Proton NMR comparison of the Saccharomyces cerevisiae ferricytochrome c isozyme-1 monomer and covalent disulfide dimer

Proton NMR studies of Saccharomyces cerevisiae (bakers yeast) isozyme-1 monomer and dimer ferricytochrome c have been carried out. The dimer is formed via a disulfide bridge between the Cys-102 residues of monomer proteins. Nuclear Overhauser effect (NOE) experiments have led to resonance assignments for many of the heme and axial ligand (Met-80; His-18) protons in both protein forms. Resonances of the following amino acids have also been assigned in both forms: Phe-10; Pro-30; Phe-82; Trp-59; Leu-68. The proton NOE connectivity patterns of the monomer of yeast isozyme-1 ferricytochrome c are similar to those of horse, tuna, and yeast isozyme-2 ferricytochromes c, even though the observed hyperfine resonance spectra are significantly different for the various cytochromes. The pattern of dimer proton hyperfine resonances is distinct from the isozyme-1 monomer pattern, which indicates that the formation of a disulfide bridge via Cys-102 is detected at the heme site, approximately 10 A distant. It appears that a specific structural change is induced upon dimerization, which, in turn, causes specific perturbations in the vicinity of the heme. However, the general features of the NOE connectivity pattern in the dimer are the same as for the monomer indicating that dimerization does not result in drastic structural disruption. Furthermore, the 1H NMR spectrum of the dimer can be mimicked by the monomer form that results when the -SH group of Cys-102 is chemically modified with certain types of bulky, or hydrophilic reagents (i.e. 5,5'-dithiobis[2-nitrobenzoate], indicating that perturbations of the yeast isozyme-1 ferricytochrome c proton resonance spectrum observed upon dimerization are essentially due to changes in intramolecular, rather than intermolecular, interactions. These results suggest that a possible regulatory site for yeast isozyme-1 cytochrome c exists at position 102, which could conceivably have a physiological role in altering the conformation of the molecule.     

Syntaxin 1A modulates the voltage-gated L-type calcium channel (Ca(v)1.2) in a cooperative manner

Syntaxin 1A (Sx1A) modifies the activity of voltage-gated Ca2+ channels acting via the cytosolic and the two vicinal cysteines (271 and 272) at the transmembrane domain. Here we show that Sx1A modulates the Lc-type Ca2+ channel, Cav1.2, in a cooperative manner, and we explore whether channel clustering or the Sx1A homodimer is responsible for this activity. Sx1A formed homodimers but, when mutated at the two vicinal transmembrane domain cysteines, was unable to either dimerize or modify the channel activity suggesting disulfide bond formation. Moreover, applying global molecular dynamic search established a theoretical prospect of generating a disulfide bond between two Sx1A transmembrane helices. Nevertheless, Sx1A activity was not correlated with Sx1A homodimer. Application of a vicinal thiol reagent, phenylarsine oxide, abolished Sx1A action indicating the accessibility of Cys-271,272 thiols. Sx1A inhibition of channel activity was restored by phenylarsine oxide antidote, 2,3-dimercaptopropanol, consistent with thiol interaction of Sx1A. In addition, the supralinear mode of channel inhibition was correlated to the monomeric form of Sx1A and was apparent only when the three channel subunits alpha11.2/alpha2delta1/beta2a were present. This functional demonstration of cooperativity suggests that the three-subunit channel responds as a cluster, and Sx1A monomers associate with a dimer (or more) of a three-subunit Ca2+ channel. Consistent with channel cluster linked to Sx1A, a conformational change driven by membrane depolarization and Ca2+ entry would rapidly be transduced to the exocytotic machinery. As shown herein, the supralinear relationship between Sx1A and the voltage-gated Ca2+ channel within the cluster could convey the cooperativity that distinguishes the process of neurotransmitter release.     

