Accessible Cysteine Residues Research Articles

A microdomain formed by the extracellular ends of the transmembrane domains promotes activation of the G protein-coupled alpha-factor receptor.

The alpha-factor receptor (Ste2p) that promotes mating in Saccharomyces cerevisiae is similar to other G protein-coupled receptors (GPCRs) in that it contains seven transmembrane domains. Previous studies suggested that the extracellular ends of the transmembrane domains are important for Ste2p function, so a systematic scanning mutagenesis was carried out in which 46 residues near the ends of transmembrane domains 1, 2, 3, 4, and 7 were replaced with cysteine. These mutants complement mutations constructed previously near the ends of transmembrane domains 5 and 6 to analyze all the extracellular ends. Eight new mutants created in this study were partially defective in signaling (V45C, N46C, T50C, A52C, L102C, N105C, L277C, and A281C). Treatment with 2-([biotinoyl] amino) ethyl methanethiosulfonate, a thiol-specific reagent that reacts with accessible cysteine residues but not membrane-embedded cysteines, identified a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. An unusual prolonged zone of intermediate reactivity near the extracellular end of transmembrane domain 2 suggests that this region may adopt a special structure. Interestingly, residues implicated in ligand binding were mainly accessible, whereas residues involved in the subsequent step of promoting receptor activation were mainly inaccessible. These results define a receptor microdomain that provides an important framework for interpreting the mechanisms by which functionally important residues contribute to ligand binding and activation of Ste2p and other GPCRs.

The Interaction of the γ-Aminobutyric Acid Transporter GAT-1 with the Neurotransmitter Is Selectively Impaired by Sulfhydryl Modification of a Conformationally Sensitive Cysteine Residue Engineered into Extracellular Loop IV

The (Na+ + Cl-)-coupled gamma-aminobutyric acid (GABA) transporter GAT-1 keeps synaptic levels of this neurotransmitter low and thereby enables efficient GABA-ergic transmission. Extracellular loops (III, IV, and V) have been shown to contain determinants for GABA selectivity and affinity. Here we analyze the role of extracellular loop IV in transport by cysteine scanning mutagenesis. Fourteen residues of this loop have been replaced by cysteine. GABA transport by eight of the fourteen mutants is markedly more sensitive to inhibition by membrane-impermeant methane thiosulfate reagents than wild-type. Mutant A364C has high activity and is potently inhibited by the sulfhydryl reagent. GABA transport by the A364C/C74A double mutant, where the only externally accessible cysteine residue of the wild-type has been replaced by alanine, is also highly sensitive to the sulfhydryl reagents. Maximal sensitivity is observed in the presence of the cosubstrates sodium and chloride. A marked protection is afforded by GABA, provided sodium is present. This protection is also observed at 4 degrees C. The non-transportable analogue SKF100330A also protects the double mutant against sulfhydryl modification in the presence of sodium but has the opposite effect in its absence. Electrophysiological analysis shows that upon sulfhydryl modification of this mutant, GABA can no longer induce transport currents. The voltage dependence of the transient currents indicates an increased apparent affinity for sodium. Moreover, GABA is unable to suppress the transient currents. Our results indicate that part of extracellular loop IV is conformationally sensitive, and its modification selectively abolishes the interaction of the transporter with GABA.

Accessibility of cysteine residues in a cytoplasmic loop of CitS of Klebsiella pneumoniae is controlled by the catalytic state of the transporter.

