Amino Acid Residues Within the Sequence Region α55–74 of Torpedo Nicotinic Acetylcholine Receptor Interacting with Antibodies to the Main Immunogenic Region and with Snake α-Neurotoxins

Abstract

The sequence region 55–74 of the α-subunit of the acetylcholine receptor (AChR) from Torpedo californica electroplax comprises the amino-terminal end of a sequence segment—residues α67–76—forming the main immunogenic region (MIR), which is most frequently recognized by anti-AChR autoantibodies in myasthenia gravis. The synthetic sequence α55–74 of Torpedo AChR binds α-bungarotoxin (αBTX), suggesting that amino acid residues within this sequence region may contribute to formation of an αBTX binding site.

Using single-residue substituted synthetic analogues of the sequence α55–74 of Torpedo AChR, in which each residue was sequentially substituted by either glycine or alanine, we sought identification of the amino acids involved in interaction with α-neurotoxins and with three different anti-MIR monoclonal antibodies (mAbs 6, 22, and 198). Substitution of Arg₅₅, Arg₅₇, Trp₆₀, Arg₆₄, Leu₆₅, Arg₆₆, Trp₆₇, or Asn₆₈ strongly inhibited α-toxin binding, whereas substitutions of Ile₆₁, Val₆₃, Pro₆₉, Ala₇₀, Asp₇₁, or Tyr₇₂ had marginal effects. Substitutions within the region α68–72 significantly diminished binding of anti-MIR mAbs, although residue preferences differed among mAbs. Further, substituting Trp₆₀ substantially reduced binding of mAb 198, and moderately affected binding of mAb 6, and substitution of Asp₆₂ slightly but consistently affected binding of mAbs 6 and 22.     

Main Immunogenic Region of Torpedo Electroplax and Human Muscle Acetylcholine Receptor: Localization and Microheterogeneity Revealed by the Use of Synthetic Peptides

Most anti-nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR alpha-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo alpha-subunit sequence and to a segment of human muscle alpha-subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti-AChR mAbs directed against epitopes on the alpha-subunit other than the MIR. A main constituent loop of the MIR was localized within residues alpha 67-76. Residues 70 and 75, which are different in the Torpedo and human alpha-subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop alpha 67-76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment alpha 67-76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68-71. The most stable structure predicted for this segment, in both the Torpedo and human alpha-subunits, is a hairpin loop, whose apex is a type I beta-turn and whose arms are beta-strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non-MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues alpha 331-351.     

The main immunogenic region of the nicotinic acetylcholine receptor. Identification of amino acid residues interacting with different antibodies

In myasthenia gravis a highly conserved area of the nicotinic receptor (AcChR) dominates the autoantibody response (main immunogenic region, MIR), and it is formed by residues within the sequence segment 67-76 of the AcChR alpha-subunit. We have studied the binding of eight anti-MIR mAb to synthetic peptides containing the sequence segment 67-76 of the human alpha-subunit, and peptide analogues containing single residue substitutions of this sequence. We used also a peptide where both Asp70 and Asp71 were substituted by glycine residues. The binding of six anti-MIR mAb was strongly influenced by several substitutions. All these mAb required residues Asn68, and Pro69 for binding. Five of them required also Asp71 and Tyr72. Substitution of Asp70, which is an Ala residue in Torpedo AcChR, was irrelevant for the binding of an anti-Torpedo and an anti-Electrophorus mAb, and moderately reduced the binding of an anti-human mAb (no. 203). Substitution of Trp67 moderately reduced the binding of some of these mAbs. A mAb of this group (the antihuman mAb no. 198) bound in a manner only slightly influenced by ionic strength, whereas the binding of the other five mAb of this group was very sensitive to the ionic strength. Two anti-Electrophorus MIR mAb bound similarly to all peptide analogues in low ionic strength. At high ionic strength only the peptide analogue where Asp 70 was changed to a Gly residue bound significantly. This may indicate that the Electrophorus MIR has an uncharged residue at this position, as does Torpedo AcChR. Residues at position 73, 74, 75, and 76 were of little or no importance for the binding of all anti-MIR mAb. A free amino terminus was essential for the binding of most mAb. The results of competition experiments between different peptides and native AcChR for mAb binding were consistent with those obtained in direct binding experiments.     

Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor.

The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible.     

Two-dimensional 1H-NMR study of antigen-antibody interactions: binding of synthetic decapeptides to an anti-acetylcholine receptor monoclonal antibody

Two-dimensional NMR experiments [correlated spectroscopy (COSY) and two-dimensional transferred nuclear Overhauser enhancement spectroscopy (TR-NOESY)] have been applied to study the interactions of a monoclonal antibody (mAb) directed to the main immunogenic region (MIR) of the acetylcholine receptor (AChR), and four synthetic decapeptides from the MIR. The decapeptides were the Torpedo AChR alpha 67-76 fragment (W67-N68-P69-A70-D71-Y72-G73-+ ++G74-I75-K76) and its three [A69], [A73], and [A76] analogues. The results led to the following conclusions: (1) the magnitude of the TR-NOE cross peaks does not depend only on the structuration of the peptide in the bound state, but also on restrictions of the mobility, i.e., on the correlation time tau c, which can be different for every residue; (2) the binding capacity of the synthetic peptides to mAbs measured by radioimmunoassay is directly correlated to the NOE magnitude; and (3) the combined interpretation of the COSY and TR-NOESY experiments gives a qualitative information about the nature and the overall conformation of the sequence which is in contact with the mAb binding site.     

Nine Residues Influence the Binding of α-Bungarotoxin in α-Subunit Region 185–200 of Human Muscle Acetylcholine Receptor

Abstract: Identification of residues in the skeletal muscle nicotinic acetylcholine receptor (AChR) that bind snake venom a-neurotoxin antagonists of acetylcholine [e.g., α-bungarotoxin (α-BTx)] provides structural information about the neurotransmitter binding region of the receptor. Using synthetic peptides of the human AChR α-subunit region 177–208, we previously localized a pharmacologically specific binding site for α-BTx in segment 185–199. To define in more detail the residues that influence the binding of α-BTx to this region, we prepared 16 peptide analogues of the α-subunit segment 185–200, with the amino acid Lalanine sequentially replacing each native amino acid. Circular dichroism spectroscopy did not reveal changes in the secondary structure of the peptides except for the analogue in which Pro¹⁹⁴ was substituted with alanine. This implies that any change in α-BTx binding could be attributed to replacement of the native residue's side chain by alanine's methyl group, rather than to a change in the structure of the peptide. The influence of each substitution with alanine was determined by comparing the analogue to the parental sequence α 185–200 in solution-phase competition with native human AChR for binding of ¹²⁵I-labeled α-BTx. The binding of α-BTx by analogue peptides with alanine substituted for Tyr¹⁹⁰, Cys¹⁹², or Cys¹⁹³ was greatly diminished. Binding of α-BTx to peptides containing alanine replacements at Val¹⁸⁸, Thr¹⁸⁹, Pro¹⁹⁴, Asp¹⁹⁵, or Tyr¹⁹⁸ was also reduced significantly (p < 0.003). An unanticipated finding was that substitution of alanine for Ser¹⁹¹ significantly increased α-BTx binding (p < 0.003). The data imply that these nine amino acids influence the binding of the antagonist, α-BTx, to the nicotinic acetylcholine receptor of human skeletal muscle, and confirm previous reports for certain contact residues for α-BTX that were found in region α181-200 of the Torpedo AChR.     

Amino acid substitutions outside a preselected antigenic region in hemoglobin affect the binding to monoclonal antibodies obtained by immunization with the synthetic region

It is often assumed that amino acid substitutions outside a protein antigenic site have no effect on the reactivity of a protein variant with antibodies, especially monoclonal antibodies (mAbs). Substitutions that exert an effect on the reactivity of a protein variant with mAbs are frequently considered to be within the antigenic site of the mAb. To test this assumption, two mAbs [IgG_l(k) and IgG_2a (k)] were prepared by immunization with a synthetic peptide corresponding to region 63–78 of the chain of human hemoglobin (Hb). The peptide was used as an immunogen in its free form (i.e., without conjugation to a carrier), so that the results will not be made ambiguous by peptide modification nor by an immune response to sites spanning peptide and protein carrier. In addition to their reaction with human Hb, the mAbs were also studied with four primate Hbs which had no substitutions within region 63–78 and only a limited number of substitutions which occurred outside of, and at considerable distances in three-dimensional (3D) structure from, this region. Inhibition studies revealed substantial differences in the binding affinities of some of the primate Hbs, relative to human Hb. Some of the substitutions caused major decreases in binding, although they were at considerable distances in the 3D structure from the indicated site residues. It is concluded that substitutions in a protein, even when distant from an antigenic site, can exert major influences on the protein's reactivity with anti-site mAbs.     

