Analysis of the discrete structure of antibodies by affinity and antigen-binding capacity

Immune sera are a mixture of different groups of antibodies belonging to the same class of immunoglobulins, but differing in their affinity. The graphic analysis and numerical methods permit the determination of the size and affinity of each group of antibodies. Differences in affinity are manifested as differences in antigen-binding capacity. The antigen-binding capacity index, calculated as the product of the association constant multiplied by the concentration of the active centers of antibodies, is proposed.     

Single-chain site-specific mutations of fluorescein-amino acid contact residues in high affinity monoclonal antibody 4-4-20.

Previous crystallographic studies of high affinity anti-fluorescein monoclonal antibody 4-4-20 (Ka = 1.7 x 10(10) M-1) complexed with fluorescyl ligand resolved active site contact residues involved in binding. For better definition of the relative roles of three light chain antigen contact residues (L27dhis, L32tyr and L34arg), four site-specific mutations (L27dhis to L27lys, L32tyr to L32phe, and L34arg to L34lys and L34his) were generated and expressed in single-chain antigen binding derivatives of monoclonal antibody 4-4-20 containing two different polypeptide linkers (SCA 4-4-20/205c, 25 amino acids and SCA 4-4-20/212, 14 amino acids). Results showed that L27dhis and L32tyr were necessary for wild type binding affinities, however, were not required for near-wild type Qmax values (where Qmax is the maximum fluoroscein fluorescence quenching expressed as percent). Tyrosine L32 which hydrogen bonds with ligand was also characterized at the haptenic level through the use of 9-hydroxyphenylfluoron which lacks the carboxyl group to which L32 tyrosine forms a hydrogen bond. Results demonstrated that wild type SCA and mutant L32phe possessed similar HPF binding characteristics. Active site contact residue L34arg was important for fluorescein quenching maxima and binding affinity (L34his mutant), however, substitution of lysine for arginine at L34 did not have a significant effect on observed Qmax value. In addition, substitutions had no effect on structural and topological characteristics, since all mutants retained similar idiotypic and metatypic properties. Finally, two linkers were comparatively examined to determine relative contributions to mutant binding properties and stability. No linker effects were observed. Collectively, these results verified the importance of these light chain fluorescein contact residues in the binding pocket of monoclonal antibody 4-4-20.     

High resolution crystal structure of bovine mitochondrial EF-Tu in complex with GDP

The crystal structure of bovine mitochondrial elongation factor Tu (EF-Tu) in complex with GDP has been determined at a resolution of 1. 94 A. The structure is similar to that of EF-Tu:GDP from Escherichia coli and Thermus aquaticus, but the orientation of the GDP-binding domain 1 is changed relative to domains 2 and 3. Sixteen conserved water molecules common to EF-Tu and other G-proteins in the GDP-binding site are described. These water molecules create a network linking separated parts of the binding pocket. Mitochondrial EF-Tu binds nucleotides less tightly than prokaryotic EF-Tu possibly due to an increased mobility in regions close to the GDP-binding site. The C-terminal extension of mitochondrial EF-Tu has structural similarities with DNA recognising zinc fingers suggesting that the extension may be involved in recognition of RNA.     

High affinity of endonuclease VII for the Holliday structure containing one nick ensures productive resolution

During homologous recombination, genetic information is physically exchanged between parental DNAs via crossing single strands of the same polarity within a four-way DNA junction called a Holliday structure. This process is terminated by the endonucleolytic activity of resolvases, which convert the four-way DNA back to two double strands. To achieve productive resolution, the two subunits of the dimeric enzymes introduce two single-strand cuts positioned symmetrically in opposite strands across the DNA junction. Covalently linked dimers of endonuclease VII from phage T4, whether a homodimer with two or a heterodimer with only one functional catalytic centre, reacted with a synthetic cruciform DNA to form a DNA-enzyme complex immediately after addition of the enzyme. Analysis of the complexes from both reactions revealed that the bound junction contained one nick. While the active homodimer processed this nicked junction consecutively to duplex DNAs by making the second cut, the complex with the heterodimer stayed stable for the whole reaction time. Thus the high affinity of endonuclease VII for the junction containing one nick is part of the mechanism to ensure productive resolution of Holliday structures, by giving the enzyme time to make the second cut, whereupon the complex dissociates into the two duplex DNAs and the free enzyme.     