H2O2-induced intermolecular disulfide bond formation between receptor protein-tyrosine phosphatases

Receptor protein-tyrosine phosphatase alpha (RPTPalpha) belongs to the subfamily of receptor-like protein-tyrosine phosphatases that are characterized by two catalytic domains of which only the membrane-proximal one (D1) exhibits appreciable catalytic activity. The C-terminal catalytic domain (D2) regulates RPTPalpha catalytic activity by controlling rotational coupling within RPTPalpha dimers. RPTPalpha-D2 changes conformation and thereby rotational coupling within RPTPalpha dimers in response to changes in the cellular redox state. Here we report a decrease in motility of RPTPalpha from cells treated with H2O2 on non-reducing SDS-polyacrylamide gels to a position that corresponds to RPTPalpha dimers, indicating intermolecular disulfide bond formation. Using mutants of all individual cysteines in RPTPalpha and constructs encoding the individual protein-tyrosine phosphatase domains, we located the intermolecular disulfide bond to the catalytic Cys-723 in D2. Disulfide bond formation and dimer stabilization showed similar levels of concentration and time dependence. However, treatment of lysates with dithiothreitol abolished intermolecular disulfide bonds but not stable dimer formation. Intermolecular disulfide bond formation and rotational coupling were also found using a chimera of the extracellular domain of RPTPalpha fused to the transmembrane and intracellular domain of the leukocyte common antigen-related protein (LAR). These results suggest that H2O2 treatment leads to oxidation of the catalytic Cys in D2, which then rapidly forms a disulfide bond with the D2 catalytic Cys of the dyad-related monomer, rendering an inactive RPTP dimer. Recovery from oxidative stress first leads to the reduction of the disulfide bond followed by a slower refolding of the protein to the active conformation.     

Recoverin as a redox-sensitive protein

Recoverin is a member of the neuronal calcium sensor (NCS) family of EF-hand calcium binding proteins. In a visual cycle of photoreceptor cells, recoverin regulates activity of rhodopsin kinase in a Ca2+-dependent manner. Like all members of the NSC family, recoverin contains a conserved cysteine (Cys38) in nonfunctional EF-hand 1. This residue was shown to be critical for activation of target proteins in some members of the NCS family but not for interaction of recoverin with rhodopsin kinase. Spectrophotometric titration of Ca2+-loaded recoverin gave 7.6 for the pKa value of Cys38 thiol, suggesting partial deprotonation of the thiol in vivo conditions. An ability of recoverin to form a disulfide dimer and thiol-oxidized monomer under mild oxidizing conditions was found using SDS-PAGE in reducing and nonreducing conditions and Ellman's test. Both processes are reversible and modulated by Ca2+. Although formation of the disulfide dimer takes place only for Ca2+-loaded recoverin, accumulation of the oxidized monomer proceeds more effectively for apo-recoverin. The Ca2+ modulated susceptibility of the recoverin thiol to reversible oxidation may be of potential importance for functioning of recoverin in photoreceptor cells.     

Roles of the four cysteine residues in the function of the integral inner membrane Hg2+-binding protein, MerC

The roles of the four cysteine residues of the integral inner membrane Hg2+-binding protein, MerC, have been examined using site-directed mutagenesis. Residues Cys-22 and Cys-25 have previously been predicted to lie within the membrane. Substitution of each of these residues in turn with alanine resulted in complete abolition of specific Hg2+ uptake by vesicles. In contrast, substitution by alanine of the other two cysteine residues, Cys-127 and Cys-132, predicted to lie with within a C-terminal cytoplasmic tail, did not significantly affect Hg2+ uptake. Since previous results indicated that native MerC tends to form intermolecular disulfide-bonded dimers, the effects of these substitutions on dimer formation were also examined. Only the Cys-127 and Cys-132 variants spontaneously formed significant amounts of disulfide-bonded dimer. Further experiments using copper-1,10-phenanthroline indicated that each variant with an unpaired cysteine residue was more susceptible to dimer formation than native MerC.     

Biological disulfides: the third messenger? Modulation of phosphofructokinase activity by thiol/disulfide exchange