The citrate transporter CitS of Klebsiella pneumoniae is a secondary transporter that transports citrate in symport with two sodium ions and one proton. Treatment of CitS with the alkylating agent N-ethylmaleimide resulted in a complete loss of transport activity. Treatment of mutant proteins in which the five endogenous cysteine residues were mutated into serines in different combinations revealed that two cysteine residues located in the C-terminal cytoplasmic loop, Cys-398 and Cys-414, were responsible for the inactivation. Labeling with the membrane impermeable methanethiosulfonate derivatives MTSET and MTSES in right-side-out membrane vesicles showed that the cytoplasmic loop was accessible from the periplasmic side of the membrane. The membrane impermeable but more bulky maleimide AmdiS did not inactivate the transporter in right-side-out membrane vesicles. Inactivation by N-ethylmaleimide, MTSES, and MTSET was prevented by the presence of the co-ion Na(+). Protection was obtained upon binding 2 Na(+), which equals the transport stoichiometry. In the absence of Na(+), the substrate citrate had no effect on the inactivation by permeable or impermeable thiol reagents. In contrast, when subsaturating concentrations of Na(+) were present, citrate significantly reduced inactivation suggesting ordered binding of the substrate and co-ion; citrate is bound after Na(+). In the presence of the proton motive force, the reactivity of the Cys residues was increased significantly for the membrane permeable N-ethylmaleimide, while no difference was observed for the membrane impermeable thiol reagents. The results are discussed in the context of a model for the opening and closing of the translocation pore during turnover of the transporter.

Probing Local Dynamics of the Photosynthetic Bacterial Reaction Center with a Cysteine Specific Spin Label

A multifrequency electron paramagnetic resonance (EPR) approach was used to probe the dynamic structure of the bacterial photosynthetic reaction center (RC) protein. We have demonstrated that the cysteine specific nitroxide spin label MTSL can be covalently bound to a surface cysteine residue of Rhodobacter sphaeroides RC protein. We suggest that the MTSL nitroxide is bound to an accessible cysteine residue, H156, which is located on the surface of the protein on the H-subunit. Analysis of the multifrequency EPR spectra of the spin-labeled RC proteins suggests the restricted character of the protein dynamics. These dynamics can be described as fast libration in a cone with a correlation time faster than 10-9 s. Several dynamically nonequivalent sites were observed in the EPR spectra, which may reflect distinct conformational substates of local protein structure. This work provides a foundation for future studies with the goal of correlating protein motions with photosynthetic charge-transfer reactions.

Monitoring the cellular surface display of recombinant proteins by cysteine labeling and flow cytometry.

A general method is described that allows one to follow the surface display of recombinant proteins in Escherichia coli without having to use specific antibodies or enzymatic reactions. The method is based on cysteine-specific labeling through Michael addition to the double bond of maleimide and its derivatives, and takes advantage of the fact that naturally occurring surface proteins in E. coli contain no accessible cysteine residues. The method is easy to perform and could be simply applied to different analytic procedures including Western blot, spectral photometry, and flow cytometry. By using this new labeling method, single cells bearing a distinct protein at the surface could be selected by fluorescence-activated cell sorting. The data were obtained by using autodisplay, an efficient surface display system established for E. coli, but the method presented here represents rather a general solution for analyzing the surface display of recombinant proteins, independent of the cellular system used.

Palmitoylation Regulates Regulators of G-protein Signaling (RGS) 16 Function: I. MUTATION OF AMINO-TERMINAL CYSTEINE RESIDUES ON RGS16 PREVENTS ITS TARGETING TO LIPID RAFTS AND PALMITOYLATION OF AN INTERNAL CYSTEINE RESIDUE

