Identification of a cytoplasmic region of the Torpedo nicotinic acetylcholine receptor alpha-subunit by epitope mapping.

Analysis of the binding of monoclonal antibodies (mAbs) by Torpedo nicotinic acetylcholine receptor (AChR) has demonstrated that a region of the alpha-subunit between alpha-156 and alpha-179 is exposed on the cytoplasmic surface of the nicotinic post-synaptic membrane. A panel of mAbs was produced that recognized sodium dodecyl sulfate-denatured subunits of the Torpedo AChR. Antibodies recognizing alpha-subunit were distinguished in terms of their ability to bind alpha-subunit fragments generated by Staphylococcus aureus V8 protease: an 18-kDa fragment beginning at Val-46, a 20-kDa fragment beginning at Ser-173/Ser-162, and a 10 kDa fragment beginning at Asn-339. Three mAbs, selected for binding to each of the V8-protease alpha-subunit fragments, respectively, were characterized in detail. The location of epitopes recognized by both anti-V8-18 and anti-V8-20 mAbs was determined to be within alpha-156 to alpha-179 by isolation of small immunoreactive peptides from proteolytic digests of the alpha-subunit, while the mAb reactive to V8-10 was bound to an epitope within alpha-339 to alpha-386. Quantitative evaluation of binding of the anti-V8-18 and anti-V8-20 mAbs to overlapping synthetic peptides corresponding to alpha-147 to alpha-179 localized the epitopes to distinct portions of this region. Further screening of the panel of mAbs using these synthetic peptides revealed three additional mAbs that bind in this region. The mAbs that bound the three distinct V8-protease alpha-subunit fragments were shown to bind to native AChR by indirect immunofluorescence on frozen sections of Torpedo electric organ. Binding to the native AChR was to the cytoplasmic surface of the AChR since the mAbs could bind to AChR in native vesicles, in which the AChR is oriented right-side-out, only after permeabilization of the vesicles by alkaline treatment or after scrambling of the orientation of the AChR by solubilization and reconstitution into liposomes. The location of the mAb-binding sites at the cytoplasmic surface of the AChR was visualized directly by freeze-etch immunoelectron microscopy. The identification of alpha-156 and alpha-179 as containing a cytoplasmic exposed sequence implies the existence of two non-hydrophobic transmembrane sequences between the site of N-glycosylation (Asn-141) and Cys-192, a site alkylated by the cholinergic affinity labels.     

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.     

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.     

Structure and transmembrane nature of the acetylcholine receptor in amphibian skeletal muscle as revealed by cross-reacting monoclonal antibodies

A collection of 126 monoclonal antibodies (mAbs) made against acetylcholine receptors (AChRs) from the electric organs of Torpedo californica or Electrophorus electricus was tested for cross-reactivity with AChRs in cryostat sections of skeletal muscle from Rana pipiens and Xenopus laevis by indirect immunofluorescence. 49 mAbs (39%) cross-reacted with AChRs from Rana, and 25 mAbs (20%) cross-reacted with AChRs from Xenopus. mAbs specific for each of the four subunits of electric organ AChR (alpha, beta, gamma, delta) cross-reacted with AChRs from each amphibian species. mAbs cross-reacting with Xenopus AChRs were, with one exception, a subset of the mAbs cross-reacting with Rana AChRs. The major difference detected between the two species was in binding by mAbs specific for the main immunogenic region (MIR) of the alpha-subunit. Whereas 22 of 33 anti-MIR mAbs tested cross-reacted with Rana AChRs, only one of these mAbs cross-reacted with Xenopus AChRs. Some (32) of the cross-reacting mAbs were tested for binding to AChRs in intact muscle. 21 of these mAbs bound to AChRs only when membranes were made permeable with saponin. Electron microscopy using immunoperoxidase or colloidal gold techniques revealed that these mAbs recognize cytoplasmic determinants and that mAbs that do not require saponin in order to bind AChRs in intact muscle recognize extracellular determinants. These results suggest that AChRs in skeletal muscle of Rana and Xenopus are composed of subunits corresponding to the alpha-, beta-, gamma-, and delta-subunits of AChRs from fish electric organs. The subunit specificity of mAbs whose binding was examined by electron microscopy suggests that parts of each subunit (alpha, beta, gamma, delta) are exposed on the cytoplasmic surface and that, as in AChRs from fish electric organs and mammalian muscle, the MIR on alpha-subunits of Rana AChRs is exposed on the extracellular surface.     

