Identification of reactive cysteines in a protein using arsenic labeling and collision-induced dissociation tandem mass spectrometry

Trivalent arsenicals have high affinity for thiols (such as free cysteines) in proteins. We describe here the use of this property to develop a collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) technique for the identification of reactive cysteines in proteins. A trivalent arsenic species, dimethylarsinous acid (DMA (III)), with a residue mass (103.9607) and mass defect distinct from the normal 20 amino acids, was used to selectively label reactive cysteine residues in proteins. The CID fragment ions of the arsenic-labeled sequences shifted away from the more abundant normal fragments that would otherwise overlap with the ions of interest. Along with the internal and immonium ions, the arsenic-labeled fragment ions served as MS/MS signatures for identification of the binding sites and for assessment of the relative reactivity of individual cysteine residues in a protein. Using this method, we have identified two highly reactive binding sites in rat hemoglobin (Hb): Cys-13alpha and Cys-125beta. Cys-13alpha was bound to DMA (III) in the Hb of rats fed with arsenic, and this binding was responsible for arsenic accumulation in rat blood, while Cys-125beta was found to bind to glutathione in rat blood. This study revealed the relative reactivity of the cysteines in rat Hb in the following decreasing order: Cys-13alpha > Cys-111alpha > Cys-104alpha and Cys-13alpha > Cys-125beta > Cys-93beta. Arsenic-labeling is easy and fast for identification of active binding sites without enzymatic digestion and acid hydrolysis, and useful for characterization and identification of metal binding sites in other proteins.     

Mapping of the carboxyl terminus within the tertiary structure of transducin's alpha subunit using the heterobifunctional cross-linking reagent, 125I-N-(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) cysteine

A heterobifunctional cross-linking reagent, 125I-N-(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) cysteine (125-ACTP), has been synthesized. 125I-ACTP has been used to derivative reduced sulfhydryls of the retinal G protein, transducin (Gt), to form a mixed disulfide bond under mild, nondenaturing conditions (pH 7.4, 4 degrees C). The resulting disulfide was easily cleaved using reducing reagents. A 200-fold molar excess of 125I-ACTP relative to Gt resulted in the incorporation of 1-1.3 mol of the 125I-N-(3-iodo-4-azidophenylpropionamido)cysteine moiety of ACTP into Gt alpha. In contrast to 125I-ACTP, dithionitrobenzoate and dithiopyridone derivatized six sulfhydryls in native Gt. Incubation of a 10-fold molar excess of 125I-ACTP relative to Gt resulted in the derivatization of 0.75-0.9 and 0.1 mol of reduced sulfhydryls/mol Gt alpha and beta, respectively. Gt gamma was not derivatized by 125I-ACTP. Thus, Gt alpha was preferentially derivatized by 125I-ACTP. Tryptic digestion and amino acid sequencing of Gt alpha indicated that both Cys-347 near the carboxyl terminus and Cys-210 between the second and third consensus sequences forming the GTP-binding site were derivatized by 125I-ACTP in a ratio of approximately 70 and 30%, respectively. Thus, both Cys-210 and Cys-347 are labeled, even though derivatization by 125I-ACTP does not exceed 1 mol of SH/mol Gt alpha. It appears that derivatization of one sulfhydryl, either Cys-210 or Cys-347, excludes labeling of the second cysteine either by steric hindrance or induced conformational change making the second cysteine inaccessible to 125I-ACTP. Consistent with this finding was the observation that pertussis toxin-catalyzed ADP-ribosylation of Cys-347 inhibited 125I-ACTP derivatization of Cys-210. Derivatization of Gt alpha at either Cys-210 or Cys-347 by 125I-ACTP inhibited rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate binding to Gt, mimicking the effect of ADP-ribosylation of Cys-347 by pertussis toxin. ACTP contains a radioiodinated phenylazide moiety which, upon activation, can cross-link the derivatized cysteine to an adjacent polypeptide domain. Following reduction of the disulfide, the [125I] iodophenyl moiety will be transferred to the azide-inserted polypeptide. When photoactivation of the phenylazide moiety of 125I-ACTP after sulfhydryl derivatization was performed, insertion of the Cys-347 which contains Cys-210, was found.(ABSTRACT TRUNCATED AT 400 WORDS)     

