Characterization of redox state and reductase activity of protein disulfide isomerase under different redox environments using a sensitive fluorescent assay

In this study, dieosin glutathione disulfide (Di-E-GSSG) was synthesized by the reaction of eosin isothiocyanate with GSSG. Di-E-GSSG had low fluorescence which increased approximately 70-fold on reduction of its disulfide bond. The substrate was used to monitor the disulfide reductase activity of PDI. Di-E-GSSG is the most sensitive pseudo substrate for PDI reductase activity reported to date. This probe was further used as an analytical reagent to develop an end point assay for measuring the redox state of PDI. The reduction of Di-E-GSSG by reduced enzyme was studied in the absence of reducing agents and the redox state of PDI was monitored as a function of the stoichiometric changes in the amount of eosin-glutathione (EGSH) generated by the active-site dithiols of PDI. The redox state of PDI was also studied under variable [GSH]/[GSSG] ratios. The results indicate that PDI is in approximately 1/2-reduced state where the [GSH]/[GSSG] ratio is between 1:1 and 3:1, conditions similar to the lumen of endoplasmic reticulum or in the extracellular environment. On the other hand, [GSH]/[GSSG] ratios of > or =8:1, such as in cytosol, all active-site thiols would be reduced. The study was extended to utilize Di-E-GSSG to investigate the effect of variable redox ratios on the platelet surface PDI reductase activity.     

Alkylation of protein disulfide isomerase by the episulfonium ion derived from the glutathione conjugate of 1,2-dichloroethane and mass spectrometric characterization of the adducts

The reactivity of the episulfonium ion derived from S-(2-chloroethyl)glutathione (CEG), the glutathione conjugate of 1,2-dichloroethane, with the catalytic sites of protein disulfide isomerase (PDI) was investigated. The two cysteine residues of the two active sites of PDI are expected to be the major targets of alkylation. PDI was incubated with equimolar to 100-fold excess CEG. The activity of PDI was irreversibly inhibited with a concurrent loss of two thiols; however, PDI oxidative refolding activity was not completely inhibited. With mass spectrometry, sequencing PDI identified one alkylation event on each of the N-terminal cysteine residues in the two active site peptides. PDI appears robust and able to maintain some activity by steric constraint. We have established that the episulfonium ion of CEG can adduct PDI and may have important toxicologic significance for 1,2-dichloroethane toxicity.     

Studies on reduction of S-nitrosoglutathione by human carbonyl reductases 1 and 3

Human carbonyl reductases 1 and 3 (CBR1 and CBR3) are monomeric NADPH-dependent enzymes of the short-chain dehydrogenase/reductase superfamily. Despite 72% identity in primary structure they exhibit substantial differences in substrate specificity. Recently, the endogenous low molecular weight S-nitrosothiol S-nitrosoglutathione (GSNO) has been added to the broad substrate spectrum of CBR1. The current study initially addressed whether CBR3 could equally reduce GSNO which was not the case. Neither the introduction of residues which contribute to glutathione binding in CBR1, i.e. K106Q and S97V/D98A, nor the exchange C143S, which prevents a theoretical disulfide bond with C227 in CBR3, could engender activity towards GSNO. However, exchanging amino acids 236-244 in CBR3 to correspond to CBR1 was sufficient to engender catalytic activity towards GSNO. Catalytic efficiency was further improved by the exchanges Q142M, C143S, P230W and H270S. Hence, the same residues previously reported as important for reduction of carbonyl compounds appear to be key to CBR1-mediated reduction of GSNO. Furthermore, for CBR1-mediated reduction of GSNO, considerable substrate inhibition at concentrations >5 K(m) was observed. Treatment of CBR1 with GSNO followed by removal of low molecular weight compounds decreased the GSNO reducing activity, suggesting a covalent modification. Treatment with dithiothreitol, but not with ascorbic acid, could rescue the activity, indicating S-glutathionylation rather than S-nitrosation as the underlying mechanism. As C227 has previously been identified as the reactive cysteine in CBR1, the variant CBR1 C227S was generated, which, in comparison to the wild-type protein, displayed a similar k(cat), but a 30-fold higher K(m), and did not show substrate inhibition. Collectively, the results clearly argue for a physiological role of CBR1, but not for CBR3, in GSNO reduction and thus ultimately in regulation of NO signaling. Furthermore, at higher concentrations, GSNO appears to work as a suicide inhibitor for CBR1, probably through glutathionylation of C227.     