Probing the structure of the PI-SceI-DNA complex by affinity cleavage and affinity photocross-linking

The PI-SceI protein is an intein-encoded homing endonuclease that initiates the mobility of its gene by making a double strand break at a single site in the yeast genome. The PI-SceI protein splicing and endonucleolytic active sites are separately located in each of two domains in the PI-SceI structure. To determine the spatial relationship between bases in the PI-SceI recognition sequence and selected PI-SceI amino acids, the PI-SceI-DNA complex was probed by photocross-linking and affinity cleavage methods. Unique solvent-accessible cysteine residues were introduced into the two PI-SceI domains at positions 91, 97, 170, 230, 376, and 378, and the mutant proteins were modified with either 4-azidophenacyl bromide or iron (S)-1-(p-bromoacetamidobenzyl)-ethylenediaminetetraacetate (FeBABE). The phenyl azide-coupled proteins cross-linked to the PI-SceI target sequence, and the FeBABE-modified proteins cleaved the DNA proximal to the derivatized amino acid. The results suggest that an extended beta-hairpin loop in the endonuclease domain that contains residues 376 and 378 contacts the major groove near the PI-SceI cleavage site. Conversely, residues 91, 97, and 170 in the protein splicing domain are in close proximity to a distant region of the substrate. To interpret our results, we used a new PI-SceI structure that is ordered in regions of the protein that bind DNA. The data strongly support a model of the PI-SceI-DNA complex derived from this structure.     

High resolution crystal structures of the Escherichia coli lytic transglycosylase Slt70 and its complex with a peptidoglycan fragment.

The 70 kDa soluble lytic transglycosylase (Slt70) from Escherichia coli is an exo-muramidase, that catalyses the cleavage of the glycosidic bonds between N -acetylmuramic acid and N -acetylglucosamine residues in peptidoglycan, the main structural component of the bacterial cell wall. This cleavage is accompanied by the formation of a 1,6-anhydro bond between the C1 and O6 atoms in the N -acetylmuramic acid residue (anhMurNAc). Crystallographic studies at medium resolution revealed that Slt70 is a multi-domain protein consisting of a large ring-shaped alpha-superhelix with on top a catalytic domain, which resembles the fold of goose-type lysozyme. Here we report the crystal structures of native Slt70 and of its complex with a 1,6-anhydromuropeptide solved at nominal resolutions of 1.65 A and 1.90 A, respectively. The high resolution native structure reveals the details on the hydrogen bonds, electrostatic and hydrophobic interactions that stabilise the catalytic domain and the alpha-superhelix. The building-block of the alpha-superhelix is an "up-down-up-down" four-alpha-helix bundle involving both parallel and antiparallel helix pairs. Stabilisation of the fold is provided through an extensive packing of apolar atoms, mostly from leucine and alanine residues. It lacks, however, an internal consensus sequence that characterises other super-secondary helical folds like the beta-helix in pectate lyase or the (beta-alpha)-helix in the ribonuclease inhibitor. The 1, 6-anhydromuropeptide product binds in a shallow groove adjacent to the peptidoglycan-binding groove of the catalytic domain. The groove is formed by conserved residues at the interface of the catalytic domain and the alpha-superhelix. The structure of the Slt70-1, 6-anhydromuropeptide complex confirms the presence of a specific binding-site for the peptide moieties of the peptidoglycan and it substantiates the notion that Slt70 starts the cleavage reaction at the anhMurNAc end of the peptidoglycan.     

