Two Distinct Thermodynamic Gradients for Cellular Metalation of Vitamin B12.

The acquisition of CoII by the corrin component of vitamin B12 follows one of two distinct pathways, referred to as early or late CoII insertion. The late insertion pathway exploits a CoII metallochaperone (CobW) from the COG0523 family of G3E GTPases, while the early insertion pathway does not. This provides an opportunity to contrast the thermodynamics of metalation in a metallochaperone-requiring and a metallochaperone-independent pathway. In the metallochaperone-independent route, sirohydrochlorin (SHC) associates with the CbiK chelatase to form CoII-SHC. CoII-buffered enzymatic assays indicate that SHC binding enhances the thermodynamic gradient for CoII transfer from the cytosol to CbiK. In the metallochaperone-dependent pathway, hydrogenobyrinic acid a,c-diamide (HBAD) associates with the CobNST chelatase to form CoII-HBAD. Here, CoII-buffered enzymatic assays indicate that CoII transfer from the cytosol to HBAD-CobNST must somehow traverse a highly unfavorable thermodynamic gradient for CoII binding. Notably, there is a favorable gradient for CoII transfer from the cytosol to the MgIIGTP-CobW metallochaperone, but further transfer of CoII from the GTP-bound metallochaperone to the HBAD-CobNST chelatase complex is thermodynamically unfavorable. However, after nucleotide hydrolysis, CoII transfer from the chaperone to the chelatase complex is calculated to become favorable. These data reveal that the CobW metallochaperone can overcome an unfavorable thermodynamic gradient for CoII transfer from the cytosol to the chelatase by coupling this process to GTP hydrolysis.

Protein metalation in a nutshell.

Metalation, the acquisition of metals by proteins, must avoid mis-metalation with tighter binding metals. This is illustrated by four selected proteins that require different metals: all show similar ranked orders of affinity for bioavailable metals, as described in a universal affinity series (the Irving-Williams series). Crucially, cellular protein metalation occurs in competition with other metal binding sites. The strength of this competition defines the intracellular availability of each metal: its magnitude has been estimated by calibrating a cells' set of DNA-binding, metal-sensing, transcriptional regulators. This has established that metal availabilities (as free energies for forming metal complexes) are maintained to the inverse of the universal series. The tightest binding metals are least available. With these availabilities, correct metalation is achieved.

Metalation calculators for E. coli strain JM109 (DE3): aerobic, anaerobic, and hydrogen peroxide exposed cells cultured in LB media.

Three Web-based calculators, and three analogous spreadsheets, have been generated that predict in vivo metal occupancies of proteins based on known metal affinities. The calculations exploit estimates of the availabilities of the labile buffered pools of different metals inside a cell. Here, metal availabilities have been estimated for a strain of Escherichia coli that is commonly used in molecular biology and biochemistry research, e.g. in the production of recombinant proteins. Metal availabilities have been examined for cells grown in Luria-Bertani (LB) medium aerobically, anaerobically, and in response to H2O2 by monitoring the abundance of a selected set of metal-responsive transcripts by quantitative polymerase chain reaction (qPCR). The selected genes are regulated by DNA-binding metal sensors that have been thermodynamically characterized in related bacterial cells enabling gene expression to be read out as a function of intracellular metal availabilities expressed as free energies for forming metal complexes. The calculators compare these values with the free energies for forming complexes with the protein of interest, derived from metal affinities, to estimate how effectively the protein can compete with exchangeable binding sites in the intracellular milieu. The calculators then inter-compete the different metals, limiting total occupancy of the site to a maximum stoichiometry of 1, to output percentage occupancies with each metal. In addition to making these new and conditional calculators available, an original purpose of this article was to provide a tutorial that discusses constraints of this approach and presents ways in which such calculators might be exploited in basic and applied research, and in next-generation manufacturing.

Protein metalation in biology.