Autopalmitoylation of tubulin

Pure rat brain tubulin is readily palmitoylated in vitro using [3H]palmitoyl CoA but no added enzymes. A maximum of approximately six palmitic acids are added per dimer in 2-3 h at 36-37 degrees C under native conditions. Both alpha and beta tubulin are labeled, and 63-73% of the label was hydroxylamine-labile, presumed thioesters. Labeling increases with increasing pH and temperature, and with low concentrations of guanidine HCl or KCl (but not with urea) to a maximum of approximately 13 palmitates/dimer. High SDS and guanidine HCl concentrations are inhibitory. At no time could all 20 cysteine residues of the dimer be palmitoylated. Polymerization to microtubules, or use of tubulin S, markedly decreases the accessibility of the palmitoylation sites. Palmitoylation increases the electrophoretic mobility of a portion of alpha tubulin toward the beta band. Palmitoylated tubulin binds a colchicine analogue normally, but during three warm/cold polymerization/depolymerization cycles there is a progressive loss of palmitoylated tubulin, indicating decreased polymerization competence. We postulate that local electrostatic factors are major regulators of reactivity of tubulin cysteine residues toward palmitoyl CoA, and that the negative charges surrounding a number of the cysteines are sensitive to negative charges on palmitoyl CoA.     

Mapping the binding site of colchicinoids on beta -tubulin. 2-Chloroacetyl-2-demethylthiocolchicine covalently reacts predominantly with cysteine 239 and secondarily with cysteine 354

2-Chloroacetyl-2-demethylthiocolchicine (2CTC) and 3-chloroacetyl-3-demethylthiocolchicine (3CTC) resemble colchicine in binding to tubulin and react covalently with beta-tubulin, forming adducts with cysteine residues 239 and 354. The adducts at Cys-239 are less stable than those at Cys-354 during formic acid digestion. Extrapolating to zero time, the Cys-239 to Cys-354 adduct ratio is 77:23 for 2CTC and 27:73 for 3CTC. Using energy minimization modeling to dock colchicinoids into the electron crystallographic model of beta-tubulin in protofilaments (Nogales, E. , Wolf, S. G., and Downing, K. H. (1998) Nature 391, 199-203), we found two potential binding sites. At one, entirely encompassed within beta-tubulin, the C2- and C3-oxygen atoms of 2CTC and 3CTC overlapped poorly with those of colchicine and thiocolchicine, but distances from the reactive carbon atoms of the analogs to the sulfur atoms of the cysteine residues were qualitatively consistent with reactivity. The other potential binding site was located at the alpha/beta interface. Here, the oxygen atoms of the analogs overlapped well with those of colchicine, but relative distances of the reactive carbons to the cysteine sulfur atoms did not correlate with the observed reactivity. A significant conformational change must occur in the colchicine binding site of tubulin in the transition from the unpolymerized to the polymerized state.     

Highly reactive cysteine residues in rodent hemoglobins

Two hemoglobins with cysteine residues highly reactive toward electrophiles have been identified and characterized. Cys-125beta of guinea pig hemoglobin has a low pK(a) and forms conjugates with electrophiles more quickly than glutathione and several orders of magnitude more quickly than other protein thiols. This cysteine is capable of intercepting benzoquinone, a known carcinogenic metabolite, before other protein nucleophiles can be modified. Cys-13beta of mouse hemoglobin was observed to conjugate with electrophiles as quickly as glutathione. The structural basis of reactivity is different in the two hemoglobins and is analyzed in terms of hydrogen-bonding, solvent accessibility, and helix-dipole contributions. Complementing a previously characterized highly reactive cysteine in rat hemoglobin, identification of these cysteines suggests that the reactivity of these hemoglobins could represent a common function as a detoxification sink against carcinogens.     

