Post-translational Modification of Ribosomal Proteins: STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF RimO FROM THERMOTOGA MARITIMA, A RADICAL S-ADENOSYLMETHIONINE METHYLTHIOTRANSFERASE

Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1826–1831). This reaction is formally identical to that catalyzed by MiaB on the C2 of adenosine 37 near the anticodon of several tRNAs. We present spectroscopic evidence that Thermotoga maritima RimO, like MiaB, contains two [4Fe-4S] centers, one presumably bound to three invariant cysteines in the central radical S-adenosylmethionine (AdoMet) domain and the other to three invariant cysteines in the N-terminal UPF0004 domain. We demonstrate that holo-RimO can specifically methylthiolate the aspartate residue of a 20-mer peptide derived from S12, yielding a mixture of mono- and bismethylthio derivatives. Finally, we present the 2.0 Å crystal structure of the central radical AdoMet and the C-terminal TRAM (tRNA methyltransferase 2 and MiaB) domains in apo-RimO. Although the core of the open triose-phosphate isomerase (TIM) barrel of the radical AdoMet domain was conserved, RimO showed differences in domain organization compared with other radical AdoMet enzymes. The unusually acidic TRAM domain, likely to bind the basic S12 protein, is located at the distal edge of the radical AdoMet domain. The basic S12 protein substrate is likely to bind RimO through interactions with both the TRAM domain and the concave surface of the incomplete TIM barrel. These biophysical results provide a foundation for understanding the mechanism of methylthioation by radical AdoMet enzymes in the MiaB/RimO family.     

Two novel mutations in exon 2 of bone morphogenetic protein (BMP) 15 gene in Pakistani infertile females

ObjectiveTo determine the proportion of fertility in Pakistani infertile females and discover if there are considerable connection among BMP15 gene polymorphism, follicle maturation and hormonal regulation in Pakistani infertile females.MethodsAll selected participants were initially examined through follicle-stimulating hormones (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), Prolactin, and Trans-vaginal scan (TVS). BMP15 gene polymorphism among infertile and fertile females was done by extracted Genomic DNA from whole blood. Sanger sequencing was performed for the identification of mutation in exons-intron boundaries of the BMP15 gene. Bioinformatics tools were used to assess the protein structure.ResultsThe total five mutations including two novel missense variants of BMP15 in exon 2, whereas three previously reported i.e. two cosmic mutations (c.615delC), (c.584InsG) and one frame shift mutations (c.635delA) were also observed. The first novel mutation was found at (c.1038InsGG) (p.346Gln < Gly) in which the insertion of GG at DNA position 1038 of exon 2 resulting in a substitution of glutamine into glycine at 346th amino acid of BMP15 protein. The second novel variant (c.1049delT) (p. Ser334Pro) was also observed in exon 2 of the BMP15 gene, which substituted serine into proline at 334th amino acid of the BMP15 protein.ConclusionIt is concluded that there are various missense mutations present in exon 2 of the BMP15 gene of Pakistani infertile females, consequently expected function of protein changes due to change in codons of amino acids. Provean and SIFT suggest the two novel variants as potentially deleterious. Although three other variants were also found in Pakistani infertile females which were previously reported. These mutations may result in early blockage of folliculogenesis and ovaries become streaky. Further research is required to resolve the actual allusion of these variations in the BMP15 gene.     

Guiding protein aggregation with macromolecular crowding

Macromolecular crowding is expected to have a significant effect on protein aggregation. In the present study we analyzed the effect of macromolecular crowding on fibrillation of four proteins, bovine S-carboxymethyl-alpha-lactalbumin (a disordered form of the protein with reduced three out of four disulfide bridges), human insulin, bovine core histones, and human alpha-synuclein. These proteins are structurally different, varying from natively unfolded (alpha-synuclein and core histones) to folded proteins with rigid tertiary and quaternary structures (monomeric and hexameric forms of insulin). All these proteins are known to fibrillate in diluted solutions, however their aggregation mechanisms are very divers and some of them are able to form different aggregates in addition to fibrils. We studied how macromolecular crowding guides protein between different aggregation pathways by analyzing the effect of crowding agents on the aggregation patterns under the variety of conditions favoring different aggregated end products in diluted solutions.     