Insights into deglutathionylation reactions. Different intermediates in the glutaredoxin and protein disulfide isomerase catalyzed reactions are defined by the gamma-linkage present in glutathione

Glutaredoxins are small proteins with a conserved active site (-CXX(C/S)-) and thioredoxin fold. These thiol disulfide oxidoreductases catalyze disulfide reductions, preferring GSH-mixed disulfides as substrates. We have developed a new real-time fluorescence-based method for measuring the deglutathionylation activity of glutaredoxins using a glutathionylated peptide as a substrate. Mass spectrometric analysis showed that the only intermediate in the reaction is the glutaredoxin-GSH mixed disulfide. This specificity was solely dependent on the unusual gamma-linkage present in glutathione. The deglutathionylation activity of both wild-type Escherichia coli glutaredoxin and the C14S mutant was competitively inhibited by oxidized glutathione, with K(i) values similar to the K(m) values for the glutathionylated peptide substrate, implying that glutaredoxin primarily recognizes the substrate via the glutathione moiety. In addition, wild-type glutaredoxin showed a sigmoidal dependence on GSH concentrations, the activity being significantly decreased at low GSH concentrations. Thus, under oxidative stress conditions, where the ratio of GSH/GSSG is decreased, the activity of glutaredoxin is dramatically reduced, and it will only have significant deglutathionylation activity once the oxidative stress has been removed. Different members of the protein disulfide isomerases (PDI) family showed lower activity levels when compared with glutaredoxins; however, their deglutathionylation activities were comparable with their oxidase activities. Furthermore, in contrast to the glutaredoxin-GSH mixed disulfide intermediate, the only intermediate in the PDI-catalyzed reaction was PDI peptide mixed disulfide.     

Human quiescin-sulfhydryl oxidase, QSOX1: probing internal redox steps by mutagenesis

The flavoprotein quiescin-sulfhydryl oxidase (QSOX) rapidly inserts disulfide bonds into unfolded, reduced proteins with the concomitant reduction of oxygen to hydrogen peroxide. This study reports the first heterologous expression and enzymological characterization of a human QSOX1 isoform. Like QSOX isolated from avian egg white, recombinant HsQSOX1 is highly active toward reduced ribonuclease A (RNase) and dithiothreitol but shows a >100-fold lower k cat/ K m for reduced glutathione. Previous studies on avian QSOX led to a model in which reducing equivalents were proposed to relay through the enzyme from the first thioredoxin domain (C70-C73) to a distal disulfide (C509-C512), then across the dimer interface to the FAD-proximal disulfide (C449-C452), and finally to the FAD. The present work shows that, unlike the native avian enzyme, HsQSOX1 is monomeric. The recombinant expression system enabled construction of the first cysteine mutants for mechanistic dissection of this enzyme family. Activity assays with mutant HsQSOX1 indicated that the conserved distal C509-C512 disulfide is dispensable for the oxidation of reduced RNase or dithiothreitol. The four other cysteine residues chosen for mutagenesis, C70, C73, C449, and C452, are all crucial for efficient oxidation of reduced RNase. C452, of the proximal disulfide, is shown to be the charge-transfer donor to the flavin ring of QSOX, and its partner, C449, is expected to be the interchange thiol, forming a mixed disulfide with C70 in the thioredoxin domain. These data demonstrate that all the internal redox steps occur within the same polypeptide chain of mammalian QSOX and commence with a direct interaction between the reduced thioredoxin domain and the proximal disulfide of the Erv/ALR domain.     

Catalysis of thiol/disulfide exchange. Glutaredoxin 1 and protein-disulfide isomerase use different mechanisms to enhance oxidase and reductase activities

Glutaredoxin (Grx) and protein-disulfide isomerase (PDI) are members of the thioredoxin superfamily of thiol/disulfide exchange catalysts. Thermodynamically, rat PDI is a 600-fold better oxidizing agent than Grx1 from Escherichia coli. Despite that, Grx1 is a surprisingly good protein oxidase. It catalyzes protein disulfide formation in a redox buffer with an initial velocity that is 30-fold faster than PDI. Catalysis of protein and peptide oxidation by the individual catalytic domains of PDI and by a Grx1-PDI chimera show that differences in active site chemistry are fundamental to their oxidase activity. Mutations in the active site cysteines reveal that Grx1 needs only one cysteine to catalyze rapid substrate oxidation, whereas PDI requires both cysteines. Grx1 is a good oxidase because of the high reactivity of a Grx1-glutathione mixed disulfide, and PDI is a good oxidase because of the high reactivity of the disulfide between the two active site cysteines. As a protein disulfide reductase, Grx1 is also superior to PDI. It catalyzes the reduction of nonnative disulfides in scrambled ribonuclease and protein-glutathione mixed disulfides 30-180 times faster than PDI. A multidomain structure is necessary for PDI to catalyze effective protein reduction; however, placing Grx1 into the PDI multidomain structure does not enhance its already high reductase activity. Grx1 and PDI have both found mechanisms to enhance active site reactivity toward proteins, particularly in the kinetically difficult direction: Grx1 by providing a reactive glutathione mixed disulfide to supplement its oxidase activity and PDI by utilizing its multidomain structure to supplement its reductase activity.     

