Piotr Orlewski

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

Summary: 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 a‐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 α67–76 at the extreme synaptic end of each a‐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 cytoplas‐mic 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 noti‐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.

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.

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 α‐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 ϕ dihedral angles. Molecular dynamics simulations including dimethylsulfoxide 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 a β‐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 β / β or β / α turns result in a short helical sequence in the free MIR peptide, whereas the bound analogue is folded by an 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.

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.

Conformational studies of a benzodiazepine-like peptide in SDS micelles by circular dichroism,1H NMR and molecular dynamics simulation

The conformation of a benzodiazepine-like decapeptide corresponding to the YLGYLEQLLR fragment of a casein has been examined in a sodium dodecyl sulfate micellar medium using circular dichroism, two-dimensional1H NMR spectroscopy and restrained molecular dynamics simulation. The decapeptide adopts an amphipathic 310-helicoid structure in which the E6...R10 ionic bridge stabilizes the C-terminus.

Solution structure of an SRYD-containing sequence (250–257) of the fibronectin-like Leishmania gp63 protein by restrained molecular dynamics

The IASRYDQL synthetic octapeptide (250–257) of the Leishmania major surface glycoprotein gp63 efficiently inhibits parasite attachment to the macrophage receptors in in vitro experiments, and the SRYD-containing tetrapeptide mimics antigenically and functionally the RGDS sequence of fibronectin. The conformational properties of the octapeptide were investigated in dimethylsulfoxide (DMSO) with the combined use of NMR data (vicinal coupling constants, nuclear Overhauser effects (NOEs) and temperature coefficient values), molecular modeling by energy minimization and molecular dynamics. The structure is characterized by the high occurrence, exceeding 95%, of the Arg-Asp side-chain-side-chain ionic interaction, which plays a key role in the backbone folding through a distorted type-I β-turn involving the Gln256-NH to Arg253-CO hydrogen bond.

THREE-DIMENSIONAL STRUCTURE OF A13+ -CONTAINING PEPTIDES BY NMR AND MOLECULAR MODELING STUDY : COMPLEXATION OF A THYMIC HORMONE

Abstract The biological coordination chemistry of aluminum has received only limited attention until the last decade. To this end, the interaction between aluminum and thymulin, a linear nonapeptide of thymic origin isolated from serum, was investigated by using high resolution NMR spectroscopy. These experiments were performed in two different solvents, namely DMSO-d 6 and the so-called cryoprotective mixture (water+DMSO), i.e. a mixture that resembles water in many properties. NMR data analysis indicates the existence of one type of complex with a 1: 2 stoichiometry, associating two peptide molecules and one Al 3+ cation. In this complex the Asn 9 carboxylate C-terminal group and the Ser 4 hydroxyl group are ligated to the metal. The three-dimensional structure of the 1:2 1 3+ -thymulin complex was determined by distance geometry calculations and restrained molecular dynamics simulations. Distance geometry calculations were performed with the Biosym DG 11 program using the NOE interproton distances as constraints. Further structural analysis was carried out by restrained molecular dynamics calculations with the Biosym DISCOVER program.

TR-NOE and MD studies ofLeishmania gp63 SRYD-containing sequences bound to anti-SRYD monoclonal antibody

The I250 ASRYDQL257 synthetic octapeptide of theLeishmania major surface glycoprotein gp63, which efficiently inhibits parasite attachment to the macrophage receptors and mimics antigenically and functionally the RGDS sequence of fibronectin, was studied by 2D TR-NOESY in the presence of an anti-SRYD monoclonal antibody (mAbSRYD) that recognizes both SRYD-containing peptides and the cognate protein on intact parasites. Molecular modeling was performed using distance constraints obtained from TR-NOEs. The bound structure was compared with that of the free peptide in DMSO solution and with the crystal structure of the RYD fragment of the OPG2 Fab, an antireceptor antibody that mimics an RGD cell adhesion site.