Monoclonal antibodies to cytoplasmic domains of the acetylcholine receptor

Fourteen clonal hybridoma lines that secrete monoclonal antibodies (mabs) to the Torpedo acetylcholine receptor (AChR) have been isolated. When analyzed by an immunoreplica technique, two mabs recognized the alpha subunit, three reacted with the beta subunit, one reacted with the gamma chain, and five recognized the delta subunit. One mab failed to react with any of the subunits using this assay and two mabs recognized determinants found on both the gamma and the delta subunits. These were classified according to their reactivities with the membrane-bound Torpedo AChR. One category is comprised of mabs (including both anti-alpha mabs) that recognize extracellular epitopes. A second classification included mabs that are unable to bind the membrane-associated AChR. The third category is comprised of mabs directed against cytoplasmic epitopes of the AChR. The latter mabs, all of which recognize the gamma or delta subunits or both, bind only slightly to sealed, outside-out Torpedo vesicles. The binding is increased 10-20-fold by either alkaline extraction or treatment of the vesicles with 10 mM lithium diiodosalicylate but not by permeabilization of the vesicles with saponin. Three of the six mabs in this category react with frog muscle endplates but only if the cytoplasmic surface of the membrane is accessible.     

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.     

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)].     

Precise epitope mapping of monoclonal antibodies to the cytoplasmic side of the acetylcholine receptor alpha subunit. Dissecting a potentially myasthenogenic epitope.

The epitopes for twelve monoclonal antibodies against the cytoplasmic side of the acetylcholine receptor (AChR) alpha subunit were precisely mapped using over 300 continuously overlapping synthetic peptides attached on poly(ethylene) rods. mAb cross-reactive between Torpedo and human AChR generally bound to the homologous peptides from both species. Epitopes 4-10-residues long were identified. One mAb could bind to either arm on both sides of a beta-turn structure. Five mAb bound to a very-immunogenic cytoplasmic epitope on alpha 373-380 (VICE-alpha). Three of the mAb against VICE-alpha were earlier found to cross-react with non-AChR protein(s), present in thymomas from myasthenia gravis patients but absent in thymomas from non-myasthenics. Since VICE-alpha has a potentially crucial pathogenic role, the antigenic role of each residue within it was subsequently studied by 55 analogues, most having single amino acid substitutions. All the mAb against VICE-alpha bound similarly but not identically to the analogues, thus explaining their known binding heterogeneity. Lys373 proved indispensable for mAb binding. Ile376, Glu377, Gly378 and Lys380 were quite critical, while Ser374, Ala375 and Val379 seemed rather inactive. These data should prove instructive in searches for VICE-alpha-like epitopes carrying autoantigens with potential involvement in myasthenia gravis and should further expand the applications of the anti-(AChR) mAb in AChR studies.     

Monoclonal antibodies specific for each of the two toxin-binding sites of Torpedo acetylcholine receptor

