Biological function made crystal clear - annotation of hypothetical proteins via structural genomics.

Many of the gene products of completely sequenced organisms are 'hypothetical' - they cannot be related to any previously characterized proteins - and so are of completely unknown function. Structural studies provide one means of obtaining functional information in these cases. A 'structural genomics' project has been initiated aimed at determining the structures of 50 hypothetical proteins from Haemophilus influenzae to gain an understanding of their function. Each stage of the project - target selection, protein production, crystallization, structure determination, and structure analysis - makes use of recent advances to streamline procedures. Early results from this and similar projects are encouraging in that some level of functional understanding can be deduced from experimentally solved structures.

mAb against hen egg-white lysozyme regulate its presentation to CD4(+) T cells.

Specific antibodies increase antigen uptake and presentation by antigen-presenting cells via the B cell receptor in B cells or FcgammaR in dendritic cells. To determine whether the interaction between antibody and antigen could influence the set of peptides presented by MHC II molecules, we analyzed the presentation of different CD4(+) T cell epitopes of hen egg-white lysozyme (HEL) after the capture of immune complexes formed between HEL and seven different specific mAb. The 103-117 T cell epitope (I-E(d)) was specifically and selectively up-regulated by the D1.3 and F9.13.7 mAb that binds to proximal loops in the native structure of HEL. Furthermore, Ii-independent T cell epitopes exposed on the HEL surface (116-129 and 34-45, I-A(k) restricted) which require a mild processing involving the recycling of MHC II molecules were selectively up-regulated by mAb that overlap those T cell epitopes (D1.3 and D44.1). However, F10.6.6, somatically derived from the same germ line genes as D44.1 and exhibiting an higher affinity for HEL, was without effect on the presentation of the 34-45 epitope. An Ii-dependent T cell epitope buried into the tertiary structure of HEL (45-61, I-A(k) restricted) and requiring the neosynthesis of MHC II was up-regulated by high-affinity mAb recognizing epitopes located at the N- or C-terminus of the T cell epitope. These results strongly suggest that (i) the spatial relationship linking the T cell epitope and the B cell epitope recognized by the mAb, (ii) the intrinsic processing requirements of the T cell epitope, and (iii) the antibody affinity influences the presentation of a given T cell epitope.

Lack of significant differences in association rates and affinities of antibodies from short-term and long-term responses to hen egg lysozyme.

The affinities (Ka) and association rate constants (kon) of 23 mouse (BALB/c) anti-lysozyme mAbs obtained after short and prolonged immunizations have been measured by plasmon resonance techniques. The affinities for the 23 Abs, measured using their Fab, range from Ka = 1.1 x 10(7) to 1.4 x 10(10) M-1. There is no significant correlation between time or dose of immunization and affinity or association rates, indicating no time- or dose-dependent maturation of the response within the doses and times that were explored. IgMs are produced early and late in the response, with intrinsic affinities <10(5) M-1. Two independently derived mAbs, D44.1 (short term) and F10.6.6 (from a longer term response), result from identical or nearly identical somatic recombination events of germline gene segments. F10.6.6 has more mutations and a higher affinity constant (Ka = 1.4 x 10(10) M-1) than D44.1 (Ka = 1.1 x 10(7) M-1). Although higher affinities may result from an accumulation of mutations, they do not correlate with the length and dose of immunogenic challenge.

Crystallization and preliminary x-ray diffraction analysis of the lumazine synthase from Brucella abortus.

Lumazine synthase from Brucella abortus was overexpressed in Escherichia coli, refolded, and purified to apparent homogeneity. Crystals of lumazine synthase were grown by the hanging drop vapor diffusion method using polyethylene glycol 8000 or ammonium sulfate as precipitants. They belong to the trigonal space group P321 with cell parameters a = b = 132.00A, c = 167.25 A. A complete diffraction data set to 3.7 A resolution has been collected using synchrotron radiation. Preliminary analysis of the quaternary structure of this protein by means of a self-rotation function calculated with the diffraction data clearly indicates 532 symmetry compatible with the presence of an icosahedral lumazine synthase particle.

