A multiply substituted G–H loop from foot-and-mouth disease virus in complex with a neutralizing antibody: a role for water molecules

Wendy F. Ochoa1, Susana G. Kalko1, Mauricio G. Mateu2, Paula Gomes3, David Andreu3, Esteban Domingo2, Ignasi Fita1 and Nuria Verdaguer1

Instituto Biología Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Jordi-Girona 18-26, 08034 Barcelona, Spain1
Centro de Biología Molecular ‘Severo Ochoa', Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, 28049 Cantoblanco, Madrid, Spain2
Departament de Química Orgánica, Universitat de Barcelona, 08028 Barcelona, Spain3

Author for correspondence: Nuria Verdaguer. Fax +34 93 2045904. e-mail nvmcri{at}cid.csic.es


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The crystal structure of a 15 amino acid synthetic peptide, corresponding to the sequence of the major antigenic site A (G–H loop of VP1) from a multiple variant of foot-and-mouth disease virus (FMDV), has been determined at 2·3  resolution. The variant peptide includes four amino acid substitutions in the loop relative to the previously studied peptide representing FMDV C-S8c1 and corresponds to the loop of a natural FMDV isolate of subtype C1. The peptide was complexed with the Fab fragment of the neutralizing monoclonal antibody 4C4. The peptide adopts a compact fold with a nearly cyclic conformation and a disposition of the receptor-recognition motif Arg–Gly–Asp that is closely related to the previously determined structure for the viral loop, as part of the virion, and for unsubstituted synthetic peptide antigen bound to neutralizing antibodies. New structural findings include the observation that well-defined solvent molecules appear to play a major role in stabilizing the conformation of the peptide and its interactions with the antibody. Structural results are supported by molecular-dynamic simulations. The multiply substituted peptide developed compensatory mechanisms to bind the antibody with a conformation very similar to that of its unsubstituted counterpart. One water molecule, which for steric reasons could not occupy the same position in the unsubstituted antigen, establishes hydrogen bonds with three peptide amino acids. The constancy of the structure of an antigenic domain despite multiple amino acid substitutions has implications for vaccine design.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Foot-and-mouth disease virus (FMDV) is the causative agent of the most economically important disease of cloven-hoofed animals worldwide (Pereira, 1981 ; Brown, 1994 ). The virus belongs to the genus Aphthovirus of the family Picornaviridae (Rueckert, 1996 ). Control of the disease has been based on large-scale vaccinations with whole-virus inactivated vaccines, limitation of animal movements and destruction of herds exposed to the virus (the ‘stamping-out’ procedure) (reviews in Bachrach, 1968 ; Brown, 1994 ; Domingo et al., 1990 ; Timoney et al., 1992 ). The available vaccines show generally good protection against infection with homologous virus and with antigenically related isolates. Difficulties facing the eradication of FMD include the antigenic diversity of FMDV in nature, which has been reflected in the identification of seven serotypes (A, O, C, SAT1, SAT2, SAT3 and Asia1), 65 subtypes, until subtyping was interrupted (Pereira, 1977 ), and multitudes of antigenic variants that often co-circulate in a given geographical area (Mateu et al., 1988 ). Antigenic variation imposes a periodic updating of vaccine strains, antigenic properties of which must match those of the circulating viruses. Furthermore, some FMD outbreaks have been traced to vaccine strains of the virus (Beck & Strohmaier, 1987 ). For these reasons, it would be highly desirable to develop effective, synthetic FMD vaccines.

Neutralizing antibodies are important determinants of protection against FMD and other picornavirus diseases (Mateu, 1995 ; McCullough et al., 1992 ; Misbah et al., 1992 ). An understanding of the types of interactions between antibodies and viruses that lead to virus neutralization is essential for vaccine design. One of the major antigenic sites of FMDV is located in the G–H loop of capsid protein VP1 (Bittle et al., 1982 ; Pfaff et al., 1982 ; Strohmaier et al., 1982 ). This loop is disordered on the surface of FMDV particles (Acharya et al., 1989 ; Curry et al., 1996 ; Lea et al., 1994 , 1995 ; Logan et al., 1993 ). For FMDV of serotype C, this antigenic site has been termed site A, and it behaves as an independent unit, with very limited influence of other capsid residues regarding the interaction of site A with antibodies (Hewat et al., 1997 ; Lea et al., 1994 ; Mateu, 1995 ; Verdaguer et al., 1999 ). The behaviour of this antigenic loop in FMDV particles in its interaction with antibodies can be mimicked faithfully with synthetic peptides that represent the relevant amino acid sequences found in authentic virus (Clarke et al., 1983 ; Mateu et al., 1989 , 1990 ; Mateu, 1995 ; Rowlands et al., 1983 ). Interestingly, antigenic site A includes a highly conserved Arg–Gly–Asp (RGD) triplet that serves as the recognition site of an integrin receptor (Berinstein et al., 1995 ; Fox et al., 1989 ; Hernández et al., 1996 ; Jackson et al., 1997 ; Mason et al., 1994 ; Neff et al., 1998 ).

