Novel surface-based methodologies for investigating GH11 xylanase-lignin derivative interactions.

The recalcitrance of lignocellulose to bioprocessing represents the core problem and remains the limiting factor in creating an economy based on lignocellulosic ethanol production. Lignin is responsible for unproductive interactions with enzymes, and understanding how lignin impairs the susceptibility of biomass to enzymatic hydrolysis represents a significant aim in optimising the biological deconstruction of lignocellulose. The objective of this study was to develop methodologies based on surface plasmon resonance (SPR), which provide novel insights into the interactions between xylanase (Tx-xyn11) and phenolic compounds or lignin oligomers. In a first approach, Tx-xyn11 was fixed onto sensor surfaces, and phenolic molecules were applied in the liquid phase. The results demonstrated weak affinity and over-stoichiometric binding, as several phenolic molecules bound to each xylanase molecule. This approach, requiring the use of soluble molecules in the liquid phase, is not applicable to insoluble lignin oligomers, such as the dehydrogenation polymer (DHP). An alternative approach was developed in which a lignin oligomer was fixed onto a sensor surface. Due to their hydrophobic properties, the preparation of stable lignin layers on the sensor surfaces represented a considerable challenge. Among the various chemical and physico-chemical approaches assayed, two approaches (physisorption via the Langmuir-Blodgett technique onto self-assembled monolayer (SAM)-modified gold and covalent coupling to a carboxylated dextran matrix) led to stable lignin layers, which allowed the study of its interactions with Tx-xyn11 in the liquid phase. Our results indicated the presence of weak and non-specific interactions between Tx-xyn11 and DHP.

A conformation of cyclosporin A in aqueous environment revealed by the X-ray structure of a cyclosporin-Fab complex.

The conformation of the immunosuppressive drug cyclosporin A (CsA) in a complex with a Fab molecule has been established by crystallographic analysis to 2.65 angstrom resolution. This conformation of CsA is similar to that recently observed in the complex with the rotamase cyclophilin, its binding protein in vivo, and totally different from its conformation in an isolated form as determined from x-ray and nuclear magnetic resonance analysis. Because the surfaces of CsA interacting with cyclophilin or with the Fab are not identical, these results suggest that the conformation of CsA observed in the bound form preexists in aqueous solution and is not produced by interaction with the proteins.

Conformational polymorphism of cyclosporin A.

BACKGROUND: Cyclosporin A (CsA) is a cyclic undecapeptide fungal metabolite with immunosuppressive properties, widely used in transplant surgery. It forms a tight complex with the ubiquitous 18 kDa cytosolic protein cyclophilin A (CypA). The conformation of CsA in this complex, as studied by NMR or X-ray crystallography, is very different from that of free CsA. Another, different conformation of CsA has been found in a complex with an antibody fragment (Fab). RESULTS: A detailed comparison of the conformations of experimentally determined structures of protein-bound CsA is presented. The X-ray and NMR structures of CsA-CypA complexes are similar. The Fab-bound conformation of CsA, as determined by X-ray crystallography, is significantly different from the cyclophilin-bound conformation. The protein-CsA interactions in both the Fab and CypA complexes involve five hydrogen bonds, and the buried CsA surface areas are 395 A2 and 300 A2, respectively. However, the CsA-protein interactions involve rather different side chain contacts in the two complexes. CONCLUSIONS: The structural results presented here are consistent with CypA recognizing and binding a population of CsA molecules which are in the required CypA-binding conformation. In contrast, the X-ray structures of the Fab complex with CsA suggest that in this case there is mutual adaptation of both receptor and ligand during complex formation.

Functional mapping of conserved residues located at the VL and VH domain interface of a Fab.

The interface between the VL and VH domains of antibodies is highly conserved. To investigate the influence of conserved interface residues on Fab function, 13 interface residues were subjected to codon-based combinatorial alanine scanning mutagenesis in Fab 57P, specific for peptide 134 to 151 of the coat protein of tobacco mosaic virus. The 13 single mutants were analysed by Western blot to determine the effect of interface modifications on Fab expression. The kinetic rate constants of peptide-Fab mutant interactions were measured using the biosensor technology. Alanine replacements did not prevent assembly of the mutated Fabs and led to a modification of their binding properties in every case. Twelve of the 13 target residues correspond to homologous positions in the VL and VH domains, which have similar folds. Mutation at homologous positions mostly had different effects on antigen binding affinity. The replacement of bulky side-chains had the most drastic effect on binding. When smaller side-chains were replaced by alanine, the binding properties of Fab mutants differed slightly (by less than a factor of two), but significantly from that of Fab 57P. Modification of some of these residues, which are located 9 to 12 A away from the base of CDR loops, is unlikely to alter loop conformation. They may affect antigen binding indirectly by influencing the relative position of the VL and VH domains. Our results demonstrate that residues situated at the VL-VH interface and which are remote from the paratope are able to influence the antigen binding properties of antibodies.

