Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit.     

A type kappa Bence Jones protein containing a cysteinyl residue in the variable region.

The proteins precipitated with ammonium sulfate from the urine of a patient (Mat) with multiple myeloma were separated into three components by ion-exchange and gel chromatographies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, amino acid analyses, immunochemical tests, and measurement of circular dichroism showed that these components were a dimer with a disulfide bond, a stable monomer, and a variable fragment, respectively. All three protein components reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) in Tris-HCl buffer at pH 8.0, indicating that they contained free sulhydryl groups. Partial reduction with dithiothreitol in the absence of denaturants yielded two SH groups per molecule from both the monomer and the dimer, and one SH group per molecule from the fragment. This indicates that the monomer of Mat protein contains a cysteinyl residue in the variable region in addition to a cysteinyl residue at the COOH terminus.The reactivities of the two SH groups of the partially reduced monomer toward iodoacetamide and iodoacetic acid were studied by polyacrylamide gel electrophoresis. The two SH groups had similar reactivities with iodacetamide, but the SH group at the COOH terminus was more reactive with iodoacetic acid than that in the variable region. The extrinsic Cotton effects of an azobenzene-2-sulfenyl group introduced into the SH group in the variable region were different from those of dye attached to the COOH terminal SH group, indicating that the two SH groups had different environments. The states of the SH groups of the intact monomer are discussed on the basis of these findings.     

Three possible disulfides in the acetylcholine receptor alpha-subunit

The cysteinyl residues of the acetylcholine receptor alpha-subunit of Torpedo californica were analyzed. All seven cysteines could be accounted for. Three possible disulfide bridges and one unpaired cysteine were indicated. The disulfide linkages were as follows: Cys128 to Cys142; Cys192 to Cys193; Cys412 to Cys418 (Cys222 is unpaired). The identification of cysteinyl residues was accomplished by a modified protein blot procedure. Cysteinyl residues of intact nicotinic acetylcholine receptor were selectively biotinylated with 3-(N-maleimidopropionyl)biocytin and subsequently detected by the 125I-labeled avidin overlay of blotted Staphylococcus aureus V8 proteolyzed alpha-subunits. Two pairs of cysteines (Cys128/Cys142 and Cys412/Cys418) could be demonstrated only after Na(BH4) reduction of the acetylcholine receptor. Cysteine residues 192 and 193 are particularly sensitive to reduction; 0.1 mM dithiothreitol is sufficient.     

Stabilization of quaternary structure of water-soluble quinoprotein glucose dehydrogenase

Water-soluble quinoprotein glucose dehydrogease (PQQGDH-B) is a dimeric enzyme whose application for glucose sensing is the focus of much attention. We attempted to increase the thermal stability of PQQGDH-B by introducing a disulfide bond at the dimer interface. The Ser residue at position 415 was selected for substitution with Cys, as structural information revealed that its side chains face each other at the dimer interface of PQQGDH-B. PQQGDH-B with Ser415Cys showed 30-fold greater thermal stability at 55°C than did the wild-type enzyme without any decrease in catalytic activity. After incubation at 70°C for 10 min, Ser415Cys retained 90% of the GDH activity of the wild-type enzyme. Disulfide bond formation between the mutant subunits was confirmed by analyses with sodium dodecylsulfate-polyacrylamide gel electrophoresis in the presence and absence of reductants. Our results indicate that the introduction of one Cys residue in each monomer of PQQGDH-B resulted in formation of a disulfide bond at the dimer interface and thus achieved a large increase in the thermal stability of the enzyme.     

Agonist binding site of Torpedo electric tissue nicotinic acetylcholine receptor. A negatively charged region of the delta subunit within 0.9 nm of the alpha subunit binding site disulfide