High resolution crystal structures of human Rab4a in its active and inactive conformations

The Ras-related human GTPase Rab4a is involved in the regulation of endocytosis through the sorting and recycling of early endosomes. Towards further insight, we have determined the three-dimensional crystal structure of human Rab4a in its GppNHp-bound state to 1.6 Angstroms resolution and in its GDP-bound state to 1.8 Angstroms resolution, respectively. Despite the similarity of the overall structure with other Rab proteins, Rab4a displays significant differences. The structures are discussed with respect to the recently determined structure of human Rab5a and its complex with the Rab5-binding domain of the bivalent effector Rabaptin-5. The Rab4 specific residue His39 modulates the nucleotide binding pocket giving rise to a reduced rate for nucleotide hydrolysis and exchange. In comparison to Rab5, Rab4a has a different GDP-bound conformation within switch 1 region and displays shifts in position and orientation of the hydrophobic triad. The observed differences at the S2-L3-S3 region represent a new example of structural plasticity among Rab proteins and may provide a structural basis to understand the differential binding of similar effector proteins.     

Refined structure of alpha-lytic protease at 1.7 A resolution. Analysis of hydrogen bonding and solvent structure

The structure of alpha-lytic protease, a serine protease produced by the bacterium Lysobacter enzymogenes, has been refined at 1.7 A resolution. The conventional R-factor is 0.131 for the 14,996 reflections between 8 and 1.7 A resolution with I greater than or equal to 2 sigma (I). The model consists of 1391 protein atoms, two sulfate ions and 156 water molecules. The overall root-meansquare error is estimated to be about 0.14 A. The refined structure was compared with homologous enzymes alpha-chymotrypsin and Streptomyces griseus protease A and B. A new sequence numbering was derived based on the alignment of these structures. The comparison showed that the greatest structural homology is around the active site residues Asp102, His57 and Ser195, and that basic folding pathways are maintained despite chemical changes in the hydrophobic cores. The hydrogen bonds in the structure were tabulated and the distances and angles of interaction are similar to those found in small molecules. The analysis also revealed the presence of close intraresidue interactions. There are only a few direct intermolecular hydrogen bonds. Most intermolecular interactions involve bridging solvent molecules. The structural importance of hydrogen bonds involving the side-chain of Asx residues is discussed. All the negatively charged groups have a counterion nearby, while the excess positively charged groups are exposed to the solvent. One of the sulfate ions is located near the active site, whereas the other is close to the N terminus. Of the 156 water molecules, only seven are not involved in a hydrogen bond. Six of these have polar groups nearby, while the remaining one is in very weak density. There are nine internal water molecules, consisting of two monomers, two dimers and one trimer. No significant second shell of solvent is observed.     

Putting two and two together: crystal structure of the FGF-receptor complex

The recently determined crystal structure of an FGF-receptor complex reveals a surprising architecture and a novel mode of receptor dimerization. The structure also elucidates the role of heparan sulfate proteoglycans in receptor activation, showing significant differences from previously proposed models.     

Substitution and solvent effects in the chalcones isomerization barrier of flavylium photochromic systems.

Useful application of photochromic compounds as optical memories implies the existence of a large kinetic barrier between the forms interconverted by light. In the case of flavylium salts, the ground state isomerization barrier between the photoisomerizable chalcone isomers is shown to correlate with the electron donating ability of the substituents, measured by their effects in the (1)H NMR chemical shifts of the aromatic protons. Substitution with electron donating groups in ring A lowers the barrier while substitution at ring B has the opposite effect. However, in water, the observed increase is higher than expected in the case of compound 4',9-dihydroxychalcone when compared with the analogous 4'-dimethylamino-9-hydroxychalcone, containing a better electron donating group in the same position. Our interpretation is that the water network is providing an efficient pathway to form tautomers. In acetonitrile, unlike water, the expected order is indeed observed: E(a)(4',9-dihydroxychalcone) = 60 kJ mol(-1) < E(a) (4'-dimethylamino-9-hydroxychalcone) = 69 kJ mol(-1).     

