Probing degeneracy in antigen-antibody recognition at the immunodominant site of foot-and-mouth disease virus.

Antigen-antibody binding is regarded as one of the most representative examples of specific molecular recognition in nature. The simplistic view of antigenic recognition in terms of a lock-and-key mechanism is obsolete, as it is evident that both antigens and antibodies are flexible and can undergo substantial mutual adaptation. This flexibility is the source of complexities such as degeneracy and nonadditivity in antigenic recognition. We have used surface plasmon resonance to study the effects of combining multiple amino acid replacements within the sequence of the antigenic GH loop of foot-and-mouth disease virus. Our aim was 2-fold: to explore the extent to which antigenic degeneracy can be extended in this particular case, and to search for potential nonadditive effects in introducing multiple amino acid replacements. Combined analysis of one such multiply substituted peptide by SPR, solution NMR and X-ray diffraction shows that antigenic degeneracy can be expected as long as residues directly interacting with the paratope are conserved and the peptide bioactive folding is unaltered.

Structure of the C2 domain from novel protein kinase Cepsilon. A membrane binding model for Ca(2+)-independent C2 domains.

Protein kinase Cepsilon (PKCepsilon) is a member of the novel PKCs which are activated by acidic phospholipids, diacylglycerol and phorbol esters, but lack the calcium dependence of classical PKC isotypes. The crystal structures of the C2 domain of PKCepsilon, crystallized both in the absence and in the presence of the two acidic phospholipids, 1,2-dicaproyl-sn-phosphatidyl-l-serine (DCPS) and 1,2-dicaproyl-sn-phosphatidic acid (DCPA), have now been determined at 2.1, 1.7 and 2.8 A resolution, respectively. The central feature of the PKCepsilon-C2 domain structure is an eight-stranded, antiparallel, beta-sandwich with a type II topology similar to that of the C2 domains from phospholipase C and from novel PKCdelta. Despite the similar topology, important differences are found between the structures of C2 domains from PKCs delta and epsilon, suggesting they be considered as different PKC subclasses. Site-directed mutagenesis experiments and structural changes in the PKCepsilon-C2 domain from crystals with DCPS or DCPA indicate, though phospholipids were not visible in these structures, that loops joining strands beta1-beta2 and beta5-beta6 participate in the binding to anionic membranes. The different behavior in membrane-binding and activation between PKCepsilon and classical PKCs appears to originate in localized structural changes, which include a major reorganization of the region corresponding to the calcium binding pocket in classical PKCs. A mechanism is proposed for the interaction of the PKCepsilon-C2 domain with model membranes that retains basic features of the docking of C2 domains from classical, calcium-dependent, PKCs.

Crystallization and preliminary X-ray analysis of clade I catalases from Pseudomonas syringae and Listeria seeligeri.

Haem-containing catalases are homotetrameric molecules that degrade hydrogen peroxide. Phylogenetically, the haem-containing catalases can be grouped into three main lines or clades. The crystal structures of seven catalases have been determined, all from clades II and III. In order to obtain a structure of an enzyme from clade I, which includes all plant, algae and some bacterial enzymes, two bacterial catalases, CatF from Pseudomonas syringae and Kat from Listeria seeligeri, have been crystallized by the hanging-drop vapour-diffusion technique, using PEG and ammonium sulfate as precipitants, respectively. Crystals of P. syringae CatF, with a plate-like morphology, belong to the monoclinic space group P2(1), with unit-cell parameters a = 60.6, b = 153.9, c = 109.2 A, beta = 102.8 degrees. From these crystals a diffraction data set to 1.8 A resolution with 98% completeness was collected using synchrotron radiation. Crystals of L. seeligeri Kat, with a well developed bipyramidal morphology, belong to space group I222 (or I2(1)2(1)2(1)), with unit-cell parameters a = 74.4, b = 121.3, c = 368.5 A. These crystals diffracted beyond 2.2 A resolution when using synchrotron radiation, but presented anisotropic diffraction, with the weakest direction perpendicular to the long c axis.

