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.     

Carrier subunit of plasma membrane transporter is required for oxidative folding of its helper subunit

We study the amino acid transport system b(0,+) as a model for folding, assembly, and early traffic of membrane protein complexes. System b(0,+) is made of two disulfide-linked membrane subunits: the carrier, b(0,+) amino acid transporter (b(0,+)AT), a polytopic protein, and the helper, related to b(0,+) amino acid transporter (rBAT), a type II glycoprotein. rBAT ectodomain mutants display folding/trafficking defects that lead to type I cystinuria. Here we show that, in the presence of b(0,+)AT, three disulfides were formed in the rBAT ectodomain. Disulfides Cys-242-Cys-273 and Cys-571-Cys-666 were essential for biogenesis. Cys-673-Cys-685 was dispensable, but the single mutants C673S, and C685S showed compromised stability and trafficking. Cys-242-Cys-273 likely was the first disulfide to form, and unpaired Cys-242 or Cys-273 disrupted oxidative folding. Strikingly, unassembled rBAT was found as an ensemble of different redox species, mainly monomeric. The ensemble did not change upon inhibition of rBAT degradation. Overall, these results indicated a b(0,+)AT-dependent oxidative folding of the rBAT ectodomain, with the initial and probably cotranslational formation of Cys-242-Cys-273, followed by the oxidation of Cys-571-Cys-666 and Cys-673-Cys-685, that was completed posttranslationally.     

Disulfide structure of the pheromone binding protein from the silkworm moth, Bombyx mori

Disulfide bond formation is the only known posttranslational modification of insect pheromone binding proteins (PBPs). In the PBPs from moths (Lepidoptera), six cysteine residues are highly conserved at positions 19, 50, 54, 97, 108 and 117, but to date nothing is known about their respective linkage or redox status. We used a multiple approach of enzymatic digestion, chemical cleavage, partial reduction with Tris-(2-carboxyethyl)phosphine, followed by digestion with endoproteinase Lys-C to determine the disulfide connectivity in the PBP from Bombyx mori (BmPBP). Identification of the reaction products by on-line liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) and protein sequencing supported the assignment of disulfide bridges at Cys-19-Cys-54, Cys-50-Cys-108 and Cys-97-Cys-117. The disulfide linkages were identical in the protein obtained by periplasmic expression in Escherichia coli and in the native BmPBP.     

Unique disulfide bonds in epidermal growth factor (EGF) domains of β3 affect structure and function of αIIbβ3 and αvβ3 integrins in different manner

The β3 subunit of αIIbβ3 and αvβ3 integrins contains four epidermal growth factor (EGF)-like domains. Each domain harbors four disulfide bonds of which one is unique for integrins. We previously discerned a regulatory role of the EGF-4 Cys-560-Cys-583 unique bond for αIIbβ3 activation. In this study we further investigated the role of all four integrin unique bonds in both αIIbβ3 and αvβ3. We created β3 mutants harboring serine substitutions of each or both cysteines that disrupt the four unique bonds (Cys-437-Cys-457 in EGF-1, Cys-473-Cys-503 in EGF-2, Cys-523-Cys-544 in EGF-3, and Cys-560-Cys-583 in EGF-4) and transfected them into baby hamster kidney cells together with normal αv or αIIb. Flow cytometry was used to measure surface expression of αIIbβ3 and αvβ3 and their activity state by soluble fibrinogen binding. Most cysteine substitutions caused similarly reduced surface expression of both receptors. Disrupting all four unique disulfide bonds by single cysteine substitutions resulted in variable constitutive activation of αIIbβ3 and αvβ3. In contrast, whereas double C437S/C457S and C473S/C503S mutations yielded constitutively active αIIbβ3 and αvβ3, the C560S/C583S mutation did not, and the C523S/C544S mutation only yielded constitutively active αIIbβ3. Activation of C523S/C544S αvβ3 mutant by activating antibody and dithiothreitol was also impaired. Molecular dynamics of C523S/C544S β3 in αIIbβ3 but not in αvβ3 displayed an altered stable conformation. Our findings indicate that unique disulfide bonds in β3 differently affect the function of αIIbβ3 and αvβ3 and suggest a free sulfhydryl-dependent regulatory role for Cys-560-Cys-583 in both αIIbβ3 and αvβ3 and for Cys-523-Cys-544 only in αvβ3.     

