WhiD is required for the late stages of sporulation in the Gram-positive bacterium Streptomyces coelicolor. WhiD is a member of the WhiB-like family of putative transcription factors that are present throughout the actinomycetes but absent from other organisms. This family of proteins has four near-invariant cysteines, suggesting that these residues might act as ligands for a metal cofactor. Overexpressed WhiD, purified from Escherichia coli, contained substoichiometric amounts of iron and had an absorption spectrum characteristic of a [2Fe-2S] cluster. After Fe-S cluster reconstitution under anaerobic conditions, WhiD contained ϳ4 iron atoms/monomer and similar amounts of sulfide ion and gave an absorption spectrum characteristic of a [4Fe-4S] cluster. Reconstituted WhiD gave no electron paramagnetic resonance signal as prepared but, after reduction with dithionite, gave an electron paramagnetic resonance signal (g ϳ2.06, Fe-S cluster proteins are an ancient class of proteins (1) that play important roles in electron transfer and in numerous metabolic pathways across all kingdoms of life. The realization that Fe-S cluster proteins can also play vital regulatory roles is comparatively recent, and only four such proteins have been characterized to date. In mammalian cells, the [4Fe-4S] cluster form of the cytosolic isozyme of aconitase catalyzes the isomerization of citrate to isocitrate. However, in its apoform, termed iron regulatory protein, it binds specific mRNAs, in some cases to block their translation and in other cases to enhance it (2, 3). The apoforms of bacterial aconitases have recently also been found to bind specific mRNAs and function as translational regulators (4, 5). In addition, three transcription factors that have regulatory Fe-S clusters have been characterized, namely, FNR, SoxR, and IscR (reviewed in Ref.3). All three of these transcription factors were discovered in E. coli, but it is now clear that Fe-S-dependent regulatory networks involving these proteins are present in many other bacterial genera. In this report we have presented evidence that WhiD, a member of a family of putative transcription factors previously characterized only at the genetic level, also binds a regulatory Fe-S cluster.The whiD developmental gene was isolated from Streptomyces coelicolor by map-based cloning and complementation (6). Sequencing revealed that WhiD is a member of the Wbl (WhiBlike) family of putative transcription factors that are present throughout the actinomycetes but absent from all other organisms so far sequenced (6, 7). The actinomycetes include Streptomyces, the genus responsible for the production of two-thirds of the known antibiotics, as well as medically important pathogens like Mycobacterium tuberculosis and Corynebacterium diphtheriae. Although the biochemical function of Wbl proteins, including WhiD, has not been demonstrated, circumstantial evidence suggests they are transcriptional activators: WhiB3, the WhiD orthologue of M. tuberculosis, was identified in a yeast two-hybrid scree...

a Metallothioneins (MTs), small cysteine-rich proteins, present in four major isoforms, are key proteins involved in zinc and copper homeostasis in mammals. To date, only one X-ray crystal structure of a MT has been solved. It demonstrates seven bivalent metal ions bound in two structurally independent domains with M 4 S 11 (a) and M 3 S 9 (b) clusters. Recent discoveries indicate that Zn(II) ions are bound with MT2 with the range from nano-to picomolar affinity, which determines its cellular zinc buffering properties that are demonstrated by the presence of partially Zn(II)-depleted MT2 species. These forms serve as Zn(II) donors or acceptors and are formed under varying cellular free Zn(II) concentrations. Due to the lack of appropriate methods, knowledge regarding the structure of partially-depleted metallothionein is lacking. Here, we describe the Zn(II) binding mechanism in human MT2 with high resolution with respect to particular Zn(II) binding sites, and provide structural insights into Zn(II)-depleted MT species. The results were obtained by the labelling of metal-free cysteine residues with iodoacetamide and subsequent top-down electrospray ionization analysis, MALDI MS, bottom-up nanoLC-MALDI-MS/MS approaches and molecular dynamics (MD) simulations. The results show that the a-domain is formed sequentially in the first stages, followed by the formation of the b-domain, although both processes overlap, which is in contrast to the widely investigated cadmium MT. Independent ZnS 4 cores are characteristic for early stages of domain formation and are clustered in later stages. However, Zn-S network rearrangement in the b-domain upon applying the seventh Zn(II) ion explains its lower affinity. Detailed analysis showed that the weakest Zn(II) ion associates with the b-domain by coordination to Cys21, which was also found to dissociate first in the presence of the apo-form of sorbitol dehydrogenase. We found that Zn(II) binding to the isolated b-domain differs significantly from the whole protein, which explains its previously observed different Zn(II)-binding properties. MD results obtained for Zn(II) binding to the whole protein and isolated b-domain are highly convergent with mass spectrometry data. This study provides a comprehensive overview of the crosstalk of structural and zinc buffering related-to-thermodynamics properties of partially metal-saturated mammalian MT2 and sheds more light on other MT proteins and zinc homeostasis. Significance to metallomicsThe growing interest in metallomics within metal-dependent biological systems provides an opportunity to explore the structural and buffering properties of metallothioneins (MTs). The wide relevance of MTs is obvious due to their multiple biological functions such as participation in zinc and copper homeostasis. However, the MT metalloproteome is still not fully explored and is rather complicated in vivo or in vitro because of its highly dynamic structure, which lacks secondary structural elements and metamorphic behaviour. We have undertaken compreh...