The structure of a synthetic peptide representing antigenic site A of FMDV C-S8c1 [a biological clone of natural isolate C-Sta Pau Sp70 (Sobrino et al., 1983 ), a virus representative of the European subtype C1 FMDVs] in a complex with the Fab of neutralizing monoclonal antibodies (MAbs) raised against the virus has been studied by X-ray crystallography (Verdaguer et al., 1995 , 1996 , 1998 ). The synthetic peptide spanned positions 136–150 of capsid protein VP1 (peptide A15, Table 1). Two complexes were studied, involving MAbs SD6 (Verdaguer et al., 1995 , 1996 ) and 4C4 (Verdaguer et al., 1998 ). The two neutralizing MAbs were raised against two different FMDV type C isolates (Mateu et al., 1990 ): SD6 against C-S8c1 and 4C4 against C1-Brescia, a virus that differs from C-S8c1 at two positions (140 and 149) within site A (Table 1). In the two complexes, the peptide antigen acquired a very similar quasi-circular conformation and the RGD motif participated directly in the interaction with residues of the complementarity-determining regions (CDRs) of the antibodies (Verdaguer et al., 1998 ). The RGD triplet appeared in an open turn conformation, very similar to that of reduced FMDV particles of serotype O (Logan et al., 1993 ), and also similar to the conformation in RGD-containing integrin ligands (Pfaff, 1997 ). Remarkably, the Gly-142 and Asp-143 residues of the RGD triplet appear to be critical for interaction both with an integrin receptor and with neutralizing antibodies directed to site A (Mateu et al., 1996 ; Verdaguer et al., 1995 ). Both structural and biochemical evidence suggest that MAbs SD6 and 4C4 neutralize by monovalent binding to antigenic site A (Verdaguer et al., 1997 , 1998 ; review in Domingo et al., 1999 ).


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Table 1. Amino acid sequences of site A peptide variants

 
The quasi-circular shape of the peptide antigen in the complexes was stabilized by peptide–antibody interactions and also through intrapeptide hydrogen bonds and van der Waals' interactions (Verdaguer et al., 1995 , 1996 , 1998 ). If a structure similar to the one found in these complexes was also maintained with variant antigens (including one or several amino acid substitutions), the structure would gain ground as the basis for the design of new anti-FMD immunogens. Several natural isolates of FMDV of serotype C have been characterized previously with regard to the amino acid sequence at antigenic site A and reactivity with MAbs (Martínez et al., 1991 ; Mateu et al., 1988 , 1989 , 1990 ; Verdaguer et al., 1995 , 1996 , 1998 ; Villaverde et al., 1991 ). One of the isolates analysed, termed C-S30 (or C1-Barcelona Sp/81), included four replacements within antigenic site A. Two of the changes were coincident with those found in the C1-Brescia virus (Ala-140->Thr, Thr-149->Ala) and two were new (Ala-138->Thr, Leu-147->Val) (Table 1). In spite of the presence of four amino acid substitutions in the G–H loop of VP1, the reactivity of FMDV C-S30 with MAb 4C4 was indistinguishable from that of FMDV C-S8c1 (Mateu et al., 1990 ). Single substitutions at positions 138 and, especially, 140 and 149 did not have a significant effect on the interaction of synthetic peptide antigens with most MAbs, including 4C4, in ELISA (Verdaguer et al., 1998 ). However, the single replacement of residue Leu-147, highly conserved among FMDVs of serotype C, by valine diminished greatly the reactivity of synthetic peptides with most MAbs, including 4C4, in enzyme immunodot assays and competitive ELISA (Mateu et al., 1992 ; Verdaguer et al., 1998 ). That is, the four amino acid replacements found at the antigenic site A of C-S30 relative to C-S8c1 (Mateu et al., 1989 , 1990 ) appear to exert a compensatory effect that restores the affinity of C-S30 (Mateu et al., 1990 ) and of a synthetic antigen representing its antigenic site A (Mateu et al., 1992 ) for MAb 4C4. Therefore, the substituted antigen offered an opportunity to investigate the structure that a variant form of antigenic site A could acquire as a complex with antibody. The results described in this report reaffirm the quasi-circular conformation for this antigenic loop and reveal new features about the forces involved in the stability of site A and about antigen–antibody interactions, in particular, key interactions mediated by water molecules.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} MAb and synthetic peptides.
The origin and characterization of the anti-FMDV MAb 4C4 (Mateu et al., 1987 , 1989 , 1990 ) and the preparation and purification of the Fab fragment of MAb 4C4 (Verdaguer et al., 1998 ) have been reported previously. Peptides were synthesized by solid-phase procedures, purified and analysed by using procedures that have been described previously (Carreño et al., 1992 ; Mateu et al., 1996 ).

{blacksquare} Crystallization and data collection.
Crystals of the complex between the Fab fragment of the MAb 4C4 and the variant peptide A15-CS30 (Table 1) were obtained by the hanging drop-vapour diffusion technique and successive micro- and macroseedings (Stura & Wilson, 1992 ). Typically small, twined needles, obtained with 18% PEG 4K, were used for microseeding, which produced larger needles at 16% PEG 4K. Finally, these larger needles were used for macroseedings in 2 µl droplets containing 7 mg/ml Fab, 1·8 mg/ml peptide, 6·5% PEG 4K, 0·2 M LiCl with 50 mM Tris–HCl (pH 9), equilibrated against a reservoir containing 13% PEG 4K equally buffered at room temperature. Crystals were orthorhombic, space group P212121 with unit cell parameters a=48·2 , b=69·3  and c=146·5 , containing one molecule of the complex per asymmetric unit, which corresponds to a solvent content volume of 49%. A data set was collected, at 100 K with 20% glycerol as a cryoprotectant, by using a MarReseach imaging plate on a Rigaku rotating anode. Intensities were evaluated and scaled internally with programs Denzo and Scalepack, respectively (Otwinowski & Minor, 1996 ). Data were 93% complete at 2·3  resolution, with an internal agreement factor (Rsymm) of 8·5% (Table 2).