Different roles of the two disulfide bonds of the cysteine proteinase inhibitor, chicken cystatin, for the conformation of the active protein.

The Cys-71-Cys-81 disulfide bond of the cysteine proteinase inhibitor, chicken cystatin, was specifically reduced by thioredoxin or low concentrations of dithiothreitol. This cleavage, followed by S-carbamoylmethylation, induced a conformational change of the protein, as evidenced by changes in isoelectric point and circular dichroism spectra and by an increased susceptibility to digestion by nontarget proteinases. The proteinase binding ability and the immunological properties of the inhibitor, however, were not detectably altered, indicating that the conformational change was limited to the region around the disrupted bond. In contrast, reduction of both disulfide bonds of cystatin by higher concentrations of dithiothreitol and subsequent alkylation led to the slow conversion of the inhibitor into two forms lacking proteinase binding ability, indicative of more extensive conformational changes. Together, these results suggest that the less accessible Cys-95-Cys-115 disulfide bond of chicken cystatin, but not the more accessible Cys-71-Cys-81 bond, is of importance for maintaining the conformation of the inhibitor required for binding of target proteinases.     

Role of vicinal cysteine pairs in metalloid sensing by the ArsD As(III)-responsive repressor

ArsD is a 120-residue repressor that regulates expression of the arsRDABC arsenical resistance operon of plasmid R773 in Escherichia coli. ArsD is released from arsRDABC promoter DNA by binding of the compounds with the metalloids As(III) or Sb(III). ArsD has three vicinal cysteine pairs, Cys-12 and Cys-13, Cys-112 and Cys-113 and Cys-119 and Cys-120. In this study, the role of these three cysteine pairs was investigated. Mutation or deletion of Cys-119-Cys-120 had no effect on repression or metalloid responsiveness in vivo or in vitro. Mutagenesis of either the Cys-12-Cys-13 pair or the Cys-112-Cys-113 pair had no effect on repression but produced loss of inducibility, suggesting that both Cys-12-Cys-13 and Cys-112-Cys-113 may be required for As(III) or Sb(III) responsiveness. Assays of binding of wild-type and mutant ArsDs by As(III) affinity chromatography showed that each of the three vicinal cysteine pairs is capable of binding As(III) independently. The effect of As(III) or Sb(III) on intrinsic protein fluorescence was used to examine the properties of individual cysteine pairs. The fluorescence of Trp-97 was shown to be quenched by the addition of Sb(III) or As(III). The vicinal Cys-112-Cys-113 pair was required for the majority of the metalloid-dependent quenching of Trp-97 fluorescence. The data are consistent with a model in which Cys-12-Cys-13 and Cys-112-Cys-113 form independent As(III) binding sites, both of which are required for in vivo ArsD function.     

Mutational analysis of the bacteriophage phi X174 replication origin

Bacteriophage phi X174 mutants within the 30 base-pair replication origin were constructed using oligodeoxynucleotide-directed mutagenesis. A total of 18 viable base substitution mutants at 13 different positions within the origin region were obtained. The majority of these ori mutants have a plaque morphology and burst size comparable to that of wild-type phi X174. Two phi X174 ori mutants with a reduced growth ability spontaneously acquired additional mutations that enhanced the growth rate. The additional mutation was located at the same site as the original mutation or was located in the N-terminal part of the gene A protein. This latter secondary mutation is responsible for a better binding and/or recognition of the gene A protein to the mutated origin. In a Darwinian experiment wild-type phi X174 outgrows all phi X174 ori mutants, indicating the superiority of the wild-type ori sequence for the reproduction of bacteriophage phi 174. Insertions and deletions were constructed at different positions within the phi X174 replication origin cloned in a plasmid. Small insertions and deletions in the A + T-rich spacer region do not inhibit phi X174 gene A protein cleavage in vitro, but severely impair packaging of single-stranded plasmid DNA in viral coats.     

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.     

Disulphide bond assignment in human tissue inhibitor of metalloproteinases (TIMP).

