Assembly of mutant subunits of the nicotinic acetylcholine receptor lacking the conserved disulfide loop structure.

Each subunit of the nicotinic acetylcholine receptor (AChR) contains two conserved cysteine residues, which are known to form a disulfide bond, in the N-terminal extracellular domain. The role of this retained structural feature in the biogenesis of the AChR was studied by expressing site-directed mutant alpha and beta subunits together with other normal subunits from Torpedo californica AChR in Xenopus oocytes. Mutation of the cysteines at position 128 or 142 in the alpha subunit, or in the beta subunit, did not prevent subunit assembly. All Cys128 and Cys142 mutants of the alpha and beta subunits were able to associate with coexpressed other normal subunits, although associational efficiency of the mutant alpha subunits with the delta subunit was reduced. Functional studies of the mutant AChR complexes showed that the mutations in the alpha subunit abolished detectable 125I-alpha-bungarotoxin (alpha-BuTX) binding in whole oocytes, whereas the mutations in the beta subunit resulted in decreased total binding of 125I-alpha-BuTX and no detectable surface 125I-alpha-BuTX binding. Additionally, all mutant subunits, when co-expressed with the other normal subunits in oocytes, produced small acetylcholine-activated membrane currents, suggesting incorporation of only small numbers of functional mutant AChRs into the plasma membrane. The functional acetylcholine-gated ion channel formed with mutant alpha subunits, but not mutant beta subunits, could not be blocked by alpha-BuTX. Thus, a disulfide bond between Cys128 and Cys142 of the AChR alpha or beta subunits is not needed for acetylcholine-binding. However, this disulfide bond on the alpha subunit is necessary for formation of the alpha-BuTX-binding site. These results also suggest that the most significant effect caused by disrupting the conserved disulfide loop structure is intracellular retention of most of the assembled AChR complexes.     

A comparison of the cyclic nucleotide-dependent protein kinases using chemical cleavage at tryptophan and cysteine

cGMP-dependent protein kinase (G-kinase) and the regulatory subunit of type I (RI) cAMP-dependent protein kinase (A-kinase) both contain a phosphorylation site located near the NH2 terminus of each enzyme. These sites can be utilized as convenient markers for the determination of the position of an amino acid residue susceptible to either chemical or enzymatic digestion. Using the tryptophan-specific reagent, N-chlorosuccinimide, the approximate location along the polypeptide chain of six reactive tryptophans in G-kinase and three reactive residues in RI were identified. Similarly, cleavage with cyanide was used to locate free and disulfide-bonded cysteines in both proteins. The approximate positions of nine cysteines in G-kinase were determined along with the location of the interchain disulfide bond and an intrachain disulfide bond. RI was found to contain three cyanide-reactive cysteines, two of which are involved in interchain disulfide bonding. A comparison of the positions of the cysteines and tryptophans determined by chemical cleavage in G-kinase and RI, with the positions of cysteine and tryptophan in the known sequence of the type II A-kinase, support the structural relationships between these enzymes. Comparison with subsequently reported primary sequences of all three enzymes indicates the limits of precision of this chemical cleavage procedure.     

The gibberellin-induced, cysteine-rich protein GIP2 from Petunia hybrida exhibits in planta antioxidant activity

Numerous GAST-like genes have been identified in various plant species. All code for small proteins with a conserved C-terminal region in which 12 cysteines are located in exactly the same positions. We have previously identified five gibberellin (GA)-induced GAST1-like genes in petunia, GIP1-5. GIP2 is expressed in elongating zones, and its suppression in transgenic petunia plants inhibits stem elongation, suggesting a role for the protein in GA-induced cell growth. However, nothing is known about the biochemical activity of GIP2 or any other GAST-like protein. As all contain putative catalytic disulfide bonds (putative redox-active cysteines), we speculated that they might be involved in redox regulation. Expression analysis of GIP2, GIP4 and GIP5 revealed that they are induced by H(2)O(2). To study whether GIP2 modulates H(2)O(2) levels, we generated transgenic petunia plants expressing GIP2 under the regulation of the ubiquitous CaMV 35S promoter. The transgene reduced H(2)O(2) levels in leaves following wounding. It also reduced the levels of H(2)O(2) in guard cells following osmotic stress and ABA treatments, leading to the suppression of stomatal closure. In addition, the transgene promoted stem and corolla elongation. As reactive oxygen species (ROS) are involved in cell elongation, we suggest that GIP2 affects growth by regulating the levels of ROS. As all known GAST-like proteins contain putative redox-active cysteines, they may all act as antioxidants.     