Regulators of G-protein signaling (RGS) proteins down-regulate signaling by heterotrimeric G-proteins by accelerating GTP hydrolysis on the G alpha subunits. Palmitoylation, the reversible addition of palmitate to cysteine residues, occurs on several RGS proteins and is critical for their activity. For RGS16, mutation of Cys-2 and Cys-12 blocks its incorporation of [3H]palmitate and ability to turn-off Gi and Gq signaling and significantly inhibited its GTPase activating protein activity toward aG alpha subunit fused to the 5-hydroxytryptamine receptor 1A, but did not reduce its plasma membrane localization based on cell fractionation studies and immunoelectron microscopy. Palmitoylation can target proteins, including many signaling proteins, to membrane microdomains, called lipid rafts. A subpopulation of endogenous RGS16 in rat liver membranes and overexpressed RGS16 in COS cells, but not the nonpalmitoylated cysteine mutant of RGS16, localized to lipid rafts. However, disruption of lipid rafts by treatment with methyl-beta-cyclodextrin did not decrease the GTPase activating protein activity of RGS16. The lipid raft fractions were enriched in protein acyltransferase activity, and RGS16 incorporated [3H]palmitate into a peptide fragment containing Cys-98, a highly conserved cysteine within the RGS box. These results suggest that the amino-terminal palmitoylation of an RGS protein promotes its lipid raft targeting that allows palmitoylation of a poorly accessible cysteine residue that we show in the accompanying article (Osterhout, J. L., Waheed, A. A., Hiol, A., Ward, R. J., Davey, P. C., Nini, L., Wang, J., Milligan, G., Jones, T. L. Z., and Druey, K. M. (2003) J. Biol. Chem. 278, 19309-19316) was critical for RGS16 and RGS4 GAP activity.

Alkylation and inactivation of human glutathione transferase zeta (hGSTZ1-1) by maleylacetone and fumarylacetone.

Glutathione transferase zeta (GSTZ1-1) catalyzes the cis-trans isomerization of maleylacetoacetate or maleylacetone (MA) to fumarylacetoacetate or fumarylacetone (FA), respectively. GSTZ1-1 also catalyzes the glutathione-dependent biotransformation of a range of alpha-haloacids, including dichloroacetic acid. The objective of this study was to investigate the mechanism of inactivation of hGSTZ1-1 by MA and FA and to determine the covalent modification of hGSTZ1-1 by MA and FA in the presence and absence of glutathione. MA and FA (0.01-1 mM) inactivated all hGSTZ1-1 polymorphic variants in a concentration- and time-dependent manner, and this inactivation was blocked by glutathione. The C16A mutant of hGSTZ1c-1c was partially inactivated by MA and FA. Electrospray ionization-tandem mass spectrometry and SALSA (Scoring Algorithm for Spectral Analysis) analyses of tryptic digests of hGSTZ1 polymorphic variants revealed that the active site (SSCSWR) and C-terminal (LLVLEAFQVSHPCR) cysteine residues of hGSTZ1-1 were covalently modified by MA and FA. MA and FA adduction resulted in diagnostic 156-Da shifts in the masses of the modified peptide ions and in their MS-MS fragment ions. Alkylation of the active-site cysteine residues, but not of the C-terminal cysteine, was relatively less intense when hGSTZ1-1 polymorphic variants were incubated with MA or FA in the presence of S-methyl glutathione. These data indicate that MA and FA are substrate and product inactivators of hGSTZ1-1 and covalently modify hGSTZ1-1 at the active-site cysteine residue in the absence of glutathione. The observation that inactivation was blocked by glutathione indicates that binding of glutathione to the active site prevents reaction of MA or FA with the active-site cysteine residue. These data also indicate that MA and FA may covalently modify and inactivate other proteins that have accessible cysteine residues and may, thereby, contribute to dichloroacetic acid-induced or hypertyrosinemia type-I-associated toxicities.

Phenoxybenzamine Binding Reveals the Helical Orientation of the Third Transmembrane Domain of Adrenergic Receptors

