell (Ả) a= 43.87,

b = 59.26,

c = 102.42,

Resolution (A) 30-2.3

No of unique reflections Completeness

Refinement statistics

Rworking (%) toc (%)

No of nonhydrogen atoms

48 705 (12 387) 99.8% (99.9%) 19.7 for 11 959 reflections 26.0 for 11 959 reflections

Rmsd from ideal values

Bond length (A’) 0.020

X-PLOR [27] The reflection data used in the model refine- ment were in the resolution range 20-2.3 A The initial Ryork and Reee after rigid body refinement were 36.5 and 40.1%, respectively After several cycles of simulated annealing together with model rebuilding in O [28], Rywork and Rf were reduced to 19.8 and 26.7%, respectively The final model statistics for the structure were 0.020 A 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)

RESULTS AND DISCUSSION

Evidence for SAK dimer in solution The dimerization of SAK in solution was detected by SDS/ PAGE, gel filtration chromatography and MALDI-TOF mass spectroscopy In addition to the band for the SAK monomer (15 kDa), one other band with molecular mass

31 kDa corresponding to the SAK dimer was observed (Fig 1A) by SDS/PAGE The MALDI-TOF mass spec- trum of the power (Fig 1B) showed that both monomer (m = 15456 Da) and dimer (m = 30 910 Da) were present The gel filtration chromatography elution profile (Fig 1C) of the stored lyophilized SAK showed two peaks that were assigned to the dimer (shorter retention time) and the monomer (longer retention time) by SDS/PAGE analysis (Fig 1D) These results demonstrated that highly purified 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 space group (P2;2;2,;) and has two molecules in the asymmetric unit This crystal structure is

1 2 M a ib

0u

cs ue 15456 Da

2 §XxD

30910 Da

ANH cm me vế ác so V Tae Set veee oe tư?

( oo ¡92C ca“ li yom az

mA2#0

dimer

f

"¿0a af 1 ¢ 15.0 20.0 a

Fig 1 Evidence for SAK dimer in solution (A) SDS/PAGE analysis of

the lyophilized highly purified SAK (A) Lane 1 newly purified SAK;

lane 2 lyophilized purified SAK after storage at 4 °C for 1 month;

Lane M, molecular mass standards (B) The MALDI-TOF mass

spectrum of the lyophilized purified SAK; the peak at 15456 Da

indicates the SAK monomer, the peak at 30 910 Da indicates the SAK

dimer (C) The elution profile of lyophilized SAK on a Superdex75

column (HR 10/30) (D) SDS/PAGE analysis of the eluted peaks from

the gel-filtration column; lane | the peak corresponding to SAK dimer

at shorter retention time; lane 2 the peak corresponding to SAK

monomer at longer retention time; Lane M, molecular mass standards

the first 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, there are 16 residues (residue SI—S16) missing

from SAKSTAR, while the N-terminus in the SAK(S41G)

structure was more structurally defined 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, of 0.62 A for the molecule

A-molecule B pair; 0.65 A for the SAKSTAR-—mole-

cule A pair; and 1.49 A 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 ‘arm’, which had different

conformations

After examining the packing geometries between the two

molecules of an asymmetric unit in the SAK(S41G) crystal,

we identified three possible dimer geometries, designated as

a-a, head-tail, and 8-8 The o-« dimer has a diad 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 along one crystal

axis of the 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 Bsheet (Fig 2C) We then examined the crystal packing of the SAKSTAR structure [30] In this structure with one molecule in the asymmetric unit, only two dimer geometries were observed These were similar to the head-tail and B-f geometries, while the o-« packing geometry was not present (see Table 2)

The significance 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-« or head— tail geometries were more than 1000 A?, much larger than the average 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 of persisting in solution among these three dimer models The residues involved in the head-tail dimer interface were not relevant

to the known epitopes We therefore focused on the o-« dimer model, which was most likely to be biologically relevant

Characteristics of the a—a dimer interface The o-x SAK dimer interface is complementary and extensive, burying 1009 A* surface area from each mono- mer In this model, the single «helix in each monomer is juxtaposed with each other in an antiparallel manner Most

of the residues involved in dimer formation are located within the ohelix In the central region of the o-« dimer interface, the exposed polar side chains of the helices participate in an extended network of salt bridges and hydrogen bonds, which are almost completely shielded from the bulk solvent by the hydrophobic side-chains nearby (Fig 3A) The A65E-B77R salt bridge is stabilized by intramolecular hydrogen bonding with residues A78V and B65E nearby, and the network is further strengthened by two additional hydrogen bonds (A65E-B62Y, B65E-— A62Y) In the central position of the o-« dimer interface and close to B77R, B136K forms an intermolecular hydrogen bond with A61E The exposed hydrophobic side-chains A62Y, A66W, B62Y and B66W of the helix wheels face each other and are in close van der Waals contact (Fig 3B)

