The chemistry and mechanism of antibody binding to protein antigens.
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Antigen-Antibody Reactions↗
Biomedical subjects
J A Tainer
Publications and source records attributed to J A Tainer.
Selenocysteine's mechanism of incorporation and evolution revealed in cDNAs of three glutathione peroxidases.
The nonsense codon, UGA, has for the first time recently been shown to encode selenocysteine in two proteins, mouse glutathione peroxidase (GSH-Px) (EC 1.11.1.9) and bacterial formate dehydrogenase. A co-translational rather than post-translational selenium-incorporation mechanism has been implicated. Furthermore, high expression levels of GSH-Px have suggested that suppression of termination is efficient and specific. We have isolated and characterized pituitary, kidney and placenta cDNAs for bovine, human and mouse GSH-Px respectively. It is demonstrated that this novel suppression event occurs in diverse tissues, in at least three mammalian species and at the translational step. Surprisingly, GSH-Px is shown to be extramitochondrially encoded, indicating a cytosolic suppression event rather than one utilizing the mitochondria's well-documented extended codon-reading ability. Sequence analysis reveals that a simple proximal contextual pattern responsible for readthrough does not exist. Analysis of predicted secondary structures of mRNAs, however, has revealed a conformation which may be unique to selenocysteine proteins and may prove useful as a tool for artificial incorporation of selenocysteines. A human intron for GSH-Px from an unspliced mRNA has been isolated whose position indicates an ancient, divergent evolutionary relationship with thioredoxin-S2, rather than an independent convergent one.
Animals↗
Chemistry of antibody binding to a protein.
The chemistry of antibody recognition was studied by mapping the antigenicity of the protein myohemerythrin with peptide homologs of the protein sequence. The results suggest that the entire protein surface is antigenic, but the probability of there being antibodies to a given site is influenced by local stereochemistry. Although accessible to an antibody binding domain, the least reactive positions cluster in the most tightly packed and least mobile regions and are closely associated with narrow, concave grooves in the molecular surface containing bound water molecules. The most frequently recognized sites form three-dimensional superassemblies characterized by high local mobility, convex surface shape, and often by negative electrostatic potential.
Antibodies↗
Mechanisms of antibody binding to a protein.
The mechanisms of antibody binding to a protein were studied by an analysis of specific amino acid residues critical to nine antigenic sites on myohemerythrin. Rabbit antisera to the whole protein were assayed for binding to more than 1500 distinct peptide analogs differing from the protein sequence by single amino acid replacements. The results, combined with information from the three-dimensional crystallographic structure, were used to evaluate probable mechanisms of antibody binding at individual sites. The data from all sites examined indicate that initial binding to solvent-exposed amino acid residues may promote local side-chain displacements and thereby allow the participation of other, previously buried, residues.
Animals↗
Three dimensional structure of bacterial pili.
Crystallographic and associated biochemical and structural studies are in progress on the fiber-forming pilin proteins of the gonococcal pilus. Preparative scale purification procedures have been developed for the gonococcal pilin protein, which appear generally applicable to bacterial pilins. For three gonococcal pilin protein strains, we have obtained both reassembled pilus fibers and three-dimensional crystals. One needle-shaped crystal form of gonococcal C30 pilin diffracts beyond 3 A resolution using synchrotron x-ray radiation. A diffraction data set to 3.5 A resolution has been collected on these needle-shaped crystals (lattice spacings a = 125.4(3) b = 120.4(3), c = 26.61(4) A) in which the packing arrangement of the pilin subunits appears to resemble that seen in the pilus fibers using electron microscopy. X-ray diffraction data confirm our proposed model for the overall polypeptide fold of a pilin subunit, which is an antiparallel 4-alpha helix bundle similar to tobacco mosaic virus coat protein and myohemerythrin.
Bacterial Outer Membrane Proteins↗
Influence of protein flexibility and peptide conformation on reactivity of monoclonal anti-peptide antibodies with a protein alpha-helix.