Anatomy of an antibody molecule: structure, kinetics, thermodynamics and mutational studies of the antilysozyme antibody D1.3.

Using site-directed mutagenesis, x-ray crystallography, microcalorimetric, equilibrium sedimentation and surface plasmon resonance detection techniques, we have examined the structure of an antibody-antigen complex and the structural and thermodynamic consequences of removing specific hydrogen bonds and van der Waals interactions in the antibody-antigen interface. These observations show that the complex is considerably tolerant, both structurally and thermodynamically, to the truncation of antibody and antigen side chains that form contacts. Alterations in interface solvent structure for two of the mutant complexes appear to compensate for the unfavorable enthalpy changes when antibody-antigen interactions are removed. These changes in solvent structure, along with the increased mobility of side chains near the mutation site, probably contribute to the observed entropy compensation. In concert, data from structural studies, reaction rates, calorimetric measurements and site directed mutations are beginning to detail the nature of antibody-protein antigen interactions.

Reduction in the amide hydrogen exchange rates of an anti-lysozyme Fv fragment due to formation of the Fv-lysozyme complex.

The Fv fragment of the monoclonal antibody D1.3 was expressed in bacteria. Standard triple resonance techniques were used to obtain the NMR resonance assignments for 211 out of 215 backbone 15N/NH atoms for D1.3 Fv. Using these assignments, hydrogen exchange rates are measured for 82 amide hydrogen atoms in D1.3 Fv free and bound to hen egg-white lysozyme. Upon binding to antigen, exchange rates are decreased for residues throughout the Fv. Many of these residues are located remote from the site of interaction with the antigen. These changes are larger than previously observed for the antigen portion of the complex. Evidently, the beta-sheet structure of the Fv propagates the effects of binding more efficiently than the antigen. These effects are compared between the three different polypeptide chains that make up the complex. These data suggest that reduced dynamics are a general feature of antibody binding to antigen.

Characterization of anti-anti-idiotypic antibodies that bind antigen and an anti-idiotype.

Two mouse monoclonal anti-anti-idiotopic antibodies (anti-anti-Id, Ab3), AF14 and AF52, were prepared by immunizing BALB/c mice with rabbit polyclonal anti-idiotypic antibodies (anti-Id, Ab2) raised against antibody D1.3 (Ab1) specific for the antigen hen egg lysozyme. AF14 and AF52 react with an "internal image" monoclonal mouse anti-Id antibody E5.2 (Ab2), previously raised against D1.3, with affinity constants (1.0 x 10(9) M-1 and 2.4 x 10(7) M-1, respectively) usually observed in secondary responses against protein antigens. They also react with the antigen but with lower affinity (1.8 x 10(6) M-1 and 3.8 x 10(6) M-1). This pattern of affinities for the anti-Id and for the antigen also was displayed by the sera of the immunized mice. The amino acid sequences of AF14 and AF52 are very close to that of D1.3. In particular, the amino acid side chains that contribute to contacts with both antigen and anti-Id are largely conserved in AF14 and AF52 compared with D1.3. Therapeutic immunizations against different pathogenic antigens using anti-Id antibodies have been proposed. Our experiments show that a response to an anti-Id immunogen elicits anti-anti-Id antibodies that are optimized for binding the anti-Id antibodies rather than the antigen.

pH dependence of antibody/lysozyme complexation.

Association between proteins often depends on the pH and ionic strength conditions of the medium in which it takes place. This is especially true in complexation involving titratable residues at the complex interface. Continuum electrostatics methods were used to calculate the pH-dependent energetics of association of hen egg lysozyme with two closely related monoclonal antibodies raised against it and the association of these antibodies against an avian species variant. A detailed analysis of the energetic contributions reveals that even though the hallmark of association in the two complexes is the presence of conserved charged-residue interactions, the environment of these interactions significantly influences the titration behavior and concomitantly the energetics. The contributing factors include minor structural rearrangements, buried interfacial area, dielectric environment of the key titratable residues, and geometry of the residue dispositions. Modeled structures of several mutant complexes were also studied so as to further delineate the contribution of individual factors to the titration behavior.