The need for a shared database infrastructure: combining X-ray crystallography and electron microscopy.

Advances in structural biology are opening greater opportunities for understanding biological structures from the cellular to the atomic level. Particularly promising are the links that can be established between the information provided by electron microscopy and the atomic structures derived from X-ray crystallography and nuclear magnetic resonance spectroscopy. Combining such different kinds of structural data can result in novel biological information on the interaction of biomolecules in large supramolecular assemblies. As a consequence, the need to develop new databases in the field of structural biology that allow for an integrated access to data from all the experimental techniques is becoming critical. Pilot studies performed in recent years have already established a solid background as far as the basic information that an integrated macromolecular structure database should contain, as well as the basic principles for integration. These efforts started in the context of the BioImage project, and resulted in a first complete database prototype that provided a versatile platform for the linking of atomic models or X-ray diffraction data with electron microscopy information. Analysis of the requirements needed to combine data at different levels of resolution have resulted in sets of specifications that make possible the integration of all these different types in the context of a web environment. The case of a structural study linking electron microscopy and X-ray data, which is already contained within the BioImage data base and in the Protein Data Bank, is used here to illustrate the current approach, while a general discussion highlights the urgent need for integrated databases.

Structure of human rhinovirus serotype 2 (HRV2).

Human rhinoviruses are classified into a major and a minor group based on their binding to ICAM-1 or to members of the LDL-receptor family, respectively. They can also be divided into groups A and B, according to their sensitivity towards a panel of antiviral compounds. The structure of human rhinovirus 2 (HRV2), which uses the LDL receptor for cell attachment and is included in antiviral group B, has been solved and refined at 2.6 A resolution by X-ray crystallography to gain information on the peculiarities of rhinoviruses, in particular from the minor receptor group. The main structural differences between HRV2 and other rhinoviruses, including the minor receptor group serotype HRV1A, are located at the internal protein shell surface and at the external antigenic sites. In the interior, the N termini of VP1 and VP4 form a three-stranded beta-sheet in an arrangement similar to that present in poliovirus, although myristate was not visible at the amino terminus of VP4 in the HRV2 structure. The betaE-betaF loop of VP2, a linear epitope within antigenic site B recognized by monoclonal antibody 8F5, adopts a conformation considerably different from that found in the complex of 8F5 with a synthetic peptide of the same sequence. This either points to considerable structural changes impinged on this loop upon antibody binding, or to the existence of more than one single conformation of the loop when the virus is in solution. The hydrophobic pocket of VP1 was found to be occupied by a pocket factor apparently identical with that present in the major receptor group virus HRV16. Electron density, consistent with the presence of a viral RNA fragment, is seen stacked against a conserved tryptophan residue.

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

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 A 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 C(1). 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.

Ca(2+) bridges the C2 membrane-binding domain of protein kinase Calpha directly to phosphatidylserine.

The C2 domain acts as a membrane-targeting module in a diverse group of proteins including classical protein kinase Cs (PKCs), where it plays an essential role in activation via calcium-dependent interactions with phosphatidylserine. The three-dimensional structures of the Ca(2+)-bound forms of the PKCalpha-C2 domain both in the absence and presence of 1, 2-dicaproyl-sn-phosphatidyl-L-serine have now been determined by X-ray crystallography at 2.4 and 2.6 A resolution, respectively. In the structure of the C2 ternary complex, the glycerophosphoserine moiety of the phospholipid adopts a quasi-cyclic conformation, with the phosphoryl group directly coordinated to one of the Ca(2+) ions. Specific recognition of the phosphatidylserine is reinforced by additional hydrogen bonds and hydrophobic interactions with protein residues in the vicinity of the Ca(2+) binding region. The central feature of the PKCalpha-C2 domain structure is an eight-stranded, anti-parallel beta-barrel with a molecular topology and organization of the Ca(2+) binding region closely related to that found in PKCbeta-C2, although only two Ca(2+) ions have been located bound to the PKCalpha-C2 domain. The structural information provided by these results suggests a membrane binding mechanism of the PKCalpha-C2 domain in which calcium ions directly mediate the phosphatidylserine recognition while the calcium binding region 3 might penetrate into the phospholipid bilayer.