Thioredoxin-2 but not thioredoxin-1 is a substrate of thioredoxin peroxidase-1 from Drosophila melanogaster: isolation and characterization of a second thioredoxin in D. Melanogaster and evidence for distinct biological functions of Trx-1 and Trx-2

As Drosophila melanogaster does not contain glutathione reductase, the thioredoxin system has a key function for glutathione disulfide reduction in insects (Kanzok, S. M., Fechner, A., Bauer, H., Ulschmid, J. K., Müller, H. M., Botella-Munoz, J., Schneuwly, S., Schirmer, R. H., and Becker, K. (2001) Science 291, 643-646). In view of these unique conditions, the protein systems participating in peroxide metabolism and in redox signaling are of special interest. The genes for a second thioredoxin (DmTrx-2) and a thioredoxin peroxidase (DmTPx-1) were cloned and expressed, and the proteins were characterized. In its disulfide form, the 13-kDa protein thioredoxin-2 is a substrate of thioredoxin reductase-1 (K(m) = 5.2 microm, k(cat) = 14.5 s(-1)) and in its dithiol form, an electron donor for TPx-1 (K(m) = 9 microm, k(cat) = 5.4 s(-1)). DmTrx-2 is capable of reducing glutathione disulfide with a second order rate constant of 170 m(-1) s(-1) at pH 7.4 and 25 degrees C. Western blot analysis indicated that this thioredoxin represents up to 1% of the extractable protein of D. melanogaster Schneider cells or whole fruit flies. Recombinant thioredoxin peroxidase-1 (subunit molecular mass = 23 kDa) was found to be a decameric protein that can efficiently use Trx-2 but not Trx-1 as a reducing substrate. The new electron pathway found in D. melanogaster is also representative for insects that serve as vectors of disease. As a first step we have cloned and functionally expressed the gene that is the orthologue of DmTrx-2 in the malaria mosquito Anopheles gambiae.     

Autodegradation of Protein Disulfide Isomerase

Protein disulfide isomerase (PDI) and its degradation products were found in HepG2, COS-1, and CHO-K1 cells. Whether or not the products were formed through autodegradation of PDI was examined, since PDI contains the CGHC motif, which is the active center of proteolytic activity in ER-60 protease. Commercial bovine PDI was autodegraded to produce a trimmed PDI. In addition, human recombinant PDI also had autodegradation activity. Mutant recombinant PDIs with CGHC motifs of which cysteine residues were replaced with serine or alanine residues were prepared. However, they were not autodegraded, suggesting the cysteine residues of motifs are necessary for autodegradation.     

Autodegradation of protein disulfide isomerase

Protein disulfide isomerase (PDI) and its degradation products were found in HepG2, COS-1, and CHO-K1 cells. Whether or not the products were formed through autodegradation of PDI was examined, since PDI contains the CGHC motif, which is the active center of proteolytic activity in ER-60 protease. Commercial bovine PDI was autodegraded to produce a trimmed PDI. In addition, human recombinant PDI also had autodegradation activity. Mutant recombinant PDIs with CGHC motifs of which cysteine residues were replaced with serine or alanine residues were prepared. However, they were not autodegraded, suggesting the cysteine residues of motifs are necessary for autodegradation.     

Conserved residues flanking the thiol/disulfide centers of protein disulfide isomerase are not essential for catalysis of thiol/disulfide exchange.