Rabbit muscle phosphofructokinase is rapidly inactivated at pH 8.0 by incubation with low concentrations of oxidized glutathione, Coenzyme A glutathione mixed disulfide, and oxidized Coenzyme A. The inactivation is first order in disulfide concentration over the concentration ranges examined (50-200 microM), and is approximately 8-fold slower at pH 7.0 than at pH 8.0. The substrates ATP and fructose 6-phosphate protect against inactivation while effector molecules such as AMP, cAMP, and citrate do not. The oxidation of the enzyme by disulfides is fully reversible. The equilibrium constant for the reaction Ered + GSSG in equilibrium Eox + GSH at pH 8.0 is 7.1 in the absence of substrates and 2.5 in the presence of 0.1 mM ATP. For comparison, the equilibrium constant for the reaction CoASH + GSSG in equilibrium CoASSG + GSH was found to be 3.1 at pH 8.0. These equilibrium constants for thiol/disulfide exchange are such that modulation of phosphofructokinase activity by thiol/disulfide exchange in vivo is feasible. The ability of the thiol/disulfide ratio in vivo to modulate the activity of the fructose 6-phosphate/fructose 1,6-diphosphate futile cycle is discussed. The possibility is considered that modulation of the thiol/disulfide ratio in vivo may serve as a "third messenger" in response to cAMP levels, and that the activity of key enzymes of glycolysis/gluconeogenesis may be regulated in response to changing thiol/disulfide ratios.     

Thiol-disulfide exchange by thrombospondin: evidence for a thiol and a disulfide bond protected by calcium

Thrombospondin (Tsp), a protein secreted by activated platelets, forms disulfide-linked complexes with thrombin [K. J. Danishefsky, R. J. Alexander and T. C. Detwiler (1984) Biochemistry 23, 4984]. Thiols and disulfide bonds of Tsp were analyzed, and a search was made for other Tsp covalent complexes. Platelets in 1 mM EDTA were activated with ionophore A23187, and the secreted proteins were analyzed by gel electrophoresis in sodium dodecyl sulfate. One millimolar dithioerythritol (DTE) decreased the electrophoretic mobility of Tsp, indicating reduction of an intrachain disulfide bond; Ca2+ prevented this effect. Electrophoresis of single-chain Tsp prepared with 50 mM DTE in either EDTA or Ca2+ also revealed a Ca2+-stabilized intrachain disulfide bond. Ca2+ prevented the retention of Tsp on an activated thiol-Sepharose column, indicating protection of a thiol by Ca2+. Incubation at 37 degrees C for 60 min resulted in complexes with apparent mass much greater than 500 kDa. Formation of complexes was prevented by N-ethylmaleimide, by a temperature less than 25 degrees C, and by Ca2+ or Mg2+. From pH 6 to 9, complexes formed better at lower pH. Two-dimensional (nonreduced/reduced) electrophoresis revealed Tsp but no other constituents of the complexes. With 10 nM thrombin, complexes formed faster and included thrombin; Ca2+ only partially inhibited. The complex was very susceptible to dissociation by low concentrations (2.5 mM) of DTE. It is concluded that Tsp has a reactive thiol and an intrachain disulfide bond that are protected by Ca2+. When these groups are unprotected, there is intermolecular thiol-disulfide exchange.     

The reactivity of sulfhydryl groups of bovine cardiac troponin C

Bovine cardiac troponin C (cTnC) contains 2 cysteine residues, Cys-35 located in the nonfunctional Ca2+-binding loop I and Cys-84 in the N-terminal segment of the central helix. We have studied the reactivity of Cys residues in cTnC with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). The latter compound fluoresces only when reacted with the protein. The reaction with DTNB followed second order kinetics with respect to DTNB, the rate constants being 3.37 s-1 M-1 and 1.82 s-1 M-1 in the presence and absence of Ca2+, respectively. These rates are much slower than the rate of reaction with Cys-98 of skeletal TnC (sTnC) or with the urea-denatured cTnC, indicating that both Cys residues are partly buried within the structure of the protein. The increase in reactivity was induced by binding of Ca2+ to the single low affinity Ca2+ binding site (site II). The fluorescence increase upon reaction of cTnC with CPM in the absence of Ca2+ could be fitted with a single exponential equation indicating that both cysteine residues are equally available to the reagent. The reaction in the presence of Ca2+ was biphasic. Analysis of CNBr fragments of cTnC labeled with CPM under various conditions indicated that in the presence of Ca2+ the reactivity of Cys-84 is increased while that of Cys-35 is slightly decreased. This finding is consistent with the model of Herzberg et al. (Herzberg, O., Moult, J., and James, M. N. G. (1986) J. Biol. Chem. 261, 2638-2644) and the data of Ingraham and Hodges (Ingraham, R. H., and Hodges, R. S. (1988) Biochemistry 27, 5891-5898), suggesting that the Ca2+-induced conformational change in the N-terminal half of TnC involves separation of the helix C from the central helix, thereby increasing the accessibility of Cys-84. The slow overall kinetics, however, indicates that the structure in the vicinity of Cys residues is relatively compact regardless of Ca2+. We interpret the increase in reactivity towards CPM as consistent with a Ca2+-induced exposure of a hydrophobic pocket in the vicinity of Cys-84.     

