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Keywords:

  • iron/sulphur proteins;
  • essential cysteine;
  • folate;
  • MiaB;
  • YgfZ

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Proteins of the YgfZ family occur in all domains of life and are characterized by the conserved dodecapeptide motif KGC[Y/F]-x-GQE-x3-[R/K]. YgfZ proteins are known to participate in assembly or repair of iron/sulphur clusters, and to require folate for biological activity, but their mechanism of action is unknown. To assess the importance of individual residues in the conserved motif, Escherichia coli Ygf Z was expressed from a plasmid in a ΔygfZ strain and subjected to alanine-scanning mutagenesis. The impacts on YgfZ functionality were evaluated by assays of growth and of the in vivo activity of the iron/sulphur enzyme MiaB, which modifies tRNA. By these criteria, the motif's tyrosine residue (Y229) had a detectable influence but only the cysteine residue (C228) was critical, for only the C228A mutant failed to complement the growth and MiaB activity phenotypes of the ΔygfZ strain. Immunoblots confirmed that the latter result was not simply because of a low level of the C228A mutant protein. Collectively, these data demonstrate a pivotal role for the Ygf Z motif's cysteine residue and a subsidiary one for the adjacent tyrosine, and help formulate a hypothesis about the folate requirement of Ygf Z proteins.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Iron/sulphur (Fe/S) clusters are versatile cofactors with roles that include catalysis, electron transport, regulation, sulphur donation and molybdenum trafficking (Johnson et al., 2005; Dos Santos & Dean, 2008). Although Fe/S clusters are structurally simple, their assembly depends on complex machinery, the components of which are still not fully known (Johnson et al., 2005; Fontecave & Ollagnier de Choudens, 2008). One such component is the Ygf Z (COG0354) protein family, which is found in all domains of life. Ygf Z proteins have a role in assembly or maintenance of a subset of Fe/S proteins that, in Escherichia coli, includes the tRNA modification enzyme MiaB (Ote et al., 2006; Gelling et al., 2008; Waller et al., 2010). Besides reduced activity of MiaB and other Fe/S enzymes, E. coli ΔygfZ strains show various phenotypic defects, including slowed growth and sensitivity to the oxidative stress agent plumbagin (Ote et al., 2006; Lin et al., 2010; Waller et al., 2010). Bacterial, animal, protistan and plant Ygf Z proteins have all been shown to require folate for action in vivo (Waller et al., 2010, 2011), but the biochemical basis of this requirement is not understood. It has, however, been shown that the requirement is most probably for tetrahydrofolate itself, rather than for a one-carbon substituted form (Waller et al., 2010).

Ygf Z proteins are characterized by the motif KGC[Y/F]-x-GQE-x3-[R/K], of which the arginine/lysine residue initially escaped notice (Teplyakov et al., 2004). The published three-dimensional structure of E. coli Ygf Z places this motif in a surface loop of the monomer, with the cysteine residues (C228) in two molecules linked via a disulphide bridge, forming a YgfZ dimer (Teplyakov et al., 2004). There is, however, evidence that not all YgfZ is dimeric in vivo: the equivalent of C228 in a plant YgfZ protein was shown to be an in vivo target for reduction by thioredoxin (Hägglund et al., 2008). Besides being implicated in dimer formation, the conserved cysteine residue is of interest because a mutational analysis of certain motif residues in E. coli Ygf Z implicated C228 as a determinant of plumbagin sensitivity (Lin et al., 2010). To gain further insight into the function of the Ygf Z motif, this study analysed the criticality of each of its conserved residues to growth and to MiaB activity. Only C228 was found to be indispensable.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Bacterial strains and growth

Complementation studies were carried out using the ΔygfZ strain described previously (Waller et al., 2010). This strain was transformed with pBAD24 containing the wild-type E. coli ygfZ gene (EcYgfZ∷pBAD24; Waller et al., 2010) or mutants thereof, in which one of the conserved residues in the Ygf Z motif had been replaced by alanine by site-directed mutagenesis (Cormack, 2008). Cells were grown at 37 °C in Antibiotic Medium 3 (Difco), LB medium with or without 30 μM plumbagin, or M9 minimal medium plus 2 g L−1 glycerol as indicated. Media were solidified with 15 g L−1 agar; ampicillin and kanamycin were used at 50 and 50 μg mL−1, respectively. Gene expression was induced with 0.2 g L−1 l-arabinose. Growth kinetics were followed in a Bioscreen-C Automated Growth Curve Analysis System (Growth Curves USA, MA) using the following parameters: continuous shaking; reading every 30 min; culture volume, 200 μL. As inoculum, overnight cultures in LB plus ampicillin and kanamycin (washed three times with M9 medium plus glycerol before dilution) were diluted to give a final OD600 nm of 0.005. Bioscreen experiments used triplicate cultures of three independent strains.