We have isolated and characterized 12 monoclonal antibodies (mAbs) that block the binding of alpha-bungarotoxin (alpha-BuTx) to the acetylcholine receptor (AChR) of Torpedo californica. Two of the mAbs block alpha-BuTx binding completely; the other 10 inhibit only about 50% of the binding. The mAbs that partially inhibit alpha-BuTx binding can be divided into two groups by examination of the additive effect of pairs of mAbs on toxin binding, and by analysis of competition between mAbs for binding to the AChR. These two groups of mAbs, which we have termed A and B, appear to recognize different toxin-binding sites on the same receptor. A and B mAbs were used to determine the kinetic and pharmacological properties of the two sites. The site recognized by A mAbs binds alpha-BuTx with a forward rate constant of 0.98 X 10(5) M-1 s-1, d-tubocurarine (dTC) with a KD of (6.8 +/- 0.3) X 10(-8) M, and pancuronium with a KD of (1.9 +/- 1.0) X 10(-9) M. The site recognized by B mAbs binds alpha-BuTx with a forward rate constant of 9.3 X 10(5) M-1 s-1, dTC with a KD of (4.6 +/- 0.3) X 10(-6) M, and pancuronium with a KD of (9.3 +/- 0.8) X 10(-6) M. Binding of A and B mAbs to the AChR was variably inhibited by nicotinic cholinergic agonists and antagonists, and by alpha-conotoxin. The observed pattern of inhibition is consistent with the relative affinity of the two sites for antagonists as given above but also indicates that the mAbs recognize a diversity of epitopes within each site.     

Location of antigenic determinants on primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping

The binding domains of 28 monoclonal antibodies (mAbs) against the alpha, beta, and delta subunits of the Torpedo acetylcholine receptor were mapped on the primary sequences of these subunits. Small peptide fragments (2000-20,000 daltons) of the purified subunits were obtained by digestion with staphylococcal V8 protease and papain, separated on a discontinuous polyacrylamide gel electrophoretic system, and electroblotted onto diaminophenyl thioether paper. The blots were probed with the various monoclonal antibodies and also with antibodies against carboxy-terminal decapeptides of the alpha, beta, and delta subunits to identify the carboxy-terminal fragments. From inspection of the binding patterns of the various antibodies to the subunits fragments and the molecular weights of these fragments, and by using the carboxy termini of the subunits as reference points, it was possible to deduce the regions on the primary sequence of each subunit in which the antibodies bound and in some cases to order the binding sites within these sequences. mAb 148, which inhibits receptor function by cross-linking receptor molecules on the cytoplasmic side, was mapped to the sequence beta 368-406. The main immunogenic region of the native receptor, which is of pathological importance in the autoimmune disease myasthenia gravis, was mapped by using mAb 210 to within 80 amino acid residues (alpha 46-127). The overall antigenic structure of alpha subunits was examined. Synthetic peptides have been used to locate determinants responsible for 83% of the antibodies in antisera to denatured alpha subunits and 46% of the antibodies to denatured alpha subunits in antisera to intact receptor. Theoretical models of the transmembrane orientation of the subunit polypeptide chains were tested by determining whether mapped monoclonal antibodies bound to the extracellular or intracellular surface of receptor-rich membranes. Our results confirm previous reports that the carboxy termini of the subunits are exposed on the intracellular surface, as is part of the region between a putative channel-forming domain (M5) and a putative membrane-spanning region (M3). However, contrary to current theoretical models, the region between M5 and the putative membrane-spanning sequence M4 also appears to be on the intracellular surface, implying that M4 and M5 are not membrane-spanning domains.(ABSTRACT TRUNCATED AT 400 WORDS)     

Treatments that extract the 43K protein from acetylcholine receptor clusters modify the conformation of cytoplasmic domains of all subunits of the receptor.

The nicotinic acetylcholine receptor (AChR) of Torpedo electric organ and vertebrate skeletal muscle is closely associated with a Mr 43,000 protein (43K). In this study, we have examined the effects on the AChR of treatments which remove the 43K protein. We used semiquantitative fluorescence techniques to measure the binding of antibodies to clustered AChR in cultured rat myotubes. We found that labeling by antibodies to the cytoplasmic portions of each of the four receptor polypeptides increased significantly upon extraction of the 43K protein. Labeling by an antibody to an extracellular epitope of the alpha subunits was not affected by removal of the 43K protein, suggesting that changes were restricted to the cytoplasmic domains of the AChR. Increases in labeling by antibodies were more limited following protease treatment, which removes most cytoskeletal structures but leaves the 43K protein bound to the membrane. Competition between an antibody to the beta subunit and an antibody to the gamma and delta subunits suggests that the cytoplasmic portion of the AChR still retains a degree of native structure in the absence of the 43K protein. Our results suggest that, although some of these changes may be due to simply exposing additional epitopes on the AChR, the cytoplasmic portions of all the subunits of the AChR undergo significant conformational changes upon extraction of the 43K protein.     