Hydrogen bonding and solvent structure in an antigen-antibody interface. Crystal structures and thermodynamic characterization of three Fv mutants complexed with lysozyme.

Using site-directed mutagenesis, X-ray crystallography, and titration calorimetry, we have examined the structural and thermodynamic consequences of removing specific hydrogen bonds in an antigen-antibody interface. Crystal structures of three antibody FvD1.3 mutants, VLTyr50Ser (VLY50S), VHTyr32Ala (VHY32A), and VHTyr101Phe (VHY101F), bound to hen egg white lysozyme (HEL) have been determined at resolutions ranging from 1.85 to 2.10 A. In the wild-type (WT) FvD1.3-HEL complex, the hydroxyl groups of VLTyr50, VHTyr32, and VHTyr101 each form at least one hydrogen bond with the lysozyme antigen. Thermodynamic parameters for antibody-antigen association have been measured using isothermal titration calorimetry, giving equilibrium binding constants Kb (M-1) of 2.6 x 10(7) (VLY50S), 7.0 x 10(7) (VHY32A), and 4.0 x 10(6) (VHY101F). For the WT complex, Kb is 2.7 x 10(8) M-1; thus, the affinities of the mutant Fv fragments for HEL are 10-, 4-, and 70-fold lower than that of the original antibody, respectively. In all three cases entropy compensation results in an affinity loss that would otherwise be larger. Comparison of the three mutant crystal structures with the WT structure demonstrates that the removal of direct antigen-antibody hydrogen bonds results in minimal shifts in the positions of the remaining protein atoms. These observations show that this complex is considerably tolerant, both structurally and thermodynamically, to the truncation of antibody side chains that form hydrogen bonds with the antigen. Alterations in interface solvent structure for two of the mutant complexes (VLY50S and VHY32A) appear to compensate for the unfavorable enthalpy changes when protein-protein interactions are removed. These changes in solvent structure, along with the increased mobility of side chains near the mutation site, probably contribute to the observed entropy compensation. For the VHY101F complex, the nature of the large entropy compensation is not evident from a structural comparison of the WT and mutant complexes. Differences in the local structure and dynamics of the uncomplexed Fv molecules may account for the entropic discrepancy in this case.

Crystal structure of an Fv-Fv idiotope-anti-idiotope complex at 1.9 A resolution.

Anti-idiotopic antibodies react with unique antigenic features, usually associated with the combining sites, of other antibodies. They may thus mimic specific antigens that react with the same antibodies. The structural basis of this mimicry is analyzed here in detail for an anti-idiotopic antibody that mimics the antigen, hen egg-white lysozyme. The crystal structure of an anti-hen-egg-white lysozyme antibody (D1.3) complexed with an anti-idiotopic antibody (E5.2) has been determined at a nominal resolution of 1.9 A. E5.2 contacts substantially the same residues of D1.3 as lysozyme, thus mimicking its binding to D1.3. The mimicry embodies conservation of hydrogen bonding: six of the 14 protein-protein hydrogen bonds bridging D1.3-E5.2 are structurally equivalent to hydrogen bonds bridging D1.3-lysozyme. The mimicry includes a similar number of van der Waals interactions. The mimicry of E5.2 for lysozyme, however, does not extend to the topology of the non-polar surfaces of E5.2 and lysozyme, which are in contact with D1.3 as revealed by a quantitative analysis of the contacting surface similarities between E5.2 and lysozyme. The structure discussed herein shows that an anti-idiotopic antibody can provide an approximate topological and binding-group mimicry of an external antigen, especially in the case of the hydrophilic surfaces, even though there is no sequence homology between the anti-idiotope and the antigen.

Crystal structure of the complex of the variable domain of antibody D1.3 and turkey egg white lysozyme: a novel conformational change in antibody CDR-L3 selects for antigen.