One SAKSTAR variant, which has the substitution of K136A and the addition of Lys in position 137 (ad137K), has reduced antigenicity [19,20] According to the «x 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 likely will disrupt dimer formation Therefore, we can reasonably argue that the o-« dimer pattern of SAK in the crystal lattice may be the one that exists in the solution

Antigenicity and activity of the dimer The œ-œ 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 the dimer interface is given in Table 3

A

ta Pia tex Š Lia aS bd ¿ ve ads iL af +

git ? Us f ` wrt ý ry

ADS \ TY) rd dy > VN 7 way? ‘|

Š ty ở : 1 The Ve :

> 3 ¢ # Qik cà È & }) rey Ỉ 4 Ko LLB - Sy)

-2-A*., -⁄4

teed) Ỷ > oP k

ỳ > tay

( Xƒt ` : tự ú

\ bo số,

nh A dob Ly y

Fig 2 The packing of SAK molecules in the P2,2,2, crystal and the dimer model based on the crystal structure (A) The œ—% dimer has a diad and is characterized as helix—helix packing between the two monomers (B) The head-tail dimer is formed by a crystallographic translation along one crystal axis of the SAK monomer (C) 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 The figures are drawn with MotscripT [34] and rendered by RASTER3D [35]

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 added

to the van der Waals radius

A 62Tyr OH-B 65Glu OE,, A 65Glu OEz-B 77Arg NH,

A 61Glu OE,-B136Arg Nz

A 58Glu OE,-B 74Lys Nz

A 41Ser OG-B 115Asp O

135 Arg N7—-54 Thr’OG,

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 hydrogen bonding networks in the dimer interface; more details are shown in Table 2 (B) The hydrophobic residues in the dimer interface The figures are drawn with MOLSCRIPT [34] and ren- dered by RASTER3D [35]

First, we compared the locations of the buried surface areas at the dimer interface with the antigenic sites identified

in previous studies We found that one of the major epitopes, comprising residues A72-F76 and K135-K136, lies in the 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 4A), or dimerization could pull some of the major epitopes (for instance those containing residues E75, etc.) together, making a new specific B cell epitope (Fig 4B) Another possibility is that the dimer will be more likely to activate B cells because of the 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 o-« dimer formation will block cofactor—substrate binding (Fig 4B) Model rebuilding studies of the ternary enzyme-cofactor—substrate complex based on the oo dimer model and the ternary microplasmin-SAK—micro- plasmin crystal complex [32] also showed that the œ-œ dimer cannot form the ternary complex due to steric hindrance, suggesting that oo dimer formation may destroy SAK thrombolytic activity

Implications in the design of SAK variants The discovery of a previously unknown oo dimer geometry and the consequent mapping (Fig 4A,B) of the antigenic sites in relation to the dimer interface can explain many of the previous studies (summarized in Table 3) Dimer formation provides a convincing interpretation of some previous mutation studies, particularly those of the mutant

Table 3 Antigenic sites and binding sites identified by structural and protein engineering studies

Epitope HI K35, E65, E80, D82, K130, K135

Cofactor-enzyme binding [32]

Cofactor-substrate binding [32]

Variants with reduced antigenicity [19-21]

Dimer o-c interface*

Ternary complex Ternary complex SAKSTAR variant I SAKSTAR variant II

Molecule A Molecule B

K10, K11, E19, Y24, M26, N28, E38, S41, R43, Y44, E46 E46, P48, Y62, W66, A70, Y73

E65D, K79R, E80A, D82A, K130T, K135R K35A, E65Q, K74Q, D82A, S84A, T9IDA, E99D, TIOIS, EI108A,

K109A, K130T, K135R, K136A, and insertion K137 E58, E61, Y62, E65, W66, D69, R77, V78, V79, E134, K136

E58, Y62, E65, W66, D69, K74, R77, V78, K136

* The residues involved in the dimer interface are calculated using a cut-off distance of 4.5 A.

