Role of the conserved cysteine residues of the 11.5 kDa subunit in complex I catalytic properties

Mitochondrial complex I exists as a mixture of two inter-convertible forms: active (A) and de-activated (D), the latter being sensitive to SH-modifying compounds. To investigate if the conserved cysteine-rich 11.5 kDa subunit of Neurospora crassa complex I is involved in this process, we subjected the corresponding genomic DNA to site-directed mutagenesis. The four cysteine residues of the subunit were separately substituted with serine residues and the resulting proteins were independently expressed in a null-mutant strain. All of the obtained mutant strains were able to assemble a complex I with similar kinetic properties to those observed in the wild-type enzyme, indicating that none of the cysteine residues of the 11.5 kDa protein is individually relevant for the A/D transition process. Diminished amounts of assembled complex I seem to be the major effect of these specific mutations. The cysteine residues are likely important to the acquisition and stabilization of the correct 11.5 kDa protein conformation and this is reflected in the assembly/stability of complex I.     

Site directed mutagenesis of two cysteine residues in the E. coli ogt O6-alkylguanine DNA alkyltransferase protein.

The E. coli ogt O6-alkylguanine-DNA alkyltransferase has two cysteine residues positioned identically with respect to cysteines in the E. coli ada O6-alkylguanine-DNA alkyltransferase. In order to assess their function, these residues were each substituted by a glycine to generate altered forms of the ogt protein. Mutagenesis of cysteine-139, located within a 'PCHRV' region of homology, eliminated functional activity confirming that this residue is the methyl-accepting cysteine in the active site of the protein. Substitution of cysteine 102 within the sequence 'LRTIPCG' had little effect on the ogt protein activity demonstrating that this cysteine is not directly involved with the transfer of O6-methylguanine adducts.     

Functional and structural comparisons of cysteine residues in the Val108 wild type and Met108 variant of human soluble catechol O-methyltransferase

Catechol O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. This enzyme is genetically polymorphic with a wild type and a variant form. Numerous epidemiological studies have shown that the variant form is associated with an increased risk of developing estrogen-associated cancers and a wide spectrum of mental disorders. There are seven cysteine residues in human S-COMT, all of which exist as free thiols and are susceptible to electrophilic attack and/or oxidative damage leading to enzyme inactivation. Here, the seven cysteine residues were systematically replaced by alanine residues by means of site-directed mutagenesis. The native forms and cysteine/alanine mutants were assayed for enzymatic activity, thermal stability, methylation regioselectivity, and reactivity of cysteine residues to thiol reagent. Our data showed that although there is only one encoding base difference between these two COMT forms, this difference might induce structural changes in the local area surrounding some cysteine residues, which might further contribute to the different roles they might play in enzymatic activity, and to the different susceptibility to enzyme inactivation.     

Protein inhibitors of cysteine proteinases. III. Amino-acid sequence of cystatin from chicken egg white

Cystatin, the protein inhibitor of cysteine proteinases from chicken egg white was purified by a new method. The two major forms with pI 6.5 (Peak I) and 5.6 (Peak II) were separated. Molecular masses of both forms are approx. 12700 Da as determined by gel chromatography; Form A from Peak I has a molecular mass of 12191 Da as calculated from its amino-acid sequence. The complete amino-acid sequence of Form A was determined by automated solid-phase Edman degradation of the whole inhibitor and its cyanogen bromide fragments. It contains 108 amino-acid residues. Form B from Peak II represents an elongation of Form A by 8 amino-acid residues at the N-terminus. Cystatin contains four cysteine residues, presumably forming two disulphide bridges. Comparison of the amino-acid sequences and near ultraviolet circular dichroism spectra of stefin, the cysteine proteinase inhibitor from human granulocytes, and cystatin shows that the two proteins are entirely different. According to the primary structures, probably neither proteinase inhibitor is involved in a thiol-disulphide exchange mechanism in the interaction with its target enzyme.     

