An anti-urokinase plasminogen activator receptor (uPAR) antibody: crystal structure and binding epitope.
ABSTRACT Human urokinase-type plasminogen activator receptor (uPAR/CD87) is expressed at the invasive interface of the tumor-stromal microenvironment in many human cancers and interacts with a wide array of extracellular molecules. An anti-uPAR antibody (ATN615) was prepared using hybridoma technology. This antibody binds to uPAR in vitro with high affinity (K(d) approximately 1 nM) and does not interfere with uPA binding to uPAR. Here we report the crystal structure of the Fab fragment of ATN615 at 1.77 A and the analysis of ATN615-suPAR-ATF structure that was previously determined, emphasizing the ATN615-suPAR interaction. The complementarity determining regions (CDRs) of ATN615 consist of a high percentage of aromatic residues, and form a relatively flat and undulating surface. The ATN615 Fab fragment recognizes domain 3 of suPAR. The antibody-antigen recognition involves 11 suPAR residues and 12 Fab residues from five CDRs. Structural data suggest that Pro188, Asn190, Gly191, and Arg192 residues of uPAR are the key residues for the antibody recognition, while Pro189 and Arg192 render specificity of ATN615 for human uPAR. Interestingly, this antibody-antigen interface has a small contact area, mainly polar interaction with little hydrophobic character, yet has high binding strength. Furthermore, several solvent molecules (assigned as polyethylene glycols) were clearly visible in the binding interface between antibody and antigen, suggesting that solvent molecules may be important for the maximal binding between suPAR and ATN615 Fab. ATN615 undergoes small but noticeable changes in its CDR region upon antigen binding.
[show abstract] [hide abstract]
ABSTRACT: Urokinase, its receptor and the integrins are functionally associated and involved in regulation of cell signaling, migration, adhesion and proliferation. No structural information is available on this potential multimolecular complex. However, the tri-dimensional structure of urokinase, urokinase receptor and integrins is known. We have modeled the interaction of urokinase on two integrins, alphaIIbbeta3 in the open configuration and alphavbeta3 in the closed configuration. We have found that multiple lowest energy solutions point to an interaction of the kringle domain of uPA at the boundary between alpha and beta chains on the surface of the integrins. This region is not far away from peptides that have been previously shown to have a biological role in urokinase receptor/integrins dependent signaling. We demonstrated that in silico docking experiments can be successfully carried out to identify the binding mode of the kringle domain of urokinase on the scaffold of integrins in the open and closed conformation. Importantly we found that the binding mode was the same on different integrins and in both configurations. To get a molecular view of the system is a prerequisite to unravel the complex protein-protein interactions underlying urokinase/urokinase receptor/integrin mediated cell motility, adhesion and proliferation and to design rational in vitro experiments.BMC Bioinformatics 02/2008; 9 Suppl 2:S8. · 2.75 Impact Factor
An Anti-urokinase Plasminogen Activator Receptor
(uPAR) Antibody: Crystal Structure and Binding Epitope
Yongdong Li1,2, Graham Parry3, Liqing Chen4, Jennifer A. Callahan3
David E. Shaw5, Edward J. Meehan4, Andrew P. Mazar3
and Mingdong Huang1,2⁎
1State Key Laboratory of
Fujian Institute of Research
on the Structure of Matter,
Chinese Academy of Sciences,
155 Yang Qiao Xi Lu,
Fuzhou 350002, People's
Republic of China
2The Graduate School of Chinese
Academy of Sciences, Beijing,
People's Republic of China
3Attenuon L.L.C., 11535
Sorrento Valley Road, Suite 401,
San Diego, CA 92121, USA
4Laboratory for Structural
Biology, Department of
Chemistry, Graduate Programs
of Biotechnology, Chemistry and
Materials Science, University of
Alabama in Huntsville,
Huntsville, AL 35899, USA
5D.E. Shaw Research and
Development, New York,
NY 10036, USA
Human urokinase-type plasminogen activator receptor (uPAR/CD87) is
expressed at the invasive interface of the tumor-stromal microenvironment
in many human cancers and interacts with a wide array of extracellular
molecules. An anti-uPAR antibody (ATN615) was prepared using hybri-
doma technology. This antibody binds to uPAR in vitro with high affinity
(Kd∼1 nM) and does not interfere with uPA binding to uPAR. Here we
report the crystal structure of the Fab fragment of ATN615 at 1.77 Å and the
analysis of ATN615-suPAR-ATF structure that was previously determined,
emphasizing the ATN615–suPAR interaction. The complementarity deter-
mining regions (CDRs) of ATN615 consist of a high percentage of aromatic
residues, and form a relatively flat and undulating surface. The ATN615 Fab
fragment recognizes domain 3 of suPAR. The antibody–antigen recognition
involves 11 suPAR residues and 12 Fab residues from five CDRs. Structural
data suggest that Pro188, Asn190, Gly191, and Arg192 residues of uPAR are
the key residues for the antibody recognition, while Pro189 and Arg192
render specificity of ATN615 for human uPAR. Interestingly, this antibody–
antigen interface has a small contact area, mainly polar interaction with little
hydrophobic character, yet has high binding strength. Furthermore, several
solvent molecules (assigned as polyethylene glycols) were clearly visible in
the binding interface between antibody and antigen, suggesting that solvent
molecules may be important for the maximal binding between suPAR and
ATN615 Fab. ATN615 undergoes small but noticeable changes in its CDR
region upon antigen binding.
© 2006 Elsevier Ltd. All rights reserved.
Keywords: antibody; crystal structure; uPAR; ATN615; Fab
Urokinase-type plasminogen activator receptor
(uPAR), also named CD87,1,2is a cell surface protein
consisting of three cysteine-rich CD59-like domains
and is anchored to the cell surface through a
anchor (GPI). uPAR interacts with a wide array of
ligands including uPA,2,3vitronectin,4the endocytic
receptor uPARAP,5G-protein coupled receptor,6,7a
number of integrins including αMβ2,8,9αVβ5,10,11
α3β112and α5β113and others. Because of its
pleiotropic interactions with various cell surface
ligands, uPAR has been recognized to play an
important role in a number of cellular activities
including cell adhesion and migration, immune
response, wound repair, angiogenesis, inflamma-
tion, and especially tumor invasion and metas-
tasis.13–16Higher uPAR concentrations were found
in extracts of resected tumor tissue or in plasma
from patients with a variety of solid tumors, e.g.
colorectal cancer,17,18breast cancer19,20and lung
Abbreviations used: CDR, complementarity
determining region; PEG, polyethylene glycol; uPA,
urokinase-type plasminogen activator; ATF, the
amino-terminal fragment of uPA; uPAR, urokinase-type
plasminogen activator receptor; suPAR, soluble uPAR.