The crystal structure of the Fv fragment of the murine monoclonal anti-lysozyme antibody D1.3, complexed with turkey egg-white lysozyme (TEL), is presented. D1.3 (IgG1, kappa) is a secondary response antibody specific for hen egg-white lysozyme (HEL). TEL and HEL are homologous and differ in amino acid sequence in the antibody-antigen interface only at position 121. The side-chain of HEL residue Gln121 makes a pair of hydrogen bonds to main-chain atoms of the antibody light chain. In the D1.3-TEL structure, TEL residue His121 makes only one hydrogen bond with the light chain as a result of 129 degree and 145 degree change in peptide torsion angles for residues Trp92 and Ser93. Probably as a consequence of this conformational change, the D1.3-TEL association occurs at a much slower rate than the D1.3-HEL association. The D1.3-TEL complex is destabilized with respect to the D1.3-HEL interaction by the loss of two hydrogen bonds, exclusively due to the substitution of histidine for glutamine. While antibodies of secondary responses are indeed highly specific for antigen, this work demonstrates that by undergoing subtle conformational change antibodies can still recognize mutated protein antigens, albeit at a cost to affinity.

Global changes in amide hydrogen exchange rates for a protein antigen in complex with three different antibodies.

The binding of anti-lysozyme monoclonal antibodies, D44.1 or D1.3, to their antigen reduces the rate of exchange for many amide hydrogens in lysozyme. The D44.1 antibody contacts a similar region of lysozyme to the HyHEL-5 antibody, while the D1.3 antibody binds to the side of lysozyme which is opposite to the HyHEL-5 and D44.1 epitopes. We compare the effects of binding these antibodies on amide hydrogen exchange rates in lysozyme. These comparisons suggest that there are regions of lysozyme that fluctuate in a coordinated manner such that the effects of binding can be propagated to regions that are distant from the epitope. The activation enthalpies for hydrogen exchange for 36 of the 126 amide hydrogens in lysozyme and for 25 of 126 lysozyme amide hydrogens in the lysozyme-D1.3 complex are also reported. These data suggest that the reduction in amide hydrogen exchange rates upon antibody binding reflect changes in the dynamics of the antigen. These changes contribute to a reduction in the specific heat capacity upon binding.

The effect of water activity on the association constant and the enthalpy of reaction between lysozyme and the specific antibodies D1.3 and D44.1.

The reactions of lysozyme with the specific monoclonal antibody D1.3, its Fv fragment and a mutant of the Fv, were studied under conditions of reduced water activity through the addition of the cosolutes glycerol, ethanol, dioxane and methanol. Titration calorimetry, BIAcoreTM and ultracentrifugal analyses were used to determine enthalpy of reactions and affinity constants. There was a decrease in the values of the enthalpies of reactions as well as in the association constants which was proportional to the decrease in water activity. These results are consistent with a structural model in which water molecules bound to the antigen and the antibody are conserved upon complex formation and provide bonds which are important for the stability of the complex. In contrast, the reaction of lysozyme with the specific monoclonal antibody D44.1, or its Fab, showed the inverse effect: a small increase in the value of the association constant with decreasing water molarities. This is in agreement with a model in which binding of antigen to antibody D44.1 is accompanied by the release of a very small number of water molecules.

Conservation of water molecules in an antibody-antigen interaction.