The differential stimulation of brain and heart cyclic-AMP phosphodiesterase by oncomodulin

Ca2+/calmodulin dependent cyclic nucleotide phosphodiesterase, from the bovine heart and brain, purified by monoclonal antibody chromatography were tested with respect to activation by oncomodulin. The heart and brain enzymes which have previously been shown to have slightly different electrophoretic mobilities (1), were found to also differ in the oncomodulin dose-dependent activation of cAMP hydrolysis. Oncomodulin was shown to activate the heart enzyme to the same extent as calmodulin. However, this study indicates that the heart phosphodiesterase has approximately 25-fold higher affinity for oncomodulin than the brain enzyme. The oncomodulin concentration required for the half-maximal activation of the heart phosphodiesterase was estimated to be 2 X 10(-7)M. In addition, the possibility of the observed activation by oncomodulin being due to calmodulin contamination can be ruled out as the oncomodulin activation profiles were unaltered subsequent to chromatography on organomercurial agarose and the activation by oncomodulin could not be reversed by anti-calmodulin IgG.     

Physicochemical characterization of cross-linked human serum albumin dimer and its synthetic heme hybrid as an oxygen carrier

The recombinant human serum albumin (rHSA) dimer, which was cross-linked by a thiol group of Cys-34 with 1,6-bis(maleimido)hexane, has been physicochemically characterized. Reduction of the inert mixed-disulfide of Cys-34 beforehand improved the efficiency of the cross-linking reaction. The purified dimer showed a double mass and absorption coefficient, but unaltered molar ellipticity, isoelectric point (pI: 4.8) and denaturing temperature (65 degrees C). The concentration dependence of the colloid osmotic pressure (COP) demonstrated that the 8.5 g dL(-1) dimer solution has the same COP with the physiological 5 g dL(-1) rHSA. The antigenic epitopes of the albumin units are preserved after bridging the Cys-34, and the circulation lifetime of the 125I-labeled variant in rat was 18 h. A total of 16 molecules of the tetrakis[(1-methylcyclohexanamido)phenyl]porphinatoiron(II) derivative (FecycP) is incorporated into the hydrophobic cavities of the HSA dimer, giving an albumin-heme hybrid in dimeric form. It can reversibly bind and release O2 under physiological conditions (37 degrees C, pH 7.3) like hemoglobin or myoglobin. Magnetic circular dichroism (CD) revealed the formation of an O2-adduct complex and laser flash photolysis experiments showed the three-component kinetics of the O2-recombination reaction. The O2-binding affinity and the O2-association and -dissociation rate constants of this synthetic hemoprotein have also been evaluated.     

Inactivation of rabbit muscle creatine kinase by reversible formation of an internal disulfide bond induced by the fungal toxin gliotoxin

The biological activity of gliotoxin is dependent on the presence of a strained disulfide bond that can react with accessible cysteine residues on proteins. Rabbit muscle creatine kinase contains 4 cysteines per 42-kDa subunit and is active in solution as a dimer. Only Cys-282 has been identified as essential for activity. Modification of this residue results in loss of activity of the enzyme. Treatment of creatine kinase with gliotoxin resulted in a time-dependent loss of activity abrogated in the presence of reducing agents. Activity was restored when the inactivated enzyme was treated with reducing agents. Inactivation of creatine kinase by gliotoxin was accompanied by the formation of a 37-kDa form of the enzyme. This oxidized form of creatine kinase was rapidly reconverted to the 42-kDa species by the addition of reducing agents concomitant with restoration of activity. A 1:1 mixture of the oxidized and reduced monomer forms of creatine kinase as shown on polyacrylamide gel electrophoresis was equivalent to the activity of the fully reduced form of the enzyme consistent with only one reduced monomer of the dimer necessary for complete activity. Conversion of the second monomeric species of the dimer to the oxidized form by gliotoxin correlated with loss of activity. Our data are consistent with gliotoxin inducing the formation of an internal disulfide bond in creatine kinase by initially binding and possibly activating a cysteine residue on the protein, followed by reaction with a second neighboring thiol. The recently published crystal structure of creatine kinase suggests the disulfide is formed between Cys-282 and Cys-73.     