Disulphide bonds in human recombinant tissue inhibitor of metalloproteinases (TIMP) were assigned by resolving proteolytic digests of TIMP on reverse-phase h.p.l.c. and sequencing those peaks judged to contain disulphide bonds by virtue of a change in retention time on reduction. This procedure allowed the direct assignment of Cys-145-Cys-166 and the isolation of two other peptides containing two disulphide bonds each. Further peptide cleavage in conjunction with fast-atom-bombardment m.s. analysis permitted the assignments Cys-1-Cys-70, Cys-3-Cys-99, Cys-13-Cys-124 and Cys-127-Cys-174 from these peptides. The sixth bond Cys-132-Cys-137 was assigned by inference, as the native protein has no detectable free thiol groups.     

Identification of nonessential disulfide bonds and altered conformations in the v-sis protein, a homolog of the B chain of platelet-derived growth factor.

The protein encoded by v-sis, the oncogene of simian sarcoma virus, is homologous to the B chain of platelet-derived growth factor (PDGF). There are eight conserved Cys residues between PDGF-B and the v-sis protein. Both native PDGF and the v-sis protein occur as disulfide-bonded dimers, probably containing both intramolecular and intermolecular disulfide bonds. Oligonucleotide-directed mutagenesis was used to change the Cys codons to Ser codons in the v-sis gene. Four single mutants lacked detectable biological activity, indicating that Cys-127, Cys-160, Cys-171, and Cys-208 are required for formation of a biologically active v-sis protein. The other four single mutants retained biological activity as determined in transformation assays, indicating that Cys-154, Cys-163, Cys-164, and Cys-210 are dispensable for biological activity. Double and triple mutants containing three of these altered sites were constructed, some of which were transforming as well. The v-sis proteins encoded by biologically active mutants displayed significantly reduced levels of dimeric protein compared with the wild-type v-sis protein, which dimerized very efficiently. Furthermore, a mutant with a termination codon at residue 209 exhibited partial transforming activity. This study thus suggests that the minimal region required for transformation consists of residues 127 to 208. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the v-sis proteins encoded by some of the biologically active mutants exhibited an altered conformation when compared with the wild-type v-sis protein, and suggested that Cys-154 and Cys-163 participate in a nonessential disulfide bond.     

The positions of the disulfide bonds and the glycosylation site in a lectin of the acorn barnacle Megabalanus rosa

The positions of the interchain and intrachain disulfide bonds and the glycosylation site in a lectin of the acorn barnacle Megabalanus rosa were determined. The lectin (Mr 140,000) is composed of the same subunit (Mr 22,000) which is cross-linked by disulfide bonds to form a dimer. Intact lectin yielded two fragments, CB1 and CB2, by cleavage with cyanogen bromide. One intrachain and two interchain disulfide bonds were identified as Cys-53-Cys-61, Cys-14-Cys-50' and Cys-50-Cys-14', respectively, by enzymatic digestion and Edman degradation of CB1. Two intrachain disulfide bonds were determined as Cys-78-Cys-168 and Cys-144-Cys-160 by enzymatic digestion of CB2. The two intrachain disulfide bonds are well conserved through all invertebrate lectins and calcium-dependent animal lectins. S-Carboxamidomethylated lectin was digested with Staphylococcus aureus V8 proteinase and separated by reversed-phase HPLC. Glycopeptides were detected by the 4-N,N-dimethylamino-4'-azobenzene sulfonyl hyrazide method. Sequence analyses of the glycopeptides showed that a carbohydrate chain attached to Asn-39.     

Identification of a streptococcal penicillin-binding protein that reacts very slowly with penicillin.