Redox-active cysteines of a membrane electron transporter DsbD show dual compartment accessibility

The membrane-embedded domain of the unusual electron transporter DsbD (DsbDbeta) uses two redox-active cysteines to catalyze electron transfer between thioredoxin-fold polypeptides on opposite sides of the bacterial cytoplasmic membrane. How the electrons are transferred across the membrane is unknown. Here, we show that DsbDbeta displays an inherent functional and structural symmetry: first, the two cysteines of DsbDbeta can be alkylated from both the cytoplasm and the periplasm. Second, when the two cysteines are disulfide-bonded, cysteine scanning shows that the C-terminal halves of the cysteine-containing transmembrane segments 1 and 4 are exposed to the aqueous environment while the N-terminal halves are not. Third, proline residues located pseudo-symmetrically around the two cysteines are required for redox activity and accessibility of the cysteines. Fourth, mixed disulfide complexes, apparent intermediates in the electron transfer process, are detected between DsbDbeta and thioredoxin molecules on each side of the membrane. We propose a model where the two redox-active cysteines are located at the center of the membrane, accessible on both sides of the membrane to the thioredoxin proteins.     

Relevance of cysteine residues for biosynthesis and antigenicity of human hepatitis B virus e protein.

The mature form of the secretory core protein (HBe protein) of human hepatitis B virus contains four cysteines which are located at amino acid positions -7, 48, 61, and 107 relative to the HBc start methionine. In addition, there is a cysteine, Cys-183, located in the C-terminal domain of the HBe precursor, which is cleaved during HBe maturation. Here, the significance of these cysteines for biosynthesis and antigenicity of the HBe protein was examined. The cysteines at positions -7 and 61 were found to be crucial for HBe biosynthesis. As has already been described, if the Cys at position -7 is mutated, disulfide-linked HBe homodimers which have both HBe antigenicity and HBc antigenicity are expressed. Here we show that these dimers are due to Cys-61-Cys-61 disulfide bridges which are formed only if the Cys at position -7 is not present. In the wild-type protein, this dimerization appears to be inhibited by formation of intramolecular disulfide bridges between the Cys at -7 and one of the internal cysteines. Moreover, Cys-61 is important for HBe biosynthesis in general since mutation of this amino acid results in production of HBe proteins which are either only poorly secreted or possess a different antigenicity.     

Intramembranal disulfide cross-linking elucidates the super-quaternary structure of mammalian CatSpers

CatSper is a voltage-dependent calcium channel located in the plasma membrane of the sperm flagellum and is responsible for triggering hyperactive motility. A homology model for the transmembrane region was built in which the arrangement of the subunits around the pseudo-four-fold symmetry axis was deduced by the pairing of conserved transmembranal cysteines across mammals. Directly emergent of the predicted quaternary structure is an architecture in which tetramers polymerize through additional, highly conserved cysteines, creating one or more double-rows channels extending the length of the principal piece of the mammalian sperm tail. The few species that are missing these cysteines are eusocial or otherwise monogamous, suggesting that sperm competition is selective for a disulfide-crosslinked macromolecular architecture. The model suggests testable hypotheses for how CatSper channel opening might behave in response to pH, 2-arachidonoylglycerol, and mechanical force. A flippase function is hypothesized, and a source of the concomitant disulfide isomerase activity is found in CatSper-associated proteins β, δ and ε.     

Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site

BACKGROUND: Cyanase is an enzyme found in bacteria and plants that catalyzes the reaction of cyanate with bicarbonate to produce ammonia and carbon dioxide. In Escherichia coli, cyanase is induced from the cyn operon in response to extracellular cyanate. The enzyme is functionally active as a homodecamer of 17 kDa subunits, and displays half-site binding of substrates or substrate analogs. The enzyme shows no significant amino acid sequence homology with other proteins. RESULTS: We have determined the crystal structure of cyanase at 1.65 A resolution using the multiwavelength anomalous diffraction (MAD) method. Cyanase crystals are triclinic and contain one homodecamer in the asymmetric unit. Selenomethionine-labeled protein offers 40 selenium atoms for use in phasing. Structures of cyanase with bound chloride or oxalate anions, inhibitors of the enzyme, allowed identification of the active site. CONCLUSIONS: The cyanase monomer is composed of two domains. The N-terminal domain shows structural similarity to the DNA-binding alpha-helix bundle motif. The C-terminal domain has an 'open fold' with no structural homology to other proteins. The subunits of cyanase are arranged in a novel manner both at the dimer and decamer level. The dimer structure reveals the C-terminal domains to be intertwined, and the decamer is formed by a pentamer of these dimers. The active site of the enzyme is located between dimers and is comprised of residues from four adjacent subunits of the homodecamer. The structural data allow a conceivable reaction mechanism to be proposed.     

Acetylcholinesterase associates differently with its anchoring proteins ColQ and PRiMA

Acetylcholinesterase tetramers are inserted in the basal lamina of neuromuscular junctions or anchored in cell membranes through the interaction of four C-terminal t peptides with proline-rich attachment domains (PRADs) of cholinesterase-associated collagen Q (ColQ) or of the transmembrane protein PRiMA (proline-rich membrane anchor). ColQ and PRiMA differ in the length of their proline-rich motifs (10 and 15 residues, respectively). ColQ has two cysteines upstream of the PRAD, which are disulfide-linked to two AChE(T) subunits ("heavy" dimer), and the other two subunits are disulfide-linked together ("light" dimer). In contrast, PRiMA has four cysteines upstream of the PRAD. We examined whether these cysteines could be linked to AChE(T) subunits in complexes formed with PRiMA in transfected COS cells and in the mammalian brain. For comparison, we studied complexes formed with N-terminal fragments of ColQ, N-terminal fragments of PRiMA, and chimeras in which the upstream regions containing the cysteines were exchanged. We also compared the effect of mutations in the t peptides on their association with the two PRADs. We report that the two PRADs differ in their interaction with AChE(T) subunits; in complexes formed with the PRAD of PRiMA, we observed light dimers, but very few heavy dimers, even though such dimers were formed with the PQ chimera in which the N-terminal region of PRiMA was associated with the PRAD of ColQ. Complexes with PQ or with PRiMA contained heavy components, which migrated abnormally in SDS-PAGE but probably resulted from disulfide bonding of four AChE(T) subunits with the four upstream cysteines of the associated protein.     

Reduction of the periplasmic disulfide bond isomerase, DsbC, occurs by passage of electrons from cytoplasmic thioredoxin.

The Escherichia coli periplasmic protein DsbC is active both in vivo and in vitro as a protein disulfide isomerase. For DsbC to attack incorrectly formed disulfide bonds in substrate proteins, its two active-site cysteines should be in the reduced form. Here we present evidence that, in wild-type cells, these two cysteines are reduced. Further, we show that a pathway involving the cytoplasmic proteins thioredoxin reductase and thioredoxin and the cytoplasmic membrane protein DsbD is responsible for the reduction of these cysteines. Thus, reducing potential is passed from cytoplasmic electron donors through the cytoplasmic membrane to DsbC. This pathway does not appear to utilize the cytoplasmic glutathione-glutaredoxin pathway. The redox state of the active-site cysteines of DsbC correlates quite closely with its ability to assist in the folding of proteins with multiple disulfide bonds. Analysis of the activity of mutant forms of DsbC in which either or both of these cysteines have been altered further supports the role of DsbC as a disulfide bond isomerase.     