Redox sensitive cysteine residues in calbindin D28k are structurally and functionally important

Human calbindin D(28k) is a Ca(2+) binding protein that has been implicated in the protection of cells against apoptosis. In this study, the structural and functional significance of the five cysteine residues present in this protein have been investigated through a series of cysteine-to-serine mutations. The mutants were studied under relevant physiological redox potentials in which conformational changes were monitored using ANS binding. Urea-induced denaturations, as monitored by intrinsic tryptophan fluorescence, were also carried out to compare their relative stability. It was shown that the two N-terminal cysteine residues undergo a redox-driven structural change consistent with disulfide bond formation. The other cysteine residues are not by themselves sufficient at inducing structural change, but they accentuate the disulfide-dependent conformational change in a redox-dependent manner. Mass spectrometry data show that the three C-terminal cysteine residues can be modified by glutathione. Furthermore, under oxidizing conditions, the data display additional species consistent with the conversion of cysteine thiols to sulfenic acids and disulfides to disulfide-S-monoxides. The biological function of calbindin D(28k) appears to be tied to the redox state of the cysteine residues. The two N-terminal cysteine residues are required for activation of myo-inositol monophosphatase, and enzyme activation is enhanced under conditions in which these residues are oxidized. Last, oxidized calbindin D(28k) binds Ca(2+) with lower affinity than does the reduced protein.     

An approach for the assessment of the order of disruption of the elements of protein structure upon protein unfolding: A study of carbonic anhydrase B

An experimental approach named μ-analysis has been developed in order to elucidate the sequence of the loss of ordered structure by elements of a protein during the denaturation of the molecule. This approach is applicable for the analysis of proteins that fold (unfold) in a multistep process that involve the formation (destruction) of a range of intermediate states. The concept of the approach consists in systematic analysis of mutagenized forms of the protein with point substitutions of hydrophobic amino-acid residues and additional cysteine bridges. Importantly, the substitutions of the amino-acid residues must be localized to the same structural elements of the protein. Point substitutions of hydrophobic amino-acid residues mainly provide information on the structural elements of the protein that are disrupted at the final stages of protein denaturation. The addition of cysteine bridges to the surface of the protein molecule allows investigation of structural elements of the protein that are the first to unfold upon protein denaturation. Calorimetric studies of non-equilibrium melting of bovine carbonic anhydrase B yielded information on the rate constants of the unfolding of ten mutant forms of the protein. The analysis of the effects of mutations on the rates of different stages of protein unfolding allowed for elucidation of the order of disruption of structural elements of carbonic anhydrase B upon thermal denaturation.     

OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins

In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents.     

Probe of beta-galactosidase structure with iodoacetate. Differential reactivity of thiol groups in wild-type and mutant forms of beta-galactosidase.

Carboxymethylation with 14 C-labeled iodoacetate of cysteine residues in wild-type beta-galactosidase from Escherichia coli and in a defective beta-galactosidase from deletion mutant strain M15 was investigated in order to determine accessible positions in the tetrameric wild-type form and the dimeric mutant M15 protein. The extent of carboxymethylation, the effects on biological activity, antibody activation, physical stability, and the labeling of particular residues were studied. The results distinguish three groups of spatial relationships for cysteine residues in the protein, define possible regions for subunit interactions, and confirm that no cysteine residue is specifically involved in catalysis. Residue 1019 and to a lesser extent 498 are accessible in the tetrameric protein and probably represent exposed areas. In the M15 protein, these two, and three additional residues, at 76,387 and 600, were found to react significantly with reagent. One or more of the latter are suggested to be in the dimer-dimer interface. Complementation and activation by antibody are inhibited by carboxymethylation of M15 protein.     

Chemical-proteomic strategies to investigate cysteine posttranslational modifications

The unique combination of nucleophilicity and redox-sensitivity that is characteristic of cysteine residues results in a variety of posttranslational modifications (PTMs), including oxidation, nitrosation, glutathionylation, prenylation, palmitoylation and Michael adducts with lipid-derived electrophiles (LDEs). These PTMs regulate the activity of diverse protein families by modulating the reactivity of cysteine nucleophiles within active sites of enzymes, and governing protein localization between soluble and membrane-bound forms. Many of these modifications are highly labile, sensitive to small changes in the environment, and dynamic, rendering it difficult to detect these modified species within a complex proteome. Several chemical-proteomic platforms have evolved to study these modifications and enable a better understanding of the diversity of proteins that are regulated by cysteine PTMs. These platforms include: (1) chemical probes to selectively tag PTM-modified cysteines; (2) differential labeling platforms that selectively reveal and tag PTM-modified cysteines; (3) lipid, isoprene and LDE derivatives containing bioorthogonal handles; and (4) cysteine-reactivity profiling to identify PTM-induced decreases in cysteine nucleophilicity. Here, we will provide an overview of these existing chemical-proteomic strategies and their effectiveness at identifying PTM-modified cysteine residues within native biological systems.     

Topological analysis of the hepatitis B virus core particle by cysteine-cysteine cross-linking.