Phenoxybenzamine (PB), a classical alpha-adrenergic antagonist, binds irreversibly to the alpha-adrenergic receptors (ARs). Amino acid sequence alignments and the predicted helical arrangement of the seven transmembrane (TM) domains suggested an accessible cysteine residue in transmembrane 3 of the alpha(2)-ARs, in position C(3.36) (in subtypes A, B, and C corresponding to amino acid residue numbers 117/96/135, respectively), as a possible site for the PB interaction. Irreversible binding of PB to recombinant human alpha(2)-ARs (90 nm, 30 min) reduced the ligand binding capacity of alpha(2A)-, alpha(2B)-, and alpha(2C)-AR by 81, 96, and 77%. When the TM3 cysteine, Cys(117), of alpha(2A)-AR was mutated to valine (alpha(2A)-C117V), the receptor became resistant to PB (inactivation, 10%). The beta(2)-AR contains a valine in this position (V(3.36); position number 117) and a cysteine in the preceding position (Cys(116)) and was not inactivated by PB (10 microm, 30 min) (inactivation 26%). The helical orientation of TM3 was tested by exchanging the amino acids at positions 116 and 117 of the alpha(2A)-AR and beta(2)-AR. The alpha(2A)-F116C/C117V mutant was resistant to PB (inactivation, 7%), whereas beta(2)-V117C was irreversibly inactivated (inactivation, 93%), confirming that position 3.36 is exposed to receptor ligands, and position 3.35 is not exposed in the binding pocket.

Detection of disulfide bonds in bovine brain tubulin and their role in protein folding and microtubule assembly in vitro: a novel disulfide detection approach.

Cysteine residues in tubulin are actively involved in regulating ligand interactions and microtubule formation both in vivo and in vitro. These cysteine residues are sensitive reporters in determining the conformation of tubulin. Although some of the cysteines are critical in modulating drug binding and microtubule assembly, it is not clear how many of these normally exist as disulfides. The controversy regarding the disulfide bonds led us to develop a disulfide detection assay to reexamine the presence of the disulfide linkages in purified alphabeta tubulin and explore their possible biological functions in vitro. The accessible cysteine residues in alphabeta tubulin were alkylated with an excess of iodoacetamide to prevent artifactual generation of disulfide linkages in tubulin. After removal of excess iodoacetamide, tubulin was unfolded in 8 M urea. Half of the unfolded tubulin was treated with dithiothreitol to reduce any disulfide bonds present. The aliquots were then treated with iodo[(14)C]acetamide and the incorporation of radioactivity was measured. We also used the same approach to detect the disulfide linkages in the tubulin in a whole-cell extract. We found in both cases that the samples which were not treated with dithiothreitol had little or no incorporation of iodo[(14)C]acetamide, while the others that were treated with dithiothreitol had significant amounts of (14)C incorporation into tubulin. Moreover, the reduction of the disulfide linkages in tubulin resulted in inhibition of microtubule assembly (29-54%) and markedly affected refolding of the tubulin from both an intermediate and a completely unfolded state. All these data therefore suggest that tubulin has intrachain disulfide bonds in the alpha- and beta-subunits and that these disulfides assist in correct refolding of tubulin from the intermediate unfolded state or help to recover the hydrophobic domains from the completely unfolded state. These disulfides also regulate microtubule assembly and the stability of tubulin in vitro. Our results suggest that tubulin disulfides may play a role in tubulin folding and that thiol-disulfide exchange in tubulin could be a key regulator in microtubule assembly and dynamics of tubulin in vivo.

Bidirectional amyloid fiber growth for a yeast prion determinant

The polymerization of many amyloids is a two-stage process initiated by the formation of a seeding nucleus or protofibril. Soluble protein then assembles with these nuclei to form amyloid fibers. Whether fiber growth is bidirectional or unidirectional has been determined for two amyloids. In these cases, bidirectional growth was established by time lapse atomic-force microscopy. Here, we investigated the growth of amyloid fibers formed by NM, the prion-determining region of the yeast protein Sup35p. The conformational changes in NM that lead to amyloid formation in vitro serve as a model for the self-perpetuating conformational changes in Sup35p that allow this protein to serve as an epigenetic element of inheritance in vivo. To assess the directionality of fiber growth, we genetically engineered a mutant of NM so that it contained an accessible cysteine residue that was easily labeled after fiber formation. The mutant protein assembled in vitro with kinetics indistinguishable from those of the wild-type protein and propagated the heritable genetic trait [PSI(+)] with the same fidelity. In reactions nucleated with prelabeled fibers, unlabeled protein assembled at both ends. Thus, NM fiber growth is bidirectional.