The positively charged quaternary ammonium group of agonists of the nicotinic acetylcholine (ACh) receptor binds to a negative subsite at most about 1 nm from a readily reducible disulfide. This disulfide is formed by alpha Cys192 and Cys193 (Kao and Karlin, 1986). In order to identify Asp or Glu residues that may contribute to the negative subsite, we synthesized S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. Purified ACh receptor from Torpedo californica was mildly reduced and reacted with S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. The predominant product was a mixed disulfide between the 3H-N-glycylcysteamine moiety and alpha Cys192 or Cys193. In the extended conformation of [3H] N-glycylcysteamine, the distance from the glycyl amino group to the cysteamine thio group is 0.9 nm. Thus, the amino group of disulfide-linked [3H]N-glycylcysteamine could react with carboxyls within 0.9 nm of Cys192/Cys193. To promote amide bond formation between the tethered amino group and receptor carboxyls, we added 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide. The predominant sites of amide coupling were on the delta subunit, in CNBr fragment 4 (delta 164-257). This reaction was inhibited by ACh. Only the first 61 residues of delta CNBr 4 are predicted to be extracellular, and there are 11 Asp or Gly residues in this region. One or more of these residues is likely to contribute to the binding of ACh.     

Thioredoxin-dependent enzymatic activation of mercaptopyruvate sulfurtransferase. An intersubunit disulfide bond serves as a redox switch for activation

Rat 3-mercaptopyruvate sulfurtransferase (MST) contains three exposed cysteines as follows: a catalytic site cysteine, Cys(247), in the active site and Cys(154) and Cys(263) on the surface of MST. The corresponding cysteine to Cys(263) is conserved in mammalian MSTs, and Cys(154) is a unique cysteine. MST has monomer-dimer equilibrium with the assistance of oxidants and reductants. The monomer to dimer ratio is maintained at approximately 92:8 in 0.2 m potassium phosphate buffer containing no reductants under air-saturated conditions; the dimer might be symmetrical via an intersubunit disulfide bond between Cys(154) and Cys(154) and between Cys(263) and Cys(263), or asymmetrical via an intersubunit disulfide bond between Cys(154) and Cys(263). Escherichia coli reduced thioredoxin (Trx) cleaved the intersubunit disulfide bond to activate MST to 2.3- and 4.9-fold the levels of activation of dithiothreitol (DTT)-treated and DTT-untreated MST, respectively. Rat Trx also activated MST. On the other hand, reduced glutathione did not affect MST activity. E. coli C35S Trx, in which Cys(35) was replaced with Ser, formed some adducts with MST and activated MST after treatment with DTT. Thus, Cys(32) of E. coli Trx reacted with the redox-active cysteines, Cys(154) and Cys(263), by forming an intersubunit disulfide bond and a sulfenyl Cys(247). A consecutively formed disulfide bond between Trx and MST must be cleaved for the activation. E. coli C32S Trx, however, did not activate MST. Reduced Trx turns on a redox switch for the enzymatic activation of MST, which contributes to the maintenance of cellular redox homeostasis.     

Acetylcholine receptor binding site contains a disulfide cross-link between adjacent half-cystinyl residues

A conserved feature of all nicotinic receptors is the presence of a readily reducible disulfide bond adjacent to the acetylcholine binding site. Previously we showed that in intact receptor from Torpedo californica electric tissue reduction of this disulfide followed by affinity alkylation with 4-(N-maleimido)benzyltri[3H] methylammonium iodide specifically and uniquely labels the alpha subunit residues Cys-192 and Cys-193. To identify all of the half-cystinyl residues contributing to the binding site disulfide(s), we have now reduced receptor under mild conditions and alkylated with a mixture of 4-(N-maleimido)benzyltri[3H]methylammonium iodide and N-[1-14C]ethylmaleimide and find that Cys-192 and Cys-193 are labeled exclusively. Furthermore, from unreduced receptor we have isolated two cyanogen bromide peptides of alpha, one containing Cys-192 and Cys-193, and the other containing Cys-128 and Cys-142 (which are the other potential contributors to the binding site disulfide(s]. These isolated peptides incorporate iodo[1-14C]acetamide only following reduction by dithiothreitol. Our results demonstrate that: 1) the binding site disulfide is between Cys-192 and Cys-193; 2) Cys-128 is disulfide-cross-linked to Cys-142; and 3) under conditions that reduce Cys-192 and Cys-193 completely, Cys-128 and Cys-142 remain cross-linked. At the acetylcholine binding site, agonists induce a local conformational change that stabilizes the binding site disulfide against reduction. We suggest that a transition between two stable conformations of the vicinal disulfide, both involving a nonplanar cis peptide bond between Cys-192 and Cys-193, is associated with receptor activation by agonists.     