Monoclonal antibodies against an alpha-helical region of the iron-containing, oxygen-binding protein myohemerythrin were isolated following immunization of mice with either the whole protein or a peptide homolog of the helix. Three distinct epitopes within the myohemerythrin helix were identified. The individual residues within two of these epitopes that were essential for antibody binding were determined by measuring antibody binding to a set of peptides in which each amino acid of the epitope was replaced in turn by each of the other 19 amino acids. Hydrophilic residues that are exposed in the native conformation and buried, hydrophobic residues were both shown to be irreplaceable, suggesting their direct involvement in antibody binding. The influence of antigen conformation on antibody binding to these amphipathic epitopes was assessed by measuring the relative affinities of the antibodies for peptides, intact protein, and apoprotein. All of the antibodies bound to apoprotein better than to native protein, indicating that relaxation of the native structure by removal of the iron center increases antibody affinity for myohemerythrin. However, not all of the antibodies tested bound to peptides better than to protein, suggesting that increased antigen flexibility is not always sufficient to maximize antibody binding. Antibody binding to peptides appeared to also be influenced by the ability of the peptides to attain secondary structure at the epitopes, either alone or due to carrier influences.
Antibodies, Monoclonal↗
Crystallographic characterization of recombinant human CuZn superoxide dismutase.
Recombinant human CuZn superoxide dismutase as expressed in yeast has been crystallized in three different crystal forms. Hexagonal plates grow from 2.4 M ammonium sulfate, pH 7.5, and belong to the space group P6(3)22, with cell dimensions a = b = 113.5(3), c = 151.5(5) A, and Vm = 2.21 A3/dalton for two dimers per asymmetric unit. At 2.0 M ammonium sulfate, pH 7.5, chunky wedges grow in space group C222(1), a = 205.2(6), b = 166.5(4), c = 145.4(4) A with a Vm of 2.43 A3/dalton for eight dimers per asymmetric unit. With polyethylene glycol 8000, pH 7.5-8.0, hexagonal prisms are obtained with cell dimensions a = b = 197.4(6), c = 43.1(2) A, space group P6, and Vm = 2.53 A3/dalton for three dimers per asymmetric unit. All of these forms diffract to high resolution, are stable to x-rays, and appear suitable for determination of the atomic structure. Crystals of the doubly mutated enzyme (Cys6----Ala, Cys111----Ser) grown from both micro- and macroseeds of the wild type protein demonstrate the feasibility of isomorphous crystallization of site-directed mutants of the cloned parent enzyme for comparative structure-function studies.
Crystallography↗
Kinetic studies of reduction of a 1:1 cytochrome c-flavodoxin complex by free flavin semiquinones and rubredoxin.
The kinetics of reduction by free flavin semiquinones and reduced rubredoxin of the individual components of the 1:1 complex formed between horse heart cytochrome c and Clostridium pasteurianum flavodoxin have been studied. Complex formation did not affect the rate constant for reduction of flavodoxin by 5-deazariboflavin semiquinone, indicating that the accessibility of the flavin mononucleotide (FMN) of complexed flavodoxin is the same as in the free protein. Reduction of the complexed cytochrome c by the neutral flavin semiquinones of lumiflavin and riboflavin was significantly affected by complex formation (2-3-fold rate constant decrease), indicating that there are steric constraints on the accessibility of the cytochrome heme to small exogenous reductants. Reduction of complexed cytochrome c by the negatively charged semiquinones of FMN and Cl2FMN was also characterized. A repulsive electrostatic interaction between the reductants and complexed cytochrome was observed, whereas with free cytochrome an attractive interaction had previously been found. This is consistent with the presence of negative electrostatic potential at the protein interface due to uncompensated flavodoxin carboxylates, as predicted by Matthew et al. [Matthew, J. B., Weber, P. C., Salemme, F. R., & Richards, F. M. (1983) Nature (London) 301, 169-171]. Further, pseudo-first-order rate constants for the reduction of complexed cytochrome by these flavins had a nonlinear concentration dependence, rather than obeying simple second-order kinetics. This is interpreted by using a mechanism involving a rate-determining structural isomerization of the protein complex prior to the second-order electron-transfer step. The magnitude of the decrease in the rate constant for reduction of complexed cytochrome c by the negatively charged reduced rubredoxin was approximately the same as observed for free flavins. Furthermore, simple second-order kinetics were obtained, and the apparent electrostatic interaction between rubredoxin and the complex was attractive. These results suggest that flavodoxin was partially displaced from its complex with cytochrome c by a collisional interaction with rubredoxin. The effects of complexation on the kinetics have been correlated with a solvent-accessible surface representation of the computer-generated model of the flavodoxin-cytochrome c complex [Simondsen, R. P., Weber, P. C., Salemme, F. R., & Tollin, G. (1982) Biochemistry 21, 6366-6375]. The experimental observations are generally consistent with the structural model but clearly require the invocation of dynamic motions at the protein-protein interface.