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Table 2. Features of crystals of the antigen–4C4 Fab complex analysed in the present study

 
{blacksquare} Structure solution and refinement.
Crystals of the complex between peptide A15-C-S8c1/LV and the Fab of MAb 4C4 seemed related to crystals formed with the same Fab and peptide A15-C-S8c1 (the names and amino acid sequence of peptide antigens are listed in Table 1), whose structure had been solved previously (Verdaguer et al., 1998 ). However, the unit cell parameter c differed by about 10  and the structure was newly determined by molecular replacement by using the AMoRe package with the 4C4 Fab coordinates as searching model (Navaza, 1994 ). The initial solution was then optimized by allowing to move as four separated rigid bodies the variable heavy, variable light, constant heavy and constant light domains. The resulting R-factor was 0·36% in the resolution range of 15 to 4 . Examination of the electron density maps, calculated at this stage, clearly showed extra density corresponding to the oligopeptide occupying the antigen-binding site. The final model for the structure of the complex was obtained by iterative cycles of model rebuilding by using the program O (Jones et al., 1991 ) and positional refinement with XPLOR (Brünger, 1992 ), including bulk solvent correction. The refined model converged to crystallographic agreement factors R and Rfree of 24% and 26%, respectively, for 17943 reflections in the resolution shell 15·0–2·3  (Table 2).

{blacksquare} Molecular dynamics (MD) simulation.
Three sets of MD simulations, corresponding to the variable module (Fv) of the 4C4 Fab interacting with peptides A15-C-S8c1, A15-C-S8c1/LV and A15-C-S30 (Table 1), were performed by using the GROMOS96 package with its standard protein and water force fields (van Gunsteren et al., 1996 ). Crystallographic coordinates of peptides A15-C-S8c1 and A15-C-S30 (residues 137–148) were used as starting models in the corresponding simulations. Thr-148, not visible in the electron density maps of the A15-C-S8c1 complex, was added to avoid end effects in the critical Leu-147 residue, by using the information available in the A15-C-S30 structure. Starting coordinates for the A15-C-S8c1/LV peptide were then derived, with the graphic program TURBO (Rousel & Cambillau, 1989 ), by replacing Leu-147 with valine. Crystallographic water molecules determined in the present work for the complex of 4C4 Fab with the A15-C-S30 peptide were not included in the simulations.

In every simulation, the structure of the corresponding complex was placed initially at the centre of a truncated octahedron, the dimensions of which were chosen such that the minimum distance of any protein atom from the closest wall was 7 . The edge lengths of the corresponding cubic boxes were about 73 . Systems were treated as immersed into an equilibrium configuration of bulk simple point charge (SPC) water (Berendsen et al., 1986 ). Water molecules outside the box or with a distance to a solute atom of less than 2·3 were removed. The numbers of water molecules considered in the three simulations were 5271, 5272 and 5264, respectively. To relax strong water–water and water–protein non-bonded interactions, steepest-descent energy minimization was performed until stabilization was reached. After that, counter-ions were added to neutralize charged protein sites, with a subsequent energy minimization. Simulations were performed at constant volume and temperature (300 K) with periodic boundary conditions and an integration step of 2 fs. Temperature was kept constant by weak coupling to an external bath (Berendsen et al., 1984 , 1986 ). Bond lengths were constrained to equilibrium values by using the SHAKE algorithm (Rickaert et al., 1977 ). Equilibrations were achieved within 50 ps and, afterwards, the three systems were simulated for a total time of 300 ps with the total potential energy remaining essentially constant. Only amino acids from the peptides and all water molecules were allowed to move during calculations. Analysis were performed, mainly with programs contained in the GROMOS96 package, using the structures generated every 0·1 ps during the interval spanning from equilibration until the end (from about 50 to 350 ps).

{blacksquare} Coordinates.
Coordinates have been deposited in the Brookhaven protein database under accession number 1EJO.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Structure of the 4C4 Fab in complex with the A15-CS30 peptide
The final electron density maps of the complex between the 4C4 Fab and the A15-C-S30 peptide, at 2·3  resolution, allowed the positioning of the main and side chains of 431 of the total of 436 4C4 Fab residues (216 from the light chain and 220 from the heavy chain) and of 12 of the 15 peptide residues (Fig. 1A). Heavy-chain Fab residues 136–143, in the constant domain, and terminal peptide residues Ala-149, Thr-150 and the side chain of Tyr-136 were disordered, and their coordinates have not been introduced in the present model. The quality of the final maps is also reflected in the 166 well-defined solvent molecules found, nine of which participate in the interactions between the peptide and the Fab CDRs (Table 3), and three water molecules establish bridges among peptide residues (Fig. 1B).



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Fig. 1. (A) Stereo view of the (2Fo-Fc) electron density map corresponding to the antigenic peptide A15-C-S30. The peptide model and nearby solvent molecules (isolated dots) are also shown. (B) Stereo view of the Fab–peptide interactions. Only the Fab residues in direct contact with the peptide are depicted (open rods). Water molecules (W) in direct contact with the peptide or mediating the antibody recognition are also shown. Hydrogen bonds are indicated by broken lines. (C) Superposition of the A15 peptide conformations found in the 4C4 Fab complexes with the A15-C-S30 (filled) and the A15-C-S8c1 (open) peptides.

 

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Table 3. Hydrogen bonds between the A15-CS30 and A15-CS8 peptides and the 4C4 Fab

 


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Fig. 2. Contact areas for the A15 peptides in the 4C4 Fab complexes with the A15-C-S30 (filled bars) and the A15-C-S8c1 (open bars) peptides expressed as absolute values (A) or as percentages of the residue surface (B). Asterisks indicate A15-C-S30 residues that differ in A15-C-S8c1. Peptide sequences are shown in Table 1.

