(Received for publication, August 25, 1994; and in revised form, November 14, 1994)
From the
Cell surface receptors called integrins mediate diverse cell adhesion phenomena through recognition of the sequence arginine-glycine-aspartic acid (RGD) present in proteins such as fibronectin and fibrinogen. Platelet aggregation in hemostasis is mediated by the binding of fibrinogen to the gpIIb/IIIa integrin. The OPG2 antibody binds the gpIIb/IIIa receptor and acts as a ligand mimic due to the presence of an argininetyrosine-aspartic acid (RYD) sequence in the CDR3 loop of the heavy chain. The RYD loop and side chains are ordered in the 2.0-Å resolution crystal structure of the Fab fragment from this antireceptor antibody. Moreover, the RYD loop assumes two clearly defined conformations that may correspond to the orientations of the loop in the free state or bound to integrin. This molecule will serve as a tool for understanding protein-integrin recognition in platelet aggregation and other RGDmediated cell adhesion interactions.
It has been suggested that antireceptor antibodies can be used
to explore receptor-ligand interactions(1) . The antibody
combining site is composed of amino acids from
complementarity-determining regions (CDRs) within the
variable domains of heavy and light chains. In antibodies directed to
the ligand binding site of the receptor, it is possible that CDR
regions of the antireceptor antibodies are conformationally similar to
the receptor ligand. Such antibodies inhibit the binding of ligand by
the receptor, and, conversely, binding of ligand inhibits binding of
the antibody to the receptor.
The OPG2 (2) as well as the
PAC-1 (3) and LJ-CP3 (4) antibodies belong to this
class of antireceptor antibodies. These murine monoclonal antibodies
bind specifically to the receptor
(integrin) on platelets. Binding of
to fibrinogen causes platelets to aggregate, and this binding
results, in part, from recognition of an arginine-glycine-aspartic acid
(RGD) motif in fibrinogen by the platelet integrin. The OPG2, PAC-1,
and LJ-CP3 antibodies inhibit fibrinogen binding to
. Moreover, binding of these
antibodies to the platelet integrin is inhibited by RGD-containing
peptides. In this respect, we consider these related antibodies
functional ``molecular mimics'' for fibrinogen and are using
OPG2 as a tool to probe the molecular basis for
RGD-
recognition. The structural
characterization of these antibodies will have broader implications for
the study of the structure of the RGD recognition site in cell adhesion
proteins such as fibronectin and vitronectin and the interactions of
these proteins with cell surface integrins.
Cell interactions with
fibronectin and other cell adhesion proteins are mediated by a family
of receptors named integrins(5, 6) . These cell
surface receptors are membrane-bound glycoproteins composed of
and
subunits that form a noncovalent heterodimer. The
ligand-binding site of the assembled integrin is thought to be formed
by sequences from the extracellular domain of both
subunits(7, 8) . Integrins mediate a number of diverse
cell adhesion phenomena through recognition of the sequence Arg-Gly-Asp
(RGD). This recognition site was first identified in fibronectin (9, 10) but is also present in other matrix or
adhesion proteins such as fibrinogen or vitronectin. It has been
proposed that the conformation of the RGD sequence as well as amino
acids in flanking sequences (or modules) influence the recognition of
the RGD site by different
integrins(11, 12, 13) .
At least seven of
the 20 integrins characterized to date recognize the RGD sequence:
,
,
,
,
,
, and
(1, 14, 15) . Yet specific binding of
RGD-containing proteins by individual receptors mediates precise
biological effects. Apparently the conformational presentation of the
RGD sequence is critical for selective receptor recognition. The use of
antireceptor antibodies specific for the ligand-binding site of the
receptor may be particularly useful to study RGD-cognitive integrins
since the RGD sequence is located in a long flexible loop that is not
amenable to structural analysis in natural ligands such as the
fibronectin cell adhesion module III
(16, 17) and snake venom disintegrins (18, 19, 20, 21, 22, 23) .
There is great interest in RGD recognition by the platelet integrin
because of the role this integrin
plays in platelet aggregation, hemostasis, and arterial thrombotic
diseases. Consequently, the
integrin is perhaps the best characterized integrin, and much
work has been done to identify structural features recognized by this
receptor. In addition to the studies of snake venom disintegrins, which
function as
antagonists, numerous
conformationally restricted cyclic peptides have been synthesized that
are at least partially
receptor-selective(24, 25, 26, 27, 28, 29) .