Vesicle-reconstituted Low Density Lipoprotein Receptor: VISUALIZATION BY CRYOELECTRON MICROSCOPY

The low density lipoprotein (LDL) receptor is a key protein for maintaining cellular cholesterol homeostasis by binding cholesterol-rich lipoproteins through their apoB and apoE apoproteins. The LDL receptor is a transmembrane glycoprotein of M(r) approximately 115 kDa; based on its primary sequence, five distinct structural domains have been identified (Yamamoto, T., Davis, C. G., Brown, M. S., Schneider, W. J., Casey, M. L., Goldstein, J. L., and Russell, D. W. (1984) Cell 39, 27-38). As a first step toward providing a structural description of the intact LDL receptor, the receptor has been purified from bovine adrenal cortices, reconstituted into unilamellar egg yolk phosphatidylcholine vesicles, and imaged using cryoelectron microscopy (cryoEM). CryoEM has the advantage of providing images of the reconstituted LDL receptor in its frozen, fully hydrated state. LDL receptor molecules were visualized as elongated, stick-like projections from the vesicle surface with maximum dimensions approximately 120-A length by approximately 45-A width. In some of the images, a short arm (or arms) was visible at the distal end of the stick-like projections. The LDL receptor was labeled via accessible free cysteine residues, probably including that corresponding to Cys-431 of the known full-length sequence of the human LDL receptor. The accessible cysteine was demonstrated using a maleimide-biotin.streptavidin conjugate and confirmed by labeling with monomaleimido-Nanogold. Images obtained by cryoEM showed that the extracellular stick-like domain of the reconstituted LDL receptor was labeled by Nanogold. This combined cryoEM-Nanogold labeling study has provided the first low resolution structural images of the reconstituted, full-length bovine LDL receptor.

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.

“ADP Sulfurylase” from Thiobacillus denitrificansIs an Adenylylsulfate:Phosphate Adenylyltransferase and Belongs to a New Family of Nucleotidyltransferases

During AMP-dependent sulfite oxidation by some sulfur bacteria, the liberation of sulfate from adenosine-5'-phosphosulfate (APS) is catalyzed by APS:phosphate adenylyltransferase (APAT). Here we report the first biochemical and genetic characterization of APAT. We isolated this enzyme from the chemolithoautotroph Thiobacillus denitrificans and cloned the corresponding gene. The enzyme is homodimeric with 41,387-Da subunits and exhibits a specific activity of 2100 micromol min(-1) mg(-1). The K(m) values are K(m(APS)) = 300 microM and K(m(P(i))) = 12 mM. Catalysis occurs by a ping-pong mechanism with a covalently bound AMP as reaction intermediate. The arsenolysis of APS, but not of ADP, CDP, GDP, UDP, or IDP, is also catalyzed, indicating a specific and unidirectional function. The former enzyme name ADP-sulfurylase implies that the reverse reaction is catalyzed; therefore, this name should not be used any longer. Histidine modification of APAT results in complete inactivation that can be suppressed by substrate addition. APAT is highly similar to galactose-1-phosphate uridylyltransferase and also related to Ap(4)A phosphorylase. Active site residues of galactose-1-phosphate uridylyltransferase are conserved in APAT and Ap(4)A phosphorylase, suggesting a histidine as the nucleotide-binding residue in all three enzymes, which together form a new family of nucleotidyltransferases.

Inhibition of the insulin receptor kinase phosphorylation by nitric oxide: functional and structural aspects.