The CorA Mg2+ transporter is a homotetramer

CorA is a primary Mg2+ transporter for Bacteria and Archaea. The C-terminal domain of approximately 80 amino acids forms three transmembrane (TM) segments, which suggests that CorA is a homo-oligomer. A Cys residue was added to the cytoplasmic C terminus (C317) of Salmonella enterica serovar Typhimurium CorA with or without mutation of the single periplasmic Cys191 to Ser; each mutant retained function. Oxidation of the Cys191Ser Cys317 CorA gave a dimer. Oxidation of Cys317 CorA showed a dimer plus an additional band, apparently cross-linked via both Cys317 and C191. To determine oligomer order, intact cells or purified membranes were treated with formaldehyde or carbon disulfide. Higher-molecular-mass bands formed, consistent with the presence of a tetramer. Cross-linking of the Bacillus subtilis CorA expressed in Salmonella serovar Typhimurium similarly indicated a tetramer. CorA periplasmic soluble domains from both Salmonella serovar Typhimurium and the archaeon Methanococcus jannaschii were purified and shown to retain structure. Formaldehyde treatment showed formation of a tetramer. Finally, previous mutagenesis of the CorA membrane domain identified six intramembrane residues forming an apparent pore that interacts with Mg2+ during transport. Each was mutated to Cys. In mutants carrying a single intramembrane Cys residue, spontaneous disulfide bond formation that was enhanced by oxidation with Cu(II)-1,10-phenanthroline was observed between monomers, indicating that these Mg2+-interacting residues within the membrane are very close to their cognate residue on another monomer. Thus, CorA appears to be a homotetramer with a TM segment of one monomer physically close to the same TM segment of another monomer.     

Bromoacetylcholine as an affinity label of the acetylcholine receptor from Torpedo californica

Bromoacetyl[methyl-³H]choline is a highly specific label for the reduced acetylcholine binding site on the acetylcholine receptor from $\text{Torpedo californica}$. Only one of two binding sites per receptor monomer is susceptible to labeling. The labeled site is on the α chain of the receptor.     

Dimeric arrangement and structure of the membrane-bound acetylcholine receptor studied by electron microscopy.

The acetylcholine receptor protein (AChR) from the electric organ of Torpedo marmorata is studied in its membrane-bound form by electron microscopy and single-particle image averaging. About half the molecule protrudes from the membrane surface by approximately 5 nm. The low-resolution 3-D structure of this hydrated portion, including its handedness, can be deduced from averaged axial and lateral projections and from freeze-etched membrane surfaces. In native membrane fragments, a dimeric form of the AChR is observed and the relative orientation of the AChR monomers within the dimer is established. The dimers disappear upon disulfide reduction of the membrane preparations, whereas the average axial projections of the AChR monomer remain unaffected. Since the existence of disulfide bonds linking AChR monomers between their respective delta-subunits is well documented, the approximate position of the delta-subunit within the low-resolution structure of the AChR molecule can be deduced from the structure of the dimers.     

Location of disulfide bonds within the sequence of human serum cholinesterase

Human serum cholinesterase was digested with pepsin under conditions which left disulfide bonds intact. Peptides were isolated by high pressure liquid chromatography, and those containing disulfide bonds were identified by a color assay. Peptides were characterized by amino acid sequencing and composition analysis. Human serum cholinesterase contains 8 half-cystines in each subunit of 574 amino acids. Six of these form three internal disulfide bridges: between Cys65-Cys92, Cys252-Cys263, and Cys400-Cys519. A disulfide bond with Cys65 rather than Cys66 was inferred by homology with Torpedo acetylcholinesterase. Cys571 forms a disulfide bridge with Cys571 of an identical subunit. This interchain disulfide bridge is four amino acids from the carboxyl terminus. A peptide containing the interchain disulfide is readily cleaved from cholinesterase by trypsin (Lockridge, O., and La Du, B. N. (1982) J. Biol. Chem. 257, 12012-12018), suggesting that the carboxyl terminus is near the surface of the globular tetrameric protein. The disulfide bridges in human cholinesterase have exactly the same location as in Torpedo californica acetylcholinesterase. There is one potential free sulfhydryl in human cholinesterase at Cys66, but this sulfhydryl could not be alkylated. Comparison of human cholinesterase, and Torpedo and Drosophila acetylcholinesterases to the serine proteases suggests that the cholinesterases constitute a separate family of serine esterases, distinct from the trypsin family and from subtilisin.     