E-mail address of the corresponding author:
doi:10.1016/j.jmb.2006.10.059J. Mol. Biol. (2007) 365, 1117–1129
0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
cancer.21,22These high levels of uPAR are associated
with poor prognosis and an increased risk of tumor
recurrence and metastatic disease.16The close
association of uPAR to tumor invasion and metas-
tasis has led to the suggestion that inhibiting uPA
binding to uPAR or inhibiting the interaction of
uPAR with other ligands may represent a potential
method for treating metastatic cancer.23–25Antibo-
dies targeting uPAR were demonstrated to be able to
detect the presence of occult tumor metastasis in vivo
and reduce tumor growth and metastases.26,27
ATN615 is a monoclonal anti-uPAR antibody raised
against a chymotryptic fragment of soluble uPAR
(suPAR). ATN615 binds to immobilized human
suPAR with high affinity (Kd∼1 nM), but does not
recognize murine uPAR. The ATN615 Fab fragment
did not block the uPAR–uPA interaction, suggesting
that its uPAR binding epitope does not overlap with
The recent structures of antibody–protein com-
plexes have advanced our understanding of the
normal rules when antibody is bound to the antigen.
The shape of the binding interface varies extensively
from clefts, to grooves, to flatter, more undulating
surfaces and even to protrusions, depending on the
antigen. Smaller ligands seem to bind in deeper,
to flatter, more extensive surfaces.28Solvent mole-
cules, generally water molecules, have been
observed in cavities within the interface and on the
periphery,where they often form bridging hydrogen
bonds between antibody and antigen.29The interac-
conformational change in the antigen, mainly in
regions that are flexible.29There is a wide range of
variation in the extent of conformational changes
induced by a ligand in different antibody systems.
For protein antigens, the antigen-induced antibody
conformational changes have so far been of lesser
magnitude than those seen with some hapten or
peptide ligands.30Unlike the case in typical multi-
meric proteins where hydrophobic interactions are
the major determinant of the binding strength
between antibody and protein antigen,31here it is
the hydrophilic interactions that are mainly respon-
sible for the high affinity. In addition, the buriedarea
between ATN615 and suPAR is on the low side
(1182 Å2) when compared with typical antibody–
protein complexes (1284 Å2–2063 Å2, see Table 2, all
surface areas here were calculated by program
AREAIMOL32with a probe of 1.4 Å radius).
We previously determined the crystal structure of
suPAR-ATF (amino-terminal fragment of uPA) com-
plex in the presence of Fab fragment of ATN615.33
Here we go on to report the crystal structures of the
Fab fragment of ATN615 and the analysis of
ATN615-suPAR-ATF ternary complex, emphasizing
the ATN615–suPAR interaction. These results reveal
the conformational changes upon antibody–antigen
complex conformations, elucidate the binding inter-
face between suPAR and ATN615, and provide
structural interpretation on the above-mentioned
characteristics of ATN615.
Results and Discussions
Monoclonal antibody ATN615
Monoclonal antibody ATN615 was raised by
standard techniques against a chymotryptic frag-
ment of soluble uPAR (suPAR) expressed in Droso-
phila S2 cells. ATN615 bound to suPAR immobilized
on plastic with high affinity (Kd∼1 nM). Iodinated
ATN615 also specifically bound suPAR on the
surface of HeLa cells with a similar avidity
(Kd∼1.3 nM). Western blot and whole cell binding
studies showed that ATN615 was specific for human
uPARand did not cross-reactwithmouse uPAR. The
Fab fragment of ATN615 has similar affinity to the
whole molecule of ATN615 in its binding to suPAR
and to uPAR expressed on the surface of HeLa cells
(Figure 1(a) and (b)).
Overall structure and conformation of CDR
loops of ATN615
The crystal structure of the Fab fragment of
ATN615 was determined at 1.77 Å to an Rfactor
0.239 and an Rfreeof 0.273. ATN615 Fab (Figure 2)
consists of a light chain and a heavy chain; each
chain has a variable and a constant domain,
respectively. Each domain is formed by seven to
nine antiparallel β-strands covalently linked by an
intra-chain disulfide bond. The antigen-binding site
is located at one end of the variable domains, and
contains six complementarity determining regions
(CDRs) (Figures 2 and 3), three from each chain.
CDRs are numbered according to their position in
the sequence and assigned as CDR L1, L2, L3, H1,
H2, and H3 (Figures 2 and 3), and their conforma-
tions usually fall into one of the so-called structural
canonical classes.34As a whole, the antigen binding
area (CDR loops) of ATN615 forms a relatively flat
and undulating surface,35characteristic of an anti-
Antibody combining site of suPAR
Our previously reported crystal structure of the
complex between ATN615 and suPAR in the
presence of a fragment of uPA (ATF)33showed
that the binding site of suPAR with ATN615 Fab
antibody is the domain 3 of suPAR, including the
linear binding epitope 186–192 and residues 217,
220, 267, and 269. Structural sequence alignment
(Figure 4) of uPAR among different species (human,
bovine, mouse, and rat) indicates that ATN615 Fab
only recognizes the human uPAR. Among all
residues interacting with ATN615 Fab residues
186, 187, 189, 192, and 267 of human uPAR are
different from other species. Prominent differences
are located at two residues. Human Arg192 is
replaced by phenylalanine (mouse and rat) and
leucine (bovine), and Gln189 on human is replaced
by proline (mouse, rat and bovine). Both these two
residues have strong interactions with ATN615, and
Anti-uPAR Structure and Binding Epitope
thus render specificity of ATN615 to human uPAR.
This has been confirmed by Western blot of extracts
from human and mouse cells expressing uPAR (data
The ATN615-suPAR-ATF structure shows that the
uPAR binding epitope (residues 186–192, 217, 220,
267, and 269) for this Fab does not overlap with
that of uPAs. Since uPAR is capable of interacting
with multiple ligands, can this ATN615 interfere
with other uPAR ligands? In vivo, highly expressed
uPAR can interact with α5β1 integrin leading to
persistent ERK activation and tumorigenicity.36
Disrupting this interaction reduces ERK activity,
forcing cancer cells into dormancy.37–40Domain 3
of uPAR (residues 240–248) was implicated to
interact with the α5β1 integrin.41And uPAR–
uPAR ectodomain interactions were found to con-
tribute to the regulation of various cellular func-
tions of uPAR.42The similar segment (240–260)42of
uPAR was also found to be important in self
association of uPAR. The ATN615 combining site
(residues 186–192, 217, 220, 267, and 269) of suPAR
is spatially quite close to residues 240–260. As a
result, it could be deduced reasonably that the
ATN615 is likely to interrupt the α5β1binding to
uPAR and to disrupt uPAR self-association through
steric hindrance. Further functional studies are
needed to verify these conclusions.