The solvation of the antibody-antigen Fv D1.3-lysozyme complex is investigated through a study of the conservation of water molecules in crystal structures of the wild-type Fv fragment of antibody D1.3, 5 free lysozyme, the wild-type Fv D1.3-lysozyme complex, 5 Fv D1.3 mutants complexed with lysozyme and the crystal structure of an idiotope (Fv D1.3)-anti-idiotope (Fv E5.2) complex. In all, there are 99 water molecules common to the wild-type and mutant antibody-lysozyme complexes. The antibody-lysozyme interface includes 25 well-ordered solvent molecules, conserved among the wild-type and mutant Fv D1.3-lysozyme complexes, which are bound directly or through other water molecules to both antibody and antigen. In addition to contributing hydrogen bonds to the antibody-antigen interaction the solvent molecules fill many interface cavities. Comparison with x-ray crystal structures of free Fv D1.3 and free lysozyme shows that 20 of these conserved interface waters in the complex were bound to one of the free proteins. Up to 23 additional water molecules are also found in the antibody-antigen interface, however these waters do not bridge antibody and antigen and their temperature factors are much higher than those of the 25 well-ordered waters. Fifteen water molecules are displaced to form the complex, some of which are substituted by hydrophilic protein atoms, and 5 water molecules are added at the antibody- antigen interface with the formation of the complex. While the current crystal models of the D1.3-lysozyme complex do not demonstrate the increase in bound waters found in a physico-chemical study of the interaction at decreased water activities, the 25 well- ordered interface waters contribute a net gain of 10 hydrogen bonds to complex stability.

Crystal structure of a cross-reaction complex between Fab F9.13.7 and guinea fowl lysozyme.

The crystal structure of the complex between the cross-reacting antigen Guinea fowl lysozyme and the Fab from monoclonal antibody F9.13.7, raised against hen egg lysozyme, has been determined by x-ray diffraction to 3-A resolution. The antibody interacts with exposed residues of an alpha-helix and surrounding loops adjacent to the lysozyme active site cleft. The epitope of lysozyme bound by antibody F9.13.7 overlaps almost completely with that bound by antibody HyHEL10; the same 12 residues of the antigen interact with the two antibodies. The antibodies, however, have different combining sites with no sequence homology at any of their complementarity-determining regions and show a dissimilar pattern of cross-reactivity with heterologous antigens. Side chain mobility of epitope residues contributes to confer steric and electrostatic complementarity to differently shaped combining sites, allowing functional mimicry to occur. The capacity of two antibodies that have different fine specificities to bind the same area of the antigen emphasizes the operational character of the definition of an antigenic determinant. This example demonstrates that degenerate binding of the same structural motif does not require the existence of sequence homology or other chemical similarities between the different binding sites.

Molecular basis of antigen mimicry by an anti-idiotope.

Idiotopes are antigenic determinants, unique to an antibody or group of antibodies, defined by the reaction of anti-idiotopic antibodies with the antibodies bearing the idiotopes. The ensemble of idiotopes of an antibody constitutes its idiotype. Idiotypes are useful as markers to follow specific antibodies and clones of cells in immune responses and the inheritance of immunoglobulin genes. As external antigens and anti-idiotypic antibodies can competitively bind the combining site of specific antibodies, some anti-idiotypic antibodies may resemble the external antigen, thus mimicking its structure. It has been proposed that an anti-idiotypic antibody, anti-anti-X, may resemble the external antigen X and thus carry its 'internal image', but this idea is not unequivocally supported by the three-dimensional structures of anti-idiotopic antibodies, either because the structures of the external antigen or of the anti-idiotopic antibody were unknown, or because the anti-idiotopic antibodies showed no resemblance to the external antigens (reviewed in ref. 10). Functional mimicry of ligands of biological receptors by anti-idiotypic antibodies has been described in several systems (reviewed in ref. 11). But how closely can antibodies mimic antigens at the molecular level? Here we present the crystal structure of an idiotope-anti-idiotope complex between the Fv fragments of the anti-lysozyme antibody D1.3 and the anti-D1.3 antibody E5.2. D1.3 contacts the antigen, lysozyme and the anti-idiotopic E5.2 through essentially the same combining-site residues. In addition, E5.2 interacts with D1.3, making contacts similar to those between lysozyme and D1.3. Thus, the anti-idiotopic antibody E5.2 mimics lysozyme in its binding interactions with D1.3. Validating these observations, E5.2, used as an immunogen, induces an anti-lysozyme response.

Thermodynamics of antigen-antibody binding using specific anti-lysozyme antibodies.