Penicillin-binding protein (PBP) 5 of Streptococcus faecium ATCC 9790 has an unusually low affinity for penicillin (50% binding occurred at a penicillin level of 8 micrograms/ml after 60 min of incubation, and the protein only became labeled after 20 min of incubation with high concentrations of radioactive penicillin). PBPs with similar properties are carried by strains of Streptococcus durans, Streptococcus faecalis, and Streptococcus lactis but not by strains of groups A, B, C, and G streptococci or Streptococcus pneumoniae. The strains carrying the slow-reacting PBP demonstrated a sensitivity to penicillin that was several hundred times lower than that of strains not carrying it. Spontaneous mutants with minimal inhibitory concentrations of penicillin of 20, 40, and 80 micrograms/ml were isolated from S. faecium ATCC 9790. They all showed a dramatic increase in the amount of slow-reacting PBP produced. Mutants with increased penicillin resistance were also isolated from wild-type strains of S. durans, S. faecalis, and S. faecium. All of them carried a greater amount of the slow-reacting PBP than that carried by the parent. Finally, it was found that resistant S. faecium ATCC 9790 mutants grew normally in the presence of penicillin concentrations that were far above that saturating all PBPs except PBP 5. Cell growth was, on the contrary, inhibited by a penicillin concentration that saturated the slow-reacting PBP by 90%. This penicillin dose was equal to the minimal inhibitory concentration.     

Expression and purification of a soluble form of penicillin-binding protein 2 from both penicillin-susceptible and penicillin-resistant Neisseria gonorrhoeae.

Resistance to penicillin in non-beta-lactamase-producing strains of Neisseria gonorrhoeae (CMRNG strains) is mediated in part by the production of altered forms of penicillin-binding protein 2 (PBP 2) that have a decreased affinity for penicillin. The reduction in the affinity of PBP 2 is largely due to the insertion of an aspartic acid residue (Asp-345a) into the amino acid sequence of PBP 2. Truncated forms of N. gonorrhoeae PBP 2, which differed only by the insertion of Asp-345a, were constructed by placing the region of the penA genes encoding the periplasmic domain of PBP 2 (amino acids 42-581) into an ATG expression vector. When the recombinant PBP 2 molecules were overexpressed in Escherichia coli, insoluble PBP 2 inclusion bodies, which could be isolated by low-speed centrifugation of cell lysates, were formed. These insoluble aggregates were solubilized and the truncated PBP 2 polypeptides were partially purified by cation-exchange chromatography and gel filtration in the presence of denaturant prior to the refolding of the enzyme in vitro. After renaturation, gel filtration was used to separate monomeric soluble PBP 2 from improperly folded protein aggregates and other protein contaminants. A 4-liter culture of induced E. coli cells yielded 1.4 mg of soluble PBP 2 or PBP 2' (PBP 2 containing the Asp-345a insertion), both of which were estimated to be 99% pure. The affinity of soluble PBP 2' for [3H]penicillin G was decreased fourfold relative to that of soluble PBP 2, and their affinities were found to be identical to the affinities of the full-length PBP 2 enzymes that were previously determined in N. gonorrhoeae membranes. Furthermore, soluble PBP 2 displayed a rank order of affinity for several other beta-lactam antibiotics that was consistent with the rank order of affinities previously reported for the native molecules. On the basis of these results, both of these soluble PBPs should be suitable for crystallization and X-ray crystallographic analysis.     

Penicillin-binding proteins from Erwinia amylovora: mutants lacking PBP2 are avirulent.

Radiolabelled penicillin G was used to examine penicillin-binding proteins (PBPs) from Erwinia amylovora (OT1). This procedure identified seven PBPs with molecular masses ranging from 22 to 83 kDa. E. amylovora PBPs were compared with those from Escherichia coli (JM101) and from two spherical, avirulent TnphoA mutants derived from OT1. Radiolabelled penicillin G bound to only six proteins from the spherical mutants which lacked a 69-kDa PBP. The spherical mutants could be complemented by the cloned E. coli pbpA-rodA operon, which restored both cell shape and virulence to apple seedlings. This suggested that the E. amylovora 69-kDa PBP is probably the functional equivalent of the E. coli PBP2 protein. Southern blot analysis using the E. coli rodA and pbpA genes as radiolabelled probes showed that TnphoA had inserted into the E. amylovora equivalent of the E. coli rodA-pbpA operon. Southern blots to chromosomal DNAs of the two spherical mutants, using the cloned hrp and dsp genes from E. amylovora as radiolabelled probes, confirmed that the TnphoA insertions were not located in the region of the E. amylovora chromosome postulated to encode known virulence factors. Both of the spherical TnphoA mutants synthesized amounts of extracellular polysaccharide equivalent to those synthesized by the wild-type strain (OT1), were resistant to lysis in distilled water and to lysozyme, and elicited the hypersensitive response on nonhost plants. These results indicate a possible role for cell shape in the virulence of this plant pathogen.     