Inorganic metals supplement the chemical repertoire of organic molecules, especially proteins. This requires the correct metals to associate with proteins at metalation. Protein mismetalation typically occurs when excesses of unbound metals compete for a binding site ex vivo. However, in biology, excesses of metal-binding sites typically compete for limiting amounts of exchangeable metals. Here, we summarise mechanisms of metal homeostasis that sustain optimal metal availabilities in biology. We describe recent progress to understand metalation by comparing the strength of metal binding to a protein versus the strength of binding to competing sites inside cells.

Calculating metalation in cells reveals CobW acquires CoII for vitamin B12 biosynthesis while related proteins prefer ZnII.

Protein metal-occupancy (metalation) in vivo has been elusive. To address this challenge, the available free energies of metals have recently been determined from the responses of metal sensors. Here, we use these free energy values to develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells. We use the calculator to understand the function and mechanism of GTPase CobW, a predicted CoII-chaperone for vitamin B12. Upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator also reveals that related GTPases with comparable ZnII affinities to CobW, preferentially acquire ZnII due to their relatively weaker CoII affinities. The calculator is made available here for use with other proteins.

The requirement for cobalt in vitamin B12: A paradigm for protein metalation.

Vitamin B12, cobalamin, is a cobalt-containing ring-contracted modified tetrapyrrole that represents one of the most complex small molecules made by nature. In prokaryotes it is utilised as a cofactor, coenzyme, light sensor and gene regulator yet has a restricted role in assisting only two enzymes within specific eukaryotes including mammals. This deployment disparity is reflected in another unique attribute of vitamin B12 in that its biosynthesis is limited to only certain prokaryotes, with synthesisers pivotal in establishing mutualistic microbial communities. The core component of cobalamin is the corrin macrocycle that acts as the main ligand for the cobalt. Within this review we investigate why cobalt is paired specifically with the corrin ring, how cobalt is inserted during the biosynthetic process, how cobalt is made available within the cell and explore the cellular control of cobalt and cobalamin levels. The partitioning of cobalt for cobalamin biosynthesis exemplifies how cells assist metalation.

Metalation: nature's challenge in bioinorganic chemistry.

The association of proteins with metals, metalation, is challenging because the tightest binding metals are rarely the correct ones. Inside cells, correct metalation is enabled by controlled bioavailability plus extra mechanisms for tricky combinations such as iron and manganese.

Bacterial sensors define intracellular free energies for correct enzyme metalation.

There is a challenge for metalloenzymes to acquire their correct metals because some inorganic elements form more stable complexes with proteins than do others. These preferences can be overcome provided some metals are more available than others. However, while the total amount of cellular metal can be readily measured, the available levels of each metal have been more difficult to define. Metal-sensing transcriptional regulators are tuned to the intracellular availabilities of their cognate ions. Here we have determined the standard free energy for metal complex formation to which each sensor, in a set of bacterial metal sensors, is attuned: the less competitive the metal, the less favorable the free energy and hence the greater availability to which the cognate allosteric mechanism is tuned. Comparing these free energies with values derived from the metal affinities of a metalloprotein reveals the mechanism of correct metalation exemplified here by a cobalt chelatase for vitamin B12.

A unique ferredoxin acts as a player in the low-iron response of photosynthetic organisms.

Iron chronically limits aquatic photosynthesis, especially in marine environments, and the correct perception and maintenance of iron homeostasis in photosynthetic bacteria, including cyanobacteria, is therefore of global significance. Multiple adaptive mechanisms, responsive promoters, and posttranscriptional regulators have been identified, which allow cyanobacteria to respond to changing iron concentrations. However, many factors remain unclear, in particular, how iron status is perceived within the cell. Here we describe a cyanobacterial ferredoxin (Fed2), with a unique C-terminal extension, that acts as a player in iron perception. Fed2 homologs are highly conserved in photosynthetic organisms from cyanobacteria to higher plants, and, although they belong to the plant type ferredoxin family of [2Fe-2S] photosynthetic electron carriers, they are not involved in photosynthetic electron transport. As deletion of fed2 appears lethal, we developed a C-terminal truncation system to attenuate protein function. Disturbed Fed2 function resulted in decreased chlorophyll accumulation, and this was exaggerated in iron-depleted medium, where different truncations led to either exaggerated or weaker responses to low iron. Despite this, iron concentrations remained the same, or were elevated in all truncation mutants. Further analysis established that, when Fed2 function was perturbed, the classical iron limitation marker IsiA failed to accumulate at transcript and protein levels. By contrast, abundance of IsiB, which shares an operon with isiA, was unaffected by loss of Fed2 function, pinpointing the site of Fed2 action in iron perception to the level of posttranscriptional regulation.