MiaB activity analysis

Bulk nucleic acids were isolated from stationary phase cells cultured in Antibiotic Medium 3 and enriched for tRNA (Bailly et al., 2008) before Nucleobond AXR 400 column purification (Machery-Nagel). Purified tRNA was hydrolysed and analysed by liquid chromatography–tandem mass spectrometry (LC–MS) (Phillips et al., 2008).

Protein analyses

For immunoblot analysis, cells grown in LB medium to an OD600 nm of 1.0 were harvested by centrifugation, washed once in ice-cold phosphate-buffered saline and sonicated in 50 mM Tris–HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride. Extracts were centrifuged to clear. Electrophoresis and immunoblotting were as described (Turner et al., 2005); the primary antibody was anti-pentahistidine mouse monoclonal IgG (Qiagen), dilution 1 : 1000, and the secondary antibody was goat anti-mouse IgG (H + L) alkaline phosphatase conjugate (Bio-Rad), dilution 1 : 3000. Protein was estimated by the Bradford (1976) dye-binding method using bovine serum albumin as standard.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Cysteine 228 is required for normal growth and plumbagin resistance

The functional importance of the eight conserved residues in the Ygf Z motif was assessed by expressing mutant YgfZ proteins from a plasmid and testing their ability to complement various phenotypes of the ΔygfZ strain. Each residue in turn was replaced by alanine.

We first evaluated the mutant proteins qualitatively, via growth on plates containing M9 medium plus glycerol or LB medium plus plumbagin (Fig. 1a). As reported previously (Waller et al., 2010), vector-alone controls showed no growth during the assay period on either medium, and the wild-type Ygf Z protein complemented these defects. Of the mutant proteins, the C228A mutant failed to complement on either medium; all other mutants were as effective as wild type, except the Y229A mutant, which was slightly less effective on LB plus plumbagin. The data for LB medium plus plumbagin confirm and extend those of Lin et al. (2010). To detect more subtle, quantitative effects, growth in M9 plus glycerol was monitored over time in a Bioscreen system. Only the C228A mutant diverged from the wild-type growth curve (Fig. 1b and S1).

image

Figure 1. Alanine-scanning mutagenesis of the conserved motif of Escherichia coli YgfZ. Numbering of the mutated residues is shown at the top of the figure. (a) Growth on plates of the ΔygfZ strain harbouring vector alone (V), wild-type ygfZ or ygfZ genes in which the indicated residues were changed to alanine. Plates contained M9 medium with 2 g L−1 glycerol (M9 + Glyc) or LB medium plus 30 μM plumbagin (LB + PB), 0.2 g L−1 l-arabinose and appropriate antibiotics were incubated for 72 or 20 h, respectively. (b) Bioscreen growth data at 15 h (the approximate mid-point of the growth curve), in liquid M9 plus glycerol medium, for the ΔygfZ strain harbouring the same constructs as above. Data are means and SE of three biological replicates.

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The poor performance of the C228A mutant could not be attributed merely to low expression of this particular mutant protein, as immunoblot analysis showed its level in growing cells to be at least as high as that of the wild type and all other mutant proteins (Fig. 2). Together, the growth results in both liquid and solid media, and the immunoblot data thus point to C228 as the most crucial residue in the Ygf Z signature motif.

image

Figure 2. Expression of wild-type and mutant YgfZ proteins in Escherichia coli. Upper frame: Immunoblot analysis of His-tagged YgfZ constructs expressed in the ∆ygfZ strain. Gel lanes contained 10 μg protein; detection was with anti-His-tag antibody. Cells were grown in LB medium. The mutated residues are indicated in single-letter code above the lanes. V, vector alone; WT, wild-type YgfZ. Lower frame: A Ponceau S-stained blot served as a protein loading and transfer control. Molecular masses (kDa) are indicated on the right.

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Cysteine 228 is required for activity of the Fe/S enzyme MiaB

To extend these results to the biochemical level, we examined the in vivo activity of MiaB, an Fe/S enzyme that mediates the methylthiolation of N6-isopentenyladenosine (i6A) in tRNA to 2-methylthio-N6-isopentenyladenosine (ms2i6A). As MiaB activity depends upon Ygf Z activity, the ms2i6A/i6A ratio in tRNA, determined using LC-MS, is a semiquantitative measure of MiaB activity that in effect reports the activity of Ygf Z (Waller et al., 2010). As expected, ΔygfZ cells harbouring vector alone showed no detectable conversion of i6A to ms2i6A and consequently an ms2i6A/i6A ratio of zero, whereas cells expressing wild-type Ygf Z showed a ratio of 9.1 (Fig. 3). The ratio for a representative mutant with no growth phenotype (G230A) was not significantly different. The ratio of 2.7 for the Y229A mutant was modestly but significantly (< 0.05) lower than wild type, but the ratio of 0.18 for the C229A mutant was dramatically lower. These results for a biochemical phenotype thus mirror the growth data in showing C228 to be by far the most important single residue for Ygf Z function, with the neighbouring Y229 having a much smaller effect. It is possible that the minor effect of Y229 is because of its influence either on the positioning of C228 for efficient interaction with MiaB, or on the properties of C228, as electrostatic interactions between sulphur-containing residues and aromatic residues are a common structural theme in proteins (Reid et al., 1985; Tauer et al., 2005).