Molecular modeling of the complex between Torpedo acetylcholine receptor and anti-MIR Fab198

Myasthenia gravis is a neuromuscular disorder caused by an antibody-mediated autoimmune response to the muscle-type nicotinic acetylcholine receptor (AChR). The majority of monoclonal antibodies (mAbs) produced in rats immunized with intact AChR compete with each other for binding to an area of the alpha-subunit called the main immunogenic region (MIR). The availability of a complex between the AChR and Fab198 (Fab fragment of the anti-MIR mAb198) would help understand how the antigen and antibody interact and in designing improved antibody fragments that protect against the destructive activity of myasthenic antibodies. In the present study, we modeled the Torpedo AChR/Fab198 complex, based primarily on the recent 4A resolution structure of the Torpedo AChR. In order to computationally dock the two structures, we used the ZDOCK software. The total accessible surface area change of the complex compared to those of experimentally determined antigen-antibody complexes indicates an intermediate size contact surface. CDRs H3 and L3 seem to contribute most to the binding, while L2 seems to contribute least. These data suggest mutagenesis experiments aimed at validating the model and improving the binding affinity of Fab198 for the AChR.     

Localization of conserved B-cell epitopes among encapsulated and non-encapsulated Haemophilus influenzae P2 porin proteins using synthetic peptides.

The P2 outer membrane protein of Haemophilus influenzae belongs to a class of apparently ubiquitous proteins in Gram-negative bacteria that function as porins. Murine hybridomas raised to the P2 protein and synthetic peptides were used to investigate the structural and antigenic relationships among P2 proteins of encapsulated and non-encapsulated H. influenzae. Three monoclonal antibodies (mAbs), P2-17, P2-18 and P2-19, recognizing epitopes on the P2 protein, as shown by Western immunoblotting of outer membrane preparations, and purified and recombinant P2 proteins are described. The epitopes reactive with the mAbs were widely distributed among H. influenzae strains since 70-100% of strains of encapsulated and non-encapsulated isolates collected worldwide were recognized by individual mAbs. None of the mAbs reacted with H. parainfluenzae or other bacterial species. The peptide composition of P2 epitopes was determined by analysis of mAb reactivity with a series of overlapping synthetic peptides that covered the amino acid sequences of H. influenzae type b. The domains recognized by these mAbs were completely distinct. mAb P2-18, reactive with an epitope conserved among all H. influenzae P2 porin molecules which were screened, recognized a peptide corresponding to the N-terminal segment (residues 1-14). The P2-17- and P2-19-specific epitopes were located between residues 28 and 55, and 101 and 129, respectively. None of the epitopes were exposed on the cell surface since no mAbs bound to intact live bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mapping of monoclonal antibodies specific to P64k: a common antigen of several isolates of Neisseria meningitidis

P64k is a minor outer membrane protein from Neisseria meningitidis. This protein has been produced at high levels in Escherichia coli. We generated a group of monoclonal antibodies (mAbs) against recombinant P64k, which recognise four non-overlapping epitopes, as shown using competition assays with biotinylated mAbs. The P64k sequences involved in mAbs binding were mapped with synthetic overlapping peptides derived from the P64k protein, and located in the previously determined three-dimensional structure of the protein. These antibodies were also characterised by whole-cell ELISA and bactericidal tests against N. meningitidis. Only two of the recognised epitopes were exposed on the bacterial surface, and none of the mAbs showed bactericidal activity. The relationship between these results and the structural data on the epitopes bound by the mAbs is discussed.     

Epitope mapping of antibodies to acetylcholine receptor alpha subunits using peptides synthesized on polypropylene pegs.