The OPG2, PAC1, and LJ-CP3 antibodies are absolutely specific for
the integrin and do not bind to
any other integrins that recognize RGD, including the closely related
receptor. These interesting
antibodies have each incorporated a germline D-gene sequence that
includes the sequence RYD (30) in the CDR3 (H3) loop of the
heavy chain variable region. A 21-residue peptide corresponding to this
loop of the PAC1 antibody inhibits binding of PAC1 as well as
fibrinogen to agonist-stimulated platelets(3) . Moreover, when
the RYD sequence in OPG2 was replaced by RGD, (
)the
resultant protein binds with equivalent specificity and affinity as the
native OPG2, i.e. RGD-OPG2 binds specifically to
but not to
. Furthermore, substitution of Asp
Glu in the RGD site generated a mutant OPG2 with a marked loss
in binding to
. These results
clearly establish the OPG2 antibody as a true molecular mimic for
natural RGD-containing ligands. In addition, the fact that the tyrosine
can be replaced by glycine with no detectable change in specificity or
affinity suggests that the tyrosine side chain is not a critical
contact residue.
Here we report the crystal structure of the Fab fragment from the OPG2 antibody. The loop containing the RYD sequence is well ordered and reveals the precise spatial organization of this critical cell adhesion site.
The structure of the OPG2 Fab fragment was determined at
2.0-Å resolution by molecular replacement methods. The final R factor is 0.16 for 8-2.0-Å data (0.179 for
to 2.0-Å data) after refinement of protein atoms and 408
solvent atoms. The entire molecular structure was well determined at
high resolution, which is notable since many Fab crystals diffract only
to 2.7-2.8-Å resolution. The final refinement statistics
are presented in Table 2.
The OPG2 (IgG1-) assumes the
overall immunoglobulin folding pattern typical for other Fab fragments
studied to date (see Fig. 1). The elbow bend angle is 150° (i.e. the angle between V and C domain pairs). A striking
feature of the Fab structure is the H3 loop, which protrudes from the
surface of the molecule. The RYD sequence is located at the tip of this
loop at a distance of
12 Å from the location of the
combining site in other antibodies. Five other long H3 loops have been
reported in Fab crystal structures. The length of these loops ranges
from 9 to 14 amino acids, and the conformations vary from compact
involuted loops in the human Kol (36) and Pot (46) Fabs
to moderately open in R19.9(47) , to fully extended loops in
the R45-45-11 (48) and 3D6 (49) molecules. A comparison
of the OPG2 H3 loop with the structures of other extended H3 loops is
shown in Fig. 2. It is interesting to note that there is 83%
sequence homology (61% identical) between the Kol and OPG2 V
domains. In fact, because of the strong sequence homology, Kol
V
was used as probe model for molecular replacement
searches of the OPG2 crystal data. However, at an early stage in the
structure solution it was apparent that the H3 loop of OPG2 was quite
different from that of Kol, and residues 101-110 were deleted
from the model for refinement.
Figure 1:
Schematic diagram
presenting the polypeptide folding pattern of the OPG2 Fab molecule,
with the light chain colored green and the heavy chain colored yellow. V domains are at the top of the diagram. The
RYD side chains are represented by white stick models at the
tip of the CDR3 loop of the V domain. This model was
produced with the program RIBBONS(51) .
Figure 2:
Superimposed -carbon atoms of the V
domain pairs of three Fab fragments with long extended H3 loops. Note
that the overall folding patterns of these V modules are very similar.
The orientations of the H3 loops, however, are quite different
(R45-45-11, left; 3D6, center; OPG2, right).
The R45-45-11 Fab (48) is specific for cyclosporin A while the
3D6 antibody binds to the gp41 coat protein of human immunodeficiency
virus, type 1(49) .
This is the first crystal structure
of an antireceptor antibody specific for the ligand-binding site of a
receptor. Inspection of the atomic model of the OPG2 Fab reveals no
prominent antigen-combining cavity suggesting that the antibody binds
to the integrin as a ligand with a protruding loop that can interact
with the receptor binding site. In the case of OPG2, the antibody
``mimic'' and the functional cell adhesion molecule may have
marked structural similarities since the RGD sequence is also found in
a turn or loop in fibronectin(16, 17) ,
tenascin(50) , and
disintegrins(18, 19, 20, 21, 22, 23) .