The main immunogenic region (MIR) of the nicotinic acetylcholine receptor and the anti-MIR antibodies

Myasthenia gravis (MG) is caused by autoantibodies against the nicotinic acetylcholine receptor (AChR) of the neuromuscular junction. The anti-AChR antibodies are heterogeneous. However, a small region on the extracellular part of the AChR alpha subunit, called the main immunogenic region (MIR), seems to be the major target of the anti-AChR antibodies, but not of the specific T-cells, in experimental animals and possibly in MG patients. The major loop of the overlapping epitopes for all testable anti-MIR monoclonal antibodies (MAbs) was localized within residues 67-76 (WNPADYGGIK for Torpedo and WNPDDYGGVK for human AChR) of the alpha subunit. The N-terminal half of alpha 67-76 is the most critical, Asn68 and Asp71 being indispensable for binding. Yet anti-MIR antibodies are functionally and structurally quite heterogeneous. Anti-MIR MAbs do not affect channel gating, but they are very potent in mediating acceleration of AChR degradation (antigenic modulation) in cell cultures and in transferring experimental MG in animals. Fab fragments of anti-MIR MAbs bound to the AChR prevent the majority of the MG patients' antibodies from binding to and causing loss of the AChR. Whether this inhibition means that most MG antibodies bind on the same small region or is a result of broad steric/allosteric effects is under current investigation.     

Monoclonal antibodies to the main immunogenic region of the nicotinic acetylcholine receptor bind to residues 61-76 of the alpha subunit

Monoclonal antibodies (mAbs) to the main immunogenic region (MIR) bind to fusion proteins containing region 37-200 of the alpha chain of Torpedo, mouse, and chicken nicotinic acetylcholine receptor. In the case of the mouse alpha chain, these mAbs react with sequence 61-216 but not with 74-216. A synthetic peptide M1, containing residues 61-76 of the mouse alpha chain, also binds these anti-MIR mAbs, showing that all or part of their binding site is included in this region. The conformational dependence and epitope specificity of the mAbs are discussed.     

The tyrosine phosphorylation site of the acetylcholine receptor beta subunit is located in a highly immunogenic epitope implicated in channel function: antibody probes for beta subunit phosphorylation and function.

Tyrosine phosphorylation of the nicotinic acetylcholine receptor (AChR) seems to be involved in AChR desensitization and localization on the postsynaptic membrane. This study reveals a probable function of the single known beta subunit phosphorylation site (beta Tyr355) and provides suitable tools for its study. The epitopes for 15 monoclonal antibodies (mAbs) against the cytoplasmic side of the AChR beta subunit were precisely mapped using > 100 synthetic peptides attached on polyethylene rods. Eleven mAbs bound to a very immunogenic cytoplasmic epitope (VICE-beta) on Torpedo beta 352-359, which contains the beta Tyr355, and to the corresponding sequence of human AChR. The contribution of each VICE-beta residue to mAb binding was then studied by peptide analogues having single residue substitutions. Overall, each of the residues beta 354-359, including beta Tyr355, proved critical for mAb binding. Two of our four mAbs known to block the ion channel were found to bind at (mAb148) or close (mAb10) to VICE-beta. Tyrosine phosphorylation of Torpedo AChR by endogenous kinase(s) selectively reduced binding of some VICE-beta mAbs, including the channel blocking mAb148. We conclude that VICE-beta probably plays a key role in AChR function. Elucidation of this role should be facilitated by the identified mAb tools.     

Conformation of cytoplasmic segments of acetylcholine receptor alpha- and beta-subunits probed by monoclonal antibodies: sensitivity of the antibody competition approach.