Concurrent synthesis of overlapping octameric peptides corresponding to the sequence of the Torpedo acetylcholine receptor (AChR) alpha subunit has been carried out on polypropylene supports functionalized with primary amino groups according to a method developed by M. Geysen [(1987) J. Immunol. Methods 102, 259-274]. The peptides on the solid supports have been used in an enzyme-linked immunosorbent assay. Interactions of the synthetic peptides with antibodies are then detected without removing them from the solid support. By this procedure, epitopes of both antisera and monoclonal antibodies to the Torpedo acetylcholine receptor, its subunits, and synthetic peptide fragments have been mapped. Both rat and rabbit antisera to the alpha subunit show major epitopes spanning the residues 150-165, 338-345, and 355-366 on the Torpedo AChR alpha subunit. Epitopes of monoclonal antibodies to these major epitopes and to others have been rather precisely mapped by using this technique with peptides of varying lengths. The specificity of several of these mAbs are of interest because they have been used in mapping the transmembrane orientation of the AChR alpha-subunit polypeptide chain.     

Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display

Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat. Thus, understanding mechanisms of antibody-mediated viral inhibition and neutralization escape is critical. Here, a robust yeast display system for fine epitope mapping of viral surface hemagglutinin (HA)-specific antibodies is demonstrated. The full-length H5 subtype HA (HA0) was expressed on the yeast surface in a correctly folded conformation, determined by binding of a panel of extensively characterized neutralizing human monoclonal antibodies (mAbs). These mAbs target conformationally-dependent epitopes of influenza A HA, which are highly conserved across H5 clades and group 1 serotypes. By separately displaying HA1 and HA2 subunits on yeast, domain mapping of two anti-H5 mAbs, NR2728 and H5-2A, localized their epitopes to HA1. These anti-H5 mAb epitopes were further fine mapped by using a library of yeast-displayed HA1 mutants and selecting for loss of binding without prior knowledge of potential contact residues. By overlaying key mutant residues that impacted binding onto a crystal structure of HA, the NR2728 mAb was found to interact with a fully surface-exposed contiguous patch of residues at the receptor binding site (RBS), giving insight into the mechanism underlying its potent inhibition of virus binding. The non-neutralizing H5-2A mAb was similarly mapped to a highly conserved H5 strain-specific but poorly accessible location on a loop at the trimer HA interface. These data further augment our toolchest for studying HA antigenicity, epitope diversity and accessibility in response to natural and experimental influenza infection and vaccines.     

Structure-function of the putative I-domain within the integrin beta 2 subunit

The central region (residues 125-385) of the integrin beta(2) subunit is postulated to adopt an I-domain-like fold (the beta(2)I-domain) and to play a critical role in ligand binding and heterodimer formation. To understand structure-function relationships of this region of beta(2), a homolog-scanning mutagenesis approach, which entails substitution of nonconserved hydrophilic sequences within the beta(2)I-domain with their homologous counterparts of the beta(1)I-domain, has been deployed. This approach is based on the premise that beta(1) and beta(2) are highly homologous, yet recognize different ligands. Altogether, 16 segments were switched to cover the predicted outer surface of the beta(2)I-domain. When these mutant beta(2) subunits were transfected together with wild-type alpha(M) in human 293 cells, all 16 beta(2) mutants were expressed on the cell surface as heterodimers, suggesting that these 16 sequences within the beta(2)I-domain are not critically involved in heterodimer formation between the alpha(M) and beta(2) subunits. Using these mutant alpha(M)beta(2) receptors, we have mapped the epitopes of nine beta(2)I-domain specific mAbs, and found that they all recognized at least two noncontiguous segments within this domain. The requisite spatial proximity among these non-linear sequences to form the mAb epitopes supports a model of an I-domain-like fold for this region. In addition, none of the mutations that abolish the epitopes of the nine function-blocking mAbs, including segment Pro(192)-Glu(197), destroyed ligand binding of the alpha(M)beta(2) receptor, suggesting that these function-blocking mAbs inhibit alpha(M)beta(2) function allosterically. Given the recent reports implicating the segment equivalent to Pro(192)-Glu(197) in ligand binding by beta(3) integrins, these data suggest that ligand binding by the beta(2) integrins occurs via a different mechanism than beta(3). Finally, both the conformation of the beta(2)I-domain and C3bi binding activity of alpha(M)beta(2) were dependent on a high affinity Ca(2+) binding site (K(d) = 105 microm), which is most likely located within this region of beta(2).     