X-ray structure of the [FeFe]-hydrogenase maturase HydE from Thermotoga maritima

Maturation of the [FeFe]-hydrogenase active site depends on at least the expression of three gene products called HydE, HydF, and HydG. We have solved the high resolution structure of recombinant, reconstituted S-adenosine-L-methionine-dependent HydE from Thermotoga maritima. Besides the conserved [Fe(4)S(4)] cluster involved in the radical-based reaction, this HydE was reported to have a second [Fe(4)S(4)] cluster coordinated by three Cys residues. However, in our crystals, depending on the reconstitution and soaking conditions, this second cluster is either a [Fe(2)S(2)] center, with water occupying the fourth ligand site or is absent. We have carried out site-directed mutagenesis studies on the related HydE from Clostridium acetobutylicum, along with in silico docking and crystal soaking experiments, to define the active site region and three anion-binding sites inside a large, positive cavity, one of which binds SCN(-) with high affinity. Although the overall triose-phosphate isomerase-barrel structure of HydE is very similar to that of biotin synthase, the residues that line the internal cavity are significantly different in the two enzymes.     

Activation of class III ribonucleotide reductase by flavodoxin: a protein radical-driven electron transfer to the iron-sulfur center

In its active form, Escherichia coli class III ribonucleotide reductase homodimer alpha(2) relies on a protein free radical located on the Gly(681) residue of the alpha polypeptide. The formation of the glycyl radical, namely, the activation of the enzyme, involves the concerted action of four components: S-adenosylmethionine (AdoMet), dithiothreitol (DTT), an Fe-S protein called beta or "activase", and a reducing system consisting of NADPH, NADPH:flavodoxin oxidoreductase, and flavodoxin (fldx). It has been proposed that a reductant serves to generate a reduced [4Fe-4S](+) cluster absolutely required for the reductive cleavage of AdoMet and the generation of the radical. Here, we suggest that the one-electron reduced form of flavodoxin (SQ), the only detectable product of the in vitro enzymatic reduction of flavodoxin, can support the formation of the glycyl radical. However, the redox potential of the Fe-S center of the enzyme is shown to be approximately 300 mV more negative than that of the SQ/fldx couple and not shifted to a more positive value by AdoMet binding. It is also more negative than that of the HQ/SQ couple, HQ being the fully reduced form of flavodoxin. Our interpretation is that activation of ribonucleotide reductase occurs through coupling of the reduction of the Fe-S center by flavodoxin to two thermodynamically favorable reactions, the oxidation of the cluster by AdoMet, yielding methionine and the 5'-deoxyadenosyl radical, and the oxidation of the glycine residue to the corresponding glycyl radical by the 5'-deoxyadenosyl radical. The second reaction plays the major role on the basis that a Gly-to-Ala mutation results in a greatly decreased production of methionine.     

Characterization of the gene encoding the [Fe]-hydrogenase from Megasphaera elsdenii

The gene encoding the [Fe]-hydrogenase from the anaerobic bacterium Megasphaera elsdenii has been cloned and sequenced. The gene is monocistronic, in keeping with the protein being a monomer. The translated protein sequence (484 residues, M(r)=53 kDa) comprises a small 2[4Fe-4S] ferredoxin-like domain and a large domain containing the catalytic site. Comparisons with other [Fe]-hydrogenase sequences, including two of which the crystal structures are known, show that the M. elsdenii protein is among the smallest of these enzymes and provide useful indications regarding the basic structural core common to all [Fe]-hydrogenases. It is, nevertheless, to be noted that the genome of Thermotoga maritima encodes a putative [Fe]-hydrogenase that would consist of only 301 residues.     

Antileishmanial IgG and IgE antibodies recognize predominantly carbohydrate epitopes of glycosylated antigens in visceral leishmaniasis

The specificity of human antileishmanial IgG and IgE antibodies to glycosylated antigens of Leishmania chagasi was evaluated. An ELISA was performed with soluble leishmanial antigen (SLA) and a panel of 95 sera including samples from patients with subclinical infection (SC) and visceral leishmaniasis (VL), subjects cured of visceral leishmaniasis (CVL), and from healthy individuals from endemic areas (HIEA). Antileishmanial IgG were verified for 18 (40%) of 45 SC subjects (mean absorbance of 0.49 +/- 0.17). All nine sera from VL patients had such antibody (0.99 +/- 0.21), while 11 (65%) of 17 CVL individuals were seropositive (0.46 +/- 0.05). Only three (12%) of 24 HIEA controls reacted in IgG-ELISA. Antileishmanial IgE was detected in 26 (58%) of 45 SC patients (0.35 +/- 0.14), and in all VL patients (0.65 +/- 0.29). These antibodies were also detected in 13(76%) of 17 CVL subjects (0.42 +/- 0.14) while all HIEA controls were seronegative. There was no correlation between antileishmanial IgG and IgE antibody absorbances. Mild periodate oxidation at acid pH of SLA carbohydrates drastically diminished its antigenicity in both IgG and IgE-ELISA, affecting mainly the antigens of 125, 102, 94, and 63 kDa as demonstrated by western immunoblotting.     