Enzyme IIMtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: identification of the activity-linked cysteine on the mannitol carrier

The cysteines of the membrane-bound mannitol-specific enzyme II (EIIMtl) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system have been labeled with 4-vinylpyridine. After proteolytic breakdown and reversed-phase HPLC, the peptides containing cysteines 110, 384, and 571 could be identified. N-Ethylmaleimide (NEM) treatment of the native unphosphorylated enzyme results in incorporation of one NEM label per molecule and loss of enzymatic activity [Roossien, F. F., & Robillard, G. T. (1984) Biochemistry 23, 211-215]. NEM treatment and inactivation prevented 4-vinylpyridine incorporation into the Cys-384-containing peptide, identifying this residue as the activity-linked cysteine. Both oxidation and phosphorylation of the native enzyme protected the enzyme against NEM labeling of Cys-384. Positive identification of the activity-linked cysteine was accomplished by inactivation with [14C]iodoacetamide, proteolytic fragmentation, isolation of the peptide, and amino acid sequencing.     

Chloroethylclonidine and 2-aminoethyl methanethiosulfonate recognize two different conformations of the human alpha(2A)-adrenergic receptor.

The substituted cysteine-accessibility method and two sulfhydryl-specific reagents, the methane-thiosulfonate derivative 2-aminoethyl methanethiosulfonate (MTSEA) and the alpha(2)-adrenergic receptor (alpha(2)-AR) agonist chloroethylclonidine (CEC), were used to determine the relative accessibility of engineered cysteines in the fifth transmembrane domain of the human alpha(2A)-AR (Halpha2A). The second-order rate constants for the reaction of the receptor with MTSEA and CEC were determined with the wild type Halpha2A (cysteine at position 201) and receptor mutants containing accessible cysteines at other positions within the binding-site crevice (positions 197, 200, and 204). The rate of reaction of CEC was similar to that of MTSEA at residues Cys-197, Cys-201, and Cys-204. The rate of reaction of CEC with Cys-200, however, was more than 5 times that of MTSEA, suggesting that these compounds may interact with two different receptor conformations. MTSEA, having no recognition specificity for the receptor, likely reacts with the predominant inactive receptor conformation (R), whereas the agonist CEC may stabilize and react preferentially with the active receptor conformation (R*). This hypothesis was consistent with three-dimensional receptor-ligand models, which further suggest that alpha(2A)-AR activation may involve the clockwise rotation of transmembrane domain 5.     

Mapping of reactive sulfhydryls in avian liver 3-hydroxy-3-methylglutaryl coenzyme A synthase

Avian liver mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase contains seven sulfhydryls per 53 kDa subunit. Peptides that harbor these sulfhydryls can be mapped by reverse-phase HPLC separation of tryptic digests of denatured 14C-carboxymethylated enzyme. Native enzyme is inactivated by a variety of reagents that target cysteine residues. Of particular interest is the enzyme's sensitivity to reagents (e.g., CdCl2, copper phenanthroline) that target vicinal thiols. The identity of the cysteines which are modified by these reagents can be determined by peptide mapping after denaturation. 14C-carboxymethylation and trypsin digestion of the sample. While the extent of reaction of any particular cysteinyl sulfhydryl depends on the identity of the reagent employed, three of the protein's seven cysteinyl sulfhydryls are frequently modified upon inactivation of the enzyme. The peptides which contain these reactive sulfhydryls have been isolated and their sequences have been determined by Edman degradation techniques. Comparison of these sequences with the deduced primary structure of the rodent cytosolic enzyme (Gil et al. (1986) J. Biol. Chem. 261, 3710) indicates strong homologies. These homologies allow assignment of the reactive residues as Cys-129, Cys-224 and Cys-268. The sensitivity of these residues to reagents that target vicinal thiols, coupled with the fact that cys-129 is the residue involved in formation of the acyl-S-enzyme intermediate (Vollmer et al. (1988) Biochemistry 27, 4288), suggests that these three residues may be closely juxtaposed within the enzyme's catalytic domain.     