 
The A15-C-S30 peptide shows an overall conformation closely related to the one found in other site A complexes determined previously (Verdaguer et al., 1995 , 1998 ) (Fig. 1 C). The RGD motif, residues 141–143, is located in an open turn conformation preceded by an extended region, residues 136–140, and followed by a short helix, positions 144–148 (Fig. 1). All of the peptide residues incorporated into the model interact directly with the hypervariable regions of the Fab (Table 3 and Fig. 2). In particular, the entire molecular surfaces of Asp-143, from the RGD motif, and Leu-144 are in contact with the antibody, similar to what had been observed in complexes involving Fab A15-C-S8c1 (Fig. 2) (Verdaguer et al., 1995 , 1998 ). However, the substituted residues Thr-138 and, particularly, Thr-140 and Val-147 do not present extensive interactions with the antibody (Fig. 2). The fourth substitution, Ala-149, not visible in the electron density and not included in the present model, must also remain exposed to the solvent and far from the antibody.

Comparisons of the site A variant peptides A15-CS30 and A15-CS8c1
The root-mean-square deviation (RMSD) between the main-chain peptide atoms in the A15-C-S30 and A15-C-S8c1 Fab 4C4 complexes is only 0·4  for the common residues 137–147. The Arg-141 side chain has different dispositions in the two peptides (Fig. 1C), although the electron density corresponding to this residue was weak in both complexes (Fig. 1A; see also Verdaguer et al., 1995 , 1998 ). The replaced residues, Thr-138 and Val-147, show the largest main-chain deviations of 0·7 and 1·0 , respectively. Despite the high structural similarity between the two peptides A15-C-S30 and A15-C-S8c1, differences in the main-chain conformational angles are important, particularly around the RGD motif (Fig. 2). Thus, in the A15-C-S30 peptide, residues Thr-140 and Arg-141 are situated in the Ramachandran region corresponding to {alpha}-helices while, in the A15-C-S8c1 peptide, Ala-140 and Arg-141 are in the regions of {beta}-strands and left-handed helices, respectively. A third situation is found in the structure reported for the G–H loop of the reduced FMDV serotype O1 (Lea et al., 1993 , 1995 ), where the corresponding residues Leu-144 and Arg-145 are both found in the Ramachandran region corresponding to {beta}-strands. The flexibility of Gly-142 allows compensatory main-chain torsional angles that result in the overall structural similarity of the three peptides. It is important to notice the presence, in the A15-CS30 structure, of one water molecule (named w8 in Fig. 1) that is hydrogen-bonded with the side chain of Thr-138 and the main-chain oxygen atoms of residues Leu-144 and Leu-147. This water molecule cannot be present, for steric reasons, in the A15-C-S8c1 structure, where the bulkier Leu-147 side chain fills the available space (Fig. 3).



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Fig. 3. Space-filling representation of the three peptide models A15-C-S8c1 (A), A15-C-S8/LV (B) and A15-C-S30 (C). Peptide residues that contribute to intrapeptide hydrophobic interactions are highlighted in dark-blue. The water molecule found in contact with the substituted Thr-138 in the A15-C-S30 peptide is displayed in red (C).

 
MD analysis
Three MD simulations corresponding to complexes of the Fv 4C4 with peptides A15-C-S8c1, A15-C-S8c1/LV and A15-C-S30 were carried out as described in Methods. The main difference between the averaged conformations of the three peptides is the opening of the C-terminal region in the simulation of the single-substituted A15-C-S8c1/LV peptide (Fig. 4A). Other significant differences can also be seen at the amino termini and for the Arg-141 side chain. The stability of the averaged conformations was analysed by evaluating the RMSD values of the peptide fluctuations in every simulation. The lowest values correspond to the A15-C-S30 peptide, while the more constant main-chain region includes the RGD motif and Leu-144 in the three peptides, with Asp-143 having the smallest variation. The side chains of Asp-143 and Leu-144 also appear extremely invariant, probably reflecting the extent and the stability of the interactions of these residues with the antibody. The stability of the tetra-substituted peptide A15-C-S30 observed during the simulation is contributed by a hydrogen-bonded water molecule located in a site very close to the position of the bridging crystallographic water (w8) (Figs 1B and 4B). This water site presents a high occupation and a low RMSD of about 0·1 . Hydrogen-bond interactions take place between the water molecule that occupies this site and polar atoms from the substituted residues Thr-138, Leu-144 and Val-147 (Table 3). The increased hydrophobic surface exposed to the solvent appears to explain the departure from the ‘correct' site A-like conformation observed by MD with the A15-CS8/LV peptide (Fig. 3).



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Fig. 4. (A) Stereo view of the superposition of the A15-C-S8c1 (filled), A15-C-S8/LV (shaded) and A15-C-S30 (open) peptides corresponding to the averaged conformations calculated in 300 ps of MD simulations of the corresponding Fv–peptide complexes. (B) Stereo view of the superposition of the A15-C-S30 coordinates from the crystal (filled) and the MD simulation (open). The water molecule found in the crystal structure (shaded) is situated close to the water site determined in the MD simulation (open).