Topological dispositions of lysine alpha 380 and lysine gamma 486 in the acetylcholine receptor from Torpedo californica

The locations have been determined, with respect to the plasma membrane, of lysine alpha 380 and lysine gamma 486 in the alpha subunit and the gamma subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the alpha subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the gamma subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium [3H]-borohydride. Saponin was added to a portion of the vesicles prior to labeling to render them permeable to pyridoxal phosphate. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine alpha 380 and lysine gamma 486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The peptides bound and released by the immunoadsorbents were positively identified and quantified by high-pressure liquid chromatography. Modification of lysine alpha 380 in the native acetylcholine receptor in sealed vesicles increased 5-fold in the presence of saponin, while modification of lysine gamma 486 was unaffected by the presence of saponin. The conclusions that follow from these results are that lysine alpha 380 is on the inside surface of a vesicle and lysine gamma 486 is on the outside surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

A cysteine-linkable,short cleavable photoprobe with dual functionality to explore protein-protein interfaces

We developed a bifunctional photoprobe with dual functionality, that can be specifically tethered to cysteinyl residues of peptides and proteins through a short cleavable disulfide bond. Thus, an aryldiazonium moiety is positioned at approximately 8.5 A from the modified cysteinyl alpha-carbon, leading to one of the shortest cleavable linkages. In a sodium azide-containing buffer, the aryldiazonium moiety is transformed into an aryl azide. Therefore, with one bifunctional photoprobe two types of photogenerated species can be obtained: a hydrophilic and positively charged arylcation or a hydrophobic nitrene. We coupled the aryldiazonium probe, in a site-directed manner, to a nicotinic acetylcholine receptor competitive antagonist, obtained by chemical engineering of an analogue of a snake alpha-neurotoxin. In this molecule, Arg33, which is known to interact with the receptor, was replaced by a cysteine residue, where the photoprobe could be attached. Under inactinic light, this novel photosensitive snake toxin behaved as a reversible ligand on the Torpedo acetylcholine receptor. However, when irradiated at 391 nm, it generated a highly reactive arylcation which labeled mostly the receptor alpha-subunit, confirming the location of the tip of the second toxic loop near this receptor subunit. Finally, we showed that reduction of the disulfide bond, linking the ligand to the photocoupled receptor, allowed introduction of radioactivity on the labeled residue(s), opening the way to further characterization and avoiding the synthesis of a radioactive bifunctional photoprobe.     

Acetylcholine receptor dimers are stabilized by extracellular disulfide bonding

Torpedo acetylcholine receptor (AcChR) exists predominantly as dimers, formed by two monomers held together by a disulfide bridge(s). The dimers are easily cleaved to monomers by reducing agents. 2-mercaptoethanesulfonic acid is shown to be a membrane-impermeant reducing agent which cleaves receptor dimers when it is present only on the outside of intact membrane vesicles. There is no increase in the extent of cleavage when 2-mercaptoethanesulfonic acid is also loaded inside the vesicles. Therefore the disulfide bond(s) involved in the dimerization of the Torpedo acetylcholine receptor is (are) formed by cysteine residues which are exposed on the extracellular side of the membrane.     

Crystal structure of a dimeric oxidized form of human peroxiredoxin 5

Peroxiredoxin 5 is the last discovered mammalian member of an ubiquitous family of peroxidases widely distributed among prokaryotes and eukaryotes. Mammalian peroxiredoxin 5 has been recently classified as an atypical 2-Cys peroxiredoxin due to the presence of a conserved peroxidatic N-terminal cysteine (Cys47) and an unconserved resolving C-terminal cysteine residue (Cys151) forming an intramolecular disulfide intermediate in the oxidized enzyme. We have recently reported the crystal structure of human peroxiredoxin 5 in its reduced form. Here, a new crystal form of human peroxiredoxin 5 is described at 2.0 A resolution. The asymmetric unit contains three polypeptide chains. Surprisingly, beside two reduced chains, the third one is oxidized although the enzyme was crystallized under initial reducing conditions in the presence of 1 mM 1,4-dithio-dl-threitol. The oxidized polypeptide chain forms an homodimer with a symmetry-related one through intermolecular disulfide bonds between Cys47 and Cys151. The formation of these disulfide bonds is accompanied by the partial unwinding of the N-terminal parts of the alpha2 helix, which, in the reduced form, contains the peroxidatic Cys47 and the alpha6 helix, which is sequentially close to the resolving residue Cys151. In each monomer of the oxidized chain, the C-terminal part including the alpha6 helix is completely reorganized and is isolated from the rest of the protein on an extended arm. In the oxidized dimer, the arm belonging to the first monomer now appears at the surface of the second subunit and vice versa.     