Novel Residues Lining the CFTR Chloride Channel Pore Identified by Functional Modification of Introduced Cysteines

Substituted cysteine accessibility mutagenesis (SCAM) has been used widely to identify pore-lining amino acid side chains in ion channel proteins. However, functional effects on permeation and gating can be difficult to separate, leading to uncertainty concerning the location of reactive cysteine side chains. We have combined SCAM with investigation of the charge-dependent effects of methanethiosulfonate (MTS) reagents on the functional permeation properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channels. We find that cysteines substituted for seven out of 21 continuous amino acids in the eleventh and twelfth transmembrane (TM) regions can be modified by external application of positively charged [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium [2-sulfonatoethyl] MTS (MTSES). Modification of these cysteines leads to changes in the open channel current–voltage relationship at both the macroscopic and single-channel current levels that reflect specific, charge-dependent effects on the rate of Cl⁻ permeation through the channel from the external solution. This approach therefore identifies amino acid side chains that lie within the permeation pathway. Cysteine mutagenesis of pore-lining residues also affects intrapore anion binding and anion selectivity, giving more information regarding the roles of these residues. Our results demonstrate a straightforward method of screening for pore-lining amino acids in ion channels. We suggest that TM11 contributes to the CFTR pore and that the extracellular loop between TMs 11 and 12 lies close to the outer mouth of the pore.     

Cytoplasmic vestibule of the weak inward rectifier Kir6.2 potassium channel.

Intracellular application of certain charged methanethiosulfonate (MTS) reagents modified and irreversibly inhibited Kir6.2 channels when cysteine substitutions were introduced at positions Ile-210, Ile-211, or Ser-212 within the putative cytoplasmic region. Inhibition depends on the spatial dimensions of the MTS reagents. Reaction of MTS reagents, having head diameters of 7.6-8.2 A, with cysteines introduced at position Ser-212 must occur in more than two subunits of the tetrameric Kir6.2 complex to inhibit channel activity. MTS reagents with head diameters less than 6.6 A modified cysteines without causing channel inhibition. An MTS reagent with a head diameter of approximately 10 A could neither modify nor inhibit the channels. Channel inhibition is interpreted as blockage of the intracellular vestibule by MTS reagents that enter the channel vestibule and react with the cysteine residues at vestibule-lining positions. Data are consistent with the hypothesis that residues Ile-210-Ser-212 line a funnel-shaped vestibule of 20-25 A in diameter, which remains unchanged during channel gating.     

Interchain and intrachain disulphide bonds in human platelet glycoprotein IIb. Localization of the epitopes for several monoclonal antibodies.

The single interchain disulphide bond in platelet glycoprotein IIb (GPIIb) is accessible to extracellular reductants, and selective cleavage does not liberate GPIIb alpha from platelet plasma membrane, confirming that non-covalent interactions contribute to maintaining attachment of this subunit to the membrane. Eosin-maleimide labelling of isolated GPIIb after selective cleavage of this interchain disulphide bond, followed by full reduction and alkylation, CNBr cleavage, and analysis of the cleavage products allowed us to establish that this interchain disulphide bridge is formed between GPIIb beta (GPIIb beta-subunit) Cys-9 and GPIIb alpha Cys-826, and this conclusion was confirmed by independent routes. The other two cysteines of GPIIb beta (Cys-14 and Cys-19) form the single intrachain disulphide bond in this subunit. Last, the intrachain disulphides in GPIIb alpha (GPIIb alpha-subunit) are distributed in four main peptide domains which are not disulphide-bonded among themselves. The linear epitope for monoclonal antibody M1 is localized between Pro-4 and Met-24 (or Met-31) of GPIIb beta. The linear epitope for M3 is situated between Cys-826 and the C-terminus of GPIIb alpha. The M4 epitope is also linear and localized somewhere between residues 115 and 285 of GPIIb alpha. Finally, the epitopes for M5 and M6 are somewhere between Cys-608 and Met-704, within a 35 kDa membrane-bound chymotryptic product of digestion of GPIIb in whole platelets. The N-terminal amino acid sequences determined for eight different cleavage products of GPIIb alpha and GPIIb beta agree with the corresponding amino acid sequences predicted by cDNA sequence for human-erythroleukaemic-cell GPIIb [Poncz, Eisman, Heindenreich, Silver, Vilaire, Surrey, Schwartz & Bennett (1987) J. Biol. Chem. 262, 8476-8482].     