ATN615 was raised against a soluble form of
uPAR (suPAR). Thus, the implication for its interac-
tion with wild-type GPI anchored uPAR was
examined. C-terminal residue Gly283, which under-
goes GPI anchoring in the full-length uPAR, is far
away from the binding interface. Our structure also
shows that Gly283, not observed in the current
structure, will be at least 25 Å away from the nearest
ATN615 residue, and thus the GPI anchor should
not interfere with antibody recognition. These
analyses suggest that ATN615 will also bind to
full-length uPAR attached by a GPI anchor to the cell
surface, consistent with our experimental results
that ATN615 binds to uPAR on the surface of HeLa
Figure 2. (a) The overall structure of the ATN615
Fab antibody. CDRs are shown in orange (light chain)
and green (heavy chain). (b) The overall structure of
the ATN615 Fab antibody complex with suPAR in the
presence of ATF (shown in blue). Binding site of the
suPAR (shown in grey) interacts with the CDRs of the
ATN615 Fab antibody. C-terminal residue 275 was
labelled in red. Figures 2, 5, 7–9 were prepared with
Figure 1. (a) ATN615 Fab binds to purified suPAR
with high affinity. Binding of intact antibody ATN615 (○)
or ATN615 Fab (●) to suPAR immobilized on plastic was
determined using a modified ELISA assay. The affinity of
ATN615 Fab for suPAR (0.84(±0.16) nM, n=5) was
indistinguishable from that of intact ATN615 antibody
(0.94(±0.34) nM, n=5). Results of a typical experiment are
shown. (b) ATN615 Fab binds to uPAR on the surface of
HeLa cells. Binding of ATN615 antibody or ATN615 Fab to
uPAR expressed on the surface of HeLa cells was
determined using an FITC-conjugated anti-mouse second-
ary antibody and FACS. As shown in the left panel almost
all cells were specifically labeled in the presence of either
ATN615 IgG (98.7%) or ATN615 Fab (97.8%) although the
absolute mean fluorescence intensity (MFI) was lower
when ATN615 Fab was used as the probe (right panel).
Anti-uPAR Structure and Binding Epitope
Recognition of ATN615 Fab with suPAR
On the antibody side, all the CDR loops of
ATN615, except L2, are involved in the recognition
of the antigen. These CDR loops form a shallow
valley (Figure 5) shaped surface and interact with
domain 3 of suPAR. Hydrogen bonds, hydrophobic
interactions and polar interactions are observed in
this interface (Table 1). The buried surface areas of
suPAR and ATN615 Fab as a result of the complex
formation are 629 Å2and 553 Å2, respectively
(Figure 5). The framework residues of the antibody
Figure 3. Amino acid sequen-
ces of (a) the variable domain of the
light chain and (b) the variable
domain of the heavy chain of the
ATN615 Fab antibody. The residues
are numbered by the system des-
cribed by Kabat et al. CDR residues
are in bold.
Figure 4. Sequence alignment of the carboxyl region of uPAR from different species: human, bovine, mouse and rat.
Amino acid numbering is according tothe human uPAR sequence without theNH2-terminal signalsequence. The binding
site with ATN615 Fab is the residues from 186–192, 217, 220, 267, and 269, shown in bold. The underlined Gly283 is the
residue to attach glycosylphosphatidylinositol anchor.
Anti-uPAR Structure and Binding Epitope
usually contribute only a small surface area to
antigen binding,35as is the case here: only 1% of the
total buried area comes from framework residues.
The heavy chain accounts for 63% of the total
antibody–antigen contact surface, whereas the light
chain contributes only 37%. This skewed percentage
of the contact area for the light chain is typical and
lies in the middle of the observed range (21% to 49%)
for known antibody–protein complexes.30In terms
of van der Waals contacts, the light chain makes 19
contacts (all from L3 loop, Table 1 and Figure 6) to
the antigen, whereas the heavy chain contributes 27
of the total 59 contacts (five, 15, and seven from H1,
H2, and H3, respectively).
A total of 13 hydrogen bonds (ten from VHand
three from VL, Table 1, Figures 6 and 7) were
observed between suPAR and ATN615 Fab with two
main-chain–main-chain hydrogen bonds from
suPAR Gln189 O–Tyr93L N (L3) and Thr267 O–
Asp55H O (H2). This is at the higher end of typical
hydrogen bond numbers for antibody–protein com-
plexes (six to 1729), explaining the high affinity
(Kd∼1 nM) binding of suPAR and ATN615. Inter-
acting residues of the heavy chain are mainly
located in CDR H2, including residues Trp50H,
Asp55H, Asn56H, Thr57H, and Glu58H. Eight
hydrogen bonds are present between these residues
and suPAR residues Asn190, Thr267, Arg192,
Asn220, and Ser269. In CDR H1 of the heavy
chain, the main interacting residue is Tyr33H.
Tyr33H forms hydrogen bonds with the main
chain oxygen atom of Gln217 and side-chain
nitrogen of Asn220, respectively. Moreover, only
three CDR residues in the light chain, Tyr32L in
CDR L1, Asn92L and Tyr93L in CDR L3, are
Figure 5. Illustration of the interactions of ATN615 Fab with suPAR; the binding site of ATN615 was shown as surface
and suPAR as sticks. A linear epitope of suPAR, residues 186–192, is the major binding determinant for ATN615 Fab.
ATN615 Fab surface forms a valley to accommodate this linear epitope. Other residues of suPAR interacting with ATN615
Fab are 217, 220, 267, and 269. The total antibody–antigen binding area is 1182 Å2.
Table 1. Direct hydrogen bond, hydrophobic and polar
interactions between suPAR and ATN-615
The residues are numbered by the system described by Kabat
Anti-uPAR Structure and Binding Epitope
involved in hydrogen bonding with the suPAR
residues. In CDR L1, the side-chain oxygen atom of
Tyr32L of ATN615 and side-chain oxygen atom of
Asn186 of suPAR form one hydrogen bond. In CDR
L3, the side-chain nitrogen atom of Asn92L and
main-chain nitrogen atom of Tyr93L interact with
the side-chain nitrogen atom and main-chain oxy-
gen atom of suPAR Gln189, respectively. CDRs of
antibody ATN615 become more rigid as a result of
complex formation, as can be seen from the average
B factors of the CDRs (30.6 Å2) and full length Fab
(34.3 Å2) in the ATN615-suPAR-ATF complex.
Figure 6. Summary of interactions of suPAR epitope with ATN615 Fab.
Figure 7. Hydrogen bond interactions (shown in blue line) of suPAR (shown in grey sticks) with ATN615 Fab CDRs
(shown in cyan and light-blue sticks). Thirteen hydrogen bonds are shown in the Figure (distance <3.28 Å).