A Antigenic area I

⁄A

—

Molecule A

ra

Molecule B

3.4

«

Antigenic area |

B Antigenic area |

Dimer interface * ạ ¥

Fig 4 The top view of the SAK o—« dimer interface (A) and mapping of

residues involved in the antigenic sites and dimer interfaces (B) (A) The

major antigenic area I (residues 72—76, and residues 135-136) in the

close vicinity of the dimer interface is colored in red The mapping

of residues involved in the antigenic sites and dimer interfaces (B) The

œ-œ dimer interface is shown by a dashed line The mutated sites

located in the o-« dimer interface are colored in purple; the antigenic

sites are colored in red, the other o-« dimer interface residues in green

The figures are drawn with Grasp [36]

SAKSTAR (E65Q, K74Q, D82A, S84A, T90A, E99D,

T101S, E108A, K109A, K130T, K135R), and the peculiar

binding that the substitution of K136A and addition of Lys

in position 137 (ad137K) reduced antibody binding from

50% to around 30% [19,20] The insertion of residue K 137

may be sufficient to block dimer formation directly The

model also suggested that some of the antigenic sites

identified by previous studies are conformationally dimer

specific, particular epitope II [15] Based on the o-o dimer

model, we propose a promising strategy for 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 barrier 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 in 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 o-« dimer model

provides a novel basis for designing mutations aimed at further reducing the antigenicity by disruption of SAK dimer formation

ACKNOWLEDGEMENTS

We thank Dr Robert Sim and Dr L L Wong for helpful discussions This research was supported by the following grants: NSFC no

39870174 and no 39970155; Project ‘863’ no 103130306; Project ‘973’

no G1999075602, no G1999011902 and no G1998051105

REFERENCES

1 Collen, D., Schlott, B., Engelborghs, Y., Van Hoef, B., Hartmann, M., Lijnen, H.R & Behnke, D (1993) On the mechanism of the activation of human plasminogen by recombinant staphylokinase

J Biol Chem 268, 8284-8289

2 Collen, D., Van Hoef, B., Schlott, B., Hartmann, M., Guhrs, K.H

& Lijnen, H.R (1993) Mechanisms of activation of mamma- lian plasma fibrinolytic systems with streptokinase and with recombinant staphylokinase Eur J Biochem 216, 307-314

3 Silence, K., Collen, D & Lijnen, H.R (1993) Interaction between staphylokinase, plasmin(ogen), and alphaa-antiplasmin Recycling

of staphylokinase after neutralization of the plasmin-staphylo- kinase complex by alpha>-antiplasmin J Biol Chem 268, 9811—

9816

4 Lijnen, H.R (1995) Mechanism of action and thrombolytic potential of staphylokinase Verh K Acad Geneeskd Belg 57, 303-

314

5 Vanderschueren, S., Barrios, L & Kerdsinchai, P (1995) A ran- domized trial of recombinant staphylokinase versus alteplase for coronary artery patency in acute myocardial infarction The STAR Trial Group Circulation 92, 2044-2049

6 Vanderschueren, S., Stockx, L., Wilms, G., Lacroix, H., Verhae- ghe, R., Vermylen, J & Collen, D (1995) Thrombolytic therapy of peripheral arterial occlusion with recombinant staphylokinase Circulation 92, 2050-2057

7 Collen, D., Lijnen, H.R & Vanderschueren, S (1995) Staphylo- kinase: fibrinolytic properties and current experience in patients with occlusive arterial thrombosis Verh K Acad Geneeskd Belg 57,

183-196

8 Vanderschueren, S., Dens, J & Kerdsinchai, P (1997) A pilot randomized coronary patency trial of double-bolus recombinant staphylokinase versus front-loaded altteplase in acute myocardial infaraction Am Heart J 134, 213-219

9 Collen, D., Vanderschueren, S & Van de Werf, F (1996) Fibrin- selective thrombolytic therapy with recombinant staphylokinase Haemostasis Supplement 4, 294-300

10 Collen, D (1998) Staphylokinase: a potent, uniquely fibrin-selec- tive thrombolytic agent Nat Med 4, 279-284

11 Lijnen, H.R., Van Hoef, B & Collen, D (1995) Interactions

of staphylokinase with human platelets Thromb Haemost 73,

472-477

12 Abdelouahed, M., Hatmi, M., Helft, G., Emadi, S., Elalamy, I & Samama, M.M (1997) Comparative effects of recombinant staphylokinase and streptokinase on platelet aggregation Thromb Haemost 77, 815-817

13 Vanderschueren, S.M., Stassen, J.M & Collen, D (1994) On the immunogenicity of recombinant staphylokinase in patients and in animal models Thromb Haemost 72, 297-301

14 Declerck, P.J., Vanderschueren, S., Billiet, J., Moreau, H & Collen, D (1994) Prevalence and induction of circulating anti- bodies against recombinant staphylokinase Thromb Haemost 71,

129-133

15 Collen, D., Bernaerts, R & Declerck, P (1996) Recombinant staphylokinase variants with altered immunoreactivity I: Con- struction and characterization Circulation 94, 197-206.