Fine control of metal concentrations is necessary for cells to discern zinc from cobalt.

Bacteria possess transcription factors whose DNA-binding activity is altered upon binding to specific metals, but metal binding is not specific in vitro. Here we show that tight regulation of buffered intracellular metal concentrations is a prerequisite for metal specificity of Zur, ZntR, RcnR and FrmR in Salmonella Typhimurium. In cells, at non-inhibitory elevated concentrations, Zur and ZntR, only respond to Zn(II), RcnR to cobalt and FrmR to formaldehyde. However, in vitro all these sensors bind non-cognate metals, which alters DNA binding. We model the responses of these sensors to intracellular-buffered concentrations of Co(II) and Zn(II) based upon determined abundances, metal affinities and DNA affinities of each apo- and metalated sensor. The cognate sensors are modelled to respond at the lowest concentrations of their cognate metal, explaining specificity. However, other sensors are modelled to respond at concentrations only slightly higher, and cobalt or Zn(II) shock triggers mal-responses that match these predictions. Thus, perfect metal specificity is fine-tuned to a narrow range of buffered intracellular metal concentrations.

A tight tunable range for Ni(II) sensing and buffering in cells.

The metal affinities of metal-sensing transcriptional regulators co-vary with cellular metal concentrations over more than 12 orders of magnitude. To understand the cause of this relationship, we determined the structure of the Ni(II) sensor InrS and then created cyanobacteria (Synechocystis PCC 6803) in which transcription of genes encoding a Ni(II) exporter and a Ni(II) importer were controlled by InrS variants with weaker Ni(II) affinities. Variant strains were sensitive to elevated nickel and contained more nickel, but the increase was small compared with the change in Ni(II) affinity. All of the variant sensors retained the allosteric mechanism that inhibits DNA binding following metal binding, but a response to nickel in vivo was observed only when the sensitivity was set to respond in a relatively narrow (less than two orders of magnitude) range of nickel concentrations. Thus, the Ni(II) affinity of InrS is attuned to cellular metal concentrations rather than the converse.

Cost-effectiveness analysis of offering free leisure centre memberships to physically inactive members of the public receiving state benefits: a case study.

BACKGROUND: We evaluated the cost-effectiveness of the Give-it-a-Go programme, which offers free leisure centre memberships to physically inactive members of the public in a single London Borough receiving state benefits. METHODS: A decision analytic Markov model was developed to analyse lifetime costs and quality-adjusted life-years (QALYs) of 1025 people recruited to the intervention versus no intervention. In the intervention group, people were offered 4 months of free membership at a leisure centre. Physical activity levels were assessed at 0 and 4 months using the International Physical Activity Questionnaire (IPAQ). Higher levels of physical activity were assumed to decrease the risk of coronary heart disease, stroke and diabetes mellitus type II, as well as improve mental health. Costs were assessed from a National Health Service (NHS) perspective. Uncertainty was assessed using one-way and probabilistic sensitivity analyses. RESULTS: One-hundred fifty nine participants (15.5 %) completed the programme by attending the leisure centre for 4 months. Compared with no intervention, Give it a Go increased costs by £67.25 and QALYs by 0.0033 (equivalent to 1.21 days in full health) per recruited person. The incremental costs per QALY gained were £20,347. The results were highly sensitive to the magnitude of mental health gain due to physical activity and the duration of the effect of the programme (1 year in the base case analysis). When the mental health gain was omitted from the analysis, the incremental cost per QALY gained increased to almost £1.5 million. In the probabilistic sensitivity analysis, the incremental costs per QALY gained were below £20,000 in 39 % of the 5000 simulations. CONCLUSIONS: Give it a Go did not significantly increase life-expectancy, but had a positive influence on quality of life due to the mental health gain of physical activity. If the increase in physical activity caused by Give it a Go lasts for more than 1 year, the programme would be cost-effective given a willingness to pay for a QALY of £20,000.