Anti-uPAR Structure and Binding Epitope
Although many suPAR residues are involved in
the ATN615–suPAR interactions, a few key
residues43can be identified based on the ATN615-
suPAR-ATF structure. Arg192 of the suPAR epitope
makes ten contacts with the heavy chain of ATN615,
which include three hydrogen bonds, two between
its side-chain NH2 and Asn56H OD1 and Thr57H O
of CDR H2, and one between its side-chain NH1 and
Glu58H OE2 of CDR H2. Arg192 uses about 40% of
its total surface (131 out of total area 347 Å2) to
contact with CDR. Another key suPAR epitope
residue is Pro188, which is deeply buried in a pocket
formed by CDR loops L3 and H3. The contact area
between Pro188 and ATN615 is 150 Å2, which is
about three-fifths of the total surface area of a
proline residue (240 Å2). This interaction involves 11
van der Waals contacts.
Asn190 and Gly191, another two suPAR residues
buried in the CDR valley, also appear to play a key
role in antibody binding. These two residues alone
contribute 257 Å2of the total 1182 Å2antigen contact
surface area, and make ten out of the total 59
antibody–antigen contacts. The Asn190 main-chain
oxygen atom forms one hydrogen bond with the
ATN615 side-chain atom NE1 of Trp50H from CDR
H2 that is completely buried in the interface.
The whole ATN615–suPAR interface is polar and
hydrophilic in nature with little hydrophobic inter-
action, in contrast to typical multimeric proteins,
which usually have hydrophobic interfaces.31The
total contact surface between antibody–antigen is
also quite small (1182 Å2). However, the shape
complementarity (Sc, calculated by CCP4 program
SC44) between Fab and suPAR (0.74, Table 2) is
higher than typical antibody–protein complexes
(0.60–0.70, see Table 2). Therefore, it may be this
surface shape complementarity that contributes to
the high affinity binding between ATN615 and
Solvent molecules between binding site of
antibody and antigen
It is interesting to note that the buried area of
ATN615 Fab on suPAR (1182 Å2) is on the low side
when compared with typical antibody–antigen
complexes, which range from 1284 Å2to 2063 Å2
(Table 2). Regardless, this complex retains high
affinity binding between antigen and antibody.
Table 3. X-ray data collection and refinement statistics of
the ATN615 Fab
A. Data collection statistics
Total no. of observations
No. of unique reflections
a=37.2, b=84.5, c=134.0
B. Refinement statistics
Resolution range (Å)
No. of reflections for
Root mean square bonds (Å)
Root mean square angles (°)
No. of protein atoms
No. of solvent molecules
Luzzati error (Å)
Residues in disallowed region
87.4 11.8Ramachandran analysis
(% in most favor,
From Wilson plot (A2)
Mean B value (A2)
aNumbers in parentheses represent corresponding values for
the highest resolution shell of 1.77 Å.
bThese loops have poor electron density.
Table 2. Comparison of the ATN615-suPAR-ATF complex with other antibody–antigen complexes in terms of shape
complementarity (Sc, calculated by CCP4 program SC44) and antibody–antigen buried surface areas
PDB CodeFabAntigenScFab (Å2)Ag (Å2)Resol'n (Å)References
Ag, antigen; HEL, hen egg lysozyme; PEL, pheasant egg lysozyme; BEL, bobwhite quail egg white lysozyme; GEL, guineafowl egg white
lysozyme; N9, N9 neuraminidase; HPr, histidine-containing phosphocarrier protein. All surface areas here were calculated by program
AREAIMOL32with a probe of 1.4 Å radius.
Anti-uPAR Structure and Binding Epitope
Some extra electron densities were observed in the
suPAR and ATN615 Fab interface. Considering the
5% ethylene glycol used in the crystallization of
ATN615-suPAR-ATF ternary complex33and the
shape of this electron density, we modeled these
electron densities as polyethylene glycol (Figure 8).
These polyethylene glycols form a total of eight
hydrogen bonds with residues from both suPAR
and ATN615 Fab. The contact area between ATN615
Fab fragment and these PEG molecules is about
583.6 Å2, and 346.6 Å2between PEG and suPAR
side. It accounts for 85.7% and 50.9% of the total
PEG (680.47 Å2) surface area, respectively. Solvent
molecules are commonly observed in antibody–
antigen complexes. It has previously been demons-
trated35,45,46that solvent molecules, generally water
molecules, play an important role in the antibody–
antigen interaction. Sulfate ion was also observed to
stabilize the complex between the nuclear GTP
binding protein Ran and its guanine nucleotide
exchange factor, RCC1 and inhibits dissociation of
guanine nucleotide.47It is therefore likely that
solvent molecules, but not necessarily PEG, may
be a factor in the binding interface to strengthen the
affinity between the suPAR and ATN615. Further
experiments are needed to evaluate the effect of
Binding site composition of ATN615 antibody
Another characteristic of ATN615 is the amino
acid composition of its CDRs: 16 out of total 61 (26%)
CDR residues are aromatic residues, including six
tyrosine, four tryptophan and six phenylalanine
residues (Figure 3). These three amino acids are
typically present in antibody–antigen interface.48
However, the percentage that we observed (26%) is
on the high side compared with the previously
statistics (average 15.5%, calculated from Table 2 of
Mian et al.48).
The surface of the suPAR-binding site of the
antibody ATN615 comprises 12 residues. Seven
aromatic residues (four tryptophan and three
tyrosine residues) and five aliphatic residues inter-
sperse among the CDRs of ATN615 Fab. Three of the
seven aromatic residues in the binding site are found
in positions where the presence of aromatic side-
chain is highly conserved among murine antibodies
(L32, L91, and H33).48,49The CDR forms a shallow
valley on the surface of the variable domains with
Figure 8. Extra electron density observed at the interface between suPAR and ATN615 Fab was assigned as
polyethylene glycol, suggesting the importance of solvent in the maximal interaction between antibody and antigen.
Anti-uPAR Structure and Binding Epitope
aromatic side-chains. L32, L91 and L93 form one
side of the valley and other four aromatic side-
chains H33, H50, H95 and H99 form another side.
The amino acids in the suPAR epitope insert into the
valley between these two sides (lean slightly to the
first side) (Figure 5).