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.     

Studies on the reactivity of the essential cysteine of thymidylate synthetase

The reaction of one of the four cysteinyl residues of thymidylate synthetase from methotrexate-resistant Lactobacillus casei with a variety of sulfhydryl reagents results in complete inhibition of the enzyme. Kinetic studies indicate that the rates of reactivity of the reagents tested are N-ethylmaleimide > iodoacetamide > N-(iodoacetylaminoethyl)-S-naphthylamine-1-sulfonic acid > iodoacetic acid. The enzyme is also inactivated by 5-Hg-deoxyuridylate, a compound which reacts stoichiometrically with a single cysteine. Unlike the other reagents, the inhibition produced by this compound can be completely reversed by added thiols. The same cysteine appears to react with all of the sulfhydryl reagents, as shown by competition experiments and by protection against inactivation by deoxyuridylate. Even at a 100-fold excess of the alkylating agents, only one of the four cysteines in the native enzyme was reactive, attesting to the uniqueness of this residue. Carboxypeptidase A inactivation of the enzyme does not affect either the binding of deoxyuridylate to the enzyme or the reactivity of N-ethylmaleimide with the “catalytic” cysteine. Under denaturing conditions, all four cysteinyl residues react with N-ethylmaleimide or iodoacetate, as shown by identifying the reaction products by amino acid analysis. The covalent ternary complex [(+)5,10-methylenetetrahydrofolate-5-fluorodeoxyuridylate-thymidylate synthetase] (molar ratio = 2:2:1) revealed only two cysteinyl residues capable of reacting with N-ethylmaleimide or iodoacetate upon denaturation. From these data, it appears that one cysteine is involved in the binding of deoxyuridylate and that two of the enzyme's four cysteines are responsible for binding 5-fluorodeoxyuridylate in the ternary complex.     

Reactive sulfhydryl groups of alpha 39, a guanine nucleotide-binding protein from brain. Location and function

The guanine nucleotide-binding proteins which mediate hormonal inhibition of adenylate cyclase as well as hormonal regulation of other membrane functions are alpha, beta, and gamma heterotrimers which are structurally homologous to each other. In brain, the predominant guanine nucleotide-binding component is a 39-kDa protein whose physiological role is as yet unknown. We have used N-ethylmaleimide to define functionally important sulfhydryl groups on alpha 39. Three cysteine residues in the molecule are reactive in unliganded alpha 39. Alkylation of two of these is reduced when guanosine 5'-(3'-O-thio)triphosphate (GTP gamma S) is bound. We have isolated and sequenced tryptic peptides containing the three reactive cysteines. The octapeptide containing the GTP gamma S-insensitive cysteine is at a position equivalent to amino acids 106-113 of the transducin alpha subunit (Lochrie, M. A., Hurley, J. B., and Simon, M. I. (1985) Science 228, 96-99). However, the equivalent peptide in transducin does not contain a cysteine residue. Alkylation of this cysteine blocks ADP-ribosylation of cysteine 351 by pertussis toxin. However, alkylation does not prevent association of alpha with the beta X gamma subunits nor does it inhibit GTPase activity. The two GTP gamma S-sensitive cysteines are at positions equivalent to cysteines 139 and 286 of the transducin alpha subunit. Alkylation of these residues inhibits GTPase activity. Neither of these GTP gamma S-sensitive cysteines are in those regions of alpha 39 which are highly homologous to the GTP-binding site of elongation factor Tu (Jurnak, F. (1985) Science 230, 32-36). However, both are present in the brain 41-kDa guanine nucleotide-binding protein and in the two transducins. The conservation of these cysteine residues suggests that they are important for the function of the subunits.     