The nucleocapsid, or core particle, of hepatitis B virus is formed by 180 subunits of the core protein, which contains Cys at positions 48, 61, 107 and 183, the latter constituting the C terminus. Upon adventitious oxidation, some or all of these cysteine residues participate in the formation of disulphide bridges, leading to polymerization of the subunits within the particle. To utilize the cysteine residues as topological probes, we reduced the number of possible intersubunit crosslinks by replacing these residues individually, or in all combinations, by serine. A corresponding set of variants was constructed within the context of an assembly-competent core protein variant that lacks the highly basic C-terminal region. Analysis, by polyacrylamide gel electrophoresis under non-reducing conditions, of the oxidative crosslinking products formed by the wild-type and mutant proteins expressed in Escherichia coli, revealed a clear distinction between the three N-proximal, and the C-terminal Cys: N-proximal Cys formed intermolecular disulphide bonds only with other N-proximal cysteine residues, leading to dimerization. Cys48 and Cys61, in contrast to Cys107, could be crosslinked to the homologous cysteine residues in a second subunit, and are therefore located at the dimer interface. Cys 183 predominantly formed disulphide bonds with Cys183 in subunits other than those crosslinked by the N-proximal cysteine residues. Hence, the polymers generated by oxidation of the wild-type protein are S-S-linked dimeric N-terminal domains interconnected via Cys183/Cys183 disulphide bonds. The intermolecular crosslinks between the N-proximal cysteine residues were apparently the same in the C-terminally truncated and in the full-length proteins, corroborating the model in which the N-terminal domain and the C terminus of the HBV core protein form two distinct and structurally independent entities. The strong tendency of the N-terminal domain for dimeric interactions suggests that core protein dimers are the major intermediates in hepatitis B virus nucleocapsid assembly.     

Regulation of PTP1B via glutathionylation of the active site cysteine 215.

The reversible regulation of protein tyrosine phosphatase is an important mechanism in processing signal transduction and regulating cell cycle. Recent reports have shown that the active site cysteine residue, Cys215, can be reversibly oxidized to a cysteine sulfenic derivative (Denu and Tanner, 1998; Lee et al., 1998). We propose an additional modification that has implications for the in vivo regulation of protein tyrosine phosphatase 1B (PTP1B, EC 3.1.3.48): the glutathionylation of Cys215 to a mixed protein disulfide. Treatment of PTP1B with diamide and reduced glutathione or with only glutathione disulfide (GSSG) results in a modification detected by mass spectrometry in which the cysteine residues are oxidized to mixed disulfides with glutathione. The activity is recovered by the addition of dithiothreitol, presumably by reducing the cysteine disulfides. In addition, inactivated PTP1B is reactivated enzymatically by the glutathione-specific dethiolase enzyme thioltransferase (glutaredoxin), indicating that the inactivated form of the phosphatase is a glutathionyl mixed disulfide. The cysteine sulfenic derivative can easily oxidize to its irreversible sulfinic and sulfonic forms and hinder the regulatory efficiency if it is not converted to a more stable and reversible end product such as a glutathionyl derivative. Glutathionylation of the cysteine sulfenic derivative will prevent the enzyme from further oxidation to its irreversible forms, and constitutes an efficient regulatory mechanism.     

Conserved cysteine residues in the human immunodeficiency virus type 1 transmembrane envelope protein are essential for precursor envelope cleavage.

The transmembrane (TM) protein of human immunodeficiency virus type 1 has been demonstrated to be involved in viral infectivity and syncytium formation. Two highly conserved cysteine residues in the extracellular region of the TM protein are shown to be essential for processing the 160-kDa envelope precursor into the active 120- and 41-kDa mature forms.     

Profile of the disulfide bonds in acetylcholinesterase

The inter- and intrasubunit disulfide bridges for the 11 S form of acetylcholinesterase isolated from Torpedo californica have been identified. Localized within the basal lamina of the synapse, the dimensionally asymmetric forms of acetylcholinesterase contain either two (13 S) or three (17 S) sets of catalytic subunits linked to collagenous and noncollagenous structural subunits. Limited proteolysis of these molecules yields a tetramer of catalytic subunits that sediments at 11 S. Each catalytic subunit contains 8 cysteine residues which were identified following tryptic digestion of the reduced, 14C-carboxymethylated protein. The tryptic peptides were purified by gel filtration followed by reverse-phase high performance liquid chromatography (HPLC) and then sequenced. The disulfide bonding profile was determined by treating the native, nonreduced 11 S form of acetylcholinesterase with a fluorescent, sulfhydryl-specific reagent, monobromobimane, prior to tryptic digestion. Peptides again were resolved by gel filtration and reverse-phase HPLC. One fluorescent cysteine-containing peptide was identified, indicating that a single sulfhydryl residue, Cys231, was present in its reduced form. Three pairs of disulfide-bonded peptides were identified. These were localized in the polypeptide chain based on the cDNA-deduced sequence of the protein and were identified as Cys67-Cys94, Cys254-Cys265, and Cys402-Cys521. Hence, three loops are found in the secondary structure. Cys572, located in the carboxyl-terminal tryptic peptide, was disulfide-bonded to an identical peptide and most likely forms an intersubunit cross-link. Since the 6 cysteine residues in acetylcholinesterase that are involved in intrachain disulfide bonds are conserved in the sequence of the homologous protein thyroglobulin, it is likely that both proteins share a common folding pattern in their respective tertiary structures. Cys231 and the carboxyl-terminal cysteine residue Cys572 are not conserved in thyroglobulin.     