Conformational changes due to the antigen
The elbow angle (the angle between axes of
pseudo-symmetry of variable and constant domain)
of ATN615 in the ATN615-suPAR-ATF complex is
164.6° in the middle of those observed in Fab
structure (117 to 22750) and is close to that of the
unliganded Fab (159.5°). A comparison of the CDR
loops between free and bound ATN615 Fab gives an
r.m.s. deviation of 0.923 Å. The heavy chain CDRs
have slightly larger Cαr.m.s. deviations (0.638 Å)
than those of the light chain (0.488 Å). The
individual CDRs have r.m.s. deviations of 0.284,
0.244, 0.350, 0.307, 0.749, and 0.204 Å for L1, L2, L3,
H1, H2, and H3, respectively. The shift of H2 loop is
large in the individual CDRs, but still smaller than
the overall r.m.s. deviation values (2.773 Å) of the
ATN615 heavy chain. In addition, a 15° swing of the
Glu58H side-chain is critical for making two
hydrogen bonds to the suPAR side-main carbonyl
oxygen of Ser269 and side-chain amide group of
Arg192. Despite these small overall shiftsin the CDR
loops, two differences appear significant. Firstly, the
r.m.s. deviation for all 421 Cαatoms of the ATN615
Fab is 2.131 Å, indicating overall conformational
changes of ATN615 Fab upon suPAR binding.
Secondly, the largest atom shifts in the CDR loops
upon complex formation are in CDR L2 residue
Ser56L (r.m.s. deviation 2.10 Å) and CDR H2 residue
Asp55H (r.m.s. deviation 1.87 Å).
For all variable domains, r.m.s. deviations of
0.614 Å and 0.497 Å for superimposed light chain
and heavy chain of variable domain were obtained,
respectively. It can be seen from the results that the
heavy chain (superimposed light chain of variable
domain) shows a larger backbone motion than the
light chain of variable domain (superimposed heavy
of the individual constant and variable domains
between the free and bound Fab show that both the
constant domain and variable domain have r.m.s.
deviations (2.5 Å and 0.94 Å, respectively) much
larger than experimental precision (Luzzati error of
0.31 Å). This indicates that although the constant
the variable domain still has a small but detectable
conformation change induced by suPAR binging.
Stanfield et al.35observed a substantial rotation
(16°) of the variable heavy (VH) chain relative to
the variable light (VL) chain in Fab 50.1-peptide
complex. They also proposed that this VL–VH
rotation is related to the length of CDR H3 loop
and the size of the buried surface area at the VL–VH
interface. The availability of both free and suPAR-
bound ATN615 allows us to calculate the ATN615
Fab VL–VH rotation angle upon antigen binding.
ATN615 Fab has a relatively large VL–VHinterface
(1607 Å2) compared with the Fab 50.1 (1063 Å2;
Stanfield et al.50). Upon suPAR antigen binding,
ATN615 Fab undergoes only a small VL–VHrotation
(2.0°), similar to that seen in other Fab–protein
On the suPAR side, superimposition of domain 3
of suPAR in ATN615-suPAR-ATF complex33and in
suPAR-antagonist peptide51complex shows a r.m.
s. deviation of 1.805 Å. However, superimposition
of Fab interaction epitope (186–192, 217, 220, 267,
and 269) gave a r.m.s. deviation of only 0.644 Å.
These suggest that the conformational change in
suPAR is not the result of antibody binding, but the
nature of the overall conformational flexibility of
Figure 9. Comparison of the heavy chain of variable domain of ATN615 between its free and antigen-binding
conformations shows significant differences with the largest shift of about 3 Å. The variable domains of the ATN615 light
chain were superimposed and gave a r.m.s.d. of 0.614 Å for L1-L107 Cαatoms. (a) Side view; (b) view from antigen.
Anti-uPAR Structure and Binding Epitope
We report here an anti-uPAR antibody, its crystal
structure, and its binding epitope. This antibody
binds uPAR in vitro with high affinity (Kd∼1 nM)
and does not interfere with uPA binding to uPAR.
The structures show that the CDRs of ATN615 form
a relatively flat and undulating surface. ATN615 Fab
fragment recognizes the domain 3 of suPAR. Pro188,
Asn190, Gly191, and Arg192 residues of suPAR are
the key residues for the antibody recognition, while
Pro189 and Arg192 render the specificity of ATN615
for human uPAR. The antibody–antigen recognition
involves 11 suPAR residues and 12 Fab residues
from five CDRs. Interestingly, this antibody–antigen
interface has mainly polar interaction with little
hydrophobic character, the smallest contact area
(1182 Å2) known for antibody–antigen complexes,
and yet mediates a high affinity interaction. Also,
several solvent molecules (assigned as polyethylene
glycol molecules) were clearly visible in the binding
interface between antibody and antigen, suggesting
that solvent molecules may be important for the
maximal binding between suPAR and ATN615 Fab.
Antibodies like ATN615 may provide a means to
intervene with uPAR–ligand interaction and its
(patho-) physiological consequences.
Materials and Methods
Generation ofmonoclonal anti-uPARantibody ATN615
ATN615 was raised using standard techniques against a
in Drosophila S2 cells.52,53Briefly, BAlb/c mice were immu-
nized with suPAR fragments conjugated to KLH and the
magnitude of the immune response monitored by ELISA.
spleen cells with the myeloma cell line P3x63Ag8.653.
Tissue culture supernatants from these monoclonal anti-
bodies were then assayed for activity in ELISA assays and
the isotype of each antibody determined using IsoStrips
(Roche). ATN615, isotype IgG1, was identified to bind to
suPAR immobilized to plastic with a KD of ∼1 nM.
of HeLa cells with a KDof ∼1.3 nM. Western blot analysis
demonstrated that ATN615 was specific for human uPAR
and did not cross-react with mouse uPAR.
Fab fragments of ATN615 were prepared by papain di-
gestion and purified by using proteinA-Sepharose column.
Using secondary antibodies specific for the Fcor light chain
portions of the antibody we were able to demonstrate that
the Fab fragments were at least 95% pure (data not shown).
Crystallization and X-ray diffraction data collection of
The initial crystals of Fab fragment of ATN615 antibody
were grown at 25 °C by vapor diffusion methods with a
precipitant solution containing 40% (w/v) PEG 4000,
0.1 M phosphate (pH 7.2). The concentration of ATN615
Fab fragment used in crystallization trials was 7.2 mg/ml.
These preliminary crystals were small and stacked up as
butterfly clusters and diffracted poorly with an in-house
X-ray source. We found that the treatment of protein with
hydrogen peroxide and protein concentration reduction54
were the key factors in obtaining diffracting crystals.
Optimized crystals were grown with 3.5 mg/ml protein
containing 0.9% hydrogen peroxide, and with a precipi-
tant containing 28% PEG 3350, 100 mM Tris-HCl (pH 8.5).
The crystals diffracted to 1.77 Å at the APS X-ray source.
Crystals were soaked briefly in solution containing 25%
(v/v) glycerol as a cryoprotectant and frozen in a liquid
N2stream. X-ray diffraction data collection was carried
out at −173 °C on the APS SER-CAT beamline 22-ID,
Chicago, USA. The diffraction data were indexed and
processed using the HKL2000 program.55Final crystals
belong to orthorhombic P212121space group with unit cell
parameters a=37.2 Å, b=84.5 Å, c=134.0 Å, and the
asymmetric unit contains one Fab fragment.