Protein disulfide isomerase (PDI) catalyzes the oxidative folding of proteins containing disulfide bonds by increasing the rate of disulfide bond rearrangements which normally occur during the folding process. The amino acid sequences of the N- and C-terminal redox active sites (PWCGHCK) in PDI are completely conserved from yeast to man and display considerable identity with the redox-active center of thioredoxin (EWCGPCK). Available data indicate that the two thiol/disulfide centers of PDI can function independently in the isomerase reaction and that the cysteine residues in each active site are essential for catalysis. To evaluate the role of residues flanking the active-site cysteines of PDI in function, a variety of mutations were introduced into the N-terminal active site of PDI within the context of both a functional C-terminal active site and an inactive C-terminal active site in which serine residues replaced C379 and C382. Replacement of non-cysteine residues (W34 to Ser, G36 to Ala, and K39 to Arg) resulted in only a modest reduction in catalytic activity in both the oxidative refolding of RNase A and the reduction of insulin (10-27%), independent of the status of the C-terminal active site. A somewhat larger effect was observed with the H37P mutation where approximately 80% of the activity attributable to the N-terminal domain (approximately 40%) was lost. However, the H37P mutant N-terminal site expressed within the context of an inactive C-terminal domain exhibits 30% activity, approximately 70% of the activity of the N-terminal site alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of glutaredoxin enzyme activity and protein S-glutathionylation using fluorescent eosin-glutathione

Glutaredoxins catalyze glutathione-dependent disulfide oxidoreductions, particularly reduction of glutathione (GSH)-protein mixed disulfides. Mammalian glutaredoxins are present in the cytosol/nucleus as Grx1 or in mitochondria as Grx2a. Here we describe di-eosin-glutathione disulfide (Di-E-GSSG) as a new tool to study glutaredoxin (Grx) activity. Di-E-GSSG has almost no fluorescence in its disulfide form due to self-quenching, whereas the reduced form (E-GSH) has a large fluorescence emission at 545 nm after excitation at 520 nm. Di-E-GSSG was a very poor substrate for glutathione reductase, but we discovered that the molecule was an excellent substrate for glutaredoxin in a coupled assay system with GSH, nicotinamide adenine dinucleotide phosphate (NADPH), and glutathione reductase or with lipoamide, NADH, and lipoamide dehydrogenase. In addition, Di-E-GSSG was used to glutathionylate the free SH group of bovine serum albumin (BSA), yielding eosin-glutathionylated BSA (E-GS-BSA) readily observed in ultraviolet (UV) light. E-GS-BSA also displayed a quenched fluorescence, and its Grx-catalyzed reduction could be followed by the formation of E-GSH by fluorescence emission using microtiter plates. This way of measuring Grx activity provided an ultrasensitive method that detected Grx1 and Grx2 at picomolar levels. Human Grx1 was readily quantified in 40 μl of plasma and determined to be 680 ± 208 pM in healthy controls.     

Residues C123 and D58 of the 2-methylisocitrate lyase (PrpB) enzyme of Salmonella enterica are essential for catalysis

The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50 degrees C, and the reaction required Mg(2+) ions; equimolar concentrations of Mn(2+) ions were a poor substitute for Mg(2+) (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The K(m) of PrpB for 2-MIC was measured at 19 micro M, with a k(cat) of 105 s(-1). Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpB(K121A), PrpB(H125A), and PrpB(R122K) mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in k(cat) for 2-MIC lyase activity, respectively. The PrpB(D58A) and PrpB(C123A) proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is proposed to serve as the active site base, and residue D58 is critical for the coordination of a required Mg(2+) ion.     

Expression, Purification and Functional Analysis of Hexahistidine-tagged Human Glutathione S-Transferase P1-1 and its Cysteinyl Mutants

The bacterial expression and purification of human glutathione S-transferase P1-1(hGST P1-1), as a hexahistidine-tagged polypeptide was performed. Site-directed mutagenesis was used to construct mutants in which alanine replaced two (C47A/C101A), three (C14A/C47A/C101A) or all four (C14A/C47A/ C101A/C169A) cysteine residues using the plasmid for the wild type enzyme. Analysis of their catalytic activities and kinetic parameters suggested that cysteins are not essential for the catalytic activity but may contribute to some extent to the catalytic efficiency. Moreover, on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, hexahistidine-tagged hGST P1-1 (His₆-hGST P1-1) treated with 1 mM H₂O₂ showed at least three extra bands, in addition to the native His₆-hGST P1-1 subunit band. These extra bands were not detected in the cysteinyl mutants. Thus, it indicated that disulfide bonds were formed mainly within subunits between cysteine residues, causing an apparent reduction in molecular weight, only small amounts of binding between subunits being observed. These authors contributed equally to this work.     