The conformation of the cytoplasmic side of Torpedo marmorata acetylcholine receptor (AChR) was investigated by 22 monoclonal antibodies (mAbs) binding to known sites on the amino acid sequences 339-378 and 336-469 of the AChR alpha- and beta-subunits respectively. Competitions among these mAbs for binding on the intact AChR were compared with their competition for binding on the SDS-denatured subunits and with their corresponding epitopes previously determined on the primary structure of the subunits. We found the following: The three approaches correlated very well suggesting that these mAbs bind on the intact AChR at the same sequences determined by synthetic peptides and not on irrelevant discontinuous epitopes; this finding supports conclusions of Ratnam et al. (1986a) that the amphipathic helix M5 is exposed on the cytoplasmic side of the AChR. The subunit segments alpha 339-378 and beta 336-469 seem to be extended over large distances on the cytoplasmic surface of the AChR. The cytoplasmic surface of beta-subunit has a very immunogenic region. The mAb-competition technique is very sensitive since mAbs to epitopes separated by only about seven residues did not exclude each other, and mAbs to overlapping epitopes exhibited differential competitions with other mAbs.     

Affinities of phosphorylated substrates for the E. coli tryptophan synthase α-subunit: Roles of Ser-235 and helix-8′ dipole

The roles of Ser-235 and helix-8′ (residues 235–242) in the functional binding and turnover of phosphorylated substrates by the α-subunit of the E. coli tryptophan synthase (TSase) α₂β₂-holoenzyme complex are examined. Previous crystallographic analyses indicated that this region was one of several near the phosphate moiety of the physiological substrate, indole-3-glycerol phosphate (IGP). The peptidyl amido group of Ser-235 was suggested to H-bond to the phosphate group; a helix macrodipole binding role was suggested for helix-8′. The activities and substrate K_ms of mutant α-subunits altered in this region by site-specific mutagenesis are reported here. Substitutions at Ser-235 by an acidic (glutamic acid, mutant SE235), basic (lysine, mutant SK235), or a nonpeptidyl amido-containing residue (proline, mutant SP235) exhibit 40- to 180-fold K_m increases for IGP and D -glyceraldehyde-3-phosphate; no K_m defects for indole were observed. k_cat values for SP235, SE235, and SK235 are 100, 70, and 40%, respectively, of the wild-type value. Steric considerations may explain the results with the SE235 and SK235 mutant α-subunits; however, the SP235 results are consistent with the suggested phosphate binding role for the Ser-235 peptidyl amide group during catalysis. A helix-8′ dipole role was explored following proline substitutions separately at the first six (of eight) residues. Proline substitutions at positions-1 through -4 in helix-8′ have normal indole K_ms and catalytic activities in all four TSase reactions, suggesting no major global structural changes in these proteins. By these criteria, substitutions at positions-5 and -6 lead to significant structural alterations. K_m increases for phosphorylated substrates are substantial (up to 40-fold) and are dependent upon the presence of L -serine at the β-subunit active site. In the absence of L -serine, substitution only at the first position results in binding defects; in the presence of L -serine, substitutions at the first, second and third positions show binding defects of decreasing magnitude, sequentially. Substitutions at the fourth and fifth position have no effect on substrate binding. It is suggested that during catalysis a helix dipole effect on binding may be exerted but only via inter-subunit-induced conformational changes due to ligand (L -serine) binding to the β-subunit. © 1995 Wiley-Liss, Inc.     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: Demonstration with region 113–120 (antigenic site 4) of myoglobin

Immunochemical cross-reactivity of protein variants has been very frequently used to map protein antigenic sites. The approach is based on the assumption that amino acid substitutions affecting the binding of a protein to its antibody, particularly when monoclonal antibodies (mAbs) are used, must be part of the antigenic site and not far from it. The assumption was investigated in this study by determining the effects of amino acid substitutions outside the antigenic site on the reactivity of six myglobin (Mb) variants with three mAbs of predetermined specificity prepared by immunization with a free synthetic peptide representing region 113–120 (antigenic site 4) of Mb. Two of the Mb variants used had no substitutions within residues 113–120 (the region to which the specificity of the mAbs is directed) and yet exhibited markedly decreased cross-reactions and binding affinities, relative to the reference antigen, sperm-whale Mb. The other three Mb variants possessed substitutions within, as well as outside, region 113–120 and showed very little cross-reactivities. The results of this study, particularly with the Mbs that have no substitutions within the indicated antigenic site, clearly show that substitutions outside the site, and which by design are not part of the site, can influence very markedly the reactivity of the protein variant with the anti-site mAbs. The approach can, therefore, lead to serious errors if used to identify residues of protein antigenic sites.     