Two Highly Homologous Methionine Sulfoxide Reductase A from Tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>), Exhibit Distinct Catalytic Properties

E4, which is a fruit-ripening gene that is strongly induced by ethylene, has been reported to be a member of the methionine sulfoxide reductase A (MSRA) gene. In the present study, we determined for the first time the enzymatic activity and delineated the catalytic mechanism of the E4 protein via site-directed mutagenesis. The disulfide intermolecular cross-linking, kinetics parameter, thiol content titration analysis of wild-type and mutated E4 proteins revealed that the cysteine at position 37 (Cys-37) was the key catalytic residue, and Cys-194, but not Cys-180 served as the first recycling Cys in the thioredoxin (Trx)-dependent regeneration system. In addition, the SlMSRA2 protein, which was encoded by another MSRA gene, shared high similarity with the E4 protein and was truncated at the C-terminus. The wild-type and mutated SlMSRA2 enzymes had similar activities compared to the E4 protein using DTT as a reductant, but showed extremely low activities in the Trx-dependent reduction system. Our results indicated that E4 and SlMSRA2 proteins might exhibit distinct catalytic mechanisms.     

Inhibition of glutathione reductase by oncomodulin

Evidence for a specific interaction between oncomodulin and glutathione reductase is presented. Glutathione reductase (EC 1.6.4.2) isolated from either the bovine intestinal mucosa or the rat liver was bound in a Ca2(+)-dependent manner to oncomodulin which was covalently attached to Sepharose. In addition, glutathione reductase was able to catalyze the reduction of the disulfide-linked dimer of oncomodulin. The interaction of these proteins could also be indirectly demonstrated by monitoring glutathione reductase activity since oncomodulin was shown to inhibit the enzyme in a dose-dependent manner with an apparent IC50 of approximately 5 microM. The kinetic analysis of the oncomodulin-dependent effects on glutathione reductase activity indicates that oncomodulin interacts at a site other than the active site as the oncomodulin-induced inhibition was of the noncompetitive type. The in vivo inhibition of glutathione reductase appears to be an oncomodulin-specific effect as closely related members of the troponin C superfamily such as rabbit (pI 5.5) or carp (pI 4.25) parvalbumins, as well as calmodulin, failed to affect the activity of this enzyme. The present in vitro study indicating that oncomodulin can regulate the activity of glutathione reductase could be very significant with respect to the elucidation of a physiological role for oncomodulin.     

Identification of the cysteine residues in the amino-terminal extracellular domain of the human Ca(2+) receptor critical for dimerization. Implications for function of monomeric Ca(2+) receptor.

We analyzed the effect of substituting serine for each of the 19 cysteine residues within the amino-terminal extracellular domain of the human Ca(2+) receptor on cell surface expression and receptor dimerization. C129S, C131S, C437S, C449S, and C482S were similar to wild type receptor; the other 14 cysteine to serine mutants were retained intracellularly. Four of these, C60S, C101S, C358S and C395S, were unable to dimerize. A C129S/C131S double mutant failed to dimerize but was unique in that the monomeric form expressed at the cell surface. Substitution of a cysteine for serine 132 within the C129S/C131S mutant restored receptor dimerization. Mutation of residues Cys-129, Cys-131, and Ser-132, singly and in various combinations caused a left shift in Ca(2+) response compared with wild type receptor. These results identify cysteines 129 and 131 as critical in formation of intermolecular disulfide bond(s) responsible for receptor dimerization. In a "venus flytrap" model of the receptor extracellular domain, Cys-129 and Cys-131 are located within a region protruding from one lobe of the flytrap. We suggest that this region represents a dimer interface for the receptor and that mutation of residues within the interface causes important changes in Ca(2+) response of the receptor.     

Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate

The dependence on thiol pK of the second-order rate constant (kS) for reaction of thiolate anions with MMTS was shown to follow the Br?nsted equation log kS = log G + beta pK with log G = 1.44 and 3.54 and beta = 0.635 and 0.309 for aryl and alkyl thiols, respectively. The reactivity toward MMTS of the protonated thiol group was found to be negligible in comparison to that of the thiolate anion. For 2-mercaptoethanol the reactivity toward MMTS of the protonated form of the thiol group was shown to be at least 5 X 10(9) smaller than that of the thiolate anion. The pH dependence of the second-order rate constant for reaction of the thiolate group of Cys-25 at the active site of papain was determined and shown to be consistent with the previously determined low pK for Cys-25 and its electrostatic interaction with His-159. The small dependence of the reactivity of Cys-25 on thiol pK (beta approximately 0.09) suggested that the charge-charge interactions that act through space to perturb the pK of the nucleophile at the active site of papain and perhaps other enzymes may serve to increase the fraction of nucleophile present in the reactive basic form without introducing the decrease in nucleophilic reactivity seen in model systems where pK's are lowered primarily by charge-dipole interactions.     

RegB Kinase Activity Is Repressed by Oxidative Formation of Cysteine Sulfenic Acid

RegB/RegA comprise a global redox-sensing signal transduction system utilized by a wide range of proteobacteria to sense environmental changes in oxygen tension. The conserved cysteine 265 in the sensor kinase RegB was previously reported to form an intermolecular disulfide bond under oxidizing conditions that converts RegB from an active dimer into an inactive tetramer. In this study, we demonstrate that a stable sulfenic acid (-SOH) derivative also forms at Cys-265 in vitro and in vivo when RegB is exposed to oxygen. This sulfenic acid modification is reversible and stable in the air. Autophosphorylation assay shows that reduction of the SOH at Cys-265 to a free thiol (SH) can increase RegB kinase activity in vitro. Our results suggest that a sulfenic acid modification at Cys-265 performs a regulatory role in vivo and that it may be the major oxidation state of Cys-265 under aerobic conditions. Cys-265 thus functions as a complex redox switch that can form multiple thiol modifications in response to different redox signals to control the kinase activity of RegB.     

Ligand binding rapidly induces disulfide-dependent dimerization of glycoprotein VI on the platelet plasma membrane

Thrombus formation in hemostasis or thrombotic disease is initiated by adhesion of circulating platelets to damaged blood vessel walls. Exposed subendothelial collagen interacting with platelet glycoprotein (GP) VI leads to platelet activation and integrin alpha(IIb)beta(3)-mediated aggregation. We previously showed that ligand binding to GPVI also induces metalloproteinase-dependent shedding, generating an approximately 55-kDa soluble ectodomain fragment and an approximately 10-kDa membrane-associated remnant. Here, treatment of platelets with collagen or the GPVI-targeting rattlesnake toxin convulxin also induces rapid (10-30 s) formation of a high molecular weight GPVI complex (GPVIc) under nonreducing conditions, as detected by immunoblotting with anti-GPVI antibodies. The appearance of an approximately 20-kDa remnant detectable using a polyclonal antibody against the GPVI cytoplasmic tail under nonreducing, but not reducing, conditions after ectodomain shedding and nonreduced/reduced two-dimensional SDS-polyacrylamide gel analysis of biotinylated platelets confirmed that that GPVIc was a homodimer. Formation of disulfide-linked GPVIc was prolonged in the presence of metalloproteinase inhibitor GM6001 and was independent of GPVI signaling because it was unaffected by inhibitors of Src kinases, Syk, or phosphoinositide 3-kinase. To identify the thiol involved in disulfide bond formation, wild-type or mutant GPVI, where two available sulfhydryls (Cys-274 and Cys-338) were individually mutated to serine, was expressed in rat basophilic leukemia cells. Dimerization of wild-type and C274S GPVI, but not the C338S mutant, was observed after treating cells with convulxin. We conclude that (i) a subpopulation of GPVI forms a constitutive dimer on the platelet surface, facilitating rapid disulfide cross-linking, (ii) convulxin or other GPVI agonists induce disulfide-linked GPVI dimerization independent of GPVI signaling, and (iii) the penultimate residue of the GPVI cytoplasmic tail, Cys-338, mediates disulfide-dependent dimer formation.     