Correlation of co-ordinated amino acid substitutions with function in viruses related to tobacco mosaic virus.

Sequence data are available for the coat proteins of seven tobamoviruses, with homologies ranging from at least 26% to 82%, and atomic co-ordinates are known for tobacco mosaic virus (TMV) vulgare. A significant spatial relationship has been found between groups of residues with identical amino acid substitution patterns. This strongly suggest that their location is linked to a particular function, at least in viruses identical with the wild-type for these residues. The most conserved feature of TMV is the RNA binding region. Core residues are conserved in all viruses or show mutations complementary in volume. The specificity of inter-subunit contacts is achieved in different ways in the three more distantly related viruses.

Correlation of co-ordinated amino acid changes at the two-domain interface of cysteine proteases with protein stability.

The engineering of a protein containing an alternative local residue packing for a set of side-chains has proven to be a major challenge because compositional, volumetric and steric constraints must be respected. Homologous proteins should provide examples of alternative groups of residues leading to a similar functional result. The functional significance of a pair of co-ordinated changes that are observed in the cysteine proteases family has been investigated by comparing the effect of individual or double changes on secretion, stability and activity of papain. The two changes are not independent. Detrimental effects of single mutations at one of the two positions can be partly suppressed by the co-ordinated mutation that reproduces naturally occurring contacts, indicating that these changes are concerted. Single mutations at the other position produce milder effects, suggesting a pathway for evolution.

Crystallization and preliminary X-ray investigation of a complex between a Fab fragment and its antigen, cyclosporin.

Preliminary crystallographic data are given for a complex between the cyclic undecapeptide cyclosporin and the Fab fragment of an anti-cyclosporin monoclonal antibody. Crystals of the complex are orthorhombic with space group P2(1)2(1)2(1) and diffract to 2.7 A resolution. The unit cell dimensions are a = 52.6 A, b = 70.2 A and c = 118.4 A. A native data set to 2.7 A resolution has been collected.

Immunochemical studies of tobacco mosaic virus--V. Localization of four epitopes in the protein subunit by inhibition tests with synthetic peptides and cleavage peptides from three strains.

Two antigenic determinants have been located in the vicinity of residues 1-10 and 34-39 of the coat protein of tobacco mosaic virus by means of ELISA and complement fixation inhibition experiments with synthetic peptides. The determinant present in residues 1-10 is also expressed at the outer surface of the subunits when they are assembled into virions. Two other determinants expressed in the dissociated protein subunit have been located in the vicinity of residues 55-61 and 80-90 by means of inhibition tests with tryptic peptides. These results bring to seven the number of antigenic determinants of the protein subunit that have been located.

Immunochemical studies of tobacco mosaic virus--VII. Use of comparative surface accessibility of residues in antigenically related viruses for delineating epitopes recognized by monoclonal antibodies.

The binding properties of 18 monoclonal antibodies (MAbs) directed against the tobacco mosaic virus (TMV) coat protein were studied with five related tobamoviruses and seven mutant viruses, as well as with the dissociated coat proteins of these variants. Ten of the antibodies bound to both TMV and TMV protein, but these were able to discriminate between different mutants only when whole virus particles were compared in the immunoassay. Three MAbs reacted with TMV but not with dissociated viral subunits and these recognized the same residue exchanges. Five MAbs recognized synthetic peptides of 13-28 residues corresponding to parts of the protein. By comparing the common accessible surface between TMV and antigenically related viruses, it was possible to narrow down the region recognized by some of these MAbs. The linear peptide sequence recognized by these antibodies did not represent the entire epitope and residue exchanges around this region were able to affect the binding of MAbs.

Structure-activity relationships for the interaction between cyclosporin A derivatives and the Fab fragment of a monoclonal antibody.