Antigen-antibody interaction. Synthetic peptides define linear antigenic determinants recognized by monoclonal antibodies directed to the cytoplasmic carboxyl terminus of rhodopsin

The specificities of four monoclonal antibodies rho 1D4, 1C5, 3A6, and 3D6 prepared by immunization of rod outer segments containing rhodopsin have been defined using synthetic peptides. All of these antibodies interact within the 18 residues at the COOH terminus of rhodopsin and recognize linear antigenic determinants of 4-11 residues. Twenty-seven synthetic peptide analogs of varying lengths of native sequence or containing single amino acid substitutions at each position of the COOH-terminal 18 residues have provided some insight into the mechanism of antigen-antibody binding. Our results clearly demonstrate that antibodies can be highly specific at key positions as shown by the loss of binding on single amino acid substitutions in the binding site. In contrast single amino acid substitutions at other positions in the binding site only affect affinity for some antibodies. Ionic interactions can dominate immunogenic determinants. Immunogenic determinants are not restricted to highly charged hydrophilic regions on the surface of a protein and may be dominated by hydrophobic interactions. Although certain side chains can dominate the interaction of the antigen with antibody, our results are in agreement with the interpretation that the free energies of all the contact points are additive and a certain free energy must be present to achieve binding. Antibodies with different specificities directed to the same region of the protein antigen can be produced in an immune response. Peptide antigens representing regions of a protein antigen bind best to the anti-protein antibody when the sequence is shortened to contain only those residues binding to the specificity site in the antibody. Cross-reactivity between protein antigens can be explained by conservation of the critical residues in the combining site.     

Sequence determinants regulating fibrillation of human alpha-synuclein

α-Synuclein is a neural protein that comprises the fibrillar core of Lewy bodies, a histologically defining lesion of Parkinson’s disease. To investigate the role of each specific residue of the α-synuclein molecule in fibril formation, amino acid substitutions were introduced throughout the molecule. Incorporation of proline, especially in the region spanning residues 37-89, drastically retarded fibril formation. Substitutions with polar residues showed that the hydrophobicity of the central hydrophobic region is also important in fibrillation regulation. In the N-terminal repeated region, increasing the number of negative charges interfered with fibrillation. In contrast, single amino acid substitutions in the C-terminal acidic region of α-synuclein had only minimal effects on fibrillation. More than 20 different single amino acid substitutions that were sufficient to prevent fibrillation of α-synuclein were obtained, and most of them were impaired in both nucleation and fibril elongation. Identification of sequence determinants regulating fibrillation of amyloidogenic proteins may provide valuable information for designing peptide analog drugs to prevent protein amyloidosis.     

Mapping of a cholinergic binding site by means of synthetic peptides, monoclonal antibodies, and alpha-bungarotoxin

Previous studies by several laboratories have identified a narrow sequence region of the nicotinic acetylcholine receptor (AChR) alpha subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, we used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to bind alpha-bungarotoxin (alpha-BTX) and several alpha-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the alpha subunit, and bind to AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. The synthetic peptides used in this study were approximately 20 residue long, overlapped each other by 4-6 residues, and corresponded to the complete sequence of Torpedo AChR alpha subunit. Also, overlapping peptides, corresponding to the sequence segments of each Torpedo AChR subunit homologous to alpha 166-203, were synthesized. alpha-BTX bound to a peptide containing the sequence alpha 181-200 and also, albeit to a lesser extent, to a peptide containing the sequence alpha 55-74. WF6 bound to alpha 181-200 and to a lesser extent to alpha 55-74 and alpha 134-153. The two other mAbs predominantly bound to alpha 55-74, and to a lesser extent to alpha 181-200. Peptides alpha 181-200 and alpha 55-74 both inhibited binding of 125I-alpha-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunits bound alpha-BTX or WF6, or interfered with their binding. Therefore, the cholinergic binding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR alpha subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region. Such a structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody-antigen complexes [reviewed in Davies et al. (1988)].     