To evaluate this hypothesis, we superimposed the -carbon backbone
of the OPG2 V
domain on the
-carbon backbone of the
fibronectin cell adhesion module (17) to directly compare the
framework for presentation of the RGD(RYD) loops. In fact, when 46
-carbons are superimposed with a root mean square deviation of
1.42 Å, the location of the RGD(RYD) recognition loops within the
overall modular scaffold of the two proteins is the same. (
)
Secondary structure analysis (52) of the turn
containing the RYD tripeptide indicates a ``-type'' turn
with torsion angles that are not characteristic of tight
- or
-turns. The most interesting feature of this turn is the fact that
the RYD sequence assumes two alternate conformations with equal
occupancy. The electron density in this region was strong, and yet the
model could be positioned in two orientations (see Fig. 3). In
one orientation, a solvent molecule was included with the protein
atoms. No clear distinction between the two configurations could be
made from visual inspection alone. Therefore, refinement of both
conformations was performed simultaneously, as described under
``Experimental Procedures.'' At the end of this process, it
was clear that both conformations exist equally. In an effort to
consider whether one of these conformations may exist when the loop
binds to receptor, the alternate atomic configurations are discussed in
the following sections within a context of existing biochemical data
for RGD ligands and integrins. After refinement, the final model of
this loop was fitted with two conformations (Fig. 4) that differ
by a rotation of
148° around the main chain axis between
residues 104-105. A similar rotation around the axis between
residues 107-108 results in the reorientation of Asp-105 and
Asn-108 side chains as a pair. The two intervening residues are Gly-106
and Gly-107. All
-carbon atoms are located in nearly the same
position in each conformation except C
of Gly-106 and
Gly-107. As a result of this rotation, in the two conformations, the
location of the carboxyl group of Asp-105 differs by 3.75 Å. The
side chain of Arg-103 extends from the same side of the loop as Asp-105
in both conformations, while Tyr-104 is located on the opposite side of
the loop and participates in a tight hydrogen-bonded network with
Tyr-102 and two solvent atoms. This interaction may stabilize the
conformation of the RYD loop.
Figure 3: Electron density map in the region of the CDR3 loop with the RYD sequence (8.0-2.0 Å). The model of this loop is shown in stereo in the two conformations fitted to the density map with one model in green and the other in red. A solvent molecule is located within hydrogen-bonding distance of the carboxylate oxygen OD1 of Asp-105 in the conformation shown in green. A symmetry-related molecule in the crystal lattice is shown with dashedlines (green) in the density in the upper part of the figure. Note that the density is strong except at the Gly-106-Gly-107 sequence.
Figure 4:
Comparison of the alternate conformations
in the OPG2 H3 loop. The amino acid sequence of V residues
100-110 shown in the figure is PFYRYDGGNYY. Residues
101-109 extend out from the surface of the Fab. The two alternate
conformations are shown individually in ball and stick models in panels
A and B. The side chains for RYD are labeled. In panelC, a stereo diagram is presented with the two
conformations superimposed for direct comparison. The major
conformational difference results from a rotation around the
torsion angle (N-C
) of residue Asp-105 and a
compensatory rotation around the
angle of Asn-108. Note that the
positions of the
-carbons of all residues in the loop except
Gly-106 and Gly-107 are nearly identical, while the side chain
positions of Asp-105 and Asp-108 are strikingly
different.
Such stabilizing interactions are
beneficial for structural analyses of the RGD site in long loops. For
example, in the capsid protein VP1 of the foot-and-mouth disease
virus(53) , an RGD sequence was located in a short loop
adjacent to a helix and is stabilized by -sheet interactions. This
site is conformationally quite different from RGD sites in fibronectin (16, 17) or the related protein tenascin (50) but may also have cell attachment properties. It has been
suggested that viruses bind to cell surface integrins for attachment or
entry to cells, although the identity of the receptor for
foot-and-mouth disease virus is unknown.
In the leech protein
decorsin, the polypeptide backbone of the RGD loop is stabilized by 2
prolines with the sequence, Pro-Arg-Gly-Asp-Ala-Asp-Pro(54) .
Although the arginine side chain was not clearly defined, the
conformation of the loop places the side chains in nearly opposite
extremes of the loop, in contrast to OPG2 where arginine and aspartic
acid side chains emanate from the same side of the loop. The
conformational differences of RGD in OPG2 and decorsin may be related
to receptor selectivity since decorsin binds to both
and
integrins, while OPG2 is specific for
.
No salt bridges are formed
between the Arg-103 and Asp-105 residues in either conformation (see Fig. 4). Instead, Asp-105 and Asn-108 are in position to form
hydrogen bonds in both conformations (i.e. distance between
Asp-105-OD1 and Asn-108-ND1 is 2.8 or 3.2 Å in the alternate
conformers). In one conformation (see Fig. 4, panelB), Asp-105 can also form a potential hydrogen bond (i.e. 3.0 Å) with ND1 of Asn-161 in a symmetry-related
molecule in the crystal lattice. This contact is evidence that a
similar hydrogen-bonded or electrostatic interaction involving Asp-105
is possible upon binding to integrin. Previous studies with synthetic
peptides have shown that the aspartic acid residue in the RGD sequence
is critical for integrin
recognition(26, 28, 29, 55, 56, 57, 58) .
