The third-dimensional structure of the complex between an Fv antibody fragment and an analogue of the main immunogenic region of the acetylcholine receptor: a combined two-dimensional NMR, homology, and molecular modeling approach.

Binding of autoantibodies to the acetylcholine receptor (AChR) plays a major role in the autoimmune disease Myasthenia gravis (MG). In this paper, we propose a structure model of a putative immunocomplex that gives rise to the reduction of functional AChR molecules during the course of MG. The model complex consists of the [G(70), Nle(76)] decapeptide analogue of the main immunogenic region (MIR), representing the major antigenic epitope of AChR, and the single chain Fv fragment of monoclonal antibody 198, a potent MG autoantibody. The structure of the complexed decapeptide antigen [G(70), Nle(76)]MIR was determined using two-dimensional nmr, whereas the antibody structure was derived by means of homology modeling. The final complex was constructed using calculational docking and molecular dynamics. We termed this approach "directed modeling," since the known peptide structure directs the prestructured antibody binding site to its final conformation. The independently derived structures of the peptide antigen and antibody binding site already showed a high degree of surface complementarity after the initial docking calculation, during which the peptide was conformationally restrained. The docking routine was a soft algorithm, applying a combination of Monte Carlo simulation and energy minimization. The observed shape complementarity in the docking process suggested that the structure assessments already led to anti-idiotypic conformations of peptide antigen and antibody fragment. Refinement of the complex by dynamic simulation yielded improved surface adaptation by small rearrangements within antibody and antigen. The complex presented herein was analyzed in terms of antibody-antigen interactions, properties of contacting surfaces, and segmental mobility. The structural requirements for AChR complexation by autoantibodies were explored and compared with experimental data from alanine scans of the MIR peptides. The analysis revealed that the N-terminal loop of the peptide structure, which is indispensable for antibody recognition, aligns three hydrophobic groups in a favorable arrangement leading to the burial of 40% of the peptide surface in the binding cleft upon complexation. These data should be valuable in the rational design of an Fv mutant with much improved affinity for the MIR and AChR to be used in therapeutic approaches in MG.

Anatomy of the antigenic structure of a large membrane autoantigen, the muscle-type nicotinic acetylcholine receptor.

The neuromuscular junction nicotinic acetylcholine receptor (AChR), a pentameric membrane glycoprotein, is the autoantigen involved in the autoimmune disease myasthenia gravis (MG). In animals immunized with intact AChR and in human MG, the anti-AChR antibody response is polyclonal. However, a small extracellular region of the AChR alpha-subunit, the main immunogenic region (MIR), seems to be a major target for anti-AChR antibodies. A major loop containing overlapping epitopes for several anti-MIR monoclonal antibodies (mAbs) lies within residues alpha 67-76 at the extreme synaptic end of each alpha-subunit: however, anti-MIR mAbs are functionally and structurally quite heterogeneous. Anti-MIR mAbs do not affect channel gating, but are very effective in the passive transfer of MG to animals; in contrast, their Fab or Fv fragments protect the AChR from the pathogenic effects of the intact antibodies. Antibodies against the cytoplasmic region of the AChR can be elicited by immunization with denatured AChR and the precise epitopes of many such mAbs have been identified; however, it is unlikely that such antibodies are present in significant amounts in human MG. Antibodies to other extracellular epitopes on all AChR subunits are present in both experimental and human MG; these include antibodies to the acetylcholine-binding site which affect AChR function in various ways and also induce acute experimental MG. Finally, anti-AChR antibodies cross-reactive with non-AChR antigens exist, suggesting that MG may result from molecular mimicry. Despite extensive studies, many gaps remain in our understanding of the antigenic structure of the AChR; especially in relation to human MG. A thorough understanding of the antigenic structure of the AChR is required for an in-depth understanding, and for possible specific immunotherapy, of MG.

Solution structures of the fibronectin-like Leishmania gp63 SRYD-containing sequence in the free and antibody-bound states--transferred NOE and molecular dynamics studies.

