The foetal plasma protein alpha-fetoprotein (AFP) harbours a high-affinity zinc binding site that is likely involved in transport and delivery of essential zinc during foetal development. Based on a recent electron microscopy structure of AFP and aided by biophysical studies on an AFP-derived peptide, we present a refined 5-coordinate model for this site.

Food security is defined as uninterrupted access to food that meets people's dietary needs. One essential trace element of a complete diet is zinc, which is vital for various processes, including growth, development, and the immune response. The estimated global prevalence of zinc deficiency is around 30%. Meat and meat products provide an abundant and also bioavailable source of zinc. However, in developing countries, access to meat is restricted, and in developed countries, meat consumption has declined for ethical and environmental reasons. The potential for zinc deficiency arises from (i) low concentrations of this element in plant-based diets, (ii) poor zinc absorption from plant-based food in the human intestine, and (iii) the risk of uptake of toxic metals together with essential ones. This review summarises the current knowledge concerning type 4 metallothioneins, which represent promising targets for zinc biofortification. We describe their place in the zinc route from soil to seed, their expression patterns, their role in plants, and their three-dimensional protein structure and how this affects their selectivity towards zinc. This review aims to provide a comprehensive theoretical basis for the potential use of type 4 plant metallothioneins to create zinc-biofortified crops.

Interactions between proteins and metal ions and their complexes are important in many areas of the life sciences, including physiology, medicine, and toxicology. Despite the involvement of essential elements in all major processes necessary for sustaining life, metalloproteomes remain ill-defined. This is not only owing to the complexity of metalloproteomes, but also to the non-covalent character of the complexes that most essential metals form, which complicates analysis. Similar issues may also be encountered for some toxic metals. The review discusses recently developed approaches and current challenges for the study of interactions involving entire (sub-)proteomes with such labile metal ions. In the second part, transition metals from the fourth and fifth periods are examined, most of which are xenobiotic and also tend to form more stable and/or inert complexes. A large research area in this respect concerns metallodrug–protein interactions. Particular attention is paid to separation approaches, as these need to be adapted to the reactivity of the metal under consideration.

The chemical properties of toxic cadmium and essential zinc are very similar, and organisms require intricate mechanisms that drive selective handling of metals. Previously regarded as unspecific “metal sponges”, metallothioneins (MTLs) are emerging as metal selectivity filters. By utilizing C. elegans mtl-1 and mtl-2 knockout strains, metal accumulation in single worms, single copy fluorescent-tagged transgenes, isoform specific qPCR and lifespan studies it was possible to demonstrate that the handling of cadmium and zinc by the two C. elegans metallothioneins differs fundamentally: the MTL-2 protein can handle both zinc and cadmium, but when it becomes unavailable, either via a knockout or by elevated cadmium exposure, MTL-1 takes over zinc handling, leaving MTL-2 to sequester cadmium. This division of labour is reflected in the folding behaviour of the proteins: MTL-1 folded well in presence of zinc but not cadmium, the reverse was the case for MTL-2. These differences are in part mediated by a zinc-specific mononuclear His3Cys site in the C-terminal insertion of MTL-1; its removal affected the entire C-terminal domain and may shift its metal selectivity towards zinc. Overall, we uncover how metallothionein isoform-specific responses and protein properties allow C. elegans to differentiate between toxic cadmium and essential zinc.

Zinc is required for virtually all biological processes. In plasma, Zn2+ is predominantly transported by human serum albumin (HSA), which possesses two Zn2+-binding sites of differing affinities (sites A and B). Fatty acids (FAs) are also transported by HSA, with seven structurally characterized FA-binding sites (named FA1-FA7) known. FA binding inhibits Zn2+-HSA interactions, in a manner that can impact upon hemostasis and cellular zinc uptake, but the degree to which binding at specific FA sites contributes to this inhibition is unclear. Wild-type HSA and H9A, H67A, H247A, and Y150F/R257A/S287A (FA2-KO) mutant albumins were expressed in Pichia pastoris. Isothermal titration calorimetry studies revealed that the Zn2+-binding capacity at the high-affinity Zn2+ site (site A) was reduced in H67A and H247A mutants, with site B less affected. The H9A mutation decreased Zn2+ binding at the lower-affinity site, establishing His9 as a site B ligand. Zn2+ binding to HSA and H9A was compromised by palmitate, consistent with FA binding affecting site A. 13C-NMR experiments confirmed that the FA2-KO mutations prohibited FA binding at site FA2. Zn2+ binding to the FA2-KO mutant was unaffected by myristate, suggesting binding at FA2 is solely responsible for inhibition. Molecular dynamics studies identified the steric obstruction exerted by bound FA in site FA2, which impedes the conformational change from open (FA-loaded) to closed (FA-free) states, required for Zn2+ to bind at site A. The successful targeting of the FA2 site will aid functional studies exploring the interplay between circulating FA levels and plasma Zn2+ speciation in health and disease.

Abstract Zur (zinc uptake regulator) proteins are the most widespread bacterial zinc‐sensing transcriptional regulators, being encoded in the majority of sequenced bacterial genomes. They work predominantly as transcriptional repressors of zinc uptake genes when zinc in a bacterium's environment is abundant. Zinc scarcity in turn leads to de‐repression of these genes, which leads to increased zinc uptake. More recently, several Zur proteins have also been shown to work as activators of transcription, but the mechanism(s) for this type of regulation are not fully understood. Several 3D structures of Zur proteins are available, including those from the actinobacterium Streptomyces coelicolor, pathogenic Mycobacterium tuberculosis and Xanthomonas campestris , and the marine cyanobacterium Synechococcus sp. WH8102. The SynZur protein from the latter photosynthetic organism is the focus of this entry. In contrast to many other bacteria which harbor at least two zinc‐sensing regulators, SynZur is the sole zinc‐sensing transcription factor in this cyanobacterium. Like many other