The Effectors and Sensory Sites of Formaldehyde-responsive Regulator FrmR and Metal-sensing Variant.

The DUF156 family of DNA-binding transcriptional regulators includes metal sensors that respond to cobalt and/or nickel (RcnR, InrS) or copper (CsoR) plus CstR, which responds to persulfide, and formaldehyde-responsive FrmR. Unexpectedly, the allosteric mechanism of FrmR from Salmonella enterica serovar Typhimurium is triggered by metals in vitro, and variant FrmR(E64H) gains responsiveness to Zn(II) and cobalt in vivo Here we establish that the allosteric mechanism of FrmR is triggered directly by formaldehyde in vitro Sensitivity to formaldehyde requires a cysteine (Cys(35) in FrmR) conserved in all DUF156 proteins. A crystal structure of metal- and formaldehyde-sensing FrmR(E64H) reveals that an FrmR-specific amino-terminal Pro(2) is proximal to Cys(35), and these residues form the deduced formaldehyde-sensing site. Evidence is presented that implies that residues spatially close to the conserved cysteine tune the sensitivities of DUF156 proteins above or below critical thresholds for different effectors, generating the semblance of specificity within cells. Relative to FrmR, RcnR is less responsive to formaldehyde in vitro, and RcnR does not sense formaldehyde in vivo, but reciprocal mutations FrmR(P2S) and RcnR(S2P), respectively, impair and enhance formaldehyde reactivity in vitro Formaldehyde detoxification by FrmA requires S-(hydroxymethyl)glutathione, yet glutathione inhibits formaldehyde detection by FrmR in vivo and in vitro Quantifying the number of FrmR molecules per cell and modeling formaldehyde modification as a function of [formaldehyde] demonstrates that FrmR reactivity is optimized such that FrmR is modified and frmRA is derepressed at lower [formaldehyde] than required to generate S-(hydroxymethyl)glutathione. Expression of FrmA is thereby coordinated with the accumulation of its substrate.

Trans-oligomerization of duplicated aminoacyl-tRNA synthetases maintains genetic code fidelity under stress.

Aminoacyl-tRNA synthetases (aaRSs) play a key role in deciphering the genetic message by producing charged tRNAs and are equipped with proofreading mechanisms to ensure correct pairing of tRNAs with their cognate amino acid. Duplicated aaRSs are very frequent in Nature, with 25,913 cases observed in 26,837 genomes. The oligomeric nature of many aaRSs raises the question of how the functioning and oligomerization of duplicated enzymes is organized. We characterized this issue in a model prokaryotic organism that expresses two different threonyl-tRNA synthetases, responsible for Thr-tRNA(Thr) synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNA(Thr). Low zinc promotes dissociation of dimeric T1 into monomers deprived of aminoacylation activity and simultaneous induction of T2, which is active for aminoacylation under low zinc. T2 either forms homodimers or heterodimerizes with T1 subunits that provide essential proofreading activity in trans. These findings evidence that in organisms with duplicated genes, cells can orchestrate the assemblage of aaRSs oligomers that meet the necessities of the cell in each situation. We propose that controlled oligomerization of duplicated aaRSs is an adaptive mechanism that can potentially be expanded to the plethora of organisms with duplicated oligomeric aaRSs.

Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells.