The anti-SRYD monoclonal antibody (mAbSRYD) raised against the IASRYDQL synthetic octapeptide, the 250-257 sequence of the Leishmania major surface glycoprotein gp63 recognizes both SRYD-containing peptides and the whole cognate major surface protein on intact parasites. Two SRYD-containing peptides, which antigenically and functionally mimic the RGDS sequence of fibronectin and efficiently inhibit parasite attachment to the macrophage receptors, were studied by two-dimensional transferred nuclear Overhauser effect experiments in the presence of mAbSRYD. The antibody-bound IASRYDQL octapeptide solution conformation was determined on the basis of 55 interproton-distance restraints, derived from NMR measurements. Eighteen structures which were first generated using an approach combining distance geometry and molecular dynamics, converge by energy minimization toward a folded structure with an average rmsd from the experimental data of less than 0.05 nm for the overall backbone and 0.025 nm for the SRYD motif. A distorted gamma-turn was found, stabilized by the backbone-backbone D255-NH to R253-CO hydrogen bond, while the R253 and D255 side chains are pointing in opposite directions. This latter antibody-bound structure is compared with that of the free octapeptide in dimethylsulfoxide solution, and with the crystal structure of the RYD fragment in OPG2 Fab, an antireceptor antibody that mimics the RGD cell adhesion site. On this basis, a mechanism for IASRYDQL-receptor interaction is discussed.

Two-dimensional 1H-NMR and CD structural analysis in a micellar medium of a bovine alphaS1-casein fragment having benzodiazepine-like properties.

The conformation of the benzodiazepine-like decapeptide, YLGYLEQLLR, corresponding to residues 91-100 of bovine alphaS1-casein, has been examined in SDS micelles using CD, two-dimensional 1H-NMR and restrained molecular-dynamics simulation. Evidence is presented that the decapeptide adopts a rigid structure in water/SDS micellar medium, but not in water or dimethylsulfoxide. The three-dimensional structure, consistent with the proton-proton distances obtained from the quantitative analysis of the two-dimensional NOEs, was generated by restrained energy minimization and molecular-dynamics simulation. In water/SDS micellar medium, YLGYLEQLLR adopts an amphipathic helicoid structure with distinct hydrophobic and hydrophilic faces. The relative disposition of the tyrosine aromatic rings was compared with that of the aromatic rings in the benzodiazepines.

Construction and application of a new class of sequential oligopeptide carriers (SOCn) for multiple anchoring of antigenic peptides--application to the acetylcholine receptor (AChR) main immunogenic region.

A new class of sequential oligopeptide carriers (SOCn), namely (Lys-Aib-Gly)n (n = 2-7), for anchoring antigenic peptides, is presented. These SOCn have been designed in order to assume a determined structural motif, exhibiting defined spatial orientations of the Lys-N epsilon H2 anchoring groups. The NMR study showed that SOCn adopt a rigid conformation with some regularity, initiated from the C-terminus of the carrier, while molecular dynamics simulation confirmed the occurrence of a distorted 3(10)-helix. It was also demonstrated, by 1HNMR, that all the antigenic peptides bound to the SOCn retain their original, folded active, structure and that probably they do not interact to each other. It is concluded that the beneficial structural elements of the SOCn impose a favorable disposition of the anchored peptides so that potent antigens with maximum molecular recognition are generated.

Isomerization of the Xaa-Pro peptide bond induced by ionic interactions of arginine.

Inclusion of Arg or Pro residues in proteins and peptides has been proved to play an essential role in biochemical functions through ionic interactions, conformational transitions, and formation of turns as well. In this study we present the conformational properties of the Ac-Arg-Ala-Pro (1), Ac-Arg-Ala-Pro-NH2 (2), Ac-Arg-Pro-Asp-NH2 (3), and Ac-Arg-Pro-Asp (4) tripeptides, using 1H-nmr spectroscopy and molecular dynamics. These peptides were modeled with the aim of studying the role of the Arg-guanidinium to carboxylate ionic interactions on the Xaa-Pro peptide bond isomerization. It was found with 1 and 4 that arginine preferentially interacts with the C-terminal carboxylate group, even though the beta-carboxylate is also accessible. This tendency of the Arg moiety was found to induce the cis disposition of the Ala-Pro peptide bond in 1. It was also confirmed that the Arg...Asp side chain-side chain ionic interaction in 3 plays a key role in backbone folding and structural stabilization through a type I beta-turn. The nmr pattern for 3 showed a remarkable similarity with that for various Arg-Tyr-Asp containing peptides, a sequence that is crucial for the adhesion properties of the Leishmania gp63 glycoprotein.

Compared structures of the free nicotinic acetylcholine receptor main immunogenic region (MIR) decapeptide and the antibody-bound [A76]MIR analogue: a molecular dynamics simulation from two-dimensional NMR data.

