©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Crystal Structure of the OPG2 Fab
AN ANTIRECEPTOR ANTIBODY THAT MIMICS AN RGD CELL ADHESION SITE (*)

(Received for publication, August 25, 1994; and in revised form, November 14, 1994)

Ramadurgam Kodandapani (1) B. Veerapandian (1) Thomas J. Kunicki (2) Kathryn R. Ely (1)(§)

From the  (1)Cancer Research Center, La Jolla Cancer Research Foundation, La Jolla, California 92037 and the (2)Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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)^1 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 alphabeta(3) receptor (integrin) on platelets. Binding of alphabeta(3) 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 alphabeta(3). 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-alphabeta(3) 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 alpha and beta 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: alphabeta(3), alpha(5)beta(1), alpha(4)beta(1), alpha(v)beta(1), alpha(v)beta(3), alpha(v)beta(6), and alpha(v)beta(8)(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 alphabeta(3) because of the role this integrin plays in platelet aggregation, hemostasis, and arterial thrombotic diseases. Consequently, the alphabeta(3) 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 alphabeta(3) 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 alphabeta(3) integrin and do not bind to any other integrins that recognize RGD, including the closely related alpha(v)beta(3) 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, (^2)the resultant protein binds with equivalent specificity and affinity as the native OPG2, i.e. RGD-OPG2 binds specifically to alphabeta(3) but not to alpha(v)beta(3). Furthermore, substitution of Asp Glu in the RGD site generated a mutant OPG2 with a marked loss in binding to alphabeta(3). 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 alphabeta(3) 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.


EXPERIMENTAL PROCEDURES

Data Collection

The purification and crystallization of the OPG2 Fab fragment were described previously(32) . Crystals formed in the space group P2(1)2(1)2 with unit cell dimensions a = 93.1, b = 83.8, and c = 57.3 Å, and one Fab molecule/asymmetric unit. X-ray diffraction data were collected from a single crystal to 2.0-Å resolution (32) using a Rigaku RU-200 rotating anode x-ray generator and two San Diego Multiwire Systems area detectors. The data collection statistics are presented in Table 1.



Structure Solution

The molecular replacement method (33) was used for phase calculation and structure solution. A homology search was done comparing the OPG2 Fab with the protein sequences of Fab structures available in the Brookhaven Protein Data Bank(34) , and the starting probe model for rotation and translation searches was constructed as follows: V(L), C(L), and C(H)1 domains from HyHel-10 Fab (90% homology for V(L); (35) ) and V(H) domain from Kol Fab (83% homology, (36) ). Rotation searches were done with V(L)-V(H) and C(L)-C(H)1 pairs as well as with the intact probe model with variable ``elbow bend'' angles using the real space Patterson search in X-PLOR(37) , including data from 15 to 4 Å, and this search was followed by Patterson correlation refinement in X-PLOR(38) . Translation function searches were performed in X-PLOR for the orientations identified from the rotation function for V(L)-V(H) and C(L)-C(H)1 pairs calculated with 15 to 4 Å data and a search grid of 1 Å. From these searches, a full Fab model was assembled and adjusted with rigid body refinement and positional refinement in X-PLOR. Residues were numbered sequentially and numerically. Truncated side chains were replaced in the model and after simulated annealing in X-PLOR(39) , the R factor was 0.27. Residues 101-110 (H3) were deleted from this model to begin least squares refinement with PROLSQ in GPRLSA(40, 41) . Refinement alternating with model building using FRODO (42, 43) was performed beginning with 8.0-2.3 Å data. Solvent atoms (119 atoms) were added to the model. After refinement, including data to 2.0-Å resolution, B-values were refined. When the R factor had dropped to 0.22, the atoms for residues 101-110 were built into the model using 2F(o) - F(c) difference maps and OMITMAPS(44) . When the alternate conformers of the H3 loop were recognized and when the refinement of the rest of the protein was nearly complete, PROLSQ refinement was executed using a version of the program modified for refinement with alternate conformations(45) . Each conformation (residues 101-110) was assigned 50% occupancy and refinement continued until atomic B-values for both conformations were approximately equal. Uniform B-values for this segment after the refinement indicated equal occupancy for the two conformations. Finally, to complete the refinement, after simulated annealing, more solvent atoms were added to the model and the final refinement and model building process was completed.


RESULTS AND DISCUSSION

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-kappa) 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(H) domains. In fact, because of the strong sequence homology, Kol V(H) 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(H) domain. This model was produced with the program RIBBONS(51) .




Figure 2: Superimposed alpha-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 alpha-carbon backbone of the OPG2 V(H) domain on the alpha-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 alpha-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. (^3)

Secondary structure analysis (52) of the turn containing the RYD tripeptide indicates a ``beta-type'' turn with torsion angles that are not characteristic of tight beta- 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 alpha-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(H) 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 alpha-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 beta-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 alphabeta(3) and alpha(v)beta(3) integrins, while OPG2 is specific for alphabeta(3).

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 alphabeta(3) 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 alphabeta(3) 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 alphabeta(3) 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.).




FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL46979 (to T. J. K.) and by a grant from the Lucille P. Markey Foundation to the La Jolla Cancer Research Foundation (to K. R. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

§
To whom correspondence should be addressed: La Jolla Cancer Research Foundation, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-455-6480; Fax: 619-450-3432.

(^1)
The abbreviations used are: CDR, complementarity-determining region; V(L), variable region, immunoglobulin light chain; C(L), constant region, light chain; C(H)1, first constant homology region, heavy chain; V(H), variable region, heavy chain; Fab, antigen-binding fragment.

(^2)
T. J. Kunicki, K. R. Ely, and D. S. Annis, submitted for publication.

(^3)
K. R. Ely, T. J. Kunicki, and R. Kodandapani, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Chao-Zhou Ni for helpful discussions, Elya Kurktchi for help with graphics computations, and Shelly Cunningham and Elizabeth Blount for preparing the manuscript for publication. We thank our colleagues for providing coordinates of Pot (Dr. A. Edmundson), 3D6 (Dr. X.-M. He), and R45-45-11 (Dr. J. C. Thierry) Fab molecules for comparison.


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