Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins

Intramolecular disulfide bond formation is promoted in oxidizing extracellular and endoplasmic reticulum compartments and often contributes to protein stability and function. DUOX1 and DUOX2 are distinguished from other members of the NOX protein family by the presence of a unique extracellular N-terminal region. These peroxidase-like domains lack the conserved cysteines that confer structural stability to mammalian peroxidases. Sequence-based structure predictions suggest that the thiol groups present are solvent-exposed on a single protein surface and are too distant to support intramolecular disulfide bond formation. To investigate the role of these thiol residues, we introduced four individual cysteine to glycine mutations in the peroxidase-like domains of both human DUOXs and purified the recombinant proteins. The mutations caused little change in the stabilities of the monomeric proteins, supporting the hypothesis that the thiol residues are solvent-exposed and not involved in disulfide bonds that are critical for structural integrity. However, the ability of the isolated hDUOX1 peroxidase-like domain to dimerize was altered, suggesting a role for these cysteines in protein-protein interactions that could facilitate homodimerization of the peroxidase-like domain or, in the full-length protein, heterodimeric interactions with a maturation protein. When full-length hDUOX1 was expressed in HEK293 cells, the mutations resulted in decreased H₂O₂ production that correlated with a decreased amount of the enzyme localized to the membrane surface rather than with a loss of activity or with a failure to synthesize the mutant proteins. These results support a role for the cysteine residues in intermolecular disulfide bond formation with the DUOX maturation factor DUOXA1.     

Role of individual disulfide bonds in the structural maturation of a low molecular weight glutenin subunit.

Gliadins and glutenins are the major storage proteins that accumulate in wheat endosperm cells during seed development. Although gliadins are mainly monomeric, glutenins consist of very large disulfide-linked polymers made up of high molecular weight and low molecular weight subunits. These polymers are among the largest protein molecules known in nature and are the most important determinants of the viscoelastic properties of gluten. As a first step toward the elucidation of the folding and assembly pathways that lead to glutenin polymer formation, we have exploited an in vitro system composed of wheat germ extract and bean microsomes to examine the role of disulfide bonds in the structural maturation of a low molecular weight glutenin subunit. When conditions allowing the formation of disulfide bonds were established, the in vitro synthesized low molecular weight glutenin subunit was recovered in monomeric form containing intrachain disulfide bonds. Conversely, synthesis under conditions that did not favor the formation of disulfide bonds led to the production of large aggregates from which the polypeptides could not be rescued by the post-translational generation of a more oxidizing environment. These results indicate that disulfide bond formation is essential for the conformational maturation of the low molecular weight glutenin subunit and suggest that early folding steps may play an important role in this process, allowing the timely pairing of critical cysteine residues. To determine which cysteines were important to maintain the protein in monomeric form, we prepared a set of mutants containing selected cysteine to serine substitutions. Our results show that two conserved cysteine residues form a critical disulfide bond that is essential in preventing the exposure of adhesive domains and the consequent formation of aberrant aggregates.     

The susceptibility of disulfide bonds to modification in keratin fibers undergoing tensile stress

Cysteine residues perform a dual role in mammalian hairs. The majority help stabilize the overall assembly of keratins and their associated proteins, but a proportion of inter-molecular disulfide bonds are assumed to be associated with hair mechanical flexibility. Hair cortical microstructure is hierarchical, with a complex macro-molecular organization resulting in arrays of intermediate filaments at a scale of micrometres. Intermolecular disulfide bonds occur within filaments and between them and the surrounding matrix. Wool fibers provide a good model for studying various contributions of differently situated disulfide bonds to fiber mechanics. Within this context, it is not known if all intermolecular disulfide bonds contribute equally, and, if not, then do the disproportionally involved cysteine residues occur at common locations on proteins? In this study, fibers from Romney sheep were subjected to stretching or to their breaking point under wet or dry conditions to detect, through labeling, disulfide bonds that were broken more often than randomly. We found that some cysteines were labeled more often than randomly and that these vary with fiber water content (water disrupts protein-protein hydrogen bonds). Many of the identified cysteine residues were located close to the terminal ends of keratins (head or tail domains) and keratin-associated proteins. Some cysteines in the head and tail domains of type II keratin K85 were labeled in all experimental conditions. When inter-protein hydrogen bonds were disrupted under wet conditions, disulfide labeling occurred in the head domains of type II keratins, likely affecting keratin-keratin-associated protein interactions, and tail domains of the type I keratins, likely affecting keratin-keratin interactions. In contrast, in dry fibers (containing more protein-protein hydrogen bonding), disulfide labeling was also observed in the central domains of affected keratins. This central “rod” region is associated with keratin-keratin interactions between anti-parallel heterodimers in the tetramer of the intermediate filament.     