Structure determination and refinement
The crystal structure of ATN615 was solved by the
molecular replacement (MR) method using the AMoRe
program56and refined to 1.77 Å resolution to an Rfactorof
0.239 and an Rfree of 0.273 after incorporating solvent
water molecules. Homology models of ATN615 Fab
fragment were built from the protein sequence by
SwissProt.57The individual domains of the homology
model, Hv, Hc, Lv and Lc, were used as molecular
replacement models. Distinguishable AMoRe MR rotation
solutions were obtained using models from domains Hv,
Hcand Lc but not with the Lv domain. In the translation
function step of the molecular replacement, however, only
the Lc model gave a discernible AMoRe translation
function solution. By fixing the MR solution of this Lc
model, the translation function solutions of other frag-
ments were then found. All of these domains were packed
well inside a crystal lattice and formed a Fab molecule
without severe overlap with symmetry-related molecules.
Cross-validated crystallographic refinements against
maximum likelihood targets were carried out with the
CNS58program suites (version 1.1). After cycles of
crystallographic refinement and manual adjustment, the
models were fitted to the 3Fo–2Fcsigma Aweighted maps
with the graphic program O.59Then two more rounds of
interactive model building, followed by simulated anneal-
ing, energy minimization and B-factor refinement were
carried out. The crystallographic Rfactordropped to 0.239
after adding 178 solvent molecules to the model. The
electron density for most residues is well defined except
for some loop areas (H126–H129; H27–H30; H52A–H55)
in ATN615 Fab molecules. The data collection and model
refinement statistics are summarised in Table 3. This
refined ATN615 structure was then used to phase the
Protein Data Bank accession codes
The atomic coordinates and structure factors for the free
ATN615 Fab and ATN615-suPAR-ATF complex have been
deposited in the RCSB Protein Data Bank with accession
codes 2FAT and 2FD6, respectively.
This work is supported by grants from NSFC
(30430190) and State Key Laboratory of Structural
Anti-uPAR Structure and Binding Epitope
Chemistry (IB021061). Use of the Advanced Photon
Source was supported by the US Department of
Energy, Office of Science, Office of Basic Energy
Sciences, under contract No.W-31-109-Eng-38. We
thank Yujun Wang and Jing Zhou of University of
Alabama in Huntsville as well as the staff of the APS
1. Stoppelli, M. P., Corti, A., Soffientini, A., Cassani, G.,
Blasi, F. & Assoian, R. K. (1985). Differentiation-
enhanced binding of the amino-terminal fragment of
human urokinase plasminogen activator to a specific
receptor on U937 monocytes. Proc. Natl Acad.Sci. USA,
2. Vassalli, J. D., Baccino, D. & Belin, D. (1985). A cellular
binding site for the Mr 55,000 form of the human
plasminogen activator, urokinase. J. Cell Biol. 100,
3. Quigley, J. P. (1976). Association of a protease
(plasminogen activator) with a specific membrane
fraction isolated from transformed cells. J. Cell Biol. 71,
4. Wei, Y., Waltz, D. A., Rao, N., Drummond, R. J.,
Rosenberg, S. & Chapman, H. A. (1994). Identification
of the urokinase receptor as an adhesion receptor for
vitronectin. J. Biol. Chem. 269, 32380–32388.
5. Behrendt, N. (2004). The urokinase receptor (uPAR)
and the uPAR-associated protein (uPARAP/
Endo180): membrane proteins engaged in matrix
turnover during tissue remodeling. Biol. Chem. 385,
6. Resnati, M., Guttinger, M., Valcamonica, S., Sidenius,
N., Blasi, F. & Fazioli, F. (1996). Proteolytic cleavage of
the urokinase receptor substitutes for the agonist-
induced chemotactic effect. EMBO J. 15, 1572–1582.
7. Resnati, M., Pallavicini, I., Wang, J. M., Oppenheim, J.,
Serhan, C. N., Romano, M. & Blasi, F. (2002). The
fibrinolytic receptor for urokinase activates the G
protein-coupled chemotactic receptor FPRL1/LXA4R.
Proc. Natl Acad. Sci. USA, 99, 1359–1364.
8. Wei, Y., Lukashev, M., Simon, D. I., Bodary, S. C.,
Rosenberg, S., Doyle, M. V. & Chapman, H. A. (1996).
Regulation of integrin function by the urokinase
receptor. Science, 273, 1551–1555.
9. Simon, D. I., Wei, Y., Zhang, L., Rao, N. K., Xu, H.,
Chen, Z. et al. (2000). Identification of a urokinase
receptor-integrin interaction site. Promiscuous regula-
tor of integrin function. J. Biol. Chem. 275, 10228–10234.
10. Nguyen, D. H., Catling, A. D., Webb, D. J., Sankovic,
M., Walker, L. A., Somlyo, A. V. et al. (1999). Myosin
light chain kinase functions downstream of Ras/ERK
to promote migration of urokinase-type plasminogen
activator-stimulated cells in an integrin-selective
manner. J. Cell Biol. 146, 149–164.
11. Carriero, M. V., Del Vecchio, S., Capozzoli, M., Franco,
P., Fontana, L., Zannetti, A. et al. (1999). Urokinase
receptor interacts with alpha(v)beta5 vitronectin
receptor, promoting urokinase-dependent cell migra-
tion in breast cancer. Cancer Res. 59, 5307–5314.
12. Wei, Y., Eble, J. A., Wang, Z., Kreidberg, J. A. &
Chapman, H. A. (2001). Urokinase receptors promote
beta1 integrin function through interactions with
integrin alpha3beta1. Mol. Biol. Cell, 12, 2975–2986.
13. Aguirre Ghiso, J. A., Alonso, D. F., Farias, E. F.,
Gomez, D. E. & de Kier Joffe, E. B. (1999). Deregula-
tion of the signaling pathways controlling urokinase
production. Its relationship with the invasive pheno-
type. Eur. J. Biochem. 263, 295–304.
14. Blasi, F. & Carmeliet, P. (2002). uPAR: a versatile
signalling orchestrator. Nature Rev. Mol. Cell. Biol. 3,
15. Kim, J., Yu, W., Kovalski, K. & Ossowski, L. (1998).
Requirement for specific proteases in cancer cell
intravasation as revealed by a novel semiquantitative
PCR-based assay. Cell, 94, 353–362.
16. Ploug, M. (2003). Structure-function relationships in
the interaction between the urokinase-type plasmino-
gen activator and its receptor. Curr. Pharm. Des. 9,
17. Stephens, R. W., Nielsen, H. J., Christensen, I. J.,
Thorlacius-Ussing, O., Sorensen, S., Dano, K. &
Brunner, N. (1999). Plasma urokinase receptor levels
in patients with colorectal cancer: relationship to
prognosis. J. Natl Cancer Inst. 91, 869–874.