Previous studies on cultured skeletal muscle cells have indicated that the insulin-induced expression of GLUT4 transporter protein is inhibited by nitric oxide (NO). Therefore, we determined the effect of NO on the insulin-induced autophosphorylation of the insulin receptor kinase (IRK), i.e., the first step in the insulin-mediated signal transduction pathway. The experiments showed that the insulin-induced autophosphorylation of the insulin receptor beta-chain is strongly inhibited by the NO donors 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA-NO) or S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect was ameliorated in cells depleted of glutathione (GSH), suggesting the possibility that S-nitroso-glutathione may operate as an intermediate NO donor. Complementary experiments with different Cys --> Ala mutant proteins showed, surprisingly, that all mutant proteins were inhibited by DEA-NO. Three-dimensional models of the nonphosphorylated IR beta-chain nitrosylated at the accessible cysteine residues 1056, 1138, 1234, or 1245 revealed that derivatization of any of these four cysteine residues leads essentially to the same structural changes of the IRK domain. These changes involve a movement of the amino-terminal lobe against the carboxy-terminal lobe in a direction opposite to the direction of the "lobe closure" that was previously proposed to facilitate the accessibility for ATP and the expression of catalytic activity. Our findings suggest that the occurrence of several functionally relevant cysteine residues in distinct regions of the IRK protein increases the probability of regulatory redox interactions and thus the redox sensitivity of the IRK.

Cancer-Preventive Selenocompounds Induce a Specific Redox Modification of Cysteine-Rich Regions in Ca2+- Dependent Isoenzymes of Protein Kinase C

Since protein kinase C (PKC) serves as a receptor for phorbol ester type tumor promoters and oxidants and has unique redox-active cysteine-rich regions, we have determined whether various chemopreventive selenocompounds could affect this enzyme. At lower concentrations, selenite decreased the kinase activity (IC50= 0.5 μm), while at higher concentrations it decreased phorbol ester binding. However, when the catalytic and regulatory domains of PKC were separated by proteolysis, the catalytic domain retained its sensitivity to selenite, while the regulatory domain lost its sensitivity. Cysteine residues were quantitated in PKC modified with selenite by using 5,5′-dithiobis(2-nitrobenzoic acid) and also by using 2-nitro-5-thiosulfobenzoic acid after sulfitolysis. At lower concentrations, selenite induced a modification of four cysteine residues resulting in the formation of two disulfides, while at higher concentrations it induced a modification of seven to eight cysteine residues resulting in the formation of three to four disulfides. Contrary to selenite, selenocystine and selenodiglutathione (GSSeSG) readily inactivated the kinase activity, but not the phorbol ester binding. These two agents induced a two-stage modification of PKC; a limited modification at low concentrations leads to a loss of affinity for ATP, while an excessive modification at high concentrations leads to a loss ofVmax. Selenocystine and GSSeSG were 100,000-fold more potent than GSSG in inactivating PKC. The isoenzymes α, β, and γ exhibited an identical susceptibility to these selenocompounds. These results suggested that the cysteine residues present within the catalytic domain of these isoenzymes, although apart in the sequence, may be clustered in the tertiary structure to react with selenite, as well as may be in close proximity to some of the cysteines in the regulatory domain. Selenite did not affect protein kinase A, whereas GSSeSG and selenocystine inactivated the catalytic subunit after dissociation from the regulatory subunit at concentrations 100- and 800-fold, respectively, higher than that required for PKC inactivation. All three selenocompounds did not affect the activities of phosphorylase kinase and protein phosphatase 2A. Taken together, these results suggest that the accessible redox-active cysteine residues present in the PKC catalytic domain can react with certain specificity with redox-active selenocompounds such as selenite, selenocystine, and GSSeSG relative to other protein kinases tested.

The sulfhydryl groups of Cys 269 and Cys 272 are critical for the oligomeric state of chloroplast carbonic anhydrase from Pisum sativum.