Functional analysis of a disulfide bond in the cardiac Na(+)-Ca(2+) exchanger

The electrophoretic mobility of the cardiac Na(+)-Ca(2+) exchange protein is different under reducing and nonreducing conditions. This mobility shift is eliminated in a cysteine-less exchanger, suggesting that the presence or absence of an intramolecular disulfide bond alters the conformation and mobility of the exchanger. Using cysteine mutagenesis and biochemical analysis, we have identified the cysteine residues involved in the disulfide bond. Cysteine 792 in loop h of the exchanger forms a disulfide bond with either cysteine 14 or 20 near the NH(2) terminus. Because the NH(2) terminus is extracellular, the data establish that loop h must also be extracellular. A rearrangement of disulfide bonds has previously been implicated in the stimulation of exchange activity by combinations of reducing and oxidizing agents. We have investigated the role of cysteines in the stimulation of the exchanger by the combination of FeSO(4) and dithiothreitol (Fe-DTT). Using the giant excised patch technique, we find that stimulation of the wild type exchanger by Fe-DTT is primarily due to the removal of a Na(+)-dependent inactivation process. Analysis of mutated exchangers, however, indicates that cysteines are not responsible for stimulation of the exchange activity by Fe-DTT. Ca(2+) blocks modification of the exchanger by Fe-DTT. Disulfide bonds are not involved in redox stimulation of the exchanger, and the modification reaction is unknown. Modulation of Na(+)-dependent inactivation may be a general mechanism for regulation of Na(+)-Ca(2+) exchange activity and may have physiological significance.     

Disulfide bonds in rat cutaneous fatty acid-binding protein

Unlike other fatty acid-binding proteins, cutaneous (epidermal) fatty acid-binding proteins contain a large number of cysteine residues. The status of the five cysteine residues in rat cutaneous fatty acid-binding protein was examined by chemical and mass-spectrometric analyses. Two disulfide bonds were identified, between Cys-67 and Cys-87, and between Cys-120 and Cys-127, though extent of formation of the first disulfide bond was rather low in another preparation. Cys-43 was free cysteine. Homology modeling study of the protein indicated the close proximity of the sulfur atoms of these cysteine pairs, supporting the presence of the disulfide bonds. These disulfide bonds appear not to be directly involved in fatty acid-binding activity, because a recombinant rat protein expressed in Escherichia coli in which all five cysteines are fully reduced showed fatty acid-binding activity as examined by displacement of a fluorescent fatty acid analog by long-chain fatty acids. However, the fact that the evolutionarily distant shark liver fatty acid-binding protein also has a disulfide bond corresponding to the one between Cys-120 and Cys-127, and that fatty acid-binding proteins play multiple roles suggests that some functions of cutaneous fatty acid-binding protein might be regulated by the cellular redox state through formation and reduction of disulfide bonds. Although we cannot completely exclude the possibility of oxidation during preparation and analysis, it is remarkable that a protein in cytosol under normally reducing conditions appears to contain disulfide bonds.     

Structure of the Janus protein human CLIC2

Chloride intracellular channel (CLIC) proteins possess the remarkable property of being able to convert from a water-soluble state to a membrane channel state. We determined the three-dimensional structure of human CLIC2 in its water-soluble form by X-ray crystallography at 1.8-Å resolution from two crystal forms. In contrast to the previously characterized CLIC1 protein, which forms a possibly functionally important disulfide-induced dimer under oxidizing conditions, we show that CLIC2 possesses an intramolecular disulfide and that the protein remains monomeric irrespective of redox conditions. Site-directed mutagenesis studies show that removal of the intramolecular disulfide or introduction of cysteine residues in CLIC2, equivalent to those that form the intramolecular disulfide in CLIC1, does not cause dimer formation under oxidizing conditions. We also show that CLIC2 forms pH-dependent chloride channels in vitro with higher channel activity at low pH levels and that the channels are subject to redox regulation. In both crystal forms, we observed an extended loop region from the C-terminal domain, called the foot loop, inserting itself into an interdomain crevice of a neighboring molecule. The equivalent region in the structurally related glutathione transferase superfamily corresponds to the active site. This so-called foot-in-mouth interaction suggests that CLIC2 might recognize other proteins such as the ryanodine receptor through a similar interaction.     

