Molecules for remediating or recovering uranium from contaminated environmental resources are of high current interest, with protein-based ligands coming into focus recently. Metallothioneins either bind or redox-silence a range of heavy metals, conferring protection against metal stress in many organisms. Here, we report that the cyanobacterial metallothionein SmtA competes with carbonate for uranyl binding, leading to formation of heterometallic (UO2)nZn4SmtA species, without thiol oxidation, zinc loss, or compromising secondary or tertiary structure of SmtA. In turn, only metalated and folded SmtA species were found to be capable of uranyl binding. 1H NMR studies and molecular modeling identified Glu34/Asp38 and Glu12/C-terminus as likely adventitious, but surprisingly strong, bidentate binding sites. While it is unlikely that these interactions correspond to an evolved biological function of this metallothionein, their occurrence may offer new possibilities for designing novel multipurpose bacterial metallothioneins with dual ability to sequester both soft metal ions including Cu+, Zn2+, Cd2+, Hg2+, and Pb2+ and hard, high-oxidation state heavy metals such as U(VI). The concomitant protection from the chemical toxicity of uranium may be valuable for the development of bacterial strains for bio-remediation.

Marine cyanobacteria make a significant contribution to primary production whilst occupying some of the most nutrient poor regions of the world's oceans. The low bioavailability of trace metals can limit the growth of phytoplankton in ocean waters, but only scarce data are available on the requirements of marine microbes for zinc. Recent genome mining studies suggest that marine cyanobacteria have both uptake systems for zinc and proteins that utilize zinc as a cofactor. In this study, the oligotrophic strain Synechococcus sp. WH8102 was grown at different zinc concentrations. Using metalloproteomics approaches, we demonstrate that even though this organism's growth was not affected by extremely low zinc levels, cells accumulated significant quantities of zinc, which was shown to be protein-associated by 2D liquid chromatography and ICP-MS. This indicates that the mechanisms for zinc uptake in Synechococcus sp. WH8102 are extremely efficient. Significantly, expression of SYNW2224, a putative porin, was up-regulated during growth in zinc-depleted conditions. Furthermore, along with 30 other proteins, SYNW2224 was captured by immobilised zinc affinity chromatography, indicating the presence of surface-exposed site(s) with metal-binding capacity. It is proposed that this porin plays a role in high-affinity zinc uptake in this and other cyanobacteria.

The predominant species of the cyanobacterial metalloregulatory protein SmtB as observed by ESI-MS is a dimer with all four zinc binding sites occupied.

More than 30 years have passed since the discovery of the first plant metallothionein in wheat embryos, from which the emergence of a uniquely diverse metallothionein family with a fascinating array of structural nuances and molecular properties has been witnessed. Metallothioneins are not only constitutively expressed, but the production of different types of plant metallothionein is also stimulated by a myriad of endogenous and exogenous agents in both a temporally and spatially regulated manner. This ubiquitous, yet discrete expression of metallothioneins not only signifies their importance for plant survival and development, but also suggests a functional divergence for the individual plant metallothionein subfamilies. Understanding why one type of plant metallothionein has more advantageous structural and metal binding attributes over another for a given biological process is a crucial piece in the puzzle of assigning physiological functions to these proteins. In this review, we discuss how in vivo and in vitro studies have advanced our understanding of the structure–property–function relationship for the plant metallothionein family. In particular, we highlight the progress that has been made for the Type 4 plant metallothioneins.

Zinc is one of the most important micronutrients for virtually all living organisms, and hence, it is important to understand the molecular mechanisms for its homeostasis. Besides proteins involved in transmembrane transport, both extra- and intracellular zinc-binding proteins play important roles in the respective metabolic networks. Important examples for extracellular zinc transporters are mammalian serum albumins, and for intracellular zinc handling, certain metallothioneins are of relevance. The availability of protein structures including relevant metal binding sites is a fundamental prerequisite to decipher the mechanisms that govern zinc binding dynamics in these proteins, but their determination can prove to be surprisingly challenging. Due to the spectroscopic silence of Zn2 +, combinations of biophysical techniques including electrospray ionisation mass spectrometry (ESI-MS) and multinuclear NMR, isothermal titration calorimetry (ITC) and extended X-ray absorption fine structure (EXAFS) spectroscopy, coupled with site-directed mutagenesis and molecular modelling have proven to be valuable approaches to understand not only the zinc-binding properties of metallothioneins and albumins, but also the influence of other physiologically relevant competing agents. These studies have demonstrated why the bacterial metallothionein SmtA contains a site inert towards exchange with Cd2 +, why the plant metallothionein EC from wheat is partially unfolded in the presence of Cd2 +, and how fatty acids impact on the zinc-binding ability of mammalian serum albumins.Understanding the molecular mechanisms that govern zinc movements in biological systems requires the study of structures of relevant proteins, their zinc binding sites, and how metal binding and structural dynamics including conformational changes affect each other.Highlights► The bacterial metallothionein SmtA contains a zinc finger fold. ► SmtA contains a site inert towards metal exchange. ► EC MT can only form an ordered fold in the presence of Zn, but not Cd. ► Zinc binding by albumin is allosterically linked to fatty acid binding.