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
The effects of single or multiple amino acid substitutions at antigenic site A of FMDV of serotype C on the reactivity of site A-specific neutralizing MAbs have been studied extensively by immunochemical assays employing variant viruses and substituted synthetic peptides (Mateu et al., 1989 , 1990 , 1992 ; Martínez et al., 1997 ; Novella et al., 1993 ; Verdaguer et al., 1998 ). Replacements at position 147 altered the binding of most antibodies substantially, including 4C4, even though residue 147 had only marginal interactions with that antibody. In particular, substitution of Leu-147 by valine diminished the binding of most site A-specific MAbs analysed (Mateu et al., 1992 ). However, the effect of this single substitution Leu-147->Val was apparently compensated for, at least in part, by the replacement Ala-138->Thr (Mateu et al., 1992 ). These observations and the fact that natural FMDV isolate C-S30 (Martínez et al., 1991 ; Villaverde et al., 1991 ) contains these critical replacements in its VP1 G–H loop relative to clone C-S8c1 (Table 1) encouraged the present study.

In the structure of the 4C4 Fab complexed with the variant peptide A15-C-S30 reported here, the two amino acids Thr-138 and Val-147 that were replaced in the peptide show only minor interactions with the Fab (Table 2 and Fig. 2). Differences in binding affinities for peptides with substitutions in those two residues should therefore be due mainly to free-energy differences when adopting a ‘correct' site A-like loop structure, the one recognized by antibodies or by the receptor (Fig. 5). The reduced stability of that conformation in the peptide with the single substitution Leu-147->Val would then explain the reduced affinity observed for the antibody. Instead, the double-substituted peptide could recover binding affinity by re-stabilizing the ‘correct' conformation with the additional water molecule (w8) that bridges the chain hydroxyl group of the replaced Thr-138 with the main-chain oxygen atoms of residues Leu-144 and Leu-147 (Table 3 and Fig. 3C). In the MD simulation of the A15-C-S30 variant peptide, a water molecule with a low RMSD is placed in almost exactly the same location as the one found in the crystal structure (Fig. 4B). These structural results provide a rationale for how a general change in specificity to a large diversity of antibodies can be achieved by a destabilization of the original antigen structure. In those cases, further substitutions can restore the affinity for the antibodies by increasing the stability of the original antigen conformation, even when none of the residues substituted participates in direct antigen–antibody interactions (Freire, 1999 ).



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Fig. 5. A simplified thermodynamic cycle of a Fab interacting with an antigenic peptide can be described as a two-step process. In the first step, the peptide adopts individually the structure seen in the complex that is formed in the second step, so that {Delta}Gtotal={Delta}Gfolding+{Delta}Gbinding, where {Delta}Gfolding refers to the free-energy difference between the peptide structure in solution and that found in the complex (the Fab reorganization, if required, can also be included in this term) and {Delta}Gbinding refers to the free-energy difference due to the formation of the interactions between the peptide and the Fab. A ‘lock-and-key’ recognition mechanism, in which structures remain unchanged on complex formation, corresponds to {Delta}Gfolding=0. Instead, {Delta}Gfolding<0 describes an ‘induced-fit’ mechanism, where the complex would only be stabilized by a favourable {Delta}Gbinding contribution. Analysis of Fab 4C4 complexes with a number of A15 peptides, both from the available crystal structures and with MD simulations, suggest that {Delta}Gbinding(A15-C-S30){approx}{Delta}Gbinding(A15-C-S8/LV), while the experimental data on the relative stabilities of these complexes (Mateu et al., 1992 ) imply that {Delta}Gtotal(A15-C-S30)<{Delta}Gtotal(A15-C-S8/LV), which would allow us to conclude that {Delta}Gfolding(A15-C-S30)<{Delta}Gfolding(A15-C-S8/LV). The reduced stability of the ‘correct’ site A-like conformation for the A15-C-S8/LV peptide would explain the lack of affinity of most site A MAbs for this variant peptide (see text).

 
Recognition of continuous epitopes, like the VP1 G–H loop of FMDV studied in this work, depends not only on the amino acid sequence but also on the spatial conformation of the epitope, where solvent molecules often play a critical role. Interpretation of mechanisms of virus escape from antibody recognition, essential in RNA virus evolution and vaccine design, must also take into consideration the effects of amino acid replacements in connection with the solvent rearrangements imposed. In spite of difficulties in predicting the effects of amino acid replacements in antigen–antibody recognition, the present results for FMDV encourage the design of quasi-cyclic structures reproducing antigenic site A. Data for the G–H loop from a number of different antibodies, including the multiply substituted peptide reported here, suggest a robust and strongly immunogenic structure despite the mobility of this antigenic site on the virion surface (Parry et al., 1990 ; Logan et al., 1993 ). Cocktails of quasi-circular peptides representing the most frequent escape mutants found at site A should be candidates for incorporation into vaccine formulations. Such formulations should include additional B-cell and T-cell epitopes to ensure a broad immune response to minimize the selection of escape mutants and vaccine failures (Domingo & Holland, 1992 ; Taboga et al., 1997 ).


   Acknowledgments
 
We wish to thank E. Brocchi for the generous supply of MAb 4C4 and her continuous help with our research. Work in Barcelona was funded by DGICYT grants PB95-0218 and PB97-0873. Work in Madrid was supported by grants from DGES PM97-006-C02-01 and Fundación Ramón Areces. W.F.O. is a recipient of a fellowship from the Ministerio de Educación y Cultura (Spain). P.G. thanks the Fundaçao C. Gulbenkian for a PhD fellowship and the University of Porto for temporary leave.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Acharya, R., Fry, E., Stuart, D., Fox, G., Rowlands, D. & Brown, F. (1989). The three-dimensional structure of foot-and-mouth disease virus at 2·9  resolution.Nature 337, 709-716.[Medline]

Bachrach, H. L. (1968). Foot and mouth disease virus.Annual Review of Microbiology 22, 201-244.[Medline]

Beck, E. & Strohmaier, K. (1987). Subtyping of European foot-and-mouth disease virus strains by nucleotide sequence determination. Journal of Virology 61, 1621-1629.[Medline]

Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A. & Haak, J. R. (1984). Molecular dynamics with coupling to an external bath.Journal of Chemical Physics 81, 3684-3690.

