Iron binding and oxidation kinetics in frataxin CyaY of Escherichia coli

Friedreich's ataxia is associated with a deficiency in frataxin, a conserved mitochondrial protein of unknown function. Here, we investigate the iron binding and oxidation chemistry of Escherichia coli frataxin (CyaY), a homologue of human frataxin, with the aim of better understanding the functional properties of this protein. Anaerobic isothermal titration calorimetry (ITC) demonstrates that at least two ferrous ions bind specifically but relatively weakly per CyaY monomer (K(d) approximately 4 microM). Such weak binding is consistent with the hypothesis that the protein functions as an iron chaperone. The bound Fe(II) is oxidized slowly by O(2). However, oxidation occurs rapidly and completely with H(2)O(2) through a non-enzymatic process with a stoichiometry of two Fe(II)/H(2)O(2), indicating complete reduction of H(2)O(2) to H(2)O. In accord with this stoichiometry, electron paramagnetic resonance (EPR) spin trapping experiments indicate that iron catalyzed production of hydroxyl radical from Fenton chemistry is greatly attenuated in the presence of CyaY. The Fe(III) produced from oxidation of Fe(II) by H(2)O(2) binds to the protein with a stoichiometry of six Fe(III)/CyaY monomer as independently measured by kinetic, UV-visible, fluorescence, iron analysis and pH-stat titrations. However, as many as 25-26 Fe(III)/monomer can bind to the protein, exhibiting UV absorption properties similar to those of hydrolyzed polynuclear Fe(III) species. Analytical ultracentrifugation measurements indicate that a tetramer is formed when Fe(II) is added anaerobically to the protein; multiple protein aggregates are formed upon oxidation of the bound Fe(II). The observed iron oxidation and binding properties of frataxin CyaY may afford the mitochondria protection against iron-induced oxidative damage.     

Copper binding in IscA inhibits iron‐sulphur cluster assembly in Escherichia coli

Among the iron‐sulphur cluster assembly proteins encoded by gene cluster iscSUA‐hscBA‐fdx in Escherichia coli, IscA has a unique and strong iron binding activity and can provide iron for iron‐sulphur cluster assembly in proteins in vitro. Deletion of IscA and its paralogue SufA results in an E. coli mutant that fails to assemble [4Fe‐4S] clusters in proteins under aerobic conditions, suggesting that IscA has a crucial role for iron‐sulphur cluster biogenesis. Here we report that among the iron‐sulphur cluster assembly proteins, IscA also has a strong and specific binding activity for Cu(I) in vivo and in vitro. The Cu(I) centre in IscA is stable and resistant to oxidation under aerobic conditions. Mutation of the conserved cysteine residues that are essential for the iron binding in IscA abolishes the copper binding activity, indicating that copper and iron may share the same binding site in the protein. Additional studies reveal that copper can compete with iron for the metal binding site in IscA and effectively inhibits the IscA‐mediated [4Fe‐4S] cluster assembly in E. coli cells. The results suggest that copper may not only attack the [4Fe‐4S] clusters in dehydratases, but also block the [4Fe‐4S] cluster assembly in proteins by targeting IscA in cells.     

The accessibility of proteins of the Escherichia coli 30S ribosomal subunit to antibody binding

Summary The accessibility of each of the proteins on the E. coli 30S ribosomal subunit was established by investigating whether or not immunoglobulins (IgG's) and their monovalent papain fragments (Fab's), specific for each of the 21 single ribosomal proteins, bind to the 30S subunit. The interpretation of the results of five different experimental approaches, namely Ouchterlony double diffusion and immunological sandwich methods, sucrose gradient and analytical ultracentrifugation, and functional inhibition tests, indicate that all 21 proteins of the 30S subunit have determinants available for antibody binding. There were quantitative differences between the degree of accessibility of the different ribosomal proteins. An attempt was made to correlate the results with the protein stoichiometric data of the small subunit proteins.     

The genes that encode the gonococcal transferrin binding proteins,TbpB and TbpA,are differentially regulated by MisR under iron‐replete and iron‐depleted conditions

Neisseria gonorrhoeae produces two transferrin binding proteins, TbpA and TbpB, which together enable efficient iron transport from human transferrin. We demonstrate that expression of the tbp genes is controlled by MisR, a response regulator in the two‐component regulatory system that also includes the sensor kinase MisS. The tbp genes were up‐regulated in the misR mutant under iron‐replete conditions but were conversely down‐regulated in the misR mutant under iron‐depleted conditions. The misR mutant was capable of transferrin‐iron uptake at only 50% of wild‐type levels, consistent with decreased tbp expression. We demonstrate that phosphorylated MisR specifically binds to the tbpBA promoter and that MisR interacts with five regions upstream of the tbpB start codon. These analyses confirm that MisR directly regulates tbpBA expression. The MisR binding sites in the gonococcus are only partially conserved in Neisseria meningitidis, which may explain why tbpBA was not MisR‐regulated in previous studies using this related pathogen. This is the first report of a trans‐acting protein factor other than Fur that can directly contribute to gonococcal tbpBA regulation.     