Albumin transports both fatty acids and zinc in plasma. Competitive binding studied by isothermal titration calorimetry revealed that physiologically relevant levels of fatty acids modulate the Zn-binding capacity of albumin, with far-reaching implications for biological zinc speciation. The molecular mechanism for this effect is likely due to a large conformational change elicited by fatty acid binding to a high-affinity interdomain site that disrupts at least one Zn site. Albumin may be a molecular device to “translate” certain aspects of the organismal energy state into global zinc signals.

The biomarker “ischemia-modified albumin” (IMA), measured by the albumin–cobalt-binding assay (ACB assay), is the only FDA-approved biomarker for early diagnosis of myocardial ischemia. On the basis of the hypothesis that high levels of free fatty acids are directly responsible for reduction in cobalt binding by albumin, chemically defined model systems consisting of bovine serum albumin, Co2+, and myristate were studied by isothermal titration calorimetry, 111Cd NMR spectroscopy, and ACB assays. Significantly reduced Co2+ binding to albumin, as demonstrated by an increase in the absorption of the Co-dithiothreitol adduct, elicited by adding ca. 3 mol equiv of myristate, was comparable to that observed in clinical ACB assays. Levels of free fatty acids are elevated during myocardial ischemia but also in other conditions that have been correlated with high IMA values. Hence, IMA may correspond to albumin with increased levels of bound fatty acids, and all clinical observations can be rationalized by this molecular mechanism.

Bacterial metallothioneins (MTs) have been known since the mid-1980s. The only family known until recently was the BmtA family, exemplified by the zinc- and cadmium-binding SmtA from the cyanobacterium Synechococcus PCC 7942, for which a structure was determined in 2001. Only in 2008 was a second type of bacterial MT identified in mycobacteria, and the copper-binding gene product was called MymT. Many of the features of SmtA either have been unexpected or are otherwise “unusual”, for example the presence of a zinc finger fold and the kinetic inertness of one of the four zinc ions bound to the protein. The unpredictability of molecular properties of this protein exemplified the need for continued biophysical studies of novel proteins. Homologues for SmtA have been identified in a limited number of bacterial genomes from cyanobacteria, pseudomonads, alphaproteobacteria, gammaproteobacteria, and firmicutes. Except for the residues defining the zinc finger fold, these homologous protein sequences display an intriguing variety, especially in terms of metal ligand position and identity. The increased number of homologues has allowed use of hidden Markov models to look for more remote relatives of SmtA, leading to the identification of a novel family of putative hybrid LIM domain MTs. However, database searches based on sequence similarity are of limited use for mining for further “overlooked” bacterial MTs, as so far undiscovered bacterial MTs may be too diverse from any other known MTs, and other approaches are required.

Covering: 1999 to 2009

The handling of trace elements by plants plays a fundamental role in food security and safety. Therefore, there is a need to understand the mechanisms that govern metal ion trafficking in plants. The past decade has seen an immense expansion of knowledge on metal ion transport in a variety of biological systems including plants, but as for other organisms, the mechanisms for intracellular zinc trafficking remain enigmatic. The current report highlights recent advances in understanding zinc transport in plants and in identifying the biomolecules involved in this process. A particular focus is put on pinpointing determinants for zinc specificity, and we also highlight areas in need of development. Reliable experimental speciation data are a first step towards systems biology approaches to mineral nutrition in plants—but there is also a need to understand molecular details. One intriguing question, in particular in the context of predicting protein function, concerns how discrimination between metal ions in a biological system functions.

The direct observation of binding and release of spectroscopically silent metal ions such as Zn2+ and Ca2+ by proteins has been challenging before the advent of native electrospray ionisation mass spectrometry. This report highlights the powerful capability of ESI-MS to provide insight into metalloprotein speciation that is independent of any spectroscopic property. Using the zinc-binding plant metallothionein EC from wheat as a study case, we show that ESI-MS is unique amongst other techniques in capturing intermediary metallospecies that evolve during the course of metal transfer to the chelator EDTA, as a model reaction to mimic the biological function of the protein as a zinc donor. Zinc release from the two-domain protein EC appears to be extremely rapid and non-cooperative, and progresses with loss of one zinc ion from the fully loaded Zn6 species, and a transient build-up of Zn5 and Zn4 species, which further react to give species with 0-3 zinc ions bound. 1H NMR data has provided further insights into the different behaviour of the two domains upon metal depletion.