Biochemical and structural studies with neutralizing antibodies raised against foot-and-mouth disease virus.

The function of a loop exposed on the aphthovirus capsid (the G-H loop of protein VP1) has been explored by combining genetic and structural studies with viral mutants. The loop displays a dual function of receptor recognition and interaction with neutralizing antibodies. Remarkably, some amino acid residues play a critical role in both such disparate functions. Therefore residues subjected to antibody pressure for variation may nevertheless maintain a role in receptor recognition for which invariance is a requirement. Evolution of FMDV in cell culture may relax the requirements at this site and allow further increase of antigenic diversification. Essential residues at one stage of virus evolution may become dispensable at another not very distant point in the evolutionary landscape. Implications for FMDV evolution and vaccine design are discussed.

Crystallization and preliminary X-ray analysis of human rhinovirus serotype 2 (HRV2).

Human rhinoviruses, the major cause of mild recurrent infections of the upper respiratory tract, are small icosahedral particles. Over 100 different serotypes have been identified. The majority (91 serotypes) use intercellular adhesion molecule 1 as the cell-attachment site; ten serotypes (the minor group) bind to members of the low-density lipoprotein receptor. Three different crystal forms of the minor-group human rhinovirus serotype 2 (HRV2) were obtained by the hanging-drop vapour-diffusion technique using ammonium sulfate and sodium/potassium phosphate as precipitants. Monoclinic crystals, space group P2(1), diffracted at least to 2.8 A resolution, and two complete virus particles were located in the crystal asymmetric unit. A second type of crystals had a compact cubic like morphology and diffracted beyond 2.5 A resolution. These crystals belong to a primitive orthorhombic space group, with unit-cell parameters a = 309.3, b = 353.5, c = 759.6 A, and contain one virus particle in the asymmetric unit. A third type of crystals, with a prismatic shape and belonging to space group I222, was also obtained under similar crystallization conditions. These latter crystals, with unit-cell parameters a = 308.7, b = 352.2, c = 380.5 A, diffracted to high resolution (beyond 1.8 A) and contained 15 protomers per asymmetric unit; this requires that three perpendicular crystal twofold axes coincide with three of the viral particle's dyad axes.

Flexibility of the major antigenic loop of foot-and-mouth disease virus bound to a Fab fragment of a neutralising antibody: structure and neutralisation.

The interaction of foot-and-mouth disease virus (FMDV) serotype C (clone C-S8c1) with a strongly neutralising monoclonal antibody (MAb) 4C4 has been studied by combining data from cryoelectron microscopy and x-ray crystallography. The MAb 4C4 binds to the exposed flexible GH-loop of viral protein 1 (VP1), which appears to retain its flexibility, allowing movement of the bound Fab. This is in striking contrast to MAb SD6, which binds to the same GH-loop of VP1 but exhibits no movement of the bound Fab when observed under identical conditions. However, MAbs 4C4 and SD6 have very similar neutralisation characteristics. The known atomic structure of FMDV C-S8c1 and that of the 4C4 Fab cocrystallised with a synthetic peptide corresponding to the GH-loop of VP1 were fitted to the cryoelectron microscope density map. The best fit of the 4C4 Fab is compatible only with monovalent binding of the MAb in agreement with the neutralisation data on 4C4 MAbs, Fab2s, and Fabs. The position of the bound GH-loop is related to other known positions of this loop by a hinge rotation about the base of the loop. The 4C4 Fab appears to interact almost exclusively with the G-H loop of VP1, making no other contacts with the viral capsid.

Rapid selection in modified BHK-21 cells of a foot-and-mouth disease virus variant showing alterations in cell tropism.