The ribosome and YidC. New insights into the biogenesis of Escherichia coli inner membrane proteins

In the bacterium Escherichia coli, inner membrane proteins (IMPs) are generally targeted through the signal recognition particle pathway to the Sec translocon, which is capable of both linear transport into the periplasm and lateral transport into the lipid bilayer. Lateral transport seems to be assisted by the IMP YidC. In this article, we discuss recent observations that point to a key role for the ribosome in IMP targeting and to the diverse roles of YidC in IMP assembly.     

Bacillithiol has a role in Fe–S cluster biogenesis in Staphylococcus aureus

Staphylococcus aureus does not produce the low‐molecular‐weight (LMW) thiol glutathione, but it does produce the LMW thiol bacillithiol (BSH). To better understand the roles that BSH plays in staphylococcal metabolism, we constructed and examined strains lacking BSH. Phenotypic analysis found that the BSH‐deficient strains cultured either aerobically or anaerobically had growth defects that were alleviated by the addition of exogenous iron (Fe) or the amino acids leucine and isoleucine. The activities of the iron–sulfur (Fe–S) cluster‐dependent enzymes LeuCD and IlvD, which are required for the biosynthesis of leucine and isoleucine, were decreased in strains lacking BSH. The BSH‐deficient cells also had decreased aconitase and glutamate synthase activities, suggesting a general defect in Fe–S cluster biogenesis. The phenotypes of the BSH‐deficient strains were exacerbated in strains lacking the Fe–S cluster carrier Nfu and partially suppressed by multicopy expression of either sufA or nfu, suggesting functional overlap between BSH and Fe–S carrier proteins. Biochemical analysis found that SufA bound and transferred Fe–S clusters to apo‐aconitase, verifying that it serves as an Fe–S cluster carrier. The results presented are consistent with the hypothesis that BSH has roles in Fe homeostasis and the carriage of Fe–S clusters to apo‐proteins in S. aureus.     

A novel iron‐ and copper‐binding protein in the Lyme disease spirochaete

Iron and copper are transition metals that can be toxic to cells due to their abilities to react with peroxide to generate hydroxyl radical. Ferritins and metallothioneins are known to sequester intracellular iron and copper respectively. The Lyme disease pathogen Borrelia burgdorferi does not require iron, but its genome encodes a ferritin‐like Dps (D NA‐binding p rotein from s tarved bacteria) molecule, which has been shown to be important for the spirochaete's persistence in the tick and subsequent transmission to a new host. Here, we show that the c arboxyl‐terminal c ysteine‐r ich (CCR) domain of this protein functions as a copper‐binding metallothionein. This novel fusion between Dps and metallothionein is unique to and conserved in all Borrelia species. We term this molecule BicA for B orrelia i ron‐ and c opper‐binding protein A . An isogenic mutant lacking BicA had significantly reduced levels of iron and copper and was more sensitive to iron and copper toxicity than its parental strain. Supplementation of the medium with iron or copper rendered the spirochaete more susceptible to peroxide killing. These data suggest that an important function of BicA is to detoxify excess iron and copper the spirochaete may encounter during its natural life cycle through a tick vector and a vertebrate host.     

The DnaJ proteins DJA6 and DJA5 are essential for chloroplast iron–sulfur cluster biogenesis

Fe–S clusters are ancient, ubiquitous and highly essential prosthetic groups for numerous fundamental processes of life. The biogenesis of Fe–S clusters is a multistep process including iron acquisition, sulfur mobilization, and cluster formation. Extensive studies have provided deep insights into the mechanism of the latter two assembly steps. However, the mechanism of iron utilization during chloroplast Fe–S cluster biogenesis is still unknown. Here we identified two Arabidopsis DnaJ proteins, DJA6 and DJA5, that can bind iron through their conserved cysteine residues and facilitate iron incorporation into Fe–S clusters by interactions with the SUF (sulfur utilization factor) apparatus through their J domain. Loss of these two proteins causes severe defects in the accumulation of chloroplast Fe–S proteins, a dysfunction of photosynthesis, and a significant intracellular iron overload. Evolutionary analyses revealed that DJA6 and DJA5 are highly conserved in photosynthetic organisms ranging from cyanobacteria to higher plants and share a strong evolutionary relationship with SUFE1, SUFC, and SUFD throughout the green lineage. Thus, our work uncovers a conserved mechanism of iron utilization for chloroplast Fe–S cluster biogenesis.     

