Andrew W. Foster

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.

Cyanobacterial metallochaperone inhibits deleterious side reactions of copper

Copper metallochaperones supply copper to cupro-proteins through copper-mediated protein-protein-interactions and it has been hypothesized that metallochaperones thereby inhibit copper from causing damage en route. Evidence is presented in support of this latter role for cyanobacterial metallochaperone, Atx1. In cyanobacteria Atx1 contributes towards the supply of copper to plastocyanin inside thylakoids but it is shown here that in copper-replete medium, copper can reach plastocyanin without Atx1. Unlike metallochaperone-independent copper-supply to superoxide dismutase in eukaryotes, glutathione is not essential for Atx1-independent supply to plastocyanin: Double mutants missing atx1 and gshB (encoding glutathione synthetase) accumulate the same number of atoms of copper per cell in the plastocyanin pool as wild type. Critically, Δatx1ΔgshB are hypersensitive to elevated copper relative to wild type cells and also relative to ΔgshB single mutants with evidence that hypersensitivity arises due to the mislocation of copper to sites for other metals including iron and zinc. The zinc site on the amino-terminal domain (ZiaAN) of the P1-type zinc-transporting ATPase is especially similar to the copper site of the Atx1 target PacSN, and ZiaAN will bind Cu(I) more tightly than zinc. An NMR model of a substituted-ZiaAN-Cu(I)-Atx1 heterodimer has been generated making it possible to visualize a juxtaposition of residues surrounding the ZiaAN zinc site, including Asp18, which normally repulse Atx1. Equivalent repulsion between bacterial copper metallochaperones and the amino-terminal regions of P1-type ATPases for metals other than Cu(I) is conserved, again consistent with a role for copper metallochaperones to withhold copper from binding sites for other metals.

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 ΔGCNi(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 KZn(II) (5.6 × 10−13 M) is comparable to the sensory sites of ZiaR (and Zur), but ΔGCZn(II)‐InrS·DNA is less than ΔGCZn(II)‐ZiaR·DNA implying that relative to other sensors, ΔGCZn(II)‐Sensor·DNA rather than KZn(II) determines the final detection threshold for Zn(II).

Promiscuity and preferences of metallothioneins: the cell rules

Metalloproteins are essential for many cellular functions, but it has not been clear how they distinguish between the different metals to bind the correct ones. A report in BMC Biology finds that preferences of two metallothionein isoforms for two different cations are due to inherent properties of these usually less discriminating proteins. Here these observations are discussed in the context of the cellular mechanisms that regulate metal binding to proteins.See research article: http://www.biomedcentral.com/1741-7007/9/4

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)2Cu(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.

Co(ll)-detection does not follow Kco(ll) gradient: channelling in Co(ll)-sensing.

The MerR-like transcriptional activator CoaR detects surplus Co(ll) to regulate Co(ll) efflux in a cyanobacterium. This organism also has cytosolic metal-sensors from three further families represented by Zn(ll)-sensors ZiaR and Zur plus Ni(ll)-sensor InrS. Here we discover by competition with Fura-2 that CoaR has KCo(ll) weaker than 7 × 10(-8) M, which is weaker than ZiaR, Zur and InrS (KCo(ll) = 6.94 ± 1.3 × 10(-10) M; 4.56 ± 0.16 × 10(-10) M; and 7.69 ± 1.1 × 10(-9) M respectively). KCo(ll) for CoaR is also weak in the CoaR-DNA adduct. Further, Co(ll) promotes DNA-dissociation by ZiaR and DNA-association by Zur in vitro in a manner analogous to Zn(ll), as monitored by fluorescence anisotropy. After 48 h exposure to maximum non-inhibitory [Co(ll)], CoaR responds in vivo yet the two Zn(ll)-sensors do not, despite their tighter KCo(ll) and despite Co(ll) triggering allostery in ZiaR and Zur in vitro. These data imply that the two Zn(ll) sensors fail to respond because they fail to gain access to Co(ll) under these conditions in vivo. Several lines of evidence suggest that CoaR is membrane associated via a domain with sequence similarity to precorrin isomerase, an enzyme of vitamin B12 biosynthesis. Moreover, site directed mutagenesis reveals that transcriptional activation requires CoaR residues that are predicted to form hydrogen bonds to a tetrapyrrole. The Co(ll)-requiring vitamin B12 biosynthetic pathway is also membrane associated suggesting putative mechanisms by which Co(ll)-containing tetrapyrroles and/or Co(ll) ions are channelled to CoaR.

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-tRNAThr synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNAThr. 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.

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 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 to the change in Ni(II)-affinity. All of the variant-sensors retained the allosteric mechanism which inhibits DNA binding upon metal binding but a response to nickel in vivo was only observed when the sensitivity was set to respond within a relatively narrow (less than 2 orders of magnitude) range of nickel-concentrations. The Ni(II)-affinity of InrS is attuned to cellular metal concentrations rather than the converse.

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.Bacteria possess transcription factors whose DNA-binding activity is altered upon binding to specific metals, but the binding of metals is not specific in vitro. Here, Osman et al. show that tight regulation of buffered intracellular metal concentrations is a prerequisite for metal specificity.

An XAS investigation of the nickel site structure in the transcriptional regulator InrS

InrS (Internal nickel-responsive Sensor) is a transcriptional repressor of the nickel exporter NrsD and de-represses expression of the exporter upon binding Ni(II) ions. Although a crystal structure of apo-InrS has been reported, no structure of the protein with metal ions bound is available. Herein we report the results of metal site structural investigations of Ni(II) and Cu(II) complexes of InrS using X-ray absorption spectroscopy (XAS) that are complementary to data available from the apo-InrS crystal structure, and are consistent with a planar four-coordinate [Ni(His)2(Cys)2] structure, where the ligands are derived from the side chains of His21, Cys53, His78, and Cys82. Coordination of Cu(II) to InrS forms a nearly identical planar four-coordinate complex that is consistent with a simple replacement of the Ni(II) center by Cu(II).