The redox state of cysteines 201 and 317 of the erythrocyte anion exchanger is critical for ankyrin binding

Previous studies have demonstrated that modification of erythrocyte membrane cysteine residues via disulfide cross-briding or direct derivatization with thiol reagents promotes massive morphological, rheological, and structural changes in the cell. To determine whether disruption of the band 3-ankyrin interaction, the major membrane-cytoskeletal linkage, might contribute to the above lesions, we quantitatively measured the band 3-ankyrin interaction following modification of Cys-201 and/or Cys-317 of the cytoplasmic domain of band 3. It was observed that irreversible alkylating agents (e.g. N-ethylmaleimide or iodoacetamide and its derivatives), reversible derivatizing compounds (.e.g. p-chloromercuribenzenesulfonate or glutathione), and native disulfide bond formation all blocked the ankyrin interaction. Comparison of the extent of sulfhydryl modification with the degree of inhibition of ankyrin binding further confirmed that cysteine modification was directly responsible for the inhibition. However, analysis of the site of sulfhydryl derivatization revealed that inhibition of ankyrin binding could be initiated in some cases with derivatization of Cys-201, while in other cases obstruction of Cys-317 appeared to be essential. This apparent discrepancy was resolved by demonstrating that Cys-201 of one strand of the cytoplasmic domain of band 3 dimer could disulfide bond with Cys-317 of the opposite strand, thus demonstrating that all four cysteines of the band 3 dimer are clustered at the interface between subunits. We argue that derivatization or disulfide cross-linking of these cysteines can block ankyrin binding by both conformational and steric mechanisms.     

Catalysis of creatine kinase refolding by protein disulfide isomerase involves disulfide cross-link and dimer to tetramer switch

Protein disulfide isomerase (PDI) functions as an isomerase to catalyze thiol:disulfide exchange, as a chaperone to assist protein folding, and as a subunit of prolyl-4-hydroxylase and microsomal triglyceride transfer protein. At a lower concentration of 0.2 microm, PDI facilitated the aggregation of unfolded rabbit muscle creatine kinase (CK) and exhibited anti-chaperone activity, which was shown to be mainly due to the hydrophobic interactions between PDI and CK and was independent of the cross-linking of disulfide bonds. At concentrations above 1 microm, PDI acted as a protector against aggregation but an inhibitor of reactivation during CK refolding. The inhibition effect of PDI on CK reactivation was further characterized as due to the formation of PDI-CK complexes through intermolecular disulfide bonds, a process involving Cys-36 and Cys-295 of PDI. Two disulfide-linked complexes containing both PDI and CK were obtained, and the large, soluble aggregates around 400 kDa were composed of 1 molecule of tetrameric PDI and 2 molecules of inactive intermediate dimeric CK, whereas the smaller one, around 200 kDa, was formed by 1 dimeric PDI and 1 dimeric CK. To our knowledge this is the first study revealing that PDI could switch its conformation from dimer to tetramer in its functions as a foldase. According to the observations in this research and our previous study of the folding pathways of CK, a working model was proposed for the molecular mechanism of CK refolding catalyzed by PDI.     

Cysteines 431 and 1074 are responsible for inhibitory disulfide cross-linking between the two nucleotide-binding sites in human P-glycoprotein

Human wild-type and Cys-less P-glycoproteins were expressed in Pichia pastoris and purified in high yield in detergent-soluble form. Both ran on SDS gels as a single 140-kDa band in the presence of reducing agent and showed strong verapamil-stimulated ATPase activity in the presence of added lipid. The wild type showed spontaneous formation of higher molecular mass species in the absence of reducing agent, and its ATPase was activated by dithiothreitol. Oxidation with Cu(2+) generated the same higher molecular mass species, primarily at 200 and approximately 300 kDa, in high yield. Cross-linking was reversed by dithiothreitol and prevented by pretreatment with N-ethylmaleimide. Using proteins containing different combinations of naturally occurring Cys residues, it was demonstrated that an inhibitory intramolecular disulfide bond forms between Cys-431 and Cys-1074 (located in the Walker A sequences of nucleotide-binding sites 1 and 2, respectively), giving rise to the 200-kDa species. In addition, dimeric P-glycoprotein species ( approximately 300 kDa) form by intermolecular disulfide bonding between Cys-431 and Cys-1074. The ready formation of the intramolecular disulfide between Cys-431 and Cys-1074 establishes that the two nucleotide-binding sites of P-glycoprotein are structurally very close and capable of intimate functional interaction, consistent with available information on the catalytic mechanism. Formation of such a disulfide in vivo could, in principle, underlie a regulatory mechanism and might provide a means of intervention to inhibit P-glycoprotein.