The structure of rabbit extracellular superoxide dismutase differs from the human protein

The cDNA sequence encoding rabbit, mouse, and rat extracellular superoxide dismutase (EC-SOD) predicts that the protein contains five cysteine residues. Human EC-SOD contains an additional cysteine residue and folds into two forms with distinct disulfide bridge patterns. One form is enzymatically active (aEC-SOD), while the other is inactive (iEC-SOD). Due to the lack of the additional cysteine residue rabbit, mouse, and rat EC-SOD are unable to generate an inactive fold identical to human iEC-SOD. The amino acid sequences predict the formation of aEC-SOD only, but other folding variants cannot be ruled out based on the heterogeneity observed for human EC-SOD. To test this, we purified EC-SOD from rabbit plasma and determined the disulfide bridge pattern. The results revealed that the disulfide bridges are homogeneous and identical to human aEC-SOD. Four cysteine residues are involved in two intra-disulfide bonds while the C-terminal cysteine residue forms an intersubunit disulfide bond. No evidence for other folding variants was detected. These findings show that rabbit EC-SOD exists as an enzymatically active form only. The absence of iEC-SOD in rabbits suggests that the structure and aspects of the physiological function of EC-SOD differs significantly between rabbit and humans. This is an important notion to take when using these animals as model systems for oxidative stress.     

Enzymatic activity of the Arabidopsis sulfurtransferase resides in the C-terminal domain but is boosted by the N-terminal domain and the linker peptide in the full-length enzyme

Sulfurtransferases/rhodaneses are a group of enzymes widely distributed in plants, animals, and bacteria that catalyze the transfer of sulfur from a donor molecule to a thiophilic acceptor substrate. Sulfurtransferases (STs) consist of two globular domains of nearly identical size and conformation connected by a short linker sequence. In plant STs this linker sequence is exceptionally longer than in sequences from other species. The Arabidopsis ST1 protein (AJ131404) contains five cysteine residues: one residue is universally conserved in all STs and considered to be catalytically essential; a second one, closely located in the primary sequence, is conserved only in sequences from eukaryotic species. Of the remaining three cysteine residues two are conserved in the so far known plant STs and one is unique to the Arabidopsis ST1. The aim of our study was to investigate the role of the two-domain structure, of the unique plant linker sequence and of each cysteine residue. The N- and C-terminal domains of the Arabidopsis ST1, the full-length protein with a shortened linker sequence and several point-mutated proteins were overexpressed in E. coli, purified and used for enzyme activity measurements. The C-terminal domain itself displayed ST activity which could be increased by adding the separately prepared N-terminal domain. The activity of an ST1 derivative with a shortened linker sequence was reduced by more than 60% of the wild-type activity, probably because of a drastically reduced protein stability. The replacement of each cysteine residue resulted in mutant forms which differed significantly in their stability, in the specific ST activities, and in their kinetic parameters which were determined for 3-mercaptopyruvate as well as thiosulfate as sulfur substrates: mutation of the putative active site cysteine (C332) essentially abolished activity; for C339 a crucial role at least for the turnover of thiosulfate could be identified.     