The selectivity of proteins involved in metal ion homeostasis is an important part of the puzzle to understand how cells allocate the correct metal ions to the correct proteins. Due to their similar ligand-binding properties, and their frequent co-existence in soils, essential zinc and toxic cadmium are a particularly challenging couple. Thus, minimisation of competition of Cd2+ for Zn2+ sites is of crucial importance for organisms that are in direct contact with soil. Amongst these, plants have an especially critical role, due to their importance for nutrition and energy. We have studied an embryo-specific, zinc-binding metallothionein (EC) from wheat by nuclear magnetic resonance, electrospray mass spectrometry, site-directed mutagenesis, and molecular modelling. Wheat EC exploits differences in affinities of Cys4 and Cys2His2 sites for Cd2+ and Zn2+ to achieve metal-selective protein folding. We propose that this may constitute a novel mechanism to discriminate between essential Zn2+ and toxic Cd2+.

The bacterial metallothionein SmtA binds four zinc ions with high affinity and specificity in a Zn4S9N2 cluster. We have explored the reactivity of these zinc ions towards the metal-chelator EDTA. Under pseudo-first-order conditions, initial break-down of zinc-thiolate bonds is rapid, followed by several slower phases. The reaction with stoichiometric amounts of EDTA is relatively slow and has been followed by 1H NMR and mass spectrometry. Both methods reveal that partially metallated intermediates occur during the reaction. Three- and two-metal species are observed in only minor amounts, whereas the Zn1 species is dominant during the mid stages of the reaction, before complete metal depletion occurs. These results suggest that the zinc finger site in SmtA is not only inert towards metal exchange, but also more resilient towards chelating agents. The greater inertness of this site may help to maintain the protein fold during metal depletion, and allow subsequent facile metal uptake. Conversely, it is likely that the protein fold is the major contributor to the observed persistence of Zn1SmtA in this reaction. Mass spectrometric studies with His-to-Cys mutants of SmtA reveal that the primary site for EDTA attack is the His49-containing zinc site C, and that His40 has a major influence on the reactivity of three sites.The hybrid metallothionein/zinc finger SmtA, a protein from cyanobacteria, reacts with EDTA in a multiphasic ligand exchange reaction. The coordinating histidine residues tune the reactivity of the zinc ions. One particular site in the cluster is the prime target for competing ligands, and the zinc finger site is considerably less reactive than the other three metal ions.

The cyanobacterial metallothionein (MT) SmtA is the prototype for bacterial MTs and protects against elevated levels of zinc. In contrast to mammalian MTs, bacterial MTs coordinate to metal ions not only via cysteine sulfurs, but unusually for MTs, also via histidine nitrogens. To investigate whether histidine coordination in these metal–sulfur clusters provides advantages over S-coordination only, we mutated the two metal-binding histidine residues in the cyanobacterial MT SmtA from Synechococcus PCC7942 to cysteines. We show that the mutant proteins are still capable of binding up to four zinc ions as is the wild-type protein. However, the mutations perturb protein folding and metal-binding dynamics. Interestingly, several homologues of SmtA also show variations in these two residues. We conclude that histidine residues in Synechococcus PCC7942 SmtA have a stabilising effect due to electrostatic interactions that impact on protein folding and metal cluster charge, and are involved in fine-tuning the reactivity of the bound metal ions.

The predominant species of the cyanobacterial metalloregulatory protein SmtB as observed by ESI-MS is a dimer with all four zinc binding sites occupied.

The direct observation of binding and release of spectroscopically silent metal ions such as Zn2+ and Ca2+ by proteins has been challenging before the advent of native electrospray ionisation mass spectrometry. This report highlights the powerful capability of ESI-MS to provide insight into metalloprotein speciation that is independent of any spectroscopic property. Using the zinc-binding plant metallothionein EC from wheat as a study case, we show that ESI-MS is unique amongst other techniques in capturing intermediary metallospecies that evolve during the course of metal transfer to the chelator EDTA, as a model reaction to mimic the biological function of the protein as a zinc donor. Zinc release from the two-domain protein EC appears to be extremely rapid and non-cooperative, and progresses with loss of one zinc ion from the fully loaded Zn6 species, and a transient build-up of Zn5 and Zn4 species, which further react to give species with 0-3 zinc ions bound. 1H NMR data has provided further insights into the different behaviour of the two domains upon metal depletion.