High resolution crystal structure of rat long chain hydroxy acid oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1, 2, 3-thiadiazole. Implications for inhibitor specificity and drug design

Long chain hydroxy acid oxidase (LCHAO) is responsible for the formation of methylguanidine, a toxic compound with elevated serum levels in patients with chronic renal failure. Its isozyme glycolate oxidase (GOX), has a role in the formation of oxalate, which can lead to pathological deposits of calcium oxalate, in particular in the disease primary hyperoxaluria. Inhibitors of these two enzymes may have therapeutic value. These enzymes are the only human members of the family of FMN-dependent l-2-hydroxy acid-oxidizing enzymes, with yeast flavocytochrome b₂ (Fcb2) among its well studied members. We screened a chemical library for inhibitors, using in parallel rat LCHAO, human GOX and the Fcb2 flavodehydrogenase domain (FDH). Among the hits was an inhibitor, CCPST, with an IC₅₀ in the micromolar range for all three enzymes. We report here the crystal structure of a complex between this compound and LCHAO at 1.3 Å resolution. In comparison with a lower resolution structure of this enzyme, binding of the inhibitor induces a conformational change in part of the TIM barrel loop 4, as well as protonation of the active site histidine. The CCPST interactions are compared with those it forms with human GOX and those formed by two other inhibitors with human GOX and spinach GOX. These compounds differ from CCPST in having the sulfur replaced with a nitrogen in the five-membered ring as well as different hydrophobic substituents. The possible reason for the ∼100-fold difference in affinity between these two series of inhibitors is discussed. The present results indicate that specificity is an issue in the quest for therapeutic inhibitors of either LCHAO or GOX, but they may give leads for this quest.     

High resolution structure,stability, and synaptotagmin binding of a truncated neuronal SNARE complex

The structure of a truncated SNARE complex has been solved to 1.4-A resolution revealing a stabilizing salt bridge, sites of hydration, and conformational variability of the ionic central layer that were not observed in a previously published structure at 2.4-A resolution (Sutton, R. B., Fasshauer, D., Jahn, R., and Brunger, A. T. (1998) Nature 395, 347-353). The truncated complex lacks residues involved in phospholipid binding and denatures at a lower temperature than longer complexes as assessed by SDS and circular dichroism thermal melts. The truncated SNARE complex is monomeric, and it retains binding to synaptotagmin I.     

High resolution crystal structures of T4 phage beta-glucosyltransferase: induced fit and effect of substrate and metal binding

beta-Glucosyltransferase (BGT) is a DNA-modifying enzyme encoded by bacteriophage T4 that transfers glucose from uridine diphosphoglucose to 5-hydroxymethyl cytosine bases of phage T4 DNA. We report six X-ray structures of the substrate-free and the UDP-bound enzyme. Four also contain metal ions which activate the enzyme, including Mg(2+) in forms 1 and 2 and Mn(2+) or Ca(2+). The substrate-free BGT structure differs by a domain movement from one previously determined in another space group. Further domain movements are seen in the complex with UDP and the four UDP-metal complexes. Mg(2+), Mn(2+) and Ca(2+) bind near the beta-phosphate of the nucleotide, but they occupy slightly different positions and have different ligands depending on the metal and the crystal form. Whilst the metal site observed in these complexes with the product UDP is not compatible with a role in activating glucose transfer, it approximates the position of the positive charge in the oxocarbonium ion thought to form on the glucose moiety of the substrate during catalysis.     

Characterization of antigen-binding receptors in vitro. II. Antigen-binding capacity and affinity of lymphoid receptors after immunization.

We examined the kinetics and affinity of antigen binding in lymphoid populations in mice after immunization. There is increased binding capacity in lymphoid cells from animals that have undergone primary immunization. This increase would seem to be related to increased numbers of antigen-binding cells (rosette-forming cells). The serum antibody titers rise after the increasing binding capacity and numbers of BSA rosette-forming cells have increased. There is an increased amount of antigen bound per antigen-binding cell at certain times after immunization with two peaks in this capacity being demonstrable--one occurring at 4 days after immunization and the second occurring approximately 12 days after immunization and persisting for prolonged periods after that. With time, after immunization two separable peaks of increased antigen-binding cells become apparent, one very early (before Day 4) and one later (after Day 20 to 30). The affinity constants for antigen-binding cells have been measured and found to be high, and to increase with time after immunization. It appears that the heterogeneity of the affinity constants for antigen-binding cells is high early in immunity and becomes more homogeneous with time after immunization.