FrmR from Salmonella enterica serovar typhimurium (a CsoR/RcnR-like transcriptional de-repressor) is shown to repress the frmRA operator-promoter, and repression is alleviated by formaldehyde but not manganese, iron, cobalt, nickel, copper, or Zn(II) within cells. In contrast, repression by a mutant FrmRE64H (which gains an RcnR metal ligand) is alleviated by cobalt and Zn(II). Unexpectedly, FrmR was found to already bind Co(II), Zn(II), and Cu(I), and moreover metals, as well as formaldehyde, trigger an allosteric response that weakens DNA affinity. However, the sensory metal sites of the cells' endogenous metal sensors (RcnR, ZntR, Zur, and CueR) are all tighter than FrmR for their cognate metals. Furthermore, the endogenous metal sensors are shown to out-compete FrmR. The metal-sensing FrmRE64H mutant has tighter metal affinities than FrmR by approximately 1 order of magnitude. Gain of cobalt sensing by FrmRE64H remains enigmatic because the cobalt affinity of FrmRE64H is substantially weaker than that of the endogenous cobalt sensor. Cobalt sensing requires glutathione, which may assist cobalt access, conferring a kinetic advantage. For Zn(II), the metal affinity of FrmRE64H approaches the metal affinities of cognate Zn(II) sensors. Counter-intuitively, the allosteric coupling free energy for Zn(II) is smaller in metal-sensing FrmRE64H compared with nonsensing FrmR. By determining the copies of FrmR and FrmRE64H tetramers per cell, then estimating promoter occupancy as a function of intracellular Zn(II) concentration, we show how a modest tightening of Zn(II) affinity, plus weakened DNA affinity of the apoprotein, conspires to make the relative properties of FrmRE64H (compared with ZntR and Zur) sufficient to sense Zn(II) inside cells.

Camden active spaces: does the construction of active school playgrounds influence children's physical activity levels? A longitudinal quasi-experiment protocol.

INTRODUCTION: Physical activity is essential for every facet of children's health. However, physical activity levels in British children are low. The school environment is a promising setting to increase children's physical activity but limited empirical evidence exists on how a change in the outdoor physical school environment influences physical activity behaviour. The London Borough of Camden is redesigning seven existing school playgrounds to engage children to become more physically active. The primary aim of this project is to evaluate the impact of the redesigned playgrounds on children's physical activity, well-being and physical function/fitness. METHOD AND ANALYSIS: This project will use a longitudinal quasi-experimental design. Seven experimental schools and one control school will take part. One baseline data collection session and two follow-ups will be carried out. Between baseline and follow-up, the experimental school playgrounds will be redesigned. At baseline, a series of fitness tests, anthropometric and questionnaire measurements, and 7-day objective physical activity monitoring (Actigraph accelerometer) will be carried out on children (aged 5­16 years). This will be repeated at follow-up. Changes in overall physical activity levels and levels during different times of the day (eg, school breaks) will be examined. Multilevel regression modelling will be used to analyse the data. ETHICS AND DISSEMINATION: The results of this study will be disseminated through peer-review publications and scientific presentations. Ethical approval was obtained through the University College London Research Ethics Committee (Reference number: 4400/002).

Metal preferences and metallation.

The metal binding preferences of most metalloproteins do not match their metal requirements. Thus, metallation of an estimated 30% of metalloenzymes is aided by metal delivery systems, with ∼ 25% acquiring preassembled metal cofactors. The remaining ∼ 70% are presumed to compete for metals from buffered metal pools. Metallation is further aided by maintaining the relative concentrations of these pools as an inverse function of the stabilities of the respective metal complexes. For example, magnesium enzymes always prefer to bind zinc, and these metals dominate the metalloenzymes without metal delivery systems. Therefore, the buffered concentration of zinc is held at least a million-fold below magnesium inside most cells.

Encouraging data citation and discovery with the Data Citation Index.