18. Ganesh, S., Sier, C. F., Heerding, M. M., Griffioen, G.,
Lamers, C. B. & Verspaget, H. W. (1994). Urokinase
receptor and colorectal cancer survival. Lancet, 344,
19. Riisbro, R., Christensen, I. J., Piironen, T., Greenall, M.,
Larsen, B., Stephens, R. W. et al. (2002). Prognostic
significance of soluble urokinase plasminogen activa-
tor receptor in serum and cytosol of tumor tissue from
patients with primary breast cancer. Clin. Cancer Res.
20. Grondahl-Hansen, J., Peters, H. A., van Putten, W. L.,
Look, M. P., Pappot, H., Ronne, E. et al. (1995).
Prognostic significance of the receptor for urokinase
plasminogen activator in breast cancer. Clin. Cancer
Res. 1, 1079–1087.
21. Pappot, H., Hoyer-Hansen, G., Ronne, E., Hansen,
H. H., Brunner, N., Dano, K. & Grondahl-Hansen, J.
(1997). Elevated plasma levels of urokinase plasmi-
nogen activator receptor in non-small cell lung
cancer patients. Eur. J. Cancer, 33, 867–872.
22. Pedersen, H., Brunner, N., Francis, D., Osterlind, K.,
Ronne, E., Hansen, H. H. et al. (1994). Prognostic
impact of urokinase, urokinase receptor, and type 1
plasminogen activator inhibitor in squamous and
large cell lung cancer tissue. Cancer Res. 54, 4671–4675.
23. Romer, J., Nielsen, B. S. & Ploug, M. (2004). The
urokinase receptor as a potential target in cancer
therapy. Curr. Pharm. Des. 10, 2359–2376.
24. Ploug, M., Gardsvoll, H., Jorgensen, T. J., Lonborg
Hansen, L. & Dano, K. (2002).Structuralanalysis of the
interaction between urokinase-type plasminogen acti-
vator and its receptor: a potential target for anti-inva-
sive cancer therapy. Biochem. Soc. Trans. 30, 177–183.
25. Mazar, A. P. (2001). The urokinase plasminogen
activator receptor (uPAR) as a target for the diagnosis
and therapy of cancer. Anticancer Drugs, 12, 387–400.
26. Bauer, T. W., Liu, W., Fan, F., Camp, E. R., Yang, A.,
Somcio, R. J. et al. (2005). Targeting of urokinase
plasminogen activator receptor in human pancreatic
carcinoma cells inhibits c-Met-and insulin-like growth
factor-I receptor-mediated migration and invasion
and orthotopic tumor growth in mice. Cancer Res. 65,
27. Rabbani, S. A. & Gladu, J. (2002). Urokinase receptor
antibody can reduce tumor volume and detect the
presence of occult tumor metastases in vivo. Cancer
Res. 62, 2390–2397.
28. Wilson, I. A. & Stanfield, R. L. (1993). Antibody-
29. Davies, D. R. & Cohen, G. H. (1996). Interactions of
Anti-uPAR Structure and Binding Epitope
protein antigens with antibodies. Proc. Natl Acad. Sci.
USA, 93, 7–12.
30. Huang, M., Syed, R., Stura, E. A., Stone, M. J.,
Stefanko, R. S., Ruf, W. et al. (1998). The mechanism
of an inhibitory antibody on TF-initiated blood
coagulation revealed by the crystal structures of
human tissue factor, Fab 5G9 and TF, G9 complex.
J. Mol. Biol. 275, 873–894.
31. Jones, S. & Thornton, J. M. (1996). Principles of protein-
32. Collaborative Computational Project, Number 4.
(1994). The CCP4 Suite: Programs for protein crystal-
lography. Acta Crystallog. sect. D, 50, 760–763.
33. Huai, Q., Mazar, A. P., Kuo, A., Parry, G. C., Shaw,
D. E., Callahan, J. et al. (2006). Structure of human
urokinase plasminogen activator in complex with its
receptor. Science, 311, 656–659.
34. Johnson, G. & Wu, T. T. (2001). Kabat Database and its
applications: future directions. Nucl. Acids Res. 29,
35. Wilson, I. A. & Stanfield, R. L. (1994). Antibody-
antigen interactions: new structures and new confor-
mational changes. Curr. Opin. Struct. Biol. 4, 857–867.
36. Liu, D., Aguirre Ghiso, J., Estrada, Y. & Ossowski, L.
(2002). EGFR is a transducer of the urokinase receptor
initiated signal that is required for in vivo growth of a
human carcinoma. Cancer Cell, 1, 445–457.
37. Aguirre-Ghiso, J. A., Estrada, Y., Liu, D. & Ossowski,
L. (2003). ERK(MAPK) activity as a determinant of
tumor growth and dormancy; regulation by p38
(SAPK). Cancer Res. 63, 1684–1695.
38. Aguirre-Ghiso, J. A., Liu, D., Mignatti, A., Kovalski, K.
& Ossowski, L. (2001). Urokinase receptor and
fibronectin regulate the ERK(MAPK) to p38(MAPK)
activity ratios that determine carcinoma cell prolifera-
tion or dormancy in vivo. Mol. Biol. Cell, 12, 863–879.
39. Kook, Y. H., Adamski, J., Zelent, A. & Ossowski, L.
(1994). The effect of antisense inhibition of urokinase
receptor in human squamous cell carcinoma on
malignancy. EMBO J. 13, 3983–3991.
40. Yu, W., Kim, J. & Ossowski, L. (1997). Reduction in
protracted state of dormancy. J. Cell Biol. 137, 767–777.
41. Chaurasia, P., Aguirre-Ghiso, J. A., Liang, O. D.,
Gardsvoll, H., Ploug, M. & Ossowski, L. (2006). A
region in urokinase plasminogen receptor domain III
controlling a functional association with alpha5beta1
integrin and tumor growth. J. Biol. Chem. 281,
42. Liang, O. D., Bdeir, K., Matz, R. L., Chavakis, T. &
Preissner, K. T. (2003). Intermolecular contact regions
in urokinase plasminogen activator receptor. J. Bio-
chem. (Tokyo), 134, 661–666.
43. Novotny, J., Bruccoleri, R. E. & Saul, F. A. (1989). On
the attribution of binding energy in antigen-antibody
complexes McPC 603, D1.3, and HyHEL-5. Biochem-
istry, 28, 4735–4749.
44. Lawrence, M. C. & Colman, P. M. (1993). Shape
complementarity at protein/protein interfaces. J. Mol.
Biol. 234, 946–950.