With persistent foot-and-mouth disease virus (FMDV) in BHK-21 cells, there is coevolution of the cells and the resident virus; the virulence of the virus for the parental BHK-21 cells is gradually increased, and the cells become partially resistant to FMDV. Here we report that variants of FMDV C3Arg/85 were selected in a single infection of partially resistant BHK-21 cells (termed BHK-Rb cells). Indirect immunofluorescence showed that the BHK-Rb cell population was heterogeneous with regard to susceptibility to C3Arg/85 infection. Infection of BHK-Rb cells with C3Arg/85 resulted in an early phase of partial cytopathology which was followed at 6 to 10 days postinfection by the shedding of mutant FMDVs, termed C3-Rb. The selected C3-Rb variants showed increased virulence for BHK-21 cells, were able to overcome the resistance of modified BHK-21 cells to infection, and had acquired the ability to bind heparin and to infect wild-type Chinese hamster ovary (CHO) cells. A comparison of the genomic sequences of the parental and modified viruses revealed only two amino acid differences, located at the surface of the particle, at the fivefold axis of the viral capsid (Asp-9-->Ala in VP3 and either Gly-110-->Arg or His-108-->Arg in VP1). The same phenotypic and genotypic modifications occurred in a highly reproducible manner; they were seen in a number of independent infections of BHK-Rb cells with viral preparation C3Arg/85 or with clones derived from it. Neither amino acid substitutions in other structural or nonstructural proteins nor nucleotide substitutions in regulatory regions were found. These results prove that infection of partially permissive cells can promote the rapid selection of virus variants that show alterations in cell tropism and are highly virulent for the same cells.

Multiple virulence determinants of foot-and-mouth disease virus in cell culture.

Hypervirulent variants of foot-and-mouth disease virus (FMDV) of serotype C arise upon serial cytolytic or persistent infections in cell culture. A specific mutation in the internal ribosome entry site of persistent FMDV was previously associated with enhanced translation initiation activity that could contribute to the hypervirulent phenotype for BHK-21 cells. Here we report that several hypervirulent FMDV variants arising upon serial cytolytic passage show an invariant internal ribosome entry site but have a number of mutations affecting structural and nonstructural viral proteins. The construction of chimeric type O-type C infectious transcripts has allowed the mapping of a major determinant of hypervirulence to the viral capsid. Tissue culture-adapted FMDV displayed enhanced affinity for heparin, but binding to cell surface heparan sulfate moieties was not required for expression of the hypervirulent phenotype in Chinese hamster ovary (CHO) cells. Virulence was identical or even higher for glycosaminoglycan-deficient CHO cells than for wild-type CHO cells. FMDV variants with decreased affinity for heparin were selected from a high-binding parental population and analyzed. Substitutions associated with decreased heparin binding were located at positions 173 of capsid protein VP3 and 144 of capsid protein VP1. These substitutions had a moderate effect on virulence for BHK-21 cells but completely abrogated infection of CHO cells. The comparative results with several FMDV isolates show that (i) increased affinity for heparin and alterations in cell tropism may be mediated by a number of independent sites on the viral capsid and (ii) the same capsid modifications may have different effects on different cell types.

A similar pattern of interaction for different antibodies with a major antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation.

The three-dimensional structures of the Fab fragment of a neutralizing antibody raised against a foot-and-mouth disease virus (FMDV) of serotype C1, alone and complexed to an antigenic peptide representing the major antigenic site A (G-H loop of VP1), have been determined. As previously seen in a complex of the same antigen with another antibody which recognizes a different epitope within antigenic site A, the receptor recognition motif Arg-Gly-Asp and some residues from an adjacent helix participate directly in the interaction with the complementarity-determining regions of the antibody. Remarkably, the structures of the two antibodies become more similar upon binding the peptide, and both undergo considerable induced fit to accommodate the peptide with a similar array of interactions. Furthermore, the pattern of reactivities of five additional antibodies with versions of the antigenic peptide bearing amino acid replacements suggests a similar pattern of interaction of antibodies raised against widely different antigens of serotype C. The results reinforce the occurrence of a defined antigenic structure at this mobile, exposed antigenic site and imply that intratypic antigenic variation of FMDV of serotype C is due to subtle structural differences that affect antibody recognition while preserving a functional structure for the receptor binding site.