Specific reaction of alpha,beta-unsaturated carbonyl compounds such as 6-shogaol with sulfhydryl groups in tubulin leading to microtubule damage

6-Shogaol and 6-gingerol are ginger components with similar chemical structures. However, while 6-shogaol damages microtubules, 6-gingerol does not. We have investigated the molecular mechanism of 6-shogaol-induced microtubule damage and found that the action of 6-shogaol results from the structure of alpha,beta-unsaturated carbonyl compounds. alpha,beta-Unsaturated carbonyl compounds such as 6-shogaol react with sulfhydryl groups of cysteine residues in tubulin, and impair tubulin polymerization. The reaction with sulfhydryl groups depends on the chain length of alpha,beta-unsaturated carbonyl compounds. In addition, alpha,beta-unsaturated carbonyl compounds are more reactive with sulfhydryl groups in tubulin than in 2-mercaptoethanol, dithiothreitol, glutathione and papain, a cysteine protease.     

Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment.

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride channel that is regulated by phosphorylation and ATP binding. Work by others suggested that some residues in the sixth transmembrane segment (M6) might be exposed in the channel and play a role in ion conduction and selectivity. To identify the residues in M6 that are exposed in the channel and the secondary structure of M6, we used the substituted cysteine accessibility method. We mutated to cysteine, one at a time, 24 consecutive residues in and flanking the M6 segment and expressed these mutants in Xenopus oocytes. We determined the accessibility of the engineered cysteines to charged, lipophobic, sulfhydryl-specific methanethiosulfonate (MTS) reagents applied extracellularly. The cysteines substituted for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353 reacted with the MTS reagents, and we infer that they are exposed on the water-accessible surface of the protein. From the pattern of the exposed residues we infer that the secondary structure of the M6 segment includes both alpha-helical and extended regions. The diameter of the channel from the extracellular end to the level of Gln353 must be at least 6 A to allow the MTS reagents to reach these residues.     

Specific cysteines in beta3 are involved in disulfide bond exchange-dependent and -independent activation of alphaIIbbeta3

Disulfide bond exchange among cysteine residues in epidermal growth factor (EGF)-like domains of beta3 was suggested to be involved in activation of alphaIIbbeta3. To investigate the role of specific beta3 cysteines in alphaIIbbeta3 expression and activation, we expressed in baby hamster kidney cells normal alphaIIb with normal beta3 or beta3 with single or double cysteine substitutions of nine disulfide bonds in EGF-3, EGF-4, and beta-tail domains and assessed alphaIIbbeta3 surface expression and activation state by flow cytometry using P2 or PAC-1 antibodies, respectively. Most mutants displayed reduced surface expression of alphaIIbbeta3. Disruptions of disulfide bonds in EGF-3 yielded constitutively active alphaIIbbeta3, implying that these bonds stabilize the inactive alphaIIbbeta3 conformer. Mutants of the Cys-567-Cys-581 bond in EGF-4 were inactive even after exposure to alphaIIbbeta3-activating antibodies, indicating that this bond is necessary for activating alphaIIbbeta3. Disrupting Cys-560-Cys-583 in the EGF-3/EGF-4 or Cys-608-Cys-655 in beta-tail domain resulted in alphaIIbbeta3 activation only when Cys-560 or Cys-655 of each pair was mutated but not when their partners (Cys-583, Cys-608) or both cysteines were mutated, suggesting that free sulfhydryls of Cys-583 and Cys-608 participate in alphaIIbbeta3 activation by a disulfide bond exchange-dependent mechanism. The free sulfhydryl blocker dithiobisnitrobenzoic acid inhibited 70% of anti-LIBS6 antibody-induced activation of wild-type alphaIIbbeta3 and had a smaller effect on mutants, implicating disulfide bond exchange-dependent and -independent mechanisms in alphaIIbbeta3 activation. These data suggest that different disulfide bonds in beta3 EGF and beta-tail domains play variable structural and regulatory roles in alphaIIbbeta3.     