Structure-function analysis of the DNA binding domain of Saccharomyces cerevisiae ABF1.

To localize the DNA binding domain of the Saccharomyces cerevisiae Ars binding factor 1 (ABF1), a multifunctional DNA binding protein, plasmid constructs carrying point mutations and internal deletions in the ABF1 gene were generated and expressed in Escherichia coli. Normal and mutant ABF1 proteins were purified by affinity chromatography and their DNA binding activities were analyzed. The substitution of His61, Cys66 and His67 respectively, located in the zinc finger motif in the N-terminal region (amino acids 40-91), eliminated the DNA binding activity of ABF1 protein. Point mutations in the middle region of ABF1, specifically at Leu353, Leu399, Tyr403, Gly404, Phe410 and Lys434, also eliminated or reduced DNA binding activity. However, the DNA binding activity of point mutants of Ser307, Ser496 and Glu649 was the same as that of wild-type ABF1 protein and deletion mutants of amino acids 200-265, between the zinc finger region and the middle region (residues 323-496) retained DNA binding activity. As a result, we confirmed that the DNA binding domain of ABF1 appears to be bipartite and another DNA binding motif, other than the zinc finger motif, is situated between amino acid residues 323 and 496.     

The effect of cysteine mutations on recombinant deacetoxycephalosporin C synthase from S. clavuligerus

Cysteines 100, 155, and 197 of recombinant deacetoxycephalosporin C synthase were mutated to alanine residues. The C100A mutant had properties similar to those of the wild-type enzyme, but mutation of Cys-155 and Cys-197 reduced enzyme activity with penicillin N and penicillin G to different extents.     

Construction of a deletion-scanning library for the 5′-proximal promoter region of the bean β-phaseolin gene

A library of internal deletion mutants was constructed in the 5′-proximal promoter region of the gene encoding the bean seed storage protein β-phaseolin. A nick was introduced randomly in the target DNA sequence by depurination and treatment with exonuclease III, and served as the initiating point for deleting adjacent DNA sequences by S1 nuclease. A syntheticPst I linker was ligated to the blunt-ended DNA to serve as a restriction marker for mapping the approximate position of deletion mutants. Subcloning of a kanamycin marker gene into the linker site facilitated selection of plasmid DNA in which internal deletions were introduced in the target DNA sequence. Distribution of internal deletion mutants was mapped by determining the locations of thePst I site in the target sequence. DNA sequence analysis of mutants indicated that the extent of internal deletions ranged from 6 to 100 bp with a mean of 39 bp. In addition, the CAAT and TATA boxes in the promoter region of the β-phaseolin gene were effectively dissected in these mutants.     

Separation of abnormal cell wall composition from penicillin resistance through genetic transformation of Streptococcus pneumoniae.

Compared with most penicillin-susceptible isolates of Streptococcus pneumoniae, penicillin-resistant clinical isolate Hun 663 contains mosaic penicillin-binding protein (PBP) genes encoding PBPs with reduced penicillin affinities, anomalous molecular sizes, and also cell walls of unusual chemical composition. Chromosomal DNA prepared from Hun 663 was used to transform susceptible recipient cells to donor level penicillin resistance, and a resistant transformant was used next as the source of DNA in the construction of a second round of penicillin-resistant transformants. The greatly reduced penicillin affinity of the high-molecular-weight PBPs was retained in all transformants through both genetic crosses. On the other hand, PBP pattern and abnormal cell wall composition, both of which are stable, clone-specific properties of strain Hun 663, were changed: individual transformants showed a variety of new, abnormal PBP patterns. Furthermore, while the composition of cell walls resembled that of the DNA donor in the first-round transformants, it became virtually identical to that of susceptible pneumococci in the second-round transformants. The findings indicate that genetic elements encoding the low affinity of PBPs and the penicillin resistance of the bacteria are separable from determinants that are responsible for the abnormal cell wall composition that often accompanies penicillin resistance in clinical strains of pneumococci.