The isocitrate dehydrogenase kinase/phosphatase from Escherichia coli is highly sensitive to in-vitro oxidative conditions role of cysteine67 and cysteine108 in the formation of a disulfide-bonded homodimer.

Isocitrate dehydrogenase kinase/phosphatase (IDHK/P) is a homodimeric enzyme which controls the oxidative metabolism of Escherichia coli, and exibits a high intrinsic ATPase activity. When subjected to electrophoresis under nonreducing conditions, the purified enzyme migrates partially as a dimer. The proportion of the dimer over the monomer is greatly increased by treatment with cupric 1,10 phenanthrolinate or 5,5'-dithio-bis(2-nitrobenzoic acid), and fully reversed by dithiothreitol, indicating that covalent dimerization is produced by a disulfide bond. To identify the residue(s) involved in this intermolecular disulfide-bond, each of the eight cysteines of the enzyme was individually mutated into a serine. It was found that, under nonreducing conditions, the electrophoretic patterns of all corresponding mutants are identical to that of the wild-type, except for the Cys67-->Ser which migrates exclusively as a monomer and for the Cys108-->Ser which migrates preferentially as a dimer. Furthermore, in contrast to the wild-type enzyme and all the other mutants, the Cys67-->Ser mutant still migrates as a monomer after treatment with cupric 1,10 phenanthrolinate. This result indicates that the intermolecular disulfide bond involves only Cys67 in each IDHK/P wild-type monomer. This was further supported by mass spectrum analysis of the tryptic peptides derived from either the cupric 1,10 phenanthrolinate-treated wild-type enzyme or the native Cys108-->Ser mutant, which show that they both contain a Cys67-Cys67 disulfide bond. Moreover, both the cupric 1,10 phenanthrolinate-treated wild-type enzyme and the native Cys108-->Ser mutant contain another disulfide bond between Cys356 and Cys480. Previous results have shown that this additional Cys356-Cys480 disulfide bond is intramolecular [Oudot, C., Jault, J.-M., Jaquinod, M., Negre, D., Prost, J.-F., Cozzone, A.J. & Cortay, J.-C. (1998) Eur. J. Biochem. 258, 579-585].     

Conversion of acetylcholine receptor dimers to monomers upon depletion of non-receptor peripheral proteins

The influence of treatments for extracting non-receptor peripheral proteins on the oligomeric states of the acetylcholine receptor has been studied in receptor-rich membranes from Torpedo marmorata. Conventional alkaline treatment of non-alkylated membranes resulted in the extraction of peripheral proteins (30% of total membrane proteins). Concomitantly, partial conversion of the dimer into the monomer was observed in the absence of exogenous reduction. Alkaline extraction at high ionic strength resulted in a marked decrease in protein solubilization, and no conversion of the dimer to the monomer occurred. Alkaline treatment extracted only one half of the peripheral proteins (15% of total protein) from membranes previously alkylated with N-ethylmaleimide or iodoacetamide, or oxidized by sodium periodate. Conversion of dimer to monomer was totally prevented by these treatments. Similar results were obtained by treatment of the membranes with lithium 3,5-diiodosalicylate. The above effects of alkaline extraction on the acetylcholine receptor can be interpreted in the context of two mutually non-exclusive mechanisms: (a) some of the peripheral proteins may directly participate in the thiol-dependent receptor aggregational states. Their extraction destroys this dynamic control. (b) Extraction of peripheral proteins destabilizes the receptor and makes it more susceptible to inter or intramolecular sulfhydryl-disulfide exchange, leading to the endogenous reduction of a proportion of the dimers.     