Berendsen, H. J., Van Gunsteren, W. F., Zwinderman, H. R. & Geurtsen, R. G. (1986). Simulations of proteins in water.Annals of the New York Academy of Sciences 482, 269-286.[Abstract]

Berinstein, A., Roivainen, M., Hovi, T., Mason, P. W. & Baxt, B. (1995). Antibodies to the vitronectin receptor (integrin alpha V beta 3) inhibit binding and infection of foot-and-mouth disease virus to cultured cells.Journal of Virology 69, 2664-2666.[Abstract]

Bittle, J. L., Houghten, R. A., Alexander, H., Shinnick, T. M., Sutcliffe, J. G., Lerner, R. A., Rowlands, D. J. & Brown, F. (1982). Protection against foot-and-mouth disease by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence.Nature 298, 30-33.[Medline]

Brown, F. (1994). Foot-and-mouth disease. In Synthetic Vaccines, pp. 416-432. Edited by B. H. Nicholson. Oxford: Blackwell Scientific.

Brünger, A. T. (1992). XPLOR manual, version 3.0. Yale University, New Haven, CT, USA.

Carreño, C., Roig, X., Cairo, J., Camarero, J., Mateu, M. G., Domingo, E., Giralt, E. & Andreu, D. (1992). Studies on antigenic variability of C strains of foot-and-mouth disease virus by means of synthetic peptides and monoclonal antibodies.International Journal of Peptide and Protein Research 39, 41-47.[Medline]

Clarke, B. E., Carroll, A. R., Rowlands, D. J., Nicholson, B. H., Houghten, R. A., Lerner, R. A. & Brown, F. (1983). Synthetic peptides mimic subtype specificity of foot-and-mouth disease virus.FEBS Letters 157, 261-264.[Medline]

Curry, S., Fry, E., Blakemore, W., Abu-Ghazaleh, R., Jackson, T., King, A., Lea, S., Newman, J., Rowlands, D. & Stuart, D. (1996). Perturbations in the surface structure of A22 Iraq foot-and-mouth disease virus accompanying coupled changes in host cell specificity and antigenicity. Structure 4, 135-145.[Medline]

Domingo, E. & Holland, J. J. (1992). Complications of RNA heterogeneity for the engineering of virus vaccines and antiviral agents. In Genetic Engineering, Principles and Methods, pp. 13-32. Edited by J. K. Setlow. New York: Plenum.

Domingo, E., Mateu, M. G., Martínez, M. A., Dopazo, J., Moya, A. & Sobrino, F. (1990). Genetic variability and antigenic diversity of foot-and-mouth disease virus. In Applied Virology Research, pp. 233-266. Edited by E. Kurkstak, R. G. Marusyk, S. A. Murphy & M. H. V. Van Regenmortel. New York: Plenum.

Domingo, E., Verdaguer, N., Ochoa, W. F., Ruiz-Jarabo, C. M., Sevilla, N., Baranowski, E., Mateu, M. G. & Fita, I. (1999). Biochemical and structural studies with neutralizing antibodies raised against foot-and-mouth disease virus. Virus Research 62, 169-175.[Medline]

Fox, G., Parry, N. R., Barnett, P. V., McGinn, B., Rowlands, D. J. & Brown, F. (1989). The cell attachment site on foot-and-mouth disease virus includes the amino acid sequence RGD (arginine-glycine-aspartic acid). Journal of General Virology 70, 625-637.[Abstract]

Freire, E. (1999). The propagation of binding interactions to remote sites in proteins: analysis of the binding of the monoclonal antibody D1.3 to lysozyme. Proceedings of the National Academy of Sciences, USA 96, 10118-10122.[Abstract/Free Full Text]

Hernández, J., Valero, M. L., Andreu, D., Domingo, E. & Mateu, M. G. (1996). Antibody and host cell recognition of foot-and-mouth disease virus (serotype C) cleaved at the Arg-Gly-Asp (RGD) motif: a structural interpretation. Journal of General Virology 77, 257-264.[Abstract]

Hewat, E. A., Verdaguer, N., Fita, I., Blakemore, W., Brookes, S., King, A., Newman, J., Domingo, E., Mateu, M. G. & Stuart, D. I. (1997). Structure of the complex of an Fab fragment of a neutralizing antibody with foot-and-mouth disease virus: positioning of a highly mobile antigenic loop. EMBO Journal 16, 1492-1500.[Abstract/Free Full Text]

Jackson, T., Sharma, A., Abu-Ghazaleh, R., Blakemore, W. E., Ellard, F. M., Simmons, D. L., Newman, J. W., Stuart, D. I. & King, A. M. (1997). Arginine-glycine-aspartic acid-specific binding by foot-and-mouth disease viruses to the purified integrin {alpha}v{beta}3 in vitro. Journal of Virology 71, 8357-8361.[Abstract]

Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallographica A47, 110-119.[Medline]

Lea, S., Hernández, J., Blakemore, W., Brocchi, E., Curry, S., Domingo, E., Fry, E., Abu-Ghazaleh, R., King, A., Newman, J., Stuart, D. & Mateu, M. G. (1993). Structure of a major immunogenic site on foot-and-mouth disease virus. Nature 362, 566-568.[Medline]