Antimicrobial Peptides from Plants

Peptides with antimicrobial properties are present in most if not all plant species. All plant antimicrobial peptides isolated so far contain even numbers of cysteines (4, 6, or 8), which are all pairwise connected by disulfide bridges, thus providing high stability to the peptides. Based on homologies at the primary structure level, plant antimicrobial peptides can be classified into distinct families including thionins, plant defensins, lipid transfer proteins, and he vein- and knottin-type antimicrobial peptides. Detailed three-dimensional structure information has been obtained for one or more members of these peptide families. All antimicrobial peptides studied thus far appear to exert their antimicrobial effect at the level of the plasma membrane of the target microorganism, but the different peptide types are likely to act via different mechanisms. Antimicrobial peptides can occur in all plant organs. In unstressed organs, antimicrobial peptides are usually most abundant in the outer cell layer lining the organ, which is consistent with a role for the antimicrobial peptides in constitutive host defense against microbial invaders attacking from the outside. Thionins are predominantly located intracellularly but are also found in the extracellular space, whereas most plant defensins and lipid transfer proteins are deposited exclusively in the extracellular space. In a number of plant species, a strong induction of genes expressing either thionins, plant defensins, or lipid transfer proteins has been observed on infection of the leaves by microbial pathogens. Hence, antimicrobial peptides can also take part in the inducible defense response of plants. Constitutive expression in transgenic plants of heterologous antimicrobial peptide genes has been achieved, which in some cases has led to enhanced resistance to particular microbial plant pathogens.     

Two subfamilies of plant purple acid phosphatases

The cDNA sequences of two purple acid phosphatases (previously purified AcPase1 and putative AcPase2) from yellow lupin ( Lupinus luteus L) have been determined. AcPase1 mRNA is 1755 nt in length, including a single open reading frame of 1434 nt that encodes 477 amino acid residues (EMBL accession number AJ458943 ). The AcPase2 mRNA is a 170-nt molecule, including a single open reading frame of 1392 nt that encodes 463 amino acid residues (EMBL accession number AJ505579 ). Both sequences were compared with known purple acid phosphatases (PAPs). The analysis of all known PAP sequences showed that they are divided into two subfamilies. The characteristic feature of the first subfamily containing AcPase1 is the lack of cysteine residues in the C-terminal region of the polypeptide around position 350. AcPase2 belongs to the second subfamily which contains a disulphide bridge formed of cysteines present around position 350. A possible relationship between structure and substrate specificity in these two PAP subfamilies is presented. Expression of AcPase1 and AcPase2 shows that the first enzyme is produced in the early stage of germination (its mRNA in polyadenylated form is present already in dry seeds), whereas the second phosphatase is produced mainly in the roots of mature plants. The early expression of AcPase1 was confirmed by a comparative analysis of a cotranslational N-glycosylation of AcPase1 and diphosphonucleotide phosphatase/phosphodiesterase ( PPD1 ) known to be expressed in the final stage of germination.     

Cysteine cross-linking defines part of the dimer and B/C domain interface of the Escherichia coli mannitol permease

Part of the dimer and B/C domain interface of the Escherichia coli mannitol permease (EII(mtl)) has been identified by the generation of disulfide bridges in a single-cysteine EII(mtl), with only the activity linked Cys(384) in the B domain, and in a double-cysteine EII(mtl) with cysteines at positions 384 and 124 in the first cytoplasmic loop of the C domain. The disulfide bridges were formed in the enzyme in inside-out membrane vesicles and in the purified enzyme by oxidation with Cu(II)-(1,10-phenanthroline)(3), and they were visualized by SDS-polyacrylamide gel electrophoresis. Discrimination between possible disulfide bridges in the dimeric double-cysteine EII(mtl) was done by partial digestion of the protein and the formation of heterodimers, in which the cysteines were located either on different subunits or on one subunit. The disulfide bridges that were identified are an intersubunit Cys(384)-Cys(384), an intersubunit Cys(124)-Cys(124), an intersubunit Cys(384)-Cys(124), and an intrasubunit Cys(384)-Cys(124). The disulfide bridges between the B and C domain were observed with purified enzyme and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mannitol did not influence the formation of the disulfide between Cys(384) and Cys(124). The close proximity of the two cysteines 124 was further confirmed with a separate C domain by oxidation with Cu(II)-(1,10-phenanthroline)(3) or by reactions with dimaleimides of different length. The data in combination with other work show that the first cytoplasmic loop around residue 124 is located at the dimer interface and involved in the interaction between the B and C domain.     

Two cysteines in each periplasmic domain of the membrane protein DsbB are required for its function in protein disulfide bond formation.