Clostridium↗
Recognition and interactions controlling the assemblies of beta barrel domains.
We present a qualitative computer graphics approach to the characterization of forces important to the assembly of beta domains that should have general utility for examining protein interactions and assembly. In our approach, the nature of the molecular surface buried by the domain contacts, the specificity of the residue-to-residue interactions, and the identity of electrostatic, hydrophobic, and hydrophilic interactions are elucidated. These techniques are applied to the beta barrel domains of Cu, Zn superoxide dismutase (SOD), immunoglobulin Fab, and tomato bushy stunt virus coat protein (TBSV), a plant viral capsid protein. By looking at a set of proteins having different numbers of interacting beta domains, we have been able to see some of the variety and also some of the patterns common to these assembled domains. Strong beta domain interactions (identified by their biochemical integrity) are apparently due to chemical, electrostatic, and shape complementarity of the molecular surfaces buried from interaction with solvent molecules. Although the amount of hydrophobic buried surface area appears to correlate with the strength of the interaction, electrostatic forces appear to be important in both stabilizing and destabilizing specific contacts. In TBSV, analysis of electrostatic interactions may help explain mechanisms of subunit accommodation to different environments, particle expansion, and pathways of assembly. The possible molecular basis for observed differences in the stability and flexibility of the domain complexes is discussed.
Capsid↗
Identical short peptide sequences in unrelated proteins can have different conformations: a testing ground for theories of immune recognition.
The ability of antibodies raised against disordered short peptides to interact frequently with their cognate sequences in intact folded proteins has raised a major theoretical issue in protein chemistry. We propose to address this issue by using antibodies raised against peptides with identical sequences, but different conformations, in pairs of unrelated proteins of known three-dimensional structure. The general search method presented here enabled us to detect candidate sequences for such immunological studies.
Animals↗
The atomic mobility component of protein antigenicity.
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Animals↗
Non-chemokinetic neutrophil attractants derived from arachidonic acid.
Gradients of 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) and 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) were significantly and equally chemotactic for human neutrophils at 15-75 microM. However, neither eicosanoid stimulated random migration when presented isotropically to cells, indicating a total lack of chemokinetic activity. These unique non-chemokinetic chemoattractants may be useful tools for dissecting the chemokinetic phase of neutrophil activation from the chemotactic phase.
Arachidonic Acid↗
Determination and analysis of the 2 A-structure of copper, zinc superoxide dismutase.
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Binding Sites↗
Biogenesis of chemotactic molecules by the arachidonate lipoxygenase system of platelets.
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Arachidonic Acids↗
New aspects of chemotaxis. Specific target-cell attraction by lipid and lipoprotein fractions of Escherichia coli chemotactic factor.
A chemotactic factor extracted from sterile filtrates of Escherichia coli cultures was strongly chemotactic for polymorphonuclear leukocytes (PMN) and rabbit alveolar macrophages (RAM). Electrophoresis of the cytotactic material yielded five lipid fractions: one that was protein-free and active toward both PMN and RAM, and four lipid-protein complexes that were strongly chemotactic only for RAM. Thin-layer chromatography of the lipid-protein complexes resulted in an unmasking of PMN activity in a peptide-free lipid extract, while the isolated peptidic components were essentially noncytotactic. The original RAM activity was retained in the unmasked lipid, which possessed chemical and chromatographic properties similar to those of a previously reported cytotaxin synthesized from arachidonic acid. These data indicate that a class of lipids derived from bacterial and cellular sources is intrinsically cytotactic for PMN and RAM. When peptide moieties are associated with cytotactic lipids, the resultant lipid-peptide complex may exhibit cellular specificity not evident in the free lipid.