Lea, S., Hernández, J., Blakemore, W., Brocchi, E., Curry, S., Domingo, E., Fry, E., Abu-Ghazaleh, R., King, A., Newman, J., Stuart, D. & Mateu, M. G. (1994). The structure and antigenicity of a type C foot-and-mouth disease virus. Structure 2, 123-139.[Medline]

Lea, S., Abu-Ghazaleh, R., Blakemore, W., Curry, S., Fry, E., Jackson, T., King, A., Logan, D., Newman, J. & Stuart, D. (1995). Structural comparison of two strains of foot-and-mouth disease virus subtype O1 and a laboratory antigenic variant, G67. Structure 3, 571-580.[Medline]

Logan, D., Abu-Ghazaleh, R., Blakemore, W., Curry, S., Jackson, T., King, A., Lea, S., Lewis, R., Newman, J., Parry, N., Rowlands, D., Stuart, D. & Fry, E. (1993). Structure of a major immunogenic site on foot-and-mouth disease virus. Nature 362, 566-568.[Medline]

McCullough, K. C., De Simone, F., Brocchi, E., Capucci, L., Crowther, J. R. & Kihm, U. (1992). Protective immune response against foot-and-mouth disease. Journal of Virology 66, 1835-1840.[Abstract]

Martínez, M. A., Hernández, J., Piccone, M. E., Palma, E. L., Domingo, E., Knowles, N. & Mateu, M. G. (1991). Two mechanisms of antigenic diversification of foot-and-mouth disease virus. Virology 184, 695-706.[Medline]

Martínez, M. A., Verdaguer, N., Mateu, M. G. & Domingo, E. (1997). Evolution subverting essentiality: dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus . Proceedings of the National Academy of Sciences, USA 94, 6798-6802.[Abstract/Free Full Text]

Mason, P. W., Rieder, E. & Baxt, B. (1994). RGD sequence of foot-and-mouth disease virus is essential for infecting cells via the natural receptor but can be bypassed by an antibody-dependent enhancement pathway. Proceedings of the National Academy of Sciences, USA 91, 1932-1936.[Abstract]

Mateu, M. G. (1995). Antibody recognition of picornaviruses and escape from neutralization: a structural view. Virus Research 38, 1-24.[Medline]

Mateu, M. G., Rocha, E., Vicente, O., Vayreda, F., Navalpotro, C., Andreu, D., Pedroso, E., Giralt, E., Enjuanes, L. & Domingo, E. (1987). Reactivity with monoclonal antibodies of viruses from an episode of foot-and-mouth disease. Virus Research 8, 261-274.[Medline]

Mateu, M. G., Da Silva, J. L., Rocha, E., De Brum, D. L., Alonso, A., Enjuanes, L., Domingo, E. & Barahona, H. (1988). Extensive antigenic heterogeneity of foot-and-mouth disease virus of serotype C. Virology 167, 113-124.[Medline]

Mateu, M. G., Martínez, M. A., Rocha, E., Andreu, D., Parejo, J., Giralt, E., Sobrino, F. & Domingo, E. (1989). Implications of a quasispecies genome structure: effect of frequent, naturally occurring amino acid substitutions on the antigenicity of foot-and-mouth disease virus. Proceedings of the National Academy of Sciences, USA 86, 5883-5887.[Abstract]

Mateu, M. G., Martínez, M. A., Capucci, L., Andreu, D., Giralt, E., Sobrino, F., Brocchi, E. & Domingo, E. (1990). A single amino acid substitution affects multiple overlapping epitopes in the major antigenic site of foot-and-mouth disease virus of serotype C. Journal of General Virology 71, 629-637.[Abstract]

Mateu, M. G., Andreu, D., Carreño, C., Roig, X., Cairó, J. J., Camarero, J. A., Giralt, E. & Domingo, E. (1992). Non-additive effects of multiple amino acid substitutions on antigen–antibody recognition. European Journal of Immunology 22, 1385-1389.[Medline]

Mateu, M. G., Valero, M. L., Andreu, D. & Domingo, E. (1996). Systematic replacement of amino acid residues within an Arg-Gly-Asp-containing loop of foot-and-mouth disease virus and effect on cell recognition. Journal of Biological Chemistry 271, 12814-12819.[Abstract/Free Full Text]

Misbah, S. A., Spickett, G. P., Ryba, P. C., Hockaday, J. M., Kroll, J. S., Sherwood, C., Kurtz, J. B., Moxon, E. R. & Chapel, H. M. (1992). Chronic enteroviral meningoencephalitis in agammaglobulinemia: case report and literature review. Journal of Clinical Immunology 12, 266-270.[Medline]

Navaza, J. (1994). AMoRe: an automated package for molecular replacement. Acta Crystallographica A50, 157-163.

Neff, S., Sá-Carvalho, D., Rieder, E., Mason, P. W., Blystone, S. D., Brown, E. J. & Baxt, B. (1998). Foot-and-mouth disease virus virulent for cattle utilizes the integrin {alpha}v{beta}3 as its receptor. Journal of Virology 72, 3587-3594.[Abstract/Free Full Text]

Novella, I. S., Borrego, B., Mateu, M. G., Domingo, E., Giralt, E. & Andreu, D. (1993). Use of substituted and tandem-repeated peptides to probe the relevance of the highly conserved RGD tripeptide in the immune response against foot-and-mouth disease virus. FEBS Letters 330, 253-259.[Medline]

Otwinowski, Z. & Minor, W. (1996). Processing of X-ray diffraction data collected in an oscillation mode. Methods in Enzymology 35, 307-326.