Design, synthesis, and conformational study of biologically active photolabeled analogues of the main immunogenic region of the acetylcholine receptor

Photoaffinity labeling is a powerful tool for the characterization of the molecular basis of ligand binding to acceptor molecules, which provides important insights for mapping the bimolecular interfaces. The autoimmune disease myasthenia gravis is caused by autoantibodies against the acetylcholine receptor (AChR). The majority of the anti-AChR antibodies bind to the "main immunogenic region" (MIR) of the AChR. To identify the contact points between the complementarity determining regions of the anti-MIR antibodies that recognize the MIR contact sites of the AChR, we present here three photoreactive dodecapeptide MIR analogues containing the photolabel p-benzoyl-L-phenylalanine (Bpa) moiety, either in position 1 or 11. The structure of the produced 12-mers was analyzed using two-dimensional (1)H-NMR spectroscopy, whereas their binding to anti-MIR monoclonal antibodies (mAbs) was determined by immunochemical assays. In all cases the modifications resulted in conservation of the beta-turn conformation of the N-terminus, which has been proved essential for antibody recognition and increased anti-MIR binding relative to the MIR decapeptide.     

Conformational requirements for molecular recognition of acetylcholine receptor main immunogenic region (MIR) analogues by monoclonal anti-MIR antibody: A two-dimensional nuclear magnetic resonance and molecular dynamics approach

The conformational properties of two [D -A⁷⁰, A⁷⁶] and [Aib⁷⁰, A⁷⁶] analogues of the α67–76 Torpedo acetylcholine receptor fragment, with low binding capacity for the anti main immunogenic region (MIR) antibodies, were studied in DMSO by two-dimensional nmr techniques and molecular dynamics simulations. The results were compared to the free and bound conformations of the [A⁷⁶] analogue, which has twice more affinity for the anti-MIR monoclonal antibody 6 (mAb6), than the natural Torpedo sequence. It appeared that a single substitution of the A⁷⁰, at a crucial position, by the D -A⁷⁰ or Aib⁷⁰, could modify completely the conformational behavior of the peptide and reduced its recognition by the anti-MIR antibody. The WNPADY rigid structure at the N-terminal part was essential for antibody recognition. The adjacent more flexible C-terminal sequence (GGIK) gives additional stability to the monoclonal antibody–peptide complex probably due to an adequate orientation of the peptide side chains in the complex, by setting them in close contact with the antibody. © 1993 John Wiley & Sons, Inc.     

Gonococcal outer-membrane protein PIB: comparative sequence analysis and localization of epitopes which are recognized by type-specific and cross-reacting monoclonal antibodies.

Comparison of the inferred amino acid sequence of outer-membrane protein PIB from gonococcal strain P9 with those from other serovars reveals that sequence variations occur in two discrete regions of the molecule centred on residues 196 (Var1) and 237 (Var2). A series of peptides spanning the amino acid sequence of the protein were synthesized on solid-phase supports and reacted with a panel of monoclonal antibodies (mAbs) which recognize either type-specific or conserved antigenic determinants on PIB. Four type-specific mAbs reacted with overlapping peptides in Var1 between residues 192-198. Analysis of the effect of amino acid substitutions revealed that the mAb specificity is generated by differences in the effect of single amino acid changes on mAb binding, so that antigenic differences between strains are revealed by different patterns of reactivity within a panel of antibodies. The variable epitopes in Var1 recognized by the type-specific mAbs lie in a hydrophilic region of the protein exposed on the gonococcal surface, and are accessible to complement-mediated bactericidal lysis. In contrast, the epitope recognized by mAb SM198 is highly conserved but is not exposed in the native protein and the antibody is non-bactericidal. However, the conserved epitope recognized by mAb SM24 is centred on residues 198-199, close to Var1 , and is exposed for bactericidal killing.