16

17

18

19

20

21

22

23

24

25

Collen, D., De Cock, F & Demarsin, E (1997) Recombinant

staphylokinase variants with altered immunoreactivity III:

Species variability of antibody binding patterns Circulation 95,

455462

Jespers, L., Jenne, S., Lasters, I & Collen, D (1997) Epitope

mapping by negative selection of randomized antigen libraries

displayed on filamentous phage J Mol Biol 269, 704-718

Jenne, S., Brepoels, K., Collen, D & Jespers, L (1998) High res-

olution mapping of the B cell epitopes of staphylokinase in

humans using negative selection of a phage-displayed antigen

library J Immunol 161, 3161-3168

Collen, D (1998) Engineered staphylokinase variants with reduced

immunogenicity Fibrinolysis Proteolysis 12 (suppl 2), 59-65

Laroche, Y., Heymans, S., Capaert, S., De Cock, F., Demarsin, E

& Collen, D (2000) Recombinant staphylokinase variants with

reduced antigenicity due to elimination of B-lymphocyte epitopes

Blood 96, 1425-1432

Collen, D., Stockx, L., Lacroix, H., Suy, R & Vanderschueren, S

(1997) Recombinant staphylokinase variants with altered immu-

noreactivity IV: identification of variants with reduced antibody

induction but intact potency Circulation 95, 463-472

Doctor, B.P., Camp, S., Gentry, M.K., Taylor, S.S & Taylor, P

(1983) Antigenic and structural differences in the catalytic subunits

of the molecular forms of acetylcholinesterase Proc Natl Acad

Sci U S A 80, 5767-5771

Zhan, C., Wan, Z & Chang, W (1996) Crystallization and pre-

liminary X-ray diffraction studies of recombinant staphykinase

Acta Cryst D52, 564-565

Lijnen, H.R., Hoef B., De Cock, K., Ueshima, S., Matsuo, O &

Collen, D (1991) On the mechanism of fibrin-specific plasminogen

activation by staphylokinase J Biol Chem 266, 11826-11832

Otwinowski, Z & Minor, W (1997) Processing of X-ray diffrac-

tion data collected in oscillation mode Methods Enzymol 276,

307-326

26

21

28

29

30

31

32

33

34

35

36

Navaza, J (1994) AMORE: an automated package for molecular replacement Acta Crystallogr A50, 157-163

Brunger, A (1993) X-PLOR, Version 3.1 A System for X-Ray Crystallography and NM R Yale University Press, New Haven, CT Jones, A.T., Zou, Y.J., Cowan, S.W & Kjeldgaard, M (1994) Improved methods for the building protein models in electron density maps and location of errors in the models Acta Crystal-

logr A42, 140-149

LaS., KowS., Ki, R.A., McArthur, M.W., Moss, D.S & Thornton, J.M (1993) PROCHECK: a program to check the stereochemical quality of protein structures J Appl Crystallogr

26, 283-291

Rabijns, A., De Bondt, H.L & De Ranter, C (1997) Three- dimensional structure of staphylokinase, a plasminogen activator Nat Struct Biol 4, 357-360

Janin, J & Rodier, F (1995) Protein —protein interaction at crystal contacts Proteins 23, 580-587

Parry, M.A., Fernandez-Catalan, C., Bergner, A., Huber, R., Hopfner, K.-P., Schlott, B., Guhrs, K.-H & Bode, W (1998) The ternary microplasmin-staphylokinase-microplasmin complex is a proteinase-cofactor-substrate complex in action Nat Struct Biol

5, 917-923

Jespers, L., Vanwetswinkel, S., Lijnen, H.R., Van Herzeele, N., Van Hoef, B., Demarsin, E., Collen, D & De Maeyer, M (1999) Structural and functional basis of plasminogen activation by staphylokinase Thromb Haemost 81, 479-485

Kraulis, P.J (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures J Appl Crystallogr 24, 946-950

Merritt, E.A & Bacon, J.A (1997) Raster3D: photorealistic molecular graphic Methods Enzymol 277, 505-524

Nicholls, A., Sharp, K.A & Honig, B (1991) Grasp-graphical representation and analysis of surface properties Proteins 11, 281-

296