Nonnative interactions between cysteines direct productive assembly of P22 tailspike protein

Nonnative disulfide bond formation can play a critical role in the assembly of disulfide bonded proteins. During the folding and assembly of the P22 tailspike protein, nonnative disulfide bonds form both in vivo and in vitro. However, the mechanism and identity of cysteine disulfide pairs remains elusive, particularly for P22 tailspike, which contains no disulfide bonds in its native, functional form. Understanding the interactions between cysteine residues is important for developing a mechanistic model for the role of nonnative cysteines in P22 tailspike assembly. Prior in vivo studies have suggested that cysteines 496, 613, and 635 are the most likely site for sulfhydryl reactivity. Here we demonstrate that these three cysteines are critical for efficient assembly of tailspike trimers, and that interactions between cysteine pairs lead to productive assembly of native tailspike.     

Human pancreas-specific protein disulfide isomerase homolog (PDIp) is redox-regulated through formation of an inter-subunit disulfide bond

Protein disulfide isomerase (PDI) and its homologs are catalysts of the formation of disulfide bonds in secretory proteins, and they also serve as molecular chaperones. In the present study, we investigated the redox-mediated regulation of the structures and functions of human pancreas-specific PDI homolog (PDIp). We found that formation of an inter-subunit disulfide bond in the recombinant human PDIp can alter not only its structure, but also its functions. PDIp exists predominantly as monomer under reducing conditions, but the dimeric form is significantly increased following the removal of the reducing agent, due to the formation of an inter-subunit disulfide bond. The oxidized PDIp (with an inter-subunit disulfide bond) appears to expose more hydrophobic patches and is more sensitive to protease digestion compared to the reduced form. Along with these structural changes, the oxidized PDIp also exhibits an enhanced chaperone activity. The formation of the inter-subunit disulfide bond in PDIp is mainly contributed by its non-active cysteine residue (cysteine-4), which is only present in human and primate PDIp, but not in rodent PDIp. In addition, we observed that the formation of the inter-subunit disulfide bond in PDIp is redox-dependent and is favored under oxidizing conditions, and that PDIp can function as a chaperone to form stable complexes with various non-native cellular proteins, particularly under oxidizing conditions. In light of these observations, it is concluded that the structures and functions of human PDIp are redox-regulated through formation of an inter-subunit disulfide bond between two cysteine-4 residues.     

ATP analogue binding to the A subunit induces conformational changes in the E subunit that involves a disulfide bond formation in plant V-ATPase.

Vacuolar H+-ATPase (V-ATPase) consists of a catalytic head, a stalk part and a membrane domain. We indirectly investigated the interaction between the A subunit (catalytic head) and the E subunit (stalk part) using an ATP analogue, adenosine 5'-[beta,gamma-imino]triphosphate (AMP-PNP), which holds the enzyme in the substrate-binding state. AMP-PNP treatment caused a mobility shift of the E subunit with a faster migration in SDS/polyacrylamide gel electrophoresis without a reductant, while ATP treatment did not. A mobility shift of the E subunit has been detected in several plants. As polypeptides with intramolecular disulfide bonds migrate faster than those without disulfide bonds, the mobility shift may be due to the formation of an intramolecular disulfide bond by two cysteine residues conserved among several plant species. The mobility shift may be involved in the binding of AMP-PNP to the ATP-binding site, which exists in the A and B subunits, as it was inhibited by the addition of ATP. Pretreatment with 2'-3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), which modifies the ATP-binding site of the B subunit under UV illumination, did not inhibit the mobility shift of the E subunit caused by AMP-PNP treatment. The response of V-ATPase following the AMP-PNP binding may cause a conformational change in the E subunit into a form that is susceptible to oxidation of cysteine residues. This is the first demonstration of interaction between the A and E subunits in the substrate-binding state of a plant V-ATPase.     

Disulfide bonding in influenza virus proteins as revealed by polyacrylamide gel electrophoresis.