Membrane-deforming proteins play distinct roles in actin pedestal biogenesis by enterohemorrhagic Escherichia coli

Many bacterial pathogens reorganize the host actin cytoskeleton during the course of infection, including enterohemorrhagic Escherichia coli (EHEC), which utilizes the effector protein EspF(U) to assemble actin filaments within plasma membrane protrusions called pedestals. EspF(U) activates N-WASP, a host actin nucleation-promoting factor that is normally auto-inhibited and found in a complex with the actin-binding protein WIP. Under native conditions, this N-WASP/WIP complex is activated by the small GTPase Cdc42 in concert with several different SH3 (Src-homology-3) domain-containing proteins. In the current study, we tested whether SH3 domains from the F-BAR (FCH-Bin-Amphiphysin-Rvs) subfamily of membrane-deforming proteins are involved in actin pedestal formation. We found that three F-BAR proteins: CIP4, FBP17, and TOCA1 (transducer of Cdc42-dependent actin assembly), play different roles during actin pedestal biogenesis. Whereas CIP4 and FBP17 inhibited actin pedestal assembly, TOCA1 stimulated this process. TOCA1 was recruited to pedestals by its SH3 domain, which bound directly to proline-rich sequences within EspF(U). Moreover, EspF(U) and TOCA1 activated the N-WASP/WIP complex in an additive fashion in vitro, suggesting that TOCA1 can augment actin assembly within pedestals. These results reveal that EspF(U) acts as a scaffold to recruit multiple actin assembly factors whose functions are normally regulated by Cdc42.     

Altering the binding activity and specificity of the leucine binding proteins of Escherichia coli

Two leucine-binding proteins with overlapping specificities for the branched-chain amino acids are present in Escherichia coli. In order to study the basis of specificity for the very similar hydrophobic ligands, we have constructed a series of site-directed mutants of both proteins based on inspection of the leucine-isoleucine-valine-binding protein crystal structure reported by Sack et al. (Sack, J. S., Saper, M. A., and Quiocho, F. A. (1989) J. Mol. Biol. 206, 171-191). Each of the mutant proteins was overexpressed and purified, and their binding activity for a wide variety of potential ligands was measured. By introducing a common restriction endonuclease cleavage site in the two proteins, two hybrid binding proteins consisting of the amino-terminal third of one binding protein fused to the carboxyl-terminal two-thirds of the other were created. The results of these studies indicated that the binding site of the leucine-isoleucine-valine binding protein can accommodate a branch at the beta-carbon of the ligand and that hydrophilic groups on the ligand can be accommodated only in certain orientations. None of the single amino acid substitutions resulted in complete switches in specificity between the two proteins, suggesting that additional residues are involved in leucine binding and discrimination among the branched-chain amino acid substrates.     

The chaperonin GroEL and other heat-shock proteins, besides DnaK, participate in ribosome biogenesis in Escherichia coli

It has been shown that in Escherichia coli the chaperone DnaK is necessary for the late stages of 50S and 30S ribosomal subunit assembly in vivo. Here we focus on the roles of other HSPs (heat-shock proteins), including the chaperonin GroEL, in addition to DnaK, in ribosome biogenesis at high temperature. GroEL is shown to be required for the very late 45S-->50S step in the biogenesis of the large ribosome subunit, but not for 30S assembly. Interestingly, overproduction of GroES/GroEL can partially compensate for a lack of DnaK/DnaJ at 44 degrees C.     

Partial conservation of functions between eukaryotic frataxin and the Escherichia coli frataxin homolog CyaY

Frataxin is a mitochondrial protein structurally conserved from bacteria to humans. Eukaryotic frataxins are known to be involved in the maintenance of mitochondrial iron balance via roles in iron delivery and iron detoxification. The prokaryotic frataxin homolog, CyaY, has been shown to bind and donate iron for the assembly of [2Fe-2S] clusters in vitro. However, in contrast to the severe phenotypes associated with the partial or complete loss of frataxin in humans and other eukaryotes, deletion of the cyaY gene does not cause any obvious alteration of iron balance in bacterial cells, an effect that probably reflects functional redundancy between CyaY and other bacterial proteins. To study CyaY function in a nonredundant setting, we have expressed a mitochondria-targeted form of CyaY in a Saccharomyces cerevisiae strain depleted of the endogenous yeast frataxin protein (yfh1Delta). We show that in this strain CyaY complements to a large extent the loss of iron-sulfur cluster enzyme activities and heme synthesis, and thereby maintains a nearly normal respiratory growth. In addition, CyaY effectively protects yfh1Delta from oxidative damage during treatment with hydrogen peroxide but is less efficient in detoxifying excess labile iron during aerobic growth.     

Ribosome biogenesis and the translation process in Escherichia coli.