DsbB is a protein component of the pathway that leads to disulfide bond formation in periplasmic proteins of Escherichia coli. Previous studies have led to the hypothesis that DsbB oxidizes the periplasmic protein DsbA, which in turn oxidizes the cysteines in other periplasmic proteins to make disulfide bonds. Gene fusion approaches were used to show that (i) DsbB is a membrane protein which spans the membrane four times and (ii) both the N- and C-termini of the protein are in the cytoplasm. Mutational analysis shows that of the six cysteines in DsbB, four are necessary for proper DsbB function in vivo. Each of the periplasmic domains of the protein has two essential cysteines. The two cysteines in the first periplasmic domain are in a Cys-X-Y-Cys configuration that is characteristic of the active site of other proteins involved in disulfide bond formation, including DsbA and protein disulfide isomerase.     

Depolarization induces intersubunit cross-linking in a S4 cysteine mutant of the Shaker potassium channel

Voltage-gated potassium (K(v)) channels are integral membrane proteins, composed of four subunits, each comprising six (S1-S6) transmembrane segments. S1-S4 comprise the voltage-sensing domain, and S5-S6 with the linker P-loop forms the ion conducting pore domain. During activation, S4 undergoes structural rearrangements that lead to the opening of the channel pore and ion conduction. To obtain details of these structural changes we have used the engineered disulfide bridge approach. For this we have introduced the L361C mutation at the extracellular end of S4 of the Shaker K channel and expressed the mutant channel in Xenopus oocytes. When exposed to mild oxidizing conditions (ambient oxygen or copper phenanthroline), Cys-361 formed an intersubunit disulfide bridge as revealed by the appearance of a dimeric band on Western blotting. As a consequence, the mutant channel suffered a significant loss in conductance (measured by two-electrode voltage clamp). Removal of native cysteines failed to prevent the disulfide formation, indicating that Cys-361 forms a disulfide with its counterpart in the neighboring subunit. The effect was voltage-dependent and occurred during channel activation after Cys-361 has been exposed to the extracellular phase. Although the disulfide bridge reduced the maximal conductance, it caused a hyperpolarizing shift in the conductance-voltage relationship and reduced the deactivation kinetics of the channel. The latter two effects suggest stabilization of the open state of the channel. In conclusion, we report that during activation the intersubunit distance between the N-terminal ends of the S4 segments of the L361C mutant Shaker K channel is reduced.     

The C-terminal t peptide of acetylcholinesterase forms an alpha helix that supports homomeric and heteromeric interactions.

Acetylcholinesterase subunits of type T (AChET) possess an alternatively spliced C-terminal peptide (t peptide) which endows them with amphiphilic properties, the capacity to form various homo-oligomers and to associate, as a tetramer, with anchoring proteins containing a proline rich attachment domain (PRAD). The t peptide contains seven conserved aromatic residues. By spectroscopic analyses of the synthetic peptides covering part or all of the t peptide of Torpedo AChET, we show that the region containing the aromatic residues adopts an alpha helical structure, which is favored in the presence of lipids and detergent micelles: these residues therefore form a hydrophobic cluster in a sector of the helix. We also analyzed the formation of disulfide bonds between two different AChET subunits, and between AChET subunits and a PRAD-containing protein [the N-terminal fragment of the ColQ protein (QN)] possessing two cysteines upstream or downstream of the PRAD. This shows that, in the complex formed by four T subunits with QN (T4-QN), the t peptides are not folded on themselves as hairpins but instead are all oriented in the same direction, antiparallel to that of the PRAD. The formation of disulfide bonds between various pairs of cysteines, introduced by mutagenesis at various positions in the t peptides, indicates that this complex possesses a surprising flexibility.     

The eight-cysteine motif, a versatile structure in plant proteins.

A number of protein sequences deduced from the molecular analysis of plant cDNA or genomic libraries can be grouped in relation to a defined number of cysteine residues located in distinct positions of their sequences. This is the case for a group of around 500 polypeptides from different species that contain a small domain (less than 100 amino acids residues) displaying a pattern of eight-cysteines in a specific order. The plant sequences containing this motif belong to proteins having different functions, ranging from storage, protection, enzyme inhibition and lipid transfer, to cell wall structure. The eight-cysteine motif (8CM) appears to be a structural scaffold of conserved helical regions connected by variable loops, as observed by three-dimensional structure analysis. It is proposed that the cysteine residues would form a network of disulfide bridges necessary, for the maintenance of the tertiary structure of the molecule together with the central helical core, while the variable loops would provide the sequences required for the specific functions of the proteins.     