WhiD, a member of the WhiB-like (Wbl) family of iron-sulfur proteins found exclusively within the actinomycetes, is required for the late stages of sporulation in Streptomyces coelicolor. Like all other Wbl proteins, WhiD has not so far been purified in a soluble form that contains a significant amount of cluster and characterization has relied on cluster-reconstituted protein. Thus, a major goal in Wbl research is to obtain and characterize native protein containing iron-sulfur clusters. Here we report the analysis of S. coelicolor WhiD purified anaerobically from E. coli as a soluble protein containing a single [4Fe-4S] 2+ cluster ligated by four cysteines. Upon exposure to oxygen, spectral features associated with the [4Fe-4S] cluster were lost in a slow reaction that unusually yielded apo-WhiD directly without significant concentrations of cluster intermediates. This process was found to be highly pH dependent with an optimal stability observed between pH 7.0 and 8.0. Low molecular weight thiols, including a mycothiol analogue and thioredoxin, exerted a small but significant protective effect against WhiD cluster loss, an activity that could be of physiological importance.[4Fe-4S] 2+ WhiD was found to react much more rapidly with superoxide than with either oxygen or hydrogen peroxide, which may also be of physiological significance. Loss of the [4Fe-4S] cluster to form apo-protein destabilized the protein fold significantly, but did not lead to complete unfolding. Finally, apo-WhiD exhibited negligible activity in an insulin-based disulfide reductase assay demonstrating that it does not function as a general protein disulfide reductase.WhiB-like (Wbl) proteins are found exclusively in the actinomycetes, a phylum of Grampositive bacteria that includes Streptomyces, the most abundant source of clinically important antibiotics and other bioactive molecules, and medically important pathogens such as Mycobacterium tuberculosis and Corynebacterium diphtheriae (1,2). Disruption of wbl genes has shown that Wbl proteins play critical roles in the biology of both Streptomyces and Mycobacterium (1,3). In Streptomyces coelicolor, WhiB, the founding member of the Wbl 1To whom correspondence should be addressed: A. J. Thomson Table S1; The reactivity of the [4Fe-4S] cluster monitored by CD spectroscopy is shown in Figure S1; The reactivity of the [4Fe-4S] cluster with superoxide is shown in Figure S2; Analysis of apo-and as isolated WhiD by SDS-PAGE is shown in Figure S3; EPR spectroscopy data following oxidation of WhiD is shown in Figure S4. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 December 29. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript family, is required for the initiation of sporulation septation, and WhiD, the subject of this report, is required for the late stages of sporulation (1,3,4). In M. tuberculosis, Wbl proteins have been implicated in...

The minimal zinc hook peptide of Rad50 and its alanine mutants form highly stable Zn(II) complexes. These peptides were successfully used as a small, efficient tag for reversible Zn(II)-mediated protein homodimerization. The high stability, its biological consequences and potential applications in protein engineering are discussed.

Background: Knowledge regarding UDP-N-acetylglucosamine transporter (NGT; SLC35A3) is incomplete due to the lack of NGT-deficient model cell lines. Results: The siRNA approach showed that NGT silencing reduces branching of complex N-glycans and keratan sulfate synthesis. Conclusion: NGT function may be coupled to the specific glycosylation pathway(s) of particular macromolecules. Significance: Our results add to the understanding of glycosylation, one of the basic posttranslational modifications.