Structure and supramolecular packing features of the dipeptide Arg-Val acetate.

The title compound crystallizes in the zwitterionic form. The crystal forms a supramolecular structure with the peptide molecules organized in head-to-tail columns in the b direction. The arginine side-chains and acetate ions interact with neighbor peptides in the c direction. Infinite hydrophobic columns are present in the a direction; they involve the valine side-chains, the acetate methyl groups and the methylene groups of the arginine side-chains. This three-dimensional organization is similar to that found in Lys-Val hydrochloride.

Efficient neutralization of foot-and-mouth disease virus by monovalent antibody binding.

Neutralization of an aphthovirus by monovalent binding of an antibody is reported. Foot-and-mouth disease virus (FMDV) clone C-S8c1 was neutralized by monoclonal antibody (MAb) SD6, which was directed to a continuous epitope within a major antigenic site of the G-H loop of capsid protein VP1. On a molar basis, the Fab fragment was at most fivefold less active in neutralization than the intact antibody, and both blocked virus attachment to cells. Neither the antibody nor the Fab fragment caused aggregation of virions, as evidenced by sucrose gradient sedimentation studies of the antibody-virus complex formed at antibody to virion ratios of 1:50 to 1:10,000. The results of neutralization of infectivity and of ultracentrifugation are fully consistent with structural data based on X-ray crystallographic and cryoelectron microscopy studies, which showed monovalent interaction of the antibody with a critical receptor binding motif Arg-Gly-Asp. The conclusions of these neutralization studies are that (i) bivalent binding of antibody is not a requisite for strong neutralization of aphthoviruses and (ii) aggregation of viral particles, which has been proposed to be the dominant neutralization mechanism of antibodies that bind monovalently to virions, is not necessary for the neutralization of FMDV C-S8c1 by MAb SD6.

Structural variability of A-DNA in crystals of the octamer d(pCpCpCpGpCpGpGpG)

We have determined the structure of the synthetic DNA octamer d(pCpCpCpGpCpGpGpG) in five different crystal forms by single crystal X-ray diffraction. One crystal belongs to the space group P4(3)2(1)2 with a = b = 41.77, c = 25.15 A, whereas all others have the space group P2(1)2(1)2(1) with progressively decreasing unit cell volumes. In all crystals the octamer forms duplexes of A-DNA and all crystals display a similar packing mode, typical for A-DNA. The structure of the duplex varies from loose to very compact when going from one crystal form to another. The most compact form exhibits a volume of 995 A3 per base pair. Such a high density has never been found in A-DNA, being more characteristic of Z-DNA crystals. A comparison of the most with the least compact forms gives a RMS value of 1.7 A, with the distance between the phosphate centers through the major groove being almost twice shorter in the compact form. The phosphate-phosphate separation across the major groove in the compact form is extremely small, 0.7 A. The helical parameters also vary significantly in the various crystal forms. Differences in the helical twist can reach 13 degrees in the same step of the octamer in different crystal forms. The results prove that A-DNA is structurally very variable and demonstrate that the local structure of the same DNA fragment can strongly depend on the crystal environment.

Evolution subverting essentiality: dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus.