Biogenesis of Thylakoid Membranes in Chlamydomonas reinhardtii y1 (A Kinetic Study of Initial Greening)

Initiation of thylakoid membrane assembly was examined in degreened cells of Chlamydomonas reinhardtii y1 cells depleted of thylakoid membranes and photosynthetic activity by growth in the dark for 3 to 4 d. Photoreductive activities of photosystem II (PSII) and photosystem I (PSI) increased with no apparent lag when degreened cells were exposed to light at 38[deg]C. However, fluorescence transients induced by actinic light, which reflect the functional state of PSII, changed only slightly during the first 2 h of greening. When these cells were treated with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) or saturating light, fluorescence increased commensurate with the cellular content of chlorophyll. In similar experiments with greening cells of C. reinhardtii CC-2341 (ac-u-g-2.3), a PSI-minus strain, fluorescence increased with chlorophyll without treatment with DCMU. These data suggested that fluorescence of initial PSII centers in greening y1 cells was quenched by activity of PSI. Continuous monitoring of fluorescence in the presence or absence of DCMU showed that assembly of quenched PSII centers occurred within seconds after exposure of y1 cells to light. These results are consistent with initial assembly of PSI and PSII within localized domains, where their proximity allows efficient energy coupling.     

Site-directed mutagenesis of glutathione S-transferase YaYa: functional studies of histidine, cysteine, and tryptophan mutants.

The rat cytosolic glutathione S-transferase Ya subunit contains three histidine residues (at positions 8, 143, and 159), two cysteine residues (at positions 18 and 112), and a single tryptophan residue (at position 21). Histidine, cysteine, and tryptophan have been proposed to be present either near or at the active site of other glutathione S-transferase subunits. The functional role of these amino acids at each of the positions was evaluated by site-directed mutagenesis in which valine or asparagine, alanine, and phenylalanine were substituted for histidine, cysteine, and tryptophan, respectively. Mutant enzymes H8V, H143V, H159N, C112A, and W21F retained either full or better catalytic efficiencies (k(cat)/Km) toward 1-chloro-2,4-dinitrobenzene and glutathione. Lower but significant k(cat)/Km values were observed for H159V and C18A toward 1-chloro-2,4-dinitrobenzene. Some mutants displayed different thermal stabilities and intrinsic fluorescence intensities, but all retained the ability to bind heme. These results indicate that histidine, cysteine, and tryptophan in the glutathione S-transferase Ya subunit are not essential for catalysis nor are they involved in the binding of heme to the YaYa homodimer.     

Studies on the function of yeast protein disulfide isomerase in renaturation of proteins.

Renaturation of two enzymes lacking disulfide bonds, citrate synthase (CS), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and another protein containing disulfide bonds, lysozyme (LZM), were studied in order to dissect the possible chaperone function from the isomerase function of yeast protein disulfide isomerase (PDI). Our findings suggest no independent chaperone activity of yeast PDI with respect to the two enzymes lacking disulfide bonds, GAPDH and CS, since neither of these enzymes required PDI for renaturation. In contrast, a high level of renaturation of LZM was observed in the presence of PDI. Renaturation of LZM involved formation and rearrangement of disulfide bonds. Additional studies using LZM as a substrate were done to examine the role of cysteine residues in the two active sites of PDI. Studies with a series of cysteine to serine mutants and truncation mutants of yeast PDI revealed that the two active sites of PDI were not equal in activity. An intramolecular disulfide bond in at least one active site of PDI was required for the oxidation of reduced LZM. The first cysteine in each active site was necessary for disulfide bond rearrangement, i.e., isomerization, in LZM, while the second cysteine was not.     

Active-site residues governing high steroid isomerase activity in human glutathione transferase A3-3