Arachidonic Acids↗
Unraveling the effect of changes in conformation and compactness at the antibody V(L)-V(H) interface upon antigen binding.
We have analyzed conformational changes that occur at the interface between the light (V(L)) and heavy (V(H)) chains in antibody variable fragments upon binding to antigens. We wrote and applied the Tiny Probe program that computes the buried atomic contact surface area of three-dimensional structures to evaluate changes in compactness of the V(L)-V(H) interface between bound and unbound antibodies. We found three categories of these changes, which correlated with the size of the antigen. Upon binding, medium-sized nonprotein antigens cause an opening of the V(L)-V(H) interface (less compact), small antigens or haptens cause a closure of the interface (more compact), whereas large protein antigens have little effect on the compactness of the V(L)-V(H) interface. The largest changes in the atomic buried contact surface area at the V(L)-V(H) interface occur in residue pairs providing two 'shock absorbers' between the edge beta-strands of the V(L) and V(H) beta-sheets forming the antibody binding site. Importantly, the correlation between the size of antigens and conformational changes indicates that the V(L)-V(H) interface in antibodies plays a significant role in the antigen binding process. Furthermore, as the energy involved in such a motion is significant (up to 3 kcal/mol), these results provide a general mechanism for how residues distant from the combining site can significantly alter the affinity of an antibody for its antigen. Thus, mutations introduced at the V(L)-V(H) interface can be used to change antibody binding affinity with antigens. Due to the tightly packed V(L)-V(H) interface, the introduction of random mutations is not advisable. Rather our analysis suggests that concerted mutations of residues preceding CDRL2 and following CDRH3 or residues preceding CDRH2 and at the end of CDRL3 are most likely to alter or improve antigen binding affinity.
Antigen-Antibody Reactions↗
Structure and mechanism of copper, zinc superoxide dismutase.
Copper, zinc superoxide dismutase (SOD) catalyses the very rapid two-step dismutation of the toxic superoxide radical (O-2) to molecular oxygen and hydrogen peroxide through the alternate reduction and oxidation of the active-site copper. We report here that after refitting and further refinement of the previous 2 A structure of SOD2, analysis of the new model and its calculated molecular surface shows an extensive surface topography of sequence-conserved residues stabilized by underlying tight packing and H-bonding. There is a single, highly complementary position for O-2 to bind to both the Cu(II) and activity-important Arg 141 with correct geometry; two water molecules form a ghost of the superoxide in this position. The geometry and molecular surface of the active site, together with biochemical data, suggest a specific model for the enzyme mechanism.
Binding Sites↗
Electrostatic recognition between superoxide and copper, zinc superoxide dismutase.
Electrostatic forces have been implicated in a variety of biologically important molecular interactions including drug orientation by DNA, protein folding and assembly, substrate binding and catalysis and macromolecular complementarity with inhibitors, drugs and hormones. To examine enzyme-substrate interactions in copper, zinc superoxide dismutase (SOD), we developed a method for the visualization and analysis of an enzyme's three-dimensional electrostatic vector field that allows the contributions of specific residues to be identified. We report here that the arrangement of electrostatic charges in SOD promotes productive enzyme-substrate interaction through substrate guidance and charge complementarity: sequence-conserved residues create an extensive electrostatic field that directs the negatively charged superoxide (O-2) substrate to the highly positive catalytic binding site at the bottom of the active-site channel. Dissection of the electrostatic potential gradient indicated the relative contributions of individual charged residues: Lys 134 and Glu 131 seem to have important roles in directing the long-range approach of O-2, while Arg 141 has local orienting effects. The reported methods of analysis may have general application for the elucidation of intermolecular recognition processes.
Binding Sites↗