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:
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.
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Ó FEBS 2002 A SAK dimer model: implications in SAK antigenicity (Eur. J. Biochem. 269) 711