An overview of the Data Citation Index is provided. Thomson Reuters developed this resource in response to a stated desire among members of the research community for increased attribution of non-traditional scholarly output. Launched in October of 2012 on the Web of science research platform, its aims include linking published research articles to their underlying data sets and tracking the citation of the data, as well as encouraging bibliographic citation of data. Cross-disciplinary search capabilities in the Index enable new possibilities for data discovery and synthesis. Data repositories are evaluated with respect to various selection criteria, with particular attention to their relevance to scientific and scholarly research. Index content reflects current data deposition practices. As data citation standards and practices continue to move toward widespread formalization and adoption, the initiative seeks to address issues of data citation, reuse, and author credit in a developing climate.

A chemical potentiator of copper-accumulation used to investigate the iron-regulons of Saccharomyces cerevisiae.

The extreme resistance of Saccharomyces cerevisiae to copper is overcome by 2-(6-benzyl-2-pyridyl)quinazoline (BPQ), providing a chemical-biology tool which has been exploited in two lines of discovery. First, BPQ is shown to form a red (BPQ)2 Cu(I) complex and promote Ctr1-independent copper-accumulation in whole cells and in mitochondria isolated from treated cells. Multiple phenotypes, including loss of aconitase activity, are consistent with copper-BPQ mediated damage to mitochondrial iron-sulphur clusters. Thus, a biochemical basis of copper-toxicity in S. cerevisiae is analogous to other organisms. Second, iron regulons controlled by Aft1/2, Cth2 and Yap5 that respond to mitochondrial iron-sulphur cluster status are modulated by copper-BPQ causing iron hyper-accumulation via upregulated iron-import. Comparison of copper-BPQ treated, untreated and copper-only treated wild-type and fra2Δ by RNA-seq has uncovered a new candidate Aft1 target-gene (LSO1) and paralogous non-target (LSO2), plus nine putative Cth2 target-transcripts. Two lines of evidence confirm that Fra2 dominates basal repression of the Aft1/2 regulons in iron-replete cultures. Fra2-independent control of these regulons is also observed but CTH2 itself appears to be atypically Fra2-dependent. However, control of Cth2-target transcripts which is independent of CTH2 transcript abundance or of Fra2, is also quantified. Use of copper-BPQ supports a substantial contribution of metabolite repression to iron-regulation.

Metal specificity of cyanobacterial nickel-responsive repressor InrS: cells maintain zinc and copper below the detection threshold for InrS.

InrS is a Ni(II)-responsive, CsoR/RcnR-like, DNA-binding transcriptional repressor of the nrsD gene, but the Ni(II) co-ordination sphere of InrS is unlike Ni(II)-RcnR. We show that copper and Zn(II) also bind tightly to InrS and in vitro these ions also impair InrS binding to the nrsD operator-promoter. InrS does not respond to Zn(II) (or copper) in vivo after 48 h, when Zn(II) sensor ZiaR responds, but InrS transiently responds (1 h) to both metals. InrS conserves only one (of two) second co-ordination shell residues of CsoR (Glu98 in InrS). The allosteric mechanism of InrS is distinct from Cu(I)-CsoR and conservation of deduced second shell residues better predicts metal specificity than do the metal ligands. The allosteric mechanism of InrS permits greater promiscuity in vitro than CsoR. The factors dictating metal-selectivity in vivo are that KNi(II) and ΔG(C)(Ni(II)-InrS·DNA) are sufficiently high, relative to other metal sensors, for InrS to detect Ni(II), while the equivalent parameters for copper may be insufficient for copper-sensing in Synechocystis (at 48 h). InrS K(Zn(II)) (5.6 × 10(-13) M) is comparable to the sensory sites of ZiaR (and Zur), but ΔG(C)(Zn(II)-InrS·DNA) is less than ΔG(C)(Zn(II)-ZiaR·DNA) implying that relative to other sensors, ΔG(C)(Zn(II)-Sensor·DNA) rather than K(Zn(II)) determines the final detection threshold for Zn(II).