Identification of sites of cysteine misincorporation during in vivo synthesis of bacteriophage T7 0.3 protein

The 0.3 protein encoded by coliphage T7 does not normally contain cysteine residues. Incorporation of [35S]cysteine can therefore be used to assay mistranslation. We have purified 0.3 protein, synthesized in the presence of [35S]cysteine, from T7 infected cells of E. coli and determined the locations of misincorporated cysteine residues. Analysis of the molecular weights (Mr) of [35S]cysteine-labeled tryptic peptides of 0.3 protein demonstrated that cysteine (encoded by UGU or UGC) is not extensively misincorporated, as might be predicted by substitution for arginine residues (encoded by CGU or CGC). Edman degradation of the amino-terminal 50 residues of [35S]cysteine-labeled 0.3 protein determined that cysteine was most frequently misincorporated at position 15, which is correctly occupied by a tyrosine residue (encoded by UAC). There are four other tyrosine codons (1 UAU; 3 UAC) in the region of the 0.3 protein studied, but these were not mistranslated. The context in which a codon is located must therefore be more important in causing mistranslation than the sequence of the codon itself. Misincorporation of [35S]cysteine was also found at positions 9 (ACC, asparagine), 16 (GAA, glutamic acid), 41 (GCC, alanine) and 42 (GAU, aspartic acid). One mistranslation event appears to increase the likelihood that the following codon will also be mistranslated. This clustering of misincorporated [35S]cysteine residues was accentuated in 0.3 protein synthesized in the presence of streptomycin.     

Substitution of serine for non-disulphide-bond-forming cysteine in grass carp (Ctenopharygodon idellus) growth hormone improves in vitro oxidative renaturation

Native grass carp (Ctenopharygodon idellus) growth hormone, has 5 cysteine amino acid residues, forms two disulphide bridges in its mature form. Recombinant grass carp growth hormone, when over-expressed in E. coli, forms inclusion bodies. In vitro oxidative renaturation of guanidine-hydrochloride dissolved recombinant grass carp growth hormone was achieved by sequential dilution and stepwise dialysis at pH 8.5. The redox potential of the refolding cocktail was maintained by glutathione disulphide/glutathione couple. The oxidative refolded protein is heterogeneous, and contains multimers, oligomers and monomers. The presence of non-disulphide-bond-forming cysteine in recombinant grass carp growth hormone enhances intermolecular disulphide bond formation and also nonnative intramolecular disulphide bond formation during protein folding. The non-disulphide-bond-forming cysteine was converted to serine by PCR-mediated site-directed mutagenesis. The resulting 4-cysteine grass carp growth hormone has improved in vitro oxidative refolding properties when studied by gel filtration and reverse phase chromatography. The refolded 4-cysteine form has less hydrophobic aggregate and has only one monomeric isoform. Both refolded 4-cysteine and 5-cysteine forms are active in radioreceptor binding assay.     

An episulfide cation (thiiranium ring) trapped in the active site of HAV 3C proteinase inactivated by peptide-based ketone inhibitors

We have solved the crystal and molecular structures of hepatitis A viral (HAV) 3C proteinase, a cysteine peptidase having a chymotrypsin-like protein fold, in complex with each of three tetrapeptidyl-based methyl ketone inhibitors to resolutions beyond 1.4 A, the highest resolution to date for a 3C or a 3C-Like (e.g. SARS viral main proteinase) peptidase. The residues of the beta-hairpin motif (residues 138-158), an extension of two beta-strands of the C-terminal beta-barrel of HAV 3C are critical for the interactions between the enzyme and the tetrapeptide portion of these inhibitors that are analogous to the residues at the P4 to P1 positions in the natural substrates of picornaviral 3C proteinases. Unexpectedly, the Sgamma of Cys172 forms two covalent bonds with each inhibitor, yielding an unusual episulfide cation (thiiranium ring) stabilized by a nearby oxyanion. This result suggests a mechanism of inactivation of 3C peptidases by methyl ketone inhibitors that is distinct from that occurring in the structurally related serine proteinases or in the papain-like cysteine peptidases. It also provides insight into the mechanisms underlying both the inactivation of HAV 3C by these inhibitors and on the proteolysis of natural substrates by this viral cysteine peptidase.     

The occurrence of disulphide bonds in purified clathrin light chains.

Three forms of clathrin light chain contain two cysteine residues. These are the predominant brain-specific forms of LCa and LCb and the non-brain form of LCb. After purification in the absence of thiols they contain intramolecular disulphide bonds. The reduced and the oxidized forms show differences in electrophoretic mobility, explaining the variable and heterogeneous patterns observed on electrophoresis. Accessibility of the thiol groups in the free light chains is greater than when they are associated with the heavy chain. In contrast the cysteine residues of the clathrin heavy chain are completely inaccessible in the absence of denaturants and are not found in disulphide bonds. The antigenic properties of the oxidized and the reduced forms of the clathrin light chains are similar, as is their capacity to bind to the clathrin heavy chain. After isolation in the presence of 10 mM-iodoacetamide, the light-chain cysteine residues are fully alkylated. The results are consistent with the reduced form being the native state and the light-chain disulphide bonds an artifact of isolation.