Aphthoviruses use a conserved Arg-Gly-Asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. Here we report that this triplet-which is also a widespread motif involved in cell-to-cell adhesion-can become dispensable upon short-term evolution of the virus harboring it. Foot-and-mouth disease virus (FMDV), which was multiply passaged in cell culture, showed an altered repertoire of antigenic variants resistant to a neutralizing monoclonal antibody. The altered repertoire includes variants with substitutions at the Arg-Gly-Asp motif. Mutants lacking this sequence replicated normally in cell culture and were indistinguishable from the parental virus. Studies with individual FMDV clones indicate that amino acid replacements on the capsid surface located around the loop harboring the Arg-Gly-Asp triplet may mediate in the dispensability of this motif. The results show that FMDV quasispecies evolving in a constant biological environment have the capability of rendering totally dispensable a receptor recognition motif previously invariant, and to ensure an alternative pathway for normal viral replication. Thus, variability of highly conserved motifs, even those that viruses have adapted from functional cellular motifs, can contribute to phenotypic flexibility of RNA viruses in nature.

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.

Data from cryo-electron microscopy and X-ray crystallography have been combined to study the interactions of foot-and-mouth disease virus serotype C (FMDV-C) with a strongly neutralizing monoclonal antibody (mAb) SD6. The mAb SD6 binds to the long flexible GH-loop of viral protein 1 (VP1) which also binds to an integrin receptor. The structure of the virus-Fab complex was determined to 30 A resolution using cryo-electron microscopy and image analysis. The known structure of FMDV-C, and of the SD6 Fab co-crystallized with a synthetic peptide corresponding to the GH-loop of VP1, were fitted to the cryo-electron microscope density map. The SD6 Fab is seen to project almost radially from the viral surface in an orientation which is only compatible with monovalent binding of the mAb. Even taking into account the mAb hinge and elbow flexibility, it is not possible to model bivalent binding without severely distorting the Fabs. The bound GH-loop is essentially in what has previously been termed the 'up' position in the best fit Fab orientation. The SD6 Fab interacts almost exclusively with the GH-loop of VP1, making very few other contacts with the viral capsid. The position and orientation of the SD6 Fab bound to FMDV-C is in accord with previous immunogenic data.

Antigenically profound amino acid substitutions occur during large population passages of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) with amino acid substitutions next to the highly conserved R-G-D motif were isolated following large population passages of the virus (N. Sevilla and E. Domingo, 1996, J. Virol., in press). Reactivity with a panel of monoclonal antibodies which recognize different epitopes within site A was abolished or highly diminished in the mutants. This provides direct evidence of a drastic antigenic change occurring in the absence of selection by antibodies. Molecular modeling studies predict only minor alterations in the conformation of the G-H loop of VP1 and the R-G-D motif in these mutants. None of these variants became dominant in many serial infections involving smaller FMDV population numbers. In addition to documenting profound antigenic variation without immune selection, the results suggest that the repertoire of antigenic variants evolving in viral quasispecies may be greatly influenced by the population size of the virus.

Crystallization and preliminary X-ray diffraction studies of the Lb proteinase from foot-and-mouth disease virus.

Different crystal forms of the C23A mutant from the leader proteinase of foot-and-mouth disease virus were obtained by the hanging drop vapor diffusion technique, using MgCl2 and PEG 6000 as precipitants. Well-developed crystals, with cubic morphology growing to approximately 1.0 mm3 in size, presented a large unit cell parameter of 274.5 A and diffracted to, at most, 5 A resolution. A second type of crystal had a tetragonal appearance and these were obtained in droplets soaked in a silica gel matrix. These crystals, with an approximate size of 0.3 X 0.3 X 0.7 mm3, diffracted to approximately 4.0 A resolution, but presented a strong anisotropic mosaicity around the longest crystal axis. Crystals with a needlelike morphology and reaching sizes of about 0.2 X 0.3 X 1.2 mm3 diffracted beyond 3.5 A resolution and were stable to X-ray radiation for approximately one day when using a conventional source at room temperature. These crystals are orthorhombic with space group I222 (or I2(1)2(1)2(1)) and unit cell dimensions a = 65.9 A, b = 104.3 A, and c = 124.0 A, and appear well suited for high-resolution studies. Density packing considerations are consistent with the presence of two molecules in the asymmetric unit and a solvent content of approximately 54%.