Chloroplast carbonic anhydrase is dependent on a reducing environment to retain its catalytic activity. To investigate the properties of the three accessible cysteine residues of pea carbonic anhydrase, mutants were made in which Ala or Ser substituted for C165, C269, and C272. The mutants at position 165 were found to be spectroscopically similarly to the wild-type. They have a high catalytic activity, and are also sensitive to oxidation. In contrast, both C269 and C272 were found to be critical both for the structure and for the catalytic activity. All mutants with substitutions at either of these two positions had to be co-overexpressed with GroES/EL chaperones to give soluble enzyme in Escherichia coli. The k(cat) values were decreased by 2 and 3 orders of magnitude for the C272A and C269A mutants, respectively, and the Km values were increased approximately 7 times. However, the binding of the inhibitor ethoxyzolamide was only slightly weakened. The near-UV CD spectra were found to be changed in both sign and intensity compared to that of the wild-type, and the far-UV spectra indicate some loss of alpha-helix structure. Moreover, the quaternary structure was changed from the wild-type octameric to tetrameric in these mutants. The results indicate that mutation of either of these cysteines causes minor structural changes around at least one of the two tryptophans of the subunit. Furthermore, the data demonstrate that C269 and C272 are involved in the interaction between subunits and are necessary for a proper structure at the tetramer-tetramer interface.

Use of the Thiol-specific Derivatizing Agent N-Iodoacetyl-3-Iiodotyrosine to Demonstrate Conformational Differences between the Unbound and hsp90-bound Glucocorticoid Receptor Hormone Binding Domain

The hormone binding domain (HBD) of the glucocorticoid receptor (GR) contains five cysteine residues, with three of them being spaced close to one another in the steroid binding pocket. The HBD also contains the contact region for the chaperone protein hsp90, which must be bound to the GR for it to have a steroid binding conformation. Binding of hsp90 to the receptor through its HBD inactivates the DNA binding domain (DBD). The DBD contains a number of cysteines essential to its DNA binding activity. Here, we assess the effects of hsp90 binding on the accessibility of cysteine residues in both the HBD and DBD to derivatization by a thiol-specific reagent. We report that N-iodoacetyltyrosine (IAT) inactivates steroid binding activity of the immunopurified, untransformed GR.hsp90 complex in a manner that is prevented by the sulfhydryl reagents cysteine and dithiothreitol but is not reversed by them. The 125I-labeled IAT derivative N-iodoacetyl-3-[125I]iodotyrosine ([125I]IAIT) covalently labels the immunopurified, hsp90-bound receptor in a thiol-specific manner. Dissociation of hsp90 leads to an approximately 2-fold increase in [125I]IAIT labeling of the full-length, 100-kDa GR. The increase in thiol labeling is related to the presence of hsp90 because it is blocked by molybdate, which prevents hsp90 dissociation. Cleavage of the [125I]IAIT-labeled receptor with trypsin yields a 15-kDa labeled fragment containing the DBD and a 30-kDa labeled fragment containing all of the cysteines in the HBD and the contact region for hsp90. Dissociation of hsp90 from the GR results in a 2.3-fold increase in [125I]IAIT labeling of the 15-kDa fragment and a 50% decrease in labeling of the 30-kDa fragment. These data are consistent with the proposal that dissociation of hsp90 from the GR produces a conformational change in the HBD such that some of the thiols that are exposed in the GR*hsp90 complex become buried and are no longer accessible to the [125I]IAIT probe. In contrast, binding of the GR to hsp90 restricts access of cysteines in the DBD to this small thiol-derivatizing agent, a restriction that is relieved as a result of unmasking or conformational change accompanying hsp90 dissociation.

Replacement of the active-site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo.