Elimination of disulfide bonds affects assembly and secretion of the human chorionic gonadotropin beta subunit

Human chorionic gonadotropin (hCG) consists of two noncovalently joined alpha and beta subunits similar to the other glycoprotein hormones. To study the function of the individual disulfide bonds in subunit assembly and secretion, site-directed mutagenesis was used to convert the 12 cysteine (Cys) residues in the beta subunit of hCG to either alanine or serine. Both cysteines of proposed disulfide pairs were also mutated. These mutant hCG beta genes were transfected alone or together with the wild-type alpha gene into Chinese hamster ovary cells. Only 3-10% assembly could be achieved with derivatives containing single Cys mutations at positions 26, 110, 72, and 90, whereas no assembly was detected with the other 8 mutants. However, double mutations of pairs 26-110 or 23-72 showed increased dimer formation (11 and 36%, respectively). The secretion rate of individual mutants varied significantly. Whereas the Cys-23 and 72 mutants were secreted normally (t1/2 = 140-190 min), the Cys-26 mutant was secreted faster (t1/2 = 70 min), and the other 9 mutants were secreted slower (t1/2 = 280-440 min); mutations of both Cys at 26 and 110 caused much faster secretion (t1/2 = 34 min). Although the secretion rate of these mutants differed, they were quantitatively recovered in the medium except for mutant Cys-88, Cys-23-72, and Cys-34-88 (40, 55, and 10% secreted, respectively). Thus, interruption of any disulfide bond in the hCG beta subunit alters the structure sufficiently to block dimer formation and in some cases slow secretion, although the stability for most of the mutant hCG beta subunits is not greatly affected. The data indicate that interruption of any hCG beta disulfide bond generates different structural forms that are unable to assemble with the alpha subunit, and that the structural requirements for stability and assembly are different.     

Structural differences between brain beta 1- and beta 2-tubulins: implications for microtubule assembly and colchicine binding.

Brain beta 1- and beta 2-tubulins are the major and minor beta-tubulin components of chordate brain tissue, respectively. Two cysteines of beta 1, but not beta 2, can be specifically cross-linked with the bifunctional sulfhydryl reagent N,N'-ethylenebis(iodoacetamide) (EBI). They are in positions 239 and 354. Although separated by 115 amino acid residues along the beta 1-chain, the two sulfur atoms are maximally 9 A apart in the beta 1 tertiary structure. The failure of beta 2 to form a similar cross-bridge is due to the absence of a cysteine in position 239. At least 10 other sequence differences are also present between beta 1 and beta 2. Positions 239 and 354 of beta 1 probably occupy a key part of the tubulin molecule. The microtubule assembly inhibitors colchicine and podophyllotoxin appear to bind on or near this site and EBI is a potent inhibitor of microtubule assembly. Furthermore, the beta 1-cysteine in position 239 appears to be the most reactive in brain tubulin under the given conditions. The marked difference between beta 1 and beta 2 in this critical region suggests that they may have different functions in brain tissue.     

Delineation of bacterial luciferase aldehyde site by bifunctional labeling reagents

Previously we have established that a highly reactive cysteinyl group on the alpha subunit is at the aldehyde site of the (alpha beta) dimeric Vibrio harveyi luciferase. Three isomeric bifunctional reagents have been synthesized and used to further delineate the luciferase aldehyde site. These probes differ in their relative positions of and distances between the two functional groups active in chemical and photochemical labelings, respectively. Each of the probes can effectively and reversibly inactivate luciferase by forming a disulfide linkage primarily to the reactive cysteinyl residue. Upon subsequent photolysis, a diazoacetate arm in each probe was activated for photochemical labeling of amino acid residues within reach. After reductive regeneration of the reactive cysteinyl residue, 0.35-0.40 probe per dimeric luciferase was found to have been photochemically incorporated, correlating well with the degree of irreversible enzyme inactivation. Low but significant amounts of the three isomeric probes initially attached to the alpha reactive cysteine through a disulfide have been found to photochemically tag certain residues on beta. The latter residues are estimated to be no more than 8-11 A away from the alpha reactive cysteine. Thus the reactive cysteinyl residue, and hence the aldehyde site, must be at or near the alpha beta subunit interface. Furthermore, the structural integrity of the microenvironment surrounding this reactive cysteinyl residue is crucial to luciferase activity. An HPLC method for the isolation of luciferase alpha and beta subunits has also been developed.