Homology modeling and molecular dynamics simulations of the N-terminal domain of wheat high molecular weight glutenin subunit 10

High molecular weight glutenin subunits (HMW-GS) are of a particular interest because of their biomechanical properties, which are important in many food systems such as breadmaking. Using fold-recognition techniques, we identified a fold compatible with the N-terminal domain of HMW-GS Dy10. This fold corresponds to the one adopted by proteins belonging to the cereal inhibitor family. Starting from three known protein structures of this family as templates, we built three models for the N-terminal domain of HMW-GS Dy10. We analyzed these models, and we propose a number of hypotheses regarding the N-terminal domain properties that can be tested experimentally. In particular, we discuss two possible ways of interaction between the N-terminal domains of the y-type HMW glutenin subunits. The first way consists in the creation of interchain disulfide bridges. According to our models, we propose two plausible scenarios: (1) the existence of an intrachain disulfide bridge between cysteines 22 and 44, leaving the three other cysteines free of engaging in intermolecular bonds; and (2) the creation of two intrachain disulfide bridges (involving cysteines 22-44 and cysteines 10-55), leaving a single cysteine (45) for creating an intermolecular disulfide bridge. We discuss these scenarios in relation to contradictory experimental results. The second way, although less likely, is nevertheless worth considering. There might exist a possibility for the N-terminal domain of Dy10, Nt-Dy10, to create oligomers, because homologous cereal inhibitor proteins are known to exist as monomers, homodimers, and heterooligomers. We also discuss, in relation to the function of the cereal inhibitor proteins, the possibility that this N-terminal domain has retained similar inhibitory functions.     

Amino terminal domains of the NMDA receptor are organized as local heterodimers

The N-methyl-D-aspartate (NMDA) receptor, an obligate heterotetrameric assembly organized as a dimer of dimers, is typically composed of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. Despite the crucial role that the NMDA receptor plays in the nervous system, the specific arrangement of subunits within the dimer-of-dimer assemblage is not conclusively known. Here we studied the organization of the amino terminal domain (ATD) of the rat GluN1/GluN2A and GluN1/GluN2B NMDA receptors by cysteine-directed, disulfide bond-mediated cross-linking. We found that GluN1 ATDs and GluN2 ATDs spontaneously formed disulfide bond-mediated dimers after introducing cysteines into the L1 interface of GluN2A or GluN2B ATD. The formation of dimer could be prevented by knocking out endogenous cysteines located near the L1 interface of GluN1. These results indicate that GluN1 and GluN2 ATDs form local heterodimers through the interactions in the L1-L1 interface and further demonstrate a dimer-of-heterodimer arrangement in GluN1/GluN2A and GluN1/GluN2B NMDA receptors.     

Purification,primary structure,and properties of <Emphasis Type="Italic">Euphorbia characias</Emphasis> latex purple acid phosphatase

A purple acid phosphatase was purified to homogeneity from Euphorbia characias latex. The native protein has a molecular mass of 130 ± 10 kDa and is formed by two apparently identical subunits, each containing one Fe(III) and one Zn(II) ion. The two subunits are connected by a disulfide bridge. The enzyme has an absorbance maximum at 540 nm, conferring a characteristic purple color due to a charge-transfer transition caused by a tyrosine residue (Tyr172) coordinated to the ferric ion. The cDNA nucleotide sequence contains an open reading frame of 1392 bp, and the deduced sequence of 463 amino acids shares a very high degree of identity (92–99%) to other purple acid phosphatases isolated from several higher plants. The enzyme hydrolyzes well p-nitrophenyl phosphate, a typical artificial substrate, and a broad range of natural phosphorylated substrates, such as ATP, ADP, glucose-6-phosphate, and phosphoenolpyruvate. The enzyme displays a pH optimum of 5.75 and is inhibited by molybdate, vanadate, and Zn²⁺, which are typical acid phosphatase inhibitors.