Protease effects on the structure of acetylcholine receptor membranes from Torpedo californica

Protease digestion of acetylcholine receptor-rich membranes derived from Torpedo californica electroplaques by homogenization and isopycnic centrifugation results in degradation of all receptor subunits without any significant effect on the appearance in electron micrographs, the toxin binding ability, or the sedimentation value of the receptor molecule. Such treatment does produce dramatic changes in the morphology of the normally 0.5- to 2-microns-diameter spherical vesicles when observed by either negative-stain or freeze-fracture electron microscopy. Removal of peripheral, apparently nonreceptor polypeptides by alkali stripping (Neubig et al. 1979, Proc. Natl. Acad. Sci. U. S. A. 76:690-694) results in increased sensitivity of the acetylcholine receptor membranes to the protease trypsin as indicated by SDS gel electrophoretic patterns and by the extent of morphologic change observed in vesicle structure. Trypsin digestion of alkali- stripped receptor membranes results in a limit degradation pattern of all four receptor subunits, whereupon all the vesicles undergo the morphological transformation to minivesicles. The protein-induced morphological transformation and the limit digestion pattern of receptor membranes are unaffected by whether the membranes are prepared so as to preserve the receptor as a disulfide bridged dimer, or prepared so as to generate monomeric receptor.     

Assembly of mutant subunits of the nicotinic acetylcholine receptor lacking the conserved disulfide loop structure.

Each subunit of the nicotinic acetylcholine receptor (AChR) contains two conserved cysteine residues, which are known to form a disulfide bond, in the N-terminal extracellular domain. The role of this retained structural feature in the biogenesis of the AChR was studied by expressing site-directed mutant alpha and beta subunits together with other normal subunits from Torpedo californica AChR in Xenopus oocytes. Mutation of the cysteines at position 128 or 142 in the alpha subunit, or in the beta subunit, did not prevent subunit assembly. All Cys128 and Cys142 mutants of the alpha and beta subunits were able to associate with coexpressed other normal subunits, although associational efficiency of the mutant alpha subunits with the delta subunit was reduced. Functional studies of the mutant AChR complexes showed that the mutations in the alpha subunit abolished detectable 125I-alpha-bungarotoxin (alpha-BuTX) binding in whole oocytes, whereas the mutations in the beta subunit resulted in decreased total binding of 125I-alpha-BuTX and no detectable surface 125I-alpha-BuTX binding. Additionally, all mutant subunits, when co-expressed with the other normal subunits in oocytes, produced small acetylcholine-activated membrane currents, suggesting incorporation of only small numbers of functional mutant AChRs into the plasma membrane. The functional acetylcholine-gated ion channel formed with mutant alpha subunits, but not mutant beta subunits, could not be blocked by alpha-BuTX. Thus, a disulfide bond between Cys128 and Cys142 of the AChR alpha or beta subunits is not needed for acetylcholine-binding. However, this disulfide bond on the alpha subunit is necessary for formation of the alpha-BuTX-binding site. These results also suggest that the most significant effect caused by disrupting the conserved disulfide loop structure is intracellular retention of most of the assembled AChR complexes.     

Evidence for a domain-swapped CD4 dimer as the coreceptor for binding to class II MHC

CD4 is a coreceptor for binding of T cells to APC and the primary receptor for HIV. The disulfide bond in the second extracellular domain (D2) of CD4 is reduced on the cell surface, which leads to formation of disulfide-linked homodimers. A large conformational change must take place in D2 to allow for formation of the disulfide-linked dimer. Domain swapping of D2 is the most likely candidate for the conformational change leading to formation of two disulfide-bonds between Cys130 in one monomer and Cys159 in the other one. Mild reduction of the extracellular part of CD4 resulted in formation of disulfide-linked dimers, which supports the domain-swapped model. The functional significance of dimer formation for coreceptor function was tested using cells expressing wild-type or disulfide-bond mutant CD4. Eliminating the D2 disulfide bond markedly impaired CD4's coreceptor function. Modeling of the complex of the TCR and domain-swapped CD4 dimer bound to class II MHC and Ag supports the domain-swapped dimer as the immune coreceptor. The known involvement of D4 residues Lys318 and Gln344 in dimer formation is also accommodated by this model. These findings imply that disulfide-linked dimeric CD4 is the preferred coreceptor for binding to APC.