Disulfide bonding in the major proteins of influenza virus A, WSN strain, was studied by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels under reducing and nonreducing conditions. The electrophoretic behavior of the proteins correlated with their localization in the virions and their chemical composition. The internal proteins of the viral particles, i.e. matrix and nucleoproteins, were shown to contain a relatively small number of cysteine residues. Electrophoresis under nonreducing conditions yielded multiple forms of the proteins which could be discriminated by small but readily observable, reproducible differences in their migration rates in the gel. the multiplicity of the protein forms was caused by the formation of intramolecular disulfide bonds in matrix and nucleoproteins that arose during or after solubilization in sodium dodecyl sulfate. On the other hand, we failed to detect native inter- and intramolecular linkages in matrix and nucleoproteins. External glycoproteins of the virions (HA and NA) had, in contrast to the internal ones, a higher number of cysteine residues and native disulfide bonds. At least three disulfide linkages were revealed in HA and NA in our experiments. In uncleaved HA all of the linkages were intramolecular. In NA at least one disulfide bond linked two identical polypeptides into a dimer. It was established that the reduction of the different disulfide linkages in HA and NA required different concentrations of the reducing agent.     

S-linked protein homocysteinylation: identifying targets based on structural, physicochemical and protein–protein interactions of homocysteinylated proteins

An elevated level of homocysteine, a thiol-containing amino acid is associated with a wide spectrum of disease conditions. A majority (>80 %) of the circulating homocysteine exist in protein-bound form. Homocysteine can bind to free cysteine residues in the protein or could cleave accessible cysteine disulfide bonds via thiol disulfide exchange reaction. Binding of homocysteine to proteins could potentially alter the structure and/or function of the protein. To date only 21 proteins have been experimentally shown to bind homocysteine. In this study we attempted to identify other proteins that could potentially bind to homocysteine based on the criteria that such proteins will have significant 3D structural homology with the proteins that have been experimentally validated and have solvent accessible cysteine residues either with high dihedral strain energy (for cysteine–cysteine disulfide bonds) or low pKa (for free cysteine residues). This analysis led us to the identification of 78 such proteins of which 68 proteins had 154 solvent accessible disulfide cysteine pairs with high dihedral strain energy and 10 proteins had free cysteine residues with low pKa that could potentially bind to homocysteine. Further, protein–protein interaction network was built to identify the interacting partners of these putative homocysteine binding proteins. We found that the 21 experimentally validated proteins had 174 interacting partners while the 78 proteins identified in our analysis had 445 first interacting partners. These proteins are mainly involved in biological activities such as complement and coagulation pathway, focal adhesion, ECM-receptor, ErbB signalling and cancer pathways, etc. paralleling the disease-specific attributes associated with hyperhomocysteinemia.     

Proteinase inhibition by proform of eosinophil major basic protein (pro-MBP) is a multistep process of intra- and intermolecular disulfide rearrangements

The metzincin metalloproteinase pregnancy-associated plasma protein A (PAPP-A, pappalysin-1) promotes cell growth by the cleavage of insulin-like growth factor-binding proteins-4 and -5, causing the release of bound insulin-like growth factors. The proteolytic activity of PAPP-A is inhibited by the proform of eosinophil major basic protein (pro-MBP), which forms a covalent 2:2 proteinase-inhibitor complex based on disulfide bonds. To understand the process of complex formation, we determined the status of cysteine residues in both of the uncomplexed molecules. A comparison of the disulfide structure of the reactants with the known disulfide structure of the PAPP-A.pro-MBP complex reveals that six cysteine residues of the pro-MBP subunit (Cys-51, Cys-89, Cys-104, Cys-107, Cys-128, and Cys-169) and two cysteine residues of the PAPP-A subunit (Cys-381 and Cys-652) change their status from the uncomplexed to the complexed states. Upon complex formation, three disulfide bonds of pro-MBP, which connect the acidic propiece with the basic, mature portion, are disrupted. In the PAPP-A.pro-MBP complex, two of these form the basis of both two interchain disulfide bonds between the PAPP-A and the pro-MBP subunits and two disulfide bonds responsible for pro-MBP dimerization, respectively. Based on the status of the reactants, we investigated the role of individual cysteine residues upon complex formation by mutagenesis of specific cysteine residues of both subunits. Our findings allow us to depict a hypothetical model of how the PAPPA.pro-MBP complex is formed. In addition, we have demonstrated that complex formation is greatly enhanced by the addition of micromolar concentrations of reductants. It is therefore possible that the activity in vivo of PAPP-A is controlled by the redox potential, and it is further tempting to speculate that such mechanism operates under pathological conditions of altered redox potential.     