Titration calorimetry measurements on the binding of hen lysozyme to the specific monoclonal IgG antibodies D1.3, D11.15, D44.1, F9.13.7, F10.6.6, their papain-cleaved antigen binding fragments (Fab) and their protein-engineered fragments consisting of non-covalently linked heavy variable chain and light variable chain domains (Fv) were performed between 6-50 degrees C in 0.15 M NaCl, 0.01 M sodium phosphate pH 7.1. The binding thermodynamic free energy change (delta G degrees b), enthalpy change (delta Hb), and entropy change (delta Sb) were the same for the whole IgG and its Fv and Fab fragments. With the exception of F9.13.7 at 13 degrees C, all the binding reactions were enthalpically driven with enthalpy changes ranging from -129 +/- 7 kJ mol-1 (D1.3 at 49.8 degrees C) to -26.2 +/- 0.6 kJ mol-1 (D44.1 at 8.0 degrees C). The heat capacity changes for the binding reaction (delta Cp) ranged from -2.72 +/- 0.16 kJ mol-1 K-1 (F9.13.7) to -0.95 +/- 0.06 kJ mol-1 K-1 (F10.6.6). The apolar surface areas buried at the binding sites estimated from the heat capacity changes indicate that the binding reactions are primarily hydrophobic, contrary to the mainly observed enthalpy-driven nature of the reactions. Conformational stabilization and the presence of water at the antigen-antibody interface may account for this discrepancy.

Protein motion and lock and key complementarity in antigen-antibody reactions.

Antibodies possess a highly complementary combining site structure to that of their specific antigens. In many instances their reactions are driven by enthalpic factors including, at least in the case of the reaction of monoclonal antibody D1.3 with lysozyme, enthalpy of solvation. They require minor structural rearrangements, and their equilibrium association constants are relatively high (10(7)-10(11) M-1). By contrast, in an idiotope--anti-idiotope (antibody-antibody) reaction, which is entropically driven, the binding equilibrium constant is only 1.5 x 10(5) M-1 at 20 degrees C. This low value results from a slow association rate (10(3) M-1 s-1) due to a selection of conformational states that allow one of the interacting molecular surfaces (the idiotope on antibody D1.3) to become complementary to that of the anti-idiotopic antibody. Thus, antibody D1.3 reacts with two different macromolecules: with its specific antigen, hen egg lysozyme, and with a specific anti-idiotopic antibody. Complementarity with lysozyme is closer to a "lock and key" model and results in high affinity (2-4 x 10(8) M-1). That with the anti-idiotopic antibody involves conformational changes at its combining site and it results in a lower association constant (1.5 x 10(5) M-1). Thus, an "induced fit" mechanism may lead to a broadening of the binding specificity but with a resulting decrease in the intrinsic binding affinity which may weaken the physiological function of antibodies.

Structural features of the reactions between antibodies and protein antigens.

Antibodies bind protein antigens over large sterically and electrostatically complementary surfaces. Van der Waals forces, hydrogen bonds, and occasionally ion pairs provide stability to antibody-antigen complexes. In addition, water molecules contribute hydrogen bonds linking antigen and antibody, and increase the complementarity of antigen-antibody interfaces. In qualification to a strict 'lock and key' mechanism, evidence of conformational changes between free and complexed antibodies indicate some accommodation to the antigen. Antibody-protein antigen reactions are enthalpically driven with varying degrees of entropic compensation, often dependent on the magnitude of the enthalpy of the reaction. In the case of two antibody-combining sites studied by X-ray diffraction, the relative arrangements of the variable domains of the light and heavy chains of the antibody change slightly from the free to the antigen-bound state. Furthermore, the contacting residues of both antibodies exhibit similar reduced mobilities when complexed to antigen, suggesting that differences in 'solvent entropy' rather than in conformational freedom may be the source of different entropic compensation factors. In concert, data from structural studies, reaction rates, calorimetric measurements, molecular dynamics simulations, and site-directed mutagenesis are beginning to detail the nature of antibody-protein antigen interactions.