image

Figure 3. MiaB activity in the ∆ygfZ strain expressing wild-type or mutant YgfZ proteins, as determined by quantification of i6A and ms2i6A, and the ms2i6A/i6A ratio, in tRNA. The ΔygfZ strain was transformed with pBAD24 alone (V) or pBAD24 encoding wild type (WT) or three mutant forms of YgfZ. Strains were grown in Antibiotic Medium 3 plus 0.2 g L−1 l-arabinose and appropriate antibiotics. The peaks for i6A and ms2i6A were separated and quantified using LC-MS. Data are means and SE for three independent cultures.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The evidence for an absolutely conserved, functionally critical, cysteine residue raises the question of what it does. There are two distinct possibilities: either the cysteine is required for covalent dimer formation, and the dimer is the active form of Ygf Z; or the cysteine thiol is required for Ygf Z action, and the monomer is the active form. As noted in the Introduction, the direct evidence available excludes neither possibility because it is clear that Ygf Z dimerizes readily, at least ex vivo (Teplyakov et al., 2004), and that at least some Ygf Z exists with a free thiol inside plant cells (Hägglund et al., 2008). It will not be possible to show definitively whether Ygf Z works as a disulphide-bonded dimer or as a thiol monomer (or both) until the action of Ygf Z can be reconstituted in vitro. However, the balance of present evidence favours the thiol monomer, as summarized in the following.

Firstly, there is reason to suspect that Ygf Z dimer formation is unphysiological. Thus, the three-dimensional structure of the dimer suggests that the intermolecular C228-C228′ disulphide bridge might not be functionally relevant because the dimer interface formed by multiple nonspecific van der Waals interactions is not extensive and contains none of the conserved dodecapeptide motif residues except C228 (Teplyakov et al., 2004). Moreover, in our pilot tests, recombinant Ygf Z isolated from E. coli was 65% monomeric even when no reductants were added (not shown).

Secondly, E. coli Ygf Z has been shown to have a redox-active cysteine, i.e. a free thiol group, in vivo (Takanishi et al., 2007). Besides C228, Ygf Z has one other cysteine residue, C63, and it was not shown which is the redox-active one (Takanishi et al., 2007). However, the crystal structure places C63 at the C-terminal end of a β-strand in domain B, which makes the sulfhydryl solvent inaccessible, and C228 in an exposed surface loop between two α-helices (α9 and α10) of the Ygf Z monomer (Teplyakov et al., 2004), suggesting that the latter is the redox-active residue.

Finally, Ygf Z belongs to the same protein family as sarcosine oxidase, dimethylglycine oxidase and the T-protein of the glycine-cleavage complex. All of these proteins use tetrahydrofolate to accept a one-carbon (formaldehyde) unit (Teplyakov et al., 2004; Scrutton & Leys, 2005), and the one structurally closest to Ygf Z – the T-protein – acts on a thiol adduct of the one-carbon unit, borne by the H-protein of the complex (Douce et al., 2001). Formaldehyde is a ubiquitous metabolite that spontaneously forms harmful adducts with reactive protein side chains (Metz et al., 2004), and it has been proposed that Ygf Z removes such inhibitory adducts from Fe/S enzymes by transferring the formaldehyde moiety to tetrahydrofolate (Waller et al., 2010). In such an enzyme repair mechanism, a cysteine thiol could logically play a go-between role, analogous to that of the active thiol in the glycine-cleavage complex, by binding formaldehyde after its removal from an Fe/S enzyme and before its transfer to tetrahydrofolate. A repair role for Ygf Z is not incompatible with the proposal that Ygf Z facilitates the breakdown of plumbagin (Lin et al., 2010) because this degradation could involve an enzyme whose activity, like that of MiaB, depends on Ygf Z.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by National Science Foundation grant number MCB-0839926 and by an endowment from the C.V. Griffin Sr. Foundation. Work in the Jez laboratory was supported by National Science Foundation grant MCB-0904215. We thank V. de Crécy-Lagard for advice.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
fml2448-sup-0001-FigureS1.TIFimage/TIF1602KFig. S1. Bioscreen growth curves for the ΔygfZ strain harbouring the pBAD24 vector alone (V) or expressing wild type (WT) or mutant forms of Ygf Z.

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