Sequence-Specific Peptides for Probing Receptors and Ion-Channel Functions Peptide and Protein Group Colloquium Organized by J. S. Davies (University of Wales, Swansea) and W. A. Gibbons (School of Pharmacy, London). Spring Meeting of the Peptide and Protein Group of the Biochemical Society/Royal Society of Chemistry held at Gregynog, Mid-Wales, 10- I 2 April I992

Interactions of an immunogenic decapeptide fragment of the neuromuscular acetylcholine receptor (AChR) with a monoclonal anti-AChR antibody Michel Marraud,* Pascal Demange, Manh-Thong Cung, Vassilios Tsikarkt Constantinos Sakarellos,t Evstratia Vatzakit and Socrates J. Tzartost CNRS-URA-494, ENSIC-INPL, BP45 I , 54001 Nancy, France, +Department of Chemistry, University of loannina, PO I 186, 45 I 10 loannina, Greece, $Hellenic Pasteur Institute, I27 Vassilissis Sofias Avenue, I I52 I Athens, Greece

Myasthenia gravis (MG) is an auto-immune disease in which auto-antibodies are raised against the postsynaptic acetylcholine receptor (AChR) and cause impairment of neuromuscular transmission. Experimental MG can be induced in animals immunized with AChRs, thus giving access to polyclonal and monoclonal anti-AChR antibodies (mAbs) [ 11. The main immunogenic region (MIR) of the AChR has been localized at the extracellular Nterminal part of the a-subunit, but it is distinct from the ionic channel allowing Na+ to enter the muscle cell, and from the binding site of snake venom atoxins as well. It has been demonstrated by mAb competition that the MIR is a very concrete and small region. By using AChR fragments and synthetic peptides, it has been observed that the smallest a-fragment to be significantly and selectively recognized by the anti-AChR mAbs, is the a67-76 decapeptide with the WNPDDYGGVK sequence in human AChR [21. However, its binding affinity is about three orders of magnitude less than that of the intact AChR. It has to be noticed that the human MIR only differs from the Torpedo

Abbreviations used: AChK, acetylcholine receptor; MG. myasthenia gravis; mAb, monoclonal anti-acetylcholine receptor antibody; MIR, main immunogenic region; MD, molecular dynamics; COSY, correlated spectroscopy; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect and exchange spectroscopy; DMSO, dimethylsulphoxide. *Towhom correspondence should be addressed.

-.

sequence by one conservative Ile and one non-conservative Aspin -,Ala substitution. In order to determine the immunogenic role of each residue in the MIR, various linear and cyclic analogues have been prepared and tested in radioimmunoassays. The N-terminal hexapeptide is strictly required for mAb recognition, but some modifications of the C-terminal tetrapeptide are accepted [3]. Furthermore, the linear [Ala'']- and [Ala7'] -analogues have twice as much affinity than the natural sequence for some of the anti-AChR mAbs. This allows the possibility of designing superactive artificial epitopes which could be used for anti-AChR antibody elimination and a better understanding of human MG. The knowledge of the MIR three-dimensional structure should help to this end. However, the secondary structure of the AChR is not known. W e therefore have carried out n.m.r. experiments and molecular dynamics (MD) simulations on both the MIR sequence and the superactive [Ala7']analogue either in the free state, or in contact with an anti-AChR mAb. W e can reasonably assume that the mAb complementary sites should fit the peptide fragment in a three-dimensional structure very similar to that it assumes in the complete AChR.

Experimental The natural Torpedo sequence (WNPADYGGIK) and its [Ala7'] -analogue (WNPADYGGIA) were prepared classically by a solid phase procedure. They were studied by 'H n.m.r. [ZD-correlated spectroscopy (COSY) and nuclear Overhauser and

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exchange spectroscopy (NOESY)] on a Briiker AM400 apparatus both in the free state [dimethylsulphoxide (DMSO) solution] and in the bound state (water solution, pH = 7.2) to the mAb6 monoclonal antibody obtained by immunizing rats against the intact Torpedo AChR. The transferred NOESY spectra of the bound peptides were scanned under the following conditions: the mAb6 concentration was adjusted to 2% of that of the pepM) in order to be in a rapid exchange tide (8 X regime allowing transfer of the nuclear Overhauser effects (NOE) from the minor bound state to the major free state [4]. The short NOE contacts were introduced as constraints in MD simulations and energy minimizations using the BIOGROMOS and SYBYI, programs on a 4D35G Silicon Graphics IRIS workstation. After relaxation of the extended geometry by several steps of energy minimization, only the backbone-backbone NOE connectivities were introduced as distance constraints for the free state. MD simulations were carried out at 300 K for a period of 40 ps. From 40 to 80 ps, the NOE constraints were smoothed by a 4-fold factor to allow the molecule more flexibility [5]. The same procedure was applied to the bound state by taking into consideration only the side-chain-side-chain NOE connectivities.