Microwave irradiation for accelerating the synthesis of acyclonucleosides of 1,2,4-triazole

The 3-(D-alditol-1-yl)-4-amino-5-mercapto-1,2,4-triazoles 4 and 5 can be successfully prepared using microwave irradiation. Condensation of 4 and 5 with p-nitrobenzaldehyde afforded Schiff bases 6 and 7, respectively. Reaction 4 and 5 with ethylchloroacetate gave the corresponding alkylated products 10 and 11. Better yields and much less time were the characteristic features of using the microwave heating over the conventional one. The structure of the prepared compounds was confirmed by 1H-NMR, 2D-NMR and mass spectra.     

Auto-transporter A protein of Neisseria meningitidis: a potent CD4+ T-cell and B-cell stimulating antigen detected by expression cloning

A meningococcal genomic expression library was screened for potent CD4+ T-cell antigens, using patients' peripheral blood lymphocytes (PBLs). One of the most promising positive clones was fully characterized. The recombinant meningococcal DNA contained a single, incomplete, open reading frame (ORF), which was fully reconstructed with reference to available genomic sequence data. The gene was designated autA (auto-transporter A) as its peptide sequence shares molecular characteristics of the auto-transporter family of proteins. Only a single copy of this gene was detected in the meningococcal, and none in the gonococcal, genomic sequence databases. The complete autA gene, when cloned into an expression vector, expressed a protein of approximately 68 kDa. Purified rAutA recalled strong secondary T-cell responses in PBLs of patients and some healthy donors, and induced strong primary T-cell responses in healthy donors. The human B-cell immunogenicity and cross-reactivity of AutA, purified under native conditions, was confirmed in dot immunoblot experiments. Immunoblots with rabbit polyclonal antibodies to rAutA demonstrated the conserved nature, antigenicity and cross-reactivity of AutA amongst meningococci of different serogroups and strains representing different hypervirulent lineages. AutA showed homology with another meningococcal and gonococcal ORF (designated AutB). AutB was cloned and expressed and used to raise an autB-specific antiserum. Immunoblot experiments indicated that AutB is not expressed in meningococci and does not cross-react with AutA. Thus, AutA, being a potent CD4+ T-cell and B-cell-stimulating antigen, which is highly conserved, deserves further investigation as a potential vaccine candidate.     

Divalent cation induced changes in structural properties of the dimeric enzyme glucose oxidase: dual effect of dimer stabilization and dissociation with loss of cooperative interactions in enzyme monomer

Glucose oxidase (GOD) from Aspergillus niger is a dimeric enzyme having high localization of negative charges on the enzyme surface and at the dimer interface. The monovalent cations induce compaction of the native conformation of GOD and enhance stability against thermal and urea denaturation [Ahmad et al. (2001) Biochemistry 40, 1947-1955]. In this paper we report the effect of the divalent cations Ca2+ and Mg2+ on the structural and stability properties of GOD. A divalent cation concentration dependent change in native conformation and subunit assembly of GOD was observed. Low concentration (up to 1 M) of CaCl2 or MgCl2 induced compaction of the native conformation of GOD, and the enzyme showed higher stability as compared to the native enzyme against urea denaturation. However, higher concentration (> or =2.0 M) of CaCl2 or MgCl2 induced dissociation of the native dimeric enzyme, resulting in stabilization of the enzyme monomer. An interesting observation was that the 3 M CaCl2-stabilized monomer of GOD retained about 70% secondary structure present in the native GOD dimer; however, there was a complete loss of cooperative interactions between these secondary structural elements present in the enzyme. Regarding the mechanism of divalent cation induced structural changes in GOD, the studies suggest that organization of water molecules by divalent cation results in stabilization of enzyme at low divalent cation concentration, whereas direct binding of these cations to the enzyme, at higher divalent cation concentration, results in dissociation and partial unfolding of the dimeric enzyme molecule.