The crystallographic structure of a complex between cyclosporin A and the Fab fragment of monoclonal antibody R45-45-11 has been solved to 2.65 A resolution (Altschuh et al., 1992a, Science 256, 92; Vix et al., 1993, Proteins 15, 339), yielding a precise three-dimensional picture of interacting surfaces. In order to evaluate the contribution of observed contacts to the energy of interaction, we have measured the effect on binding affinity of minor chemical modifications of CS. The equilibrium binding constant of the Fab fragment for a set of cyclosporin analogs was obtained by measuring in a biosensor instrument the dependence of complex formation on Fab concentration, at constant analog concentrations. Data were analysed using Scatchard plots. Differences in binding energy resulting from cyclosporin modifications discriminated between two types of contact areas. The first type displays adaptability to structural modifications of cyclosporin at the cost of a small decrease in binding energy, and contacting residues in the antibody form the periphery of the combining site. The second type does not accommodate structural changes and corresponds in cyclosporin to three residues whose modifications drastically decrease binding energy with the antibody. The corresponding contact residues in the antibody form the core of the antibody combining site.

Cooperative effects of mutations in a recombinant Fab on the kinetics of antigen binding.

Recombinant Fabs, 57P and 174P, recognizing peptide 134-151 of the coat protein of tobacco mosaic virus, differ by 15 amino acid changes in the sequence of their variable region. Kinetic analysis using BIAcore showed that they recognized five peptide variants in the same ranking order, but that Fab 174P consistently dissociated faster from the peptides compared to Fab 57P. In order to identify amino acid substitutions that are responsible for differences in dissociation rates of the two Fabs, six hybrid Fabs have been constructed by exchanging three DNA segments. Four single and five multiple mutants were obtained by site-directed mutagenesis. All Fabs recognized variant peptides in a similar ranking order. The high precision of biosensor measurements made it possible to detect small contributions to dissociation kinetics of at least five substitutions, as well as the presence of small-magnitude non-additive effects of multiple substitutions. Our results demonstrate the cooperative influence on dissociation kinetics of amino acid residues located away from each other and away from the Fab combining site.

Immunochemical studies of tobacco mosaic virus--VI. Attempts to localize viral epitopes with monoclonal antibodies.

The specificity of 18 monoclonal antibodies directed to tobacco mosaic virus (TMV) was studied by measuring their ability to bind to viral mutants, to other tobamoviruses, to dissociated viral subunits and to peptide fragments of the viral coat protein. The apparent binding specificity of the antibodies was dependent on the type of enzyme-linked immunosorbent assay used, probably because the antigens were disrupted or denatured when attached to the plastic surface of microtiter wells. The capacity of different monoclonal antibodies to detect single substitutions in the viral coat protein was used to delineate some of the topographic epitopes of TMV. By means of computer-generated images of the surface residues of the viral subunit, it was possible to identify certain clusters of residues involved in binding to some of the monoclonal antibodies. The results clearly illustrate the operational limitations encountered when monoclonal antibodies are used for elucidating the antigenic structure of proteins.

Monoclonal antipeptide antibodies: affinity and kinetic rate constants measured for the peptide and the cognate protein using a biosensor technology.

The interaction of antipeptide antibodies with the corresponding peptide and the cognate protein has been compared using a novel biosensor technology (BIAcore, Pharmacia). The peptide corresponds to residues 110-135 of the coat protein of tobacco mosaic virus, known to encompass an alpha-helical region reactive with antiprotein antibodies. A panel of 33 monoclonal antibodies raised against the peptide was studied and the epitope recognized by these antibodies was determined by the pepscan method. Further discrimination between the antibodies was performed by measurements of association and dissociation kinetic constants. Several antibodies showed an heterogeneous binding profile when reacting with the 25 residue long peptide but not with a shorter 10 residue peptide suggesting that they recognized different conformational states in the longer peptide. Equilibrium affinity constants were calculated for five of the antibodies and were found to be 10-50 times higher for the peptide than for the protein, the difference being caused mainly by a lower association rate constant.

Interaction of cyclosporin A with an Fab fragment or cyclophilin. Affinity measurements and time-dependent changes in binding.

Different conformers of the immunosuppressant cyclosporin A have been observed in structural studies of the isolated molecule and of its complex with cyclophilin or with an Fab fragment. The factors that control this conformational change are not well understood. Variations in the amount of complex formed with cyclophilin or with the antibody were measured as a function of time after adding cyclosporin to the proteins, using the Pharmacia BIAcore biosensor instrument. Up to 1 hour was needed to reach maximum complex formation in solution, which is likely to reflect the time needed for a conformational transition of cyclosporin. The equilibrium affinity constant of both proteins for cyclosporin has been measured.