Fig. I Average conformation of: ( a ) the free a67-76 Torpedo AChR fragment in DMSO, and ( b ) the [Ala76]-analoguein contact with the anti-AChR mAb6 antibody

Results Free state In water both free peptides exhibit no NOE connectivities, either indicating that they adopt a very flexible solvated conformation, or because of an unfavourable w t c value ( w, magnetic field frequency; z, correlation time). In DMSO, NOE correlations are clearly visible, but they are more numerous for the natural peptide than for the analogue, especially for the correlations due to the Ile-Ala C-terminal sequence. The most intense cross peaks are found for the short-range backbone-backbone connectivities whereas the sidechains give rise to much weaker contributions, in agreement with the quasi-free rotation of the Ca-CS bonds evidenced by the two medium C"H-CPH, vicinal coupling constants. The small temperature coefficients for the Asp7', Gly74and L y ~ / A l aN~H~ signals indicate that the corresponding N-H sites are solvent-protected, and probably involved in the intramolecular interactions. MD simulations under NOE constraints and energy minimizations show that the most probable conformation of the free natural fragment is a compact structure characterized by folding of the Proh9-

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Ala70-Asp7' and Gly73-Gly74-Ile75sequences (Fig. la). It should be noted that the average orientation of the Trph7side-chain is located in a central position, in which it is in contact with the Tyr72and side-chains. The side-chain is preferentially folded back on the peptide chain. The eight first residues of the [Ala7h]-analogue exhibit the same conformational features as those in the natural sequence. The absence of backbone-backbone NOE correlations for the Cterminal Ile-Ala dipeptide denotes a greater flexibility of the C-terminus than of the homologous part in the free natural fragment. Bound state

Considering the COSY spectra of the free peptides in aqueous solution, the presence of 2% mAb6 modifies them completely. All the cross-peaks are considerably weaker, except those for the Cterminal Ile-Lys dipeptide in the natural sequence.

Sequence-SpecificPeptides

This phenomenon, called dynamical filtering [6], expresses the fact that the correlation time t, of the bound ligand is of the same order as that of the antibody, and induces very broad signals. The sharp remaining cross-peaks for the Ile-Lys sequence denote that it is not closely bound to the antibody. All these experiments confirm that, in agreement with the biological assays, the [Ala7h]analogue is more tightly recognized than the natural sequence. The difference could be explained by better recognition of the Ile-Ala sequence compared with Ile-1,ys. We can suppose that the interaction of the positively charged 1,ys N+H, site with the peptide backbone in the natural fragment (Fig. l a ) is not in favour of an optimum recognition whereas in the intact AChR, it could interact with a complementary charge out of the fragment under consideration. We therefore have focused our attention on the transferred-NOESY spectrum of the [Ala7h]analogue in the presence of 2% mAb6. Its NOESY cross-peaks are actually more intense and more numerous than in the case of the natural fragment. However, the NOE connectivities for the amide NH protons are hardly visible probably because of their rapid exchange with the solvent at neutral pH, which corresponds to the best conditions for antigen-antibody recognition. On the contrary, the non-exchangeable protons on the side-chains give very clear NOE connectivities (Table 1). This indicates that the antigen-antibody recognition tends to freeze the rotation of the peptide side-chains. Table I

Transferred NOESY correlations between the sidechain proton signals for the [Ala76]-analogueof the a67-76 Torpedo AChR fragment in contact with the anti-AChR antibody mAb6 (molar ratio 2%) in aqueous solution (pH = 7.2) Residue

Trp6'

AsrP

m

Ala70

Asp7'

m m w

Tyr72

w

lle75

m

m

Ala76 s, strong; m. medium; w. weak

Ala'O

Asp7'

w

S

W

S

m

It is of interest to note that the free natural peptide in DMSO, and the bound [Ala"] -analogue in water, give similar NOESY patterns for the nonexchangeable protons, although the NOE correlations are weaker in the former case. This suggests that the peptide-mAb6 recognition does not include a considerable change of the backbone conformation but rather a structuration of the side-chains. The introduction of the side-chain-side-chain NOE constraints in MD simulations leads to the conformation represented in Fig. l(b). It differs from the conformation of the free natural fragment (Fig. la) by a rotation of the Ile-Ala moiety which brings the Ile side-chain closer to both the Tyr and Trp side-chains.

Conclusion The n.m.r. data on the a67-76 Torpedo AChR immunogenic fragment interacting with the antiAChR mAb6 antibody are in excellent agreement with the radioimmunoassays. The transferredNOESY correlations are more intense and numerous as its binding affinity to mAb6 increases. The best mAbWpeptide recognition is observed for the more flexible [Ala"] -analogue which probably allows a greater number of close atomic contacts. The MIR epitope seems to be composed of the WNPADY rigid core responsible for antibody recognition, and an adjacent, more flexible, sequence giving additional stability to the complex. The MIR-antibody complexation does not greatly perturb the conformation of the rigid core, but is capable of fashioning the flexible adjacent sequence. The side-chains assume a fixed orientation in the complex, and are probably in close contact with the antibody. 1. Lindstrom. J., Shelton, L). & Fugii, Y. (1988) Adv. Immunol. 42.133-284 2. Tzartos, S. J., Cung, M. T., Demange, P., Loutrari, H., Mamalaki. A., Marraud, M., Papadouli, I., Sakarellos, C. & Tsikaris. V. (1 992) Mol. Neurobiol. 5. 1-29 3. I'apadouli, I., Potamianos, S.,Hadjidakis, I., Hairaktari, E., Tsikaris, V., Sakarellos, C., Cung, M. T., Marraud, M. & Tzartos, S. J. (1990) Hiochem.J. 269,239-245 4. Clore, G. M. & Gronenborn, A. M. (1983) J. Magn. Keson. 53,423-442 5. Cung, M. T., Tsikaris, V., Demange, P., Papadouli. I., Tzartos, S. J. Sakarellos, C. & Marraud, M. (1992) I'eptide Kes. 5, 14-24 6. Weiss, M. A., Eliason. J. & States, D. J. (1984) Proc. Natl. Acad. Sci. U S A . 81, 6019-6023 Received 13 May 1992

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Interactions of an immunogenic decapeptide fragment of the neuromuscular acetylcholine receptor (AChR) with a monoclonal anti-AChR antibody.

Sequence-Specific Peptides for Probing Receptors and Ion-Channel Functions Peptide and Protein Group Colloquium Organized by J. S. Davies (University...
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