Parry, N., Fox, G., Rowlands, D., Brown, F., Fry, E., Acharya, R., Logan, D. & Stuart, D. (1990). Structural and serological evidence for a novel mechanism of antigenic variation in foot-and-mouth disease virus. Nature 347, 569-572.[Medline]

Pereira, H. G. (1977). Subtyping foot-and-mouth disease virus. Developments in Biological Standardization 35, 167-174.

Pereira, H. G. (1981). Foot-and-mouth disease virus. In Virus Diseases of Food Animals, pp. 333-363. Edited by R. P. G. Gibbs. New York: Academic Press.

Pfaff, E. (1997). Recognition sites of RGD-dependent integrins. In Integrin Ligand Interactions, pp. 101-121. Edited by J. A. Eble & K. Kühn. Austin, TX: R. G. Landes.

Pfaff, E., Mussgay, M., Bohm, H. O., Schulz, G. E. & Schaller, H. (1982). Antibodies against a preselected peptide recognize and neutralize foot and mouth disease virus. EMBO Journal 1, 869-874.[Medline]

Rickaert, J. P., Ciccotti, G. & Berendsen, H. J. C. (1977). Numerical integration of cartesian equations of motions of a system with constraints: molecular dynamics of n-alkanes. Journal of Computational Physics 23, 327-341.

Rousel, A. & Cambillau, C. (1989). TURBO-Frodo. In Silicon Graphics Geometry Partners Directory, pp. 77–79. Mountain View, CA: Silicon Graphics.

Rowlands, D. J., Clarke, B. E., Carroll, A. R., Brown, F., Nicholson, B. H., Bittle, J. L., Houghten, R. A. & Lerner, R. A. (1983). Chemical basis of antigenic variation in foot-and-mouth disease virus. Nature 306, 694-697.[Medline]

Rueckert, R. R. (1996). Picornaviridae: the viruses and their replication. In Fields Virology, pp. 609-654. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott–Raven.

Sobrino, F., Dávila, M., Ortín, J. & Domingo, E. (1983). Multiple genetic variants arise in the course of replication of foot-and-mouth disease virus in cell culture. Virology 128, 310-318.[Medline]

Strohmaier, K., Franze, R. & Adam, K.-H. (1982). Location and characterization of the antigenic portion of the FMDV immunizing protein. Journal of General Virology 59, 295-306.[Abstract]

Stura, E. A. & Wilson, I. A. (1992). Seeding techniques. In Crystallization of Nucleic Acids and Proteins. A Practical Approach, pp. 99-126. Edited by A. Ducruix & R. Giege. Oxford: IRL Press.

Taboga, O., Tami, C., Carrillo, E., Núñez, J. I., Rodríguez, A., Saíz, J. C., Blanco, E., Valero, M. L., Roig, X., Camarero, J. A., Andreu, D., Mateu, M. G., Giralt, E., Domingo, E., Sobrino, F. & Palma, E. L. (1997). A large-scale evaluation of peptide vaccines against foot-and-mouth disease: lack of solid protection in cattle and isolation of escape mutants. Journal of Virology 71, 2606-2614.[Abstract]

Timoney, J. F., Gillespie, J. H., Scott, F. N. & Barlough, J. E. (1992). Foot-and-mouth disease. In Hagan and Bruner’s Microbiology and Infectious Diseases of Domestic Animals, pp. 647–667. Ithaca, NY: Cornell University Press.

van Gunsteren, W. F., Billeter, S. R., Eising, A. A., Hünenberg, P. H., Krüger, P., Mark, A. E., Scott, W. R. P. & Tironi, I. G. (1996). Biomolecular simulation. In GROMOS96 Manual and User Guide. Zürich: Biomolecular Software.

Verdaguer, N., Mateu, M. G., Andreu, D., Giralt, E., Domingo, E. & Fita, I. (1995). Structure of the major antigenic loop of foot-and-mouth disease virus complexed with a neutralizing antibody: direct involvement of the Arg-Gly-Asp motif in the interaction. EMBO Journal 14, 1690-1696.[Abstract]

Verdaguer, N., Mateu, M. G., Bravo, J., Domingo, E. & Fita, I. (1996). Induced pocket to accommodate the cell attachment Arg-Gly-Asp motif in a neutralizing antibody against foot-and-mouth disease virus. Journal of Molecular Biology 256, 364-376.[Medline]

Verdaguer, N., Fita, I., Domingo, E. & Mateu, M. G. (1997). Efficient neutralization of foot-and-mouth disease virus by monovalent antibody binding. Journal of Virology 71, 9813-9816.[Abstract]

Verdaguer, N., Sevilla, N., Valero, M. L., Stuart, D., Brocchi, E., Andreu, D., Giralt, E., Domingo, E., Mateu, M. G. & Fita, I. (1998). A similar pattern of interaction for different antibodies with a major antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation. Journal of Virology 72, 739-748.[Abstract/Free Full Text]

Verdaguer, N., Schoehn, G., Ochoa, W. F., Fita, I., Brookes, S., King, A., Domingo, E., Mateu, M. G., Stuart, D. & Hewat, E. A. (1999). Flexibility of the major antigenic loop of foot-and-mouth disease virus bound to a Fab fragment of a neutralizing antibody: structure and neutralization. Virology 255, 260-268.[Medline]

Villaverde, A., Martínez, M. A., Sobrino, F., Dopazo, J., Moya, A. & Domingo, E. (1991). Fixation of mutations at the VP1 gene of foot-and-mouth disease virus. Can quasispecies define a transient molecular clock? Gene 103, 147-153.[Medline]

Received 2 December 1999; accepted 22 February 2000.