An engineered C‐terminal disulfide bond can partially replace the phaseolin vacuolar sorting signal

Seed storage proteins accumulate either in the endoplasmic reticulum (ER) or in vacuoles, and it would appear that polymerization events play a fundamental role in regulating the choice between the two destinies of these proteins. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N‐terminal half of the Zea mays prolamin γ‐zein forms interchain disulfide bonds that facilitate the formation of ER‐located protein bodies. Wild‐type phaseolin does not contain cysteine residues, and assembles into soluble trimers that transiently polymerize before sorting to the vacuole. These transient interactions are abolished when the C‐terminal vacuolar sorting signal AFVY is deleted, indicating that they play a role in vacuolar sorting. We reasoned that if the phaseolin interactions directly involve the C terminus of the polypeptide, a cysteine residue introduced into this region could stabilize these transient interactions. Biochemical studies of two mutated phaseolin proteins in which a single cysteine residue was inserted at the C terminus, in the presence (PHSL*) or absence (Δ418*) of the vacuolar signal AFVY, revealed that these mutated proteins form disulphide bonds. PHSL* had reduced protein solubility and a vacuolar trafficking delay with respect to wild‐type protein. Moreover, Δ418* was in part redirected to the vacuole. Our experiments strongly support the idea that vacuolar delivery of phaseolin is promoted very early in the sorting process, when polypeptides are still contained within the ER, by homotypic interactions.     

Thioredoxins and glutaredoxins as facilitators of protein folding

Thiol-disulfide oxidoreductase systems of bacterial cytoplasm and eukaryotic cytosol favor reducing conditions and protein thiol groups, while bacterial periplasm and eukaryotic endoplasmatic reticulum provide oxidizing conditions and a machinery for disulfide bond formation in the secretory pathway. Oxidoreductases of the thioredoxin fold superfamily catalyze steps in oxidative protein folding via protein-protein interactions and covalent catalysis to act as chaperones and isomerases of disulfides to generate a native fold. The active site dithiol/disulfide of thioredoxin fold proteins is CXXC where variations of the residues inside the disulfide ring are known to increase the redox potential like in protein disulfide isomerases. In the catalytic mechanism thioredoxin fold proteins bind to target proteins through conserved backbone-backbone hydrogen bonds and induce conformational changes of the target disulfide followed by nucleophilic attack by the N-terminally located low pK(a) Cys residue. This generates a mixed disulfide covalent bond which subsequently is resolved by attack from the C-terminally located Cys residue. This review will focus on two members of the thioredoxin superfamily of proteins known to be crucial for maintaining a reduced intracellular redox state, thioredoxin and glutaredoxin, and their potential functions as facilitators and regulators of protein folding and chaperone activity.     

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.     

Reversible silencing of CFTR chloride channels by glutathionylation

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and ATP-dependent chloride channel that modulates salt and water transport across lung and gut epithelia. The relationship between CFTR and oxidized forms of glutathione is of potential interest because reactive glutathione species are produced in inflamed epithelia where they may be modulators or substrates of CFTR. Here we show that CFTR channel activity in excised membrane patches is markedly inhibited by several oxidized forms of glutathione (i.e., GSSG, GSNO, and glutathione treated with diamide, a strong thiol oxidizer). Three lines of evidence indicate that the likely mechanism for this inhibitory effect is glutathionylation of a CFTR cysteine (i.e., formation of a mixed disulfide with glutathione): (a) channels could be protected from inhibition by pretreating the patch with NEM (a thiol alkylating agent) or by lowering the bath pH; (b) inhibited channels could be rescued by reducing agents (e.g., DTT) or by purified glutaredoxins (Grxs; thiol disulfide oxidoreductases) including a mutant Grx that specifically reduces mixed disulfides between glutathione and cysteines within proteins; and (c) reversible glutathionylation of CFTR polypeptides in microsomes could be detected biochemically under the same conditions. At the single channel level, the primary effect of reactive glutathione species was to markedly inhibit the opening rates of individual CFTR channels. CFTR channel inhibition was not obviously dependent on phosphorylation state but was markedly slowed when channels were first "locked open" by a poorly hydrolyzable ATP analogue (AMP-PNP). Consistent with the latter finding, we show that the major site of inhibition is cys-1344, a poorly conserved cysteine that lies proximal to the signature sequence in the second nucleotide binding domain (NBD2) of human CFTR. This region is predicted to participate in ATP-dependent channel opening and to be occluded in the nucleotide-bound state of the channel based on structural comparisons to related ATP binding cassette transporters. Our results demonstrate that human CFTR channels are reversibly inhibited by reactive glutathione species, and support an important role of the region proximal to the NBD2 signature sequence in ATP-dependent channel opening.     