For example, substitution of aspartic acid by glutamic acid, differing
by only one methyl group, abolishes activity of peptides as
antagonists(59) . This
result emphasizes the hypothesis that precise stereochemical
orientation of the Asp side chain influences integrin recognition.
Also, it has been proposed that the oxygenated carboxyl group of side
chains such as aspartic acid may coordinate calcium in the
calcium-binding sites of the integrin(60) . The conformation of
the OPG2 recognition loop shown in Fig. 4B directs the
Asp-105 side chain out from the loop and may represent the conformation
that exists when the ligand is bound to integrin. In contrast, the
other orientation of the Asp-105 carboxylate, bound to a solvent
molecule, may correspond to the free state of the ligand.
The amino
acid present in the second position (i.e. glycine) is less
critical for integrin binding. In OPG2 Fab, the side chain of tyrosine
in position 2 is oriented away from Arg-103 and Asp-105 (see Fig. 4). Apparently, the position of the tyrosine does not
impede the recognition of this sequence by the platelet integrin.
Similarly, an SRYD sequence in the major glycoprotein gp63 of Leishmania is involved in adhesion to macrophages and mimics
the RGD sequence in fibronectin(61) . As stated earlier,
substitution of Tyr Gly at residue 104 does not alter
specificity of OPG2. Rather, the tyrosine side chain in the second
position may stabilize the configuration of the Arg and Asp residues,
thereby contributing to the receptor selectivity of this ligand.
Indeed, a receptor-selective peptide has been identified with an
Arg-Cys-Asp sequence where the Cys residue in the second position forms
a disulfide bond within the cyclic peptide (62) .
Alternatively, Tyr-104 in OPG2 is located within hydrogen-bonding
distance of the side chains of Lys-142 and Asp-143 in an adjacent
molecule in the crystal lattice, suggesting that a related contact
could be made with the integrin. Interestingly, the presence of a
hydrophobic or aromatic residue proximal to the Asp residue is
associated with increased affinity for the gpIIb/IIIa integrin in the
sequence RGDW or KGDW in snake venom disintegrins (28) or in a
cyclic RGD-containing peptide(56) .
The conformational presentation of the RGD sequence is important for binding by specific receptors. The alternate conformations seen in the OPG2 Fab molecule may represent two conformations recognized by distinct or activation-dependent receptors, or the two conformations may correspond to positions that the side chains assume in the free state or when bound to integrin. Loops containing RGD sequence have been shown to be highly flexible, and, consequently, there is always concern that the loop structure observed in the free state may be quite different from the configuration that is formed when the loop is bound by receptor. Since the OPG2 ligand contains an RYD loop with far greater conformational restriction, it may indeed be possible that one of the two conformations seen in this structure is quite close to the conformation of the ligand when bound to receptor.
Conformational
adjustments are possible in CDR loops. For example, in one Fab study,
the H3 loop assumed two distinct conformations depending on whether the
Fab 17/9 was crystallized alone or in complex with a synthetic
peptide(63) . In any event, the model of the OPG2 cell adhesion
mimic provides structural evidence for one type of conformational
flexibility in the presentation of an RGD(RYD) recognition site.
Moreover, the OPG2 antireceptor Fab can provide an ``internal
image'' of the ligand-binding site of the
integrin (see Fig. 5),
which may have important implications for the design of therapeutic
agents directed to the RGD-binding site of this platelet receptor. The
interface at the ``internal image'' could be evaluated for
complementary van der Waals' and electrostatic interactions to
predict receptor contact sites. Ultimately, it will be necessary to
co-crystallize the OPG2 Fab with peptides or fragments of
integrin. Since all of the main
chain and side chain atoms are clearly defined in both conformations,
comparisons of these configurations can be made in the future when
structural data on ligand-integrin complexes become available. The
unique nature of such a complex, where the antibody provides the ligand
rather than the binding pocket, may serve as a model for other antibody
``mimics'' that can be used to study protein-receptor
recognition.
Figure 5: Graphics diagram illustrating the use of the OPG2 Fab model as an internal image of the gpIIb/IIIa receptor. The RYD loop is prominent in the center of the figure in the same orientation as Fig. 4A. To generate the image, a dot surface (31) of this region of the Fab was calculated using a 2.5-Å probe-radius to simulate the possible contact distance between the integrin and the OPG2 ligand, and then the atomic model was moved away from the dot surface. When the dot surface (green) is clipped graphically and viewed from the interior, the image represents the potential complementary surface corresponding to integrin contact sites. This diagram was generated with QUANTA (Molecular Simulations, Inc.).
The atomic coordinates have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.