Monoclonal antibodies against the main immunogenic region (MIR) of the muscle acetylcholine receptor (AChR) are capable of inducing experimental myasthenia gravis (MG) in animals. The epitope of these antibodies has been localized between residues 67 and 76 of the AChR alpha-subunit. The conformation in solution of the Torpedo californica MIR peptide and of its [A76] MIR analogue have been analyzed using molecular modeling based on nmr interproton distances and J-derived phi dihedral angles. Molecular dynamics simulations including dimethyl-sulfoxide as explicit solvent have been carried out on the free MIR peptide. Calculation of the structure of the [A76]MIR analogue bound to an anti-MIR monoclonal antibody have been performed in the presence of water molecules. A tightly folded structure appears for both peptides with alpha beta-folded N-terminal N68-P-A-D71 sequence of type I in the free state and type III in the mAb6-bound state. The C-terminal sequence is folded in two different ways according to the result in the free and bound state of the peptides: two overlapping beta/beta or beta/alpha turns result in a short helical sequence in the free MIR peptide, whereas the bound analogue is folded by uncommon hydrogen bond closing an 11-membered cycle. This structural evolution is essentially the result of the reorientation of the hydrophobic side chains that are probably directly involved in peptide--antibody recognition.

Influence of lipid peroxidation and hydrogen peroxide on muscarinic cholinergic receptors and ATP level in rat myocytes and lymphocytes.

Isolated rat neonatal cardiac myocytes and rat lymph-node lymphocytes were treated with Fe2+ cumene hydroperoxide or hydrogen peroxide. The intensity of lipid peroxidation was estimated by measuring production of malondialdehyde (MDA). The level of muscarinic cholinergic receptors was determined by [3H]-QNB binding. Cumene hydroperoxide treatment was found to induce lipid peroxidation in both myocytes and lymphocytes, the process being more pronounced in myocytes. An important decrease of muscarinic cholinergic receptor level and a significant drop of intracellular ATP level in these cells were simultaneously observed. In both cell types, hydrogen peroxide treatment decreased the ATP level while lipid peroxidation and muscarinic cholinergic receptors were unaffected. The different effect of hydrogen peroxide on lipid peroxidation and the level of muscarinic cholinergic receptors on the one hand and the ATP level on the other suggests that this substance acts mainly in the intracellular space.

Phospholipid-protein ratio in brain of suckling rabbits treated with an organophosphorus compound.

The effect of dichlorvos on phospholipids and proteins in brain of suckling rabbits has been studied by treating the animals with dichlorvos by oral gavage for 10 days starting on the 6th day of life. In 16-day-old animals a significant decrease of amino-acid concentrations in plasma was found; the concentration and synthesis of protein were lowered in their brains. The phospholipid pattern studied in the brain hippocampus exhibited changes in proportions of particular phospholipids. The ratio of phospholipids to proteins significantly increased in some brain regions. It is concluded that a phospholipid-protein imbalance in the brain of animals chronically intoxicated with organophosphorus compounds may be a primary cause of the multifocal changes in that organ.

[Usefulness of a modified screening method for the detection of phenol in the saliva in evaluating occupational exposure to this compound]. / Przydatnosc zmodyfikowanej metody skriningowej wykrywania fenolu w slinie do oceny ekspozycji przemyslowej na ten zwiazek.

So far there has not been any quick and simple method to estimate phenol exposure or phenol poisoning directly at the workstation. Our work was aiming at a modification of Lutogniewska's method of determining phenol in the saliva as an indicator of phenol exposure at the workplace. The original method has been improved and modified through essential modification, consisting in a stable borax prealkalization of blotting--paper where the saliva is deposited, which results in a more intense and fast colouring. Apart from original 2,6-dibromchinochlorimid detector, 2,6-dichlorchinochlorimid has been tested. The newly--developed technique has been used in field studies related to 19 workers in one of petrochemical plants and a control group of 40 volunteer students. The proposed method is easy to apply and convenient both for those carrying out the studies and those subjected to them. Nearly in all workers tested, after 6 hours' exposure slight traces of phenol (2 mg/100 ml saliva) have been found in the saliva. On the other hand, in those with no exposure to phenol the test was negative. The method seems to be valuable for the practical use in field studies. The findings of this tentative paper should be checked on more extensive materials.