Glutathione transferase (GST) A3-3 is the most efficient human steroid double-bond isomerase known. The activity with Delta(5)-androstene-3,17-dione is highly dependent on the phenolic hydroxyl group of Tyr-9 and the thiolate of glutathione. Removal of these groups caused an 1.1 x 10(5)-fold decrease in k(cat); the Y9F mutant displayed a 150-fold lower isomerase activity in the presence of glutathione and a further 740-fold lower activity in the absence of glutathione. The Y9F mutation in GST A3-3 did not markedly decrease the activity with the alternative substrate 1-chloro-2,4-dinitrobenzene. Residues Phe-10, Leu-111, and Ala-216 selectively govern the activity with the steroid substrate. Mutating residue 111 into phenylalanine caused a 25-fold decrease in k(cat)/K(m) for the steroid isomerization. The mutations A216S and F10S, separate or combined, affected the isomerase activity only marginally, but with the additional L111F mutation k(cat)/K(m) was reduced to 0.8% of that of the wild-type value. In contrast, the activities with 1-chloro-2,4-dinitrobenzene and phenethylisothiocyanate were not largely affected by the combined mutations F10S/L111F/A216S. K(i) values for Delta(5)-androstene-3,17-dione and Delta(4)-androstene-3,17-dione were increased by the triple mutation F10S/L111F/A216S. The pK(a) of the thiol group of active-site-bound glutathione, 6.1, increased to 6.5 in GST A3-3/Y9F. The pK(a) of the active-site Tyr-9 was 7.9 for the wild-type enzyme. The pH dependence of k(cat)/K(m) of wild-type GST A3-3 for the isomerase reaction displays two kinetic pK(a) values, 6.2 and 8.1. The basic limb of the pH dependence of k(cat) and k(cat)/K(m) disappears in the Y9F mutant. Therefore, the higher kinetic pK(a) reflects ionization of Tyr-9, and the lower one reflects ionization of glutathione. We propose a reaction mechanism for the double-bond isomerization involving abstraction of a proton from C4 in the steroid accompanied by protonation of C6, the thiolate of glutathione serving as a base and Tyr-9 assisting by polarizing the 3-oxo group of the substrate.     

Different residues in the GABA(A) receptor alpha 1T60-alpha 1K70 region mediate GABA and SR-95531 actions

Although gamma-aminobutyric acid type A receptor agonists and antagonists bind to a common site, they produce different conformational changes within the site because agonists cause channel opening and antagonists do not. We used the substituted cysteine accessibility method and two-electrode voltage clamping to identify residues within the binding pocket that are important for mediating these different actions. Each residue from alpha(1)T60 to alpha(1)K70 was mutated to cysteine and expressed with wild-type beta(2) subunits in Xenopus oocytes. Methanethiosulfonate reagents reacted with alpha(1)T60C, alpha(1)D62C, alpha(1)F64C, alpha(1)R66C, alpha(1)S68C, and alpha(1)K70C. gamma-Aminobutyric acid (GABA) slowed methanethiosulfonate modification of alpha(1)F64C, alpha(1)R66C, and alpha(1)S68C, whereas SR-95531 slowed modification of alpha(1)D62C, alpha(1)F64C, and alpha(1)R66C, demonstrating that different residues are important for mediating GABA and SR-95531 actions. In addition, methanethiosulfonate reaction rates were fastest for alpha(1)F64C and alpha(1)R66C, indicating that these residues are located in an open, aqueous environment lining the core of the binding pocket. Positively charged methanethiosulfonate reagents derivatized alpha(1)F64C and alpha(1)R66C significantly faster than a negatively charged reagent, suggesting that a negative subsite important for interacting with the ammonium group of GABA exists within the binding pocket. Pentobarbital activation of the receptor increased the rate of methanethiosulfonate modification of alpha(1)D62C and alpha(1)S68C, demonstrating that parts of the binding site undergo structural rearrangements during channel gating.     

Vulnerability of the cysteine-less proton-coupled folate transporter (PCFT-SLC46A1) to mutational stress associated with the substituted cysteine accessibility method

The proton-coupled transporter (PCFT) mediates intestinal folate absorption and folate transport from blood across the choroid plexus. The membrane topology of PCFT has been defined using the substituted cysteine accessibility method; an intramolecular disulfide bond between the Cys 66 and 298 residues, in the first and fourth extracellular loops, respectively, is present but not essential for function. The current report describes Lys 422 mutations (K422C, K422E) that have no effect on transport activity when introduced into wild-type PCFT but result in a marked loss of activity when introduced into a Cys-less PCFT which is otherwise near-fully functional. The loss of activity of both mutant PCFTs was shown to be due to impaired protein stability and expression. Additional studies were conducted with the K422C mutation in Cys-less PCFT. The impact of re-introduction of one, two, three or five, Cys residues was assessed. While there were some differences in the impact of the different Cys residues re-introduced, restoration was attributed more to a cumulative effect rather than the specific role of individual Cys residues. Preservation of the Cys66-Cys298 intramolecular disulfide bond was not required for stability of the K422C protein. These observations are relevant to studies with Cys-less transporters utilized for the characterization of proteins with the substituted cysteine accessibility method and indicate that functional defects detected in a Cys-less protein, when the tertiary structure of the molecule is stressed, are not necessarily relevant to the wild-type protein.