DsbA is a periplasmic protein of Escherichia coli that was identified genetically as being involved in the formation of disulfide bonds in secreted proteins. Its active site contains one accessible and one buried cysteine residue, separated in the primary structure by only two other residues. These cysteine residues can form a very unstable disulfide bond that is 10(3)-fold more reactive toward thiols than normal. Moreover, the mixed disulfide between the accessible cysteine residue and glutathione is 10(4)-fold more reactive than normal. Site-directed mutagenesis was carried out to replace either one or both cysteine residues by serine. Cys30 is shown to be the accessible thiol, while Cys33 is shielded from the solvent. Even though the thiol group of Cys30 is exposed and reactive, it formed a very unstable mixed disulfide with glutathione. This disulfide bond was 2.17 +/- 0.02 kcal mol-1 less stable in the native conformation than when DsbA was unfolded. If the native conformation destabilizes the mixed disulfide, the mixed disulfide must destabilize the folded conformation to the same extent. This was confirmed by demonstrating that the folded conformation of the mixed disulfide form of the mutant DsbA was 2.7 +/- 0.9 kcal mol-1 less stable than that of the reduced form; these stability effects originated almost exclusively in the folded conformation. Replacing the cysteine residues by serine destabilized the folded conformation of the reduced protein to varying extents. This suggests that the thiol groups are involved in interactions that stabilize the folded conformation, which would cause any disulfide bonds, either inter- or intramolecular, that involve these groups to be unstable.

The reactive and destabilizing disulfide bond of DsbA, a protein required for protein disulfide bond formation in vivo

The protein DsbA facilitates disulfide bond formation in the periplasm of Escherichia coli. It has only two cysteine residues that are separated in the sequence by two other residues and are shown to form a disulfide bond reversibly. Chemical modification studies demonstrate that only one of the cysteine residues has an accessible thiol group in the reduced protein. Equilibrium and kinetic characterization of thiol-disulfide exchange between DsbA and glutathione showed that the DsbA disulfide bond was 10(3)-fold more reactive than a normal protein disulfide. Similarly, the mixed disulfide between the accessible cysteine residue and glutathione was 10(4)-fold more reactive than normal. The overall equilibrium constant for DsbA disulfide bond formation from GSSG was only 8 x 10(-5) M. These properties indicate that disulfide-bonded DsbA is a potent oxidant and ideally suited for generating protein disulfide bonds. Disulfide bonds generally increase the stabilities of folded proteins, when the folded conformation reciprocally stabilizes the disulfide bonds. In contrast, the disulfide bond of DsbA was so unstable in the folded state that its stability increased by 4.5 +/- 0.1 kcal.mol-1 when the protein unfolded. This implies that the disulfide bond destabilizes the folded conformation of DsbA. This was confirmed by demonstrating that the reduced protein was 3.6 +/- 1.4 kcal.mol-1 more stable than that with the disulfide bond.

Activity of recombinant mitogillin and mitogillin immunoconjugates.

A synthetic gene for the Aspergillus protein toxin mitogillin has been synthesized and expressed in Escherichia coli. The recombinant mitogillin is a potent inhibitor of protein synthesis in vitro with an IC50 of 9.7 pM. Immunoconjugates of recombinant mitogillin derivatized with S-acetylmercaptosuccinic anhydride and 5-methyl-2-iminothiolane modified H65 antibody kill T cell lines and peripheral blood mononuclear cells expressing the human CD5 surface antigen. Native mitogillin contains 4 cysteine residues which form two disulfide pairs (Fernandez-Luna, J. L., Lopez-Otin, C., Soriano, F., and Mendez, E. (1985) Biochemistry 24, 861-867). Three derivatives of mitogillin have been assembled which substitute alanine residues for cysteine residues 5, 147, or 5 and 147. Each of these molecules retains the ability to inhibit protein synthesis in vitro with at most a 2-fold reduction in activity. The derivative mitogillinC147A can be conjugated to 5-methyl-2-iminothiolane- modified H65 antibody directly without pretreatment with S-acetylmercaptosuccinic anhydride, and the immunoconjugate is active against HSB2 cells. Genetic manipulation of toxin genes to expose an accessible cysteine residue into a recombinant product can thus be used to generate immunotoxins without initial derivatization by nonspecific cross-linking reagents.