Tobacco mosaic virus: a model antigen to study virus-antibody interactions.

For more than 50 years, tobacco mosaic virus (TMV) has been used as a model system for studying various aspects of virus-antibody interactions. Distinct epitopes called neotopes and cryptotopes have been identified in intact TMV particles and dissociated viral protein respectively and a correlation has been found to exist between the location of continuous epitopes and the extent of segmental mobility along the viral polypeptide chain. The occurrence of bivalent antibody binding was shown to influence the observed affinity of TMV antibodies and kinetic measurements of antibody binding to viral peptides made it possible to analyze the mechanism of binding of monoclonal antibodies. It seems likely that the TMV model will continue to yield a rich harvest of immunochemical data relevant to many viral systems.

Localization of antigenic determinants of a viral protein by inhibition of enzyme-linked immunosorbent assay (ELISA) with tryptic peptides.

A new method is described for localizing antigenic determinants in globular proteins by means of inhibition of enzyme-linked immunosorbent assay (ELISA) with tryptic peptides. The inhibitory activity of 10 tryptic peptides of the coat protein of 2 strains of tobacco mosaic virus (TMV) was demonstrated by inhibition of ELISA and verified by inhibition of complement fixation tests. These results bring to 7 the number of antigenic determinants of TMV protein that have been defined.

Determination of IgG and IgM levels in serum by rocket immunoelectrophoresis using yolk antibodies from immunized chickens.

A method of rocket immunoelectrophoresis based on the use of avian antiglobulin antibodies is described which permits the quantitation of IgG and IgM in human serum. The electrophoretic mobility of avian yolk immunoglobulin (IgY) and mammalian IgG is sufficiently different to obviate the need to chemically modify the antibody, for example by carbamylation. Rockets are obtained in agarose within 3-6 h in 2-(N-morpholino) ethane sulfonic acid buffer at pH 5.5 or 6.0. Avian globulins specific for human IgG and IgM were obtained easily and in large quantities from the egg yolk of hyperimmunized laying hens. The technique is also applicable to quantitation of immunoglobulins in various animal sera.

Analysis of cyclosporin interactions with antibodies and cyclophilin using the BIAcore.

The immunosuppressive cyclic undecapeptide cyclosporin A (CS) exists in various conformers in water. Up to 1 h is needed to reach maximum complex formation after mixing the drug with its receptor, cyclophilin or with a monoclonal antibody. Differences in the ability of CS and its analogs to bind to antibody or cyclophilin have been measured using the BIAcore. These experiments suggest that the rate-limiting step of complex formation is determined by the interconversion between different CS conformers existing in solution. The contribution to antibody binding of individual atomic groups of CS was evaluated by measuring the equilibrium affinity constants of analogs with the BIAcore. When the binding data were analyzed in terms of the known crystallographic structure of the CS/Fab complex, it could be shown that modifications of CS residues located in the central part of the binding site drastically affect affinity, while modifications of residues located at the periphery are more easily accommodated.

Delineation of a linear epitope by multiple peptide synthesis and phage display.

Two different approaches, the phage display technique and the Spot peptide synthesis on cellulose membranes, were used to identify sequences recognized by Fab 57P, specific for tobacco mosaic virus protein (TMVP), and define the preferred chemical composition of a functional epitope. Kinetic measurements of the interaction between peptide variants and the antibody fragment were used to further refine the molecular basis of binding activity. Our results show that the functional epitope of Fab 57P requires precise physico-chemical properties at a limited number of positions, and that residues flanking these key residues can influence binding affinity. The phage display and Spot synthesis methods allowed the straightforward localization of the binding region and the identification of residues that are essential for recognition. However, these methods yielded slightly different views of accessory factors that are able to influence antibody binding. The influence on binding activity of these factors can only be assessed through quantitative affinity measurements.

Interaction between viruses and monoclonal antibodies studied by surface plasmon resonance.

An automated biosensor system designed for measuring molecular interactions in real time and without any labelling of the reactants has been used to study the interaction of two animal viruses (vaccinia virus and poliovirus) and two plant viruses (cowpea mosaic virus and tobacco mosaic virus) with monoclonal antibodies. Using monoclonal antibodies specific for different conformational states of viral protein, it was found that the virus particles retained their conformational integrity when immobilized on the dextran matrix present on the sensor chip. Compared to conventional solid phase immunoassays, in which immobilized proteins are usually partly denatured, the biosensor system presents several advantages for studying virus-antibody interaction.