Translation, the decoding of mRNA into protein, is the third and final element of the central dogma. The ribosome, a nucleoprotein particle, is responsible and essential for this process. The bacterial ribosome consists of three rRNA molecules and approximately 55 proteins, components that are put together in an intricate and tightly regulated way. When finally matured, the quality of the particle, as well as the amount of active ribosomes, must be checked. The focus of this review is ribosome biogenesis in Escherichia coli and its cross-talk with the ongoing protein synthesis. We discuss how the ribosomal components are produced and how their synthesis is regulated according to growth rate and the nutritional contents of the medium. We also present the many accessory factors important for the correct assembly process, the list of which has grown substantially during the last few years, even though the precise mechanisms and roles of most of the proteins are not understood.     

The proteins of the messenger RNA binding site of Escherichia coli ribosomes.

Oligo(U) derivatives with [14C]-4-(N-2-chloroethyl-N-methylamino)benzaldehyde attached to 3'-end cis-diol group via acetal bond, p(Up)n-1UCHRCl as well as with [14C]-4-(N-2-chloroethyl-N-methylamino)benzylamine attached to 5'-phosphate via amide bond, ClRCH2NHpU(pU)6 were used to modify 70S E. coli ribosomes near mRNA binding centre. Within ternary complex with ribosome and tRNAPhe all reagents covalently bind to ribosome the extent of modification being 0.1-0.4 mole/mole 70S. p(Up)n-1UCHRCl alkylates either 30S (n=5,7) or both subunits (n=6,8). rRNA is preferentially modified within 30S subunit. ClRCH2NHpU(pU)6 alkylates both subunits the proteins being mainly modified. The distribution of the label among proteins differ for various reagents. S4, S5, S7, S9, S11, S13, S15, S18 and S21 are found to be alkylated within 30S subunit, proteins L1, L2, L6, L7/L12, L19, L31 and L32 are modified in the 50S subunit. Most proteins modified within 30S subunit are located at the "head" of this subunit and proteins modified within 50S subunit are located at the surface of the contact between this subunit and the "head" of 30S subunit at the model of Stoffler.     

Ribosome biogenesis is temperature‐dependent and delayed in Escherichia coli lacking the chaperones DnaK or DnaJ†

In Escherichia coli strains carrying null mutations in either the dnaK or dnaJ genes, the late stages of 30S and 50S ribosomal subunit biogenesis are slowed down in a temperature‐dependent manner. At high temperature (44°C), 32S and 45S particles (precursors to 50S subunits) and 21S particles (precursors to 30S subunits) accumulate. The latter are shown by 3′5′ rapid amplification of cDNA ends analysis to contain unprocessed or partially processed 16S ribosomal RNA at the 5′ end, but the 3′ end was never processed. This implies that maturation of 16S ribosomal RNA starts at the 5′‐terminus, and that the 3′‐terminus is only trimmed at a later step. At normal temperatures (30°C?37°C), ribosome assembly in both mutants is not arrested but is significantly delayed, as shown by pulse‐chase analysis. Assembly defects are partially compensated by an overexpression of other heat‐shock proteins, which occurs in the absence of their negative regulator DnaK, or by a plasmid‐driven overexpression of GroES/GroEL, suggesting the involvement of a network of chaperones in ribosome biogenesis.     

Chimeras of Escherichia coli and Mycobacterium tuberculosis single-stranded DNA binding proteins: characterization and function in Escherichia coli

Single stranded DNA binding proteins (SSBs) are vital for the survival of organisms. Studies on SSBs from the prototype, Escherichia coli (EcoSSB) and, an important human pathogen, Mycobacterium tuberculosis (MtuSSB) had shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. Here, we used the X-ray crystal structure data of the two SSBs to design a series of chimeric proteins (mβ1, mβ1'β2, mβ1-β5, mβ1-β6 and mβ4-β5) by transplanting β1, β1'β2, β1-β5, β1-β6 and β4-β5 regions, respectively of the N-terminal (DNA binding) domain of MtuSSB for the corresponding sequences in EcoSSB. In addition, mβ1'β2(ESWR) SSB was generated by mutating the MtuSSB specific 'PRIY' sequence in the β2 strand of mβ1'β2 SSB to EcoSSB specific 'ESWR' sequence. Biochemical characterization revealed that except for mβ1 SSB, all chimeras and a control construct lacking the C-terminal domain (ΔC SSB) bound DNA in modes corresponding to limited and unlimited modes of binding. However, the DNA on MtuSSB may follow a different path than the EcoSSB. Structural probing by protease digestion revealed that unlike other SSBs used, mβ1 SSB was also hypersensitive to chymotrypsin treatment. Further, to check for their biological activities, we developed a sensitive assay, and observed that mβ1-β6, MtuSSB, mβ1'β2 and mβ1-β5 SSBs complemented E. coli Δssb in a dose dependent manner. Complementation by the mβ1-β5 SSB was poor. In contrast, mβ1'β2(ESWR) SSB complemented E. coli as well as EcoSSB. The inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function.