45. Bhat, T. N., Bentley, G. A., Boulot, G., Greene, M. I.,
Tello, D., Dall'Acqua, W. et al. (1994). Bound water
molecules and conformational stabilization help
mediate an antigen-antibody association. Proc. Natl
Acad. Sci. USA, 91, 1089–1093.
46. Cohen, G. H., Sheriff, S. & Davies, D. R. (1996).
Refined structure of the monoclonal antibody
HyHEL-5 with its antigen hen egg-white lysozyme.
Acta Crystallog. sect. D, 52, 315–326.
47. Renault, L., Kuhlmann, J., Henkel, A. & Wittinghofer,
A. (2001). Structural basis for guanine nucleotide
exchange on Ran by the regulator of chromosome
condensation (RCC1). Cell, 105, 245–255.
48. Mian, I. S., Bradwell, A. R. & Olson, A. J. (1991).
Structure, function and properties of antibody binding
sites. J. Mol. Biol. 217, 133–151.
49. Jirholt, P., Strandberg, L., Jansson, B., Krambovitis, E.,
Soderlind, E., Borrebaeck, C. A. et al. (2001). A central
core structure in an antibody variable domain
determines antigen specificity. Protein Eng. 14, 67–74.
50. Stanfield, R. L., Zemla, A., Wilson, I. A. & Rupp, B.
(2006). Antibody elbow angles are influenced by their
light chain class. J. Mol. Biol. 357, 1566–1574.
51. Llinas, P., Le Du, M. H., Gardsvoll, H., Dano, K.,
Ploug, M., Gilquin, B. et al. (2005). Crystal structure of
the human urokinase plasminogen activator receptor
bound to an antagonist peptide. EMBO J. 24,
52. Cai Yuan, Qing Huai, Chuanbing, Bian & Mingdong,
Huang (2006). The expression, purification and
crystallization of monomeric soluble human uroki-
nase receptor. Prog. Biochem. Biophys. 33, 277–281.
53. Gardsvoll, H., Werner, F., Sondergaard, L., Dano, K. &
forms of soluble human urokinase receptor expressed
in Drosophila Schneider 2 cells after deletion of
glycosylation-sites. Protein Expr. Purif. 34, 284–295.
54. Li, Y., Shi, X., Parry, G., Chen, L., Callahan, J. A.,
Mazar, A. P. & Huang, M. (2005). Optimization of
crystals of an inhibitory antibody of urokinase
plasminogen activator receptor (uPAR) with hydro-
gen peroxide and low protein concentration. Protein
Pept. Letters, 12, 655–658.
55. Otwinowski, Z. W. (1997). Minor. Processing of X-ray
diffraction data collected in oscillation mode. Methods
Enzymol. 276, 307–326.
56. Navaza, J. (1994). AMoRe: an automated package for
molecular replacement. Acta Crystallog. sect. A, 50,
57. Guex, N. & Peitsch, M. C. (1997). SWISS-MODEL and
the Swiss-PdbViewer: an environment for compara-
tive protein modeling. Electrophoresis, 18, 2714–2723.
58. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano,
W. L., Gros, P., Grosse-Kunstleve, R. W. et al. (1998).
Crystallography and NMR system: a new software
suite for macromolecular structure determination.
Acta Crystallog. sect. D, 54, 905–921.
Improved methods for building protein models in
electron density maps and the location of errors in
these models. Acta Crystallog. sect. A, 47, 110–119.
60. Martin, A. C. (1996). Accessing the Kabat antibody
sequence database by computer. Proteins: Struct.
Funct. Genet. 25, 130–133.
61. Tulip, W. R., Varghese, J. N., Webster, R. G., Laver,
W.G. & Colman, P. M. (1992). Crystal structures of two
mutant neuraminidase-antibody complexes with
amino acid substitutions in the interface. J Mol Biol,
62. Tulip, W. R., Varghese, J. N., Laver, W.G., Webster,
R.G. & Colman, P. M. (1992). Refined crystal structure
of the influenza virus N9 neuraminidase-NC41 Fab
complex. J Mol Biol, 227, 122–148.
63. Bossart-Whitaker, P., Chang, C. Y., Novotny, J.,
Benjamin, D.C. & Sheriff, S. (1995). The crystal
structure of the antibody N10-staphylococcal nuclease
complex at 2.9 A resolution. J Mol Biol, 253, 559–575.
64. Prasad, L., Sharma, S., Vandonselaar, M., Quail, J.W.,
Anti-uPAR Structure and Binding Epitope
Lee, J. S., Waygood, E. B., Wilson, K. S., Dauter, Z. &
Delbaere, L. T. (1993). Evaluation of mutagenesis for
epitode mapping. Structure of an antibody-protein
antigen complex. J Biol Chem, 268, 10705–10708.
65. Malby, R. L., Tulip, W. R., Harley, V. R., McKimm-
Breschkin, J.L., Laver, W. G., Webster, R. G. & Colman,
P. M. (1994). The structure of a complex between the
NC10 antibody and influenza virus nueraminidase
and comparison with the overlapping binding site of
the NC41 antibody. Structure, 2, 733–746.
66. Lescar, J., Souchon, H. & Alzari, P. M. (1994). Crystal
structures of pheasant and guinea fowl egg-white
lysozymes. Protein Sci, 3, 788–798.
67. Chacko, S., Silverton, E., Kam-Morgan, L., Smith-Gill,
S., Cohen, G. & Davies, D. (1995). Structure of an
antibody-lysozyme complex unexpected effect of
conservative mutation. J Mol Biol, 245, 261–274.
68. Padlan, E. A., Silverton, E. W., Sheriff, S., Cohen, G.H.,
Smith-Gill, S. J. & Davies, D. R. (1989). Structure of an
antibody-antigen complex: crystal structure of the
HyHEL-10 Fab-lysozyme complex. Proc Natl Acad Sci
USA, 86, 5938–5942.
69. Braden, B. C., Souchon, H., Eisele, J. L., Bentley, G.A.,
Bhat, T. N., Navaza, J. & Poljak, R. J. (1994). Three-
dimensional structures of the free and the antigen-
complexed Fab from monoclonal anti-lysozyme anti-
body D44.1. J Mol Biol, 243, 767–781.
70. Chitarra, V., Alzari, P. M., Bentley, G. A., Bhat, T.N.,
Eisele, J. L., Houdusse, A., Lescar, J., Souchon, H. &
Poljak, R. J. (1993). Three-dimensional structure of a
heteroclitic antigen-antibody cross-reaction complex.
Proc Natl Acad Sci USA, 90, 7711–7715.
71. DeLano, W. L. (2002). The PyMol Molecular Graphics
system. DeLano Scientific, San Carlos, CA, USA.
Edited by I. Wilson
(Received 15 August 2006; received in revised form 11 October 2006; accepted 18 October 2006)
Available online 21 October 2006
Anti-uPAR Structure and Binding Epitope