Cross‐strand disulfides in the hydrogen bonding site of antiparallel β‐sheet (aCSDhs): Forbidden disulfides that are highly strained,easily broken

Some disulfide bonds perform important structural roles in proteins, but another group has functional roles via redox reactions. Forbidden disulfides are stressed disulfides found in recognizable protein contexts, which currently constitute more than 10% of all disulfides in the PDB. They likely have functional redox roles and constitute a major subset of all redox‐active disulfides. The torsional strain of forbidden disulfides is typically higher than for structural disulfides, but not so high as to render them immediately susceptible to reduction under physionormal conditions. Previously we characterized the most abundant forbidden disulfide in the Protein Data Bank, the aCSDn: a canonical motif in which disulfide‐bonded cysteine residues are positioned directly opposite each other on adjacent anti‐parallel β‐strands such that the backbone hydrogen‐bonded moieties are directed away from each other. Here we perform a similar analysis for the aCSDh, a less common motif in which the opposed cysteine residues are backbone hydrogen bonded. Oxidation of two Cys in this context places significant strain on the protein system, with the β‐chains tilting toward each other to allow disulfide formation. Only left‐handed aCSDh conformations are compatible with the inherent right‐handed twist of β‐sheets. aCSDhs tend to be more highly strained than aCSDns, particularly when both hydrogen bonds are formed. We discuss characterized roles of aCSDh motifs in proteins of the dataset, which include catalytic disulfides in ribonucleotide reductase and ahpC peroxidase as well as a redox‐active disulfide in P1 lysozyme, involved in a major conformation change. The dataset also includes many binding proteins.     

Disulfide bond formation during the folding of influenza virus hemagglutinin

To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.     

Paired natural cysteine mutation mapping: aid to constraining models of protein tertiary structure.

This paper discusses the benefit of mapping paired cysteine mutation patterns as a guide to identifying the positions of protein disulfide bonds. This information can facilitate the computer modeling of protein tertiary structure. First, a simple, paired natural-cysteine-mutation map is presented that identifies the positions of putative disulfide bonds in protein families. The method is based on the observation that if, during the process of evolution, a disulfide-bonded cysteine residue is not conserved, then it is likely that its counterpart will also be mutated. For each target protein, protein databases were searched for the primary amino acid sequences of all known members of distinct protein families. Primary sequence alignment was carried out using PileUp algorithms in the GCG package. To search for correlated mutations, we listed only the positions where cysteine residues were highly conserved and emphasized the mutated residues. In proteins of known three-dimensional structure, a striking pattern of paired cysteine mutations correlated with the positions of known disulfide bridges. For proteins of unknown architecture, the mutation maps showed several positions where disulfide bridging might occur.     

Essential cysteine residues for human RNase κ catalytic activity

Human RNase κ is an endoribonuclease expressed in almost all tissues and organs and belongs to a highly conserved protein family bearing representatives in all metazoans. To gain insight into the role of cysteine residues in the enzyme activity or structure, a recombinant active form of human RNase κ expressed in Pichia pastoris was treated with alkylating agents and dithiothreitol (DTT). Our results showed that the human enzyme is inactivated by DDT, while it remains fully active in the presence of alkylating agents. The unreduced recombinant protein migrates on SDS/PAGE faster than the reduced form. This observation in combination with the above findings indicated that human RNase κ does not form homodimers through disulfide bridges, and cysteine residues are not implicated in RNA catalysis but participate in the formation of intramolecular disulfide bond(s) essential for its ribonucleolytic activity. The role of the cysteine residues was further investigated by expression and study of Cys variants. Ribonucleolytic activity experiments and SDS/PAGE analysis of the wild-type and mutant proteins under reducing and non-reducing conditions demonstrated that Cys7, Cys14 and Cys85 are not essential for RNase activity. On the other hand, replacement of Cys6 or Cys69 with serine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of human RNase κ by forming a disulfide bond. Due to the absolute conservation of these cysteine residues, the Cys6-Cys69 disulfide bond is likely to exist in all RNase κ family members.