TonB induces conformational changes in surface-exposed loops of FhuA, outer membrane receptor of Escherichia coli

FhuA, outer membrane receptor of Escherichia coli, transports hydroxamate-type siderophores into the periplasm. Cytoplasmic membrane-anchored TonB transduces energy to FhuA to facilitate siderophore transport. Because the N-terminal cork domain of FhuA occludes the C-terminal beta-barrel lumen, conformational changes must occur to enable siderophore passage. To localize conformational changes at an early stage of the siderophore transport cycle, four anti-FhuA monoclonal antibodies (mAbs) were purified to homogeneity, and the epitopes that they recognize were determined by phage display. We mapped continuous and discontinuous epitopes to outer surface-exposed loops 3, 4, and 5 and to beta-barrel strand 14. To probe for conformational changes of FhuA, surface plasmon resonance measured mAb binding to FhuA in its apo- and siderophore-bound states. Changes in binding kinetics were observed for mAbs whose epitopes were mapped to outer surface-exposed loops. Further, we measured mAb binding in the absence and presence of TonB. After forming immobilized FhuA-TonB complexes, changes in kinetics of mAb binding to FhuA were even more pronounced compared with kinetics of binding in the absence of TonB. Measurement of extrinsic fluorescence of the dye MDCC conjugated to residue 336 in outer surface-exposed loop 4 revealed 33% fluorescence quenching upon ferricrocin binding and up to 56% quenching upon TonB binding. Binding of mAbs to apo- and ferricrocin-bound FhuA complemented by fluorescence spectroscopy studies showed that their cognate epitopes on loops 3, 4, and 5 undergo conformational changes upon siderophore binding. Further, our data demonstrate that TonB binding promotes conformational changes in outer surface-exposed loops of FhuA.     

Assembly of Torpedo acetylcholine receptors in Xenopus oocytes

To study pathways by which acetylcholine receptor (AChR) subunits might assemble, Torpedo alpha subunits were expressed in Xenopus oocytes alone or in combination with beta, gamma, or delta subunits. The maturation of the conformation of the main immunogenic region (MIR) on alpha subunits was measured by binding of mAbs and the maturation of the conformation of the AChR binding site on alpha subunits was measured by binding of alpha-bungarotoxin (alpha Bgt) and cholinergic ligands. The size of subunits and subunit complexes was assayed by sedimentation on sucrose gradients. It is generally accepted that native AChRs have the subunit composition alpha 2 beta gamma delta. Torpedo alpha subunits expressed alone resulted in an amorphous range of complexes with little affinity for alpha Bgt or mAbs to the MIR, rather than in a unique 5S monomeric assembly intermediate species. A previously recognized temperature-dependent failure in alpha subunit maturation may cause instability of the monomeric assembly intermediate and accumulation of aggregated denatured alpha subunits. Coexpression of alpha with beta subunits also resulted in an amorphous range of complexes. However, coexpression of alpha subunits with gamma or delta subunits resulted in the efficient formation of 6.5S alpha gamma or alpha delta complexes with high affinity for mAbs to the MIR, alpha Bgt, and small cholinergic ligands. These alpha gamma and alpha delta subunit pairs may represent normal assembly intermediates in which Torpedo alpha is stabilized and matured in conformation. Coexpression of alpha, gamma, and delta efficiently formed 8.8S complexes, whereas complexes containing alpha beta and gamma or alpha beta and delta subunits are formed less efficiently. Assembly of beta subunits with complexes containing alpha gamma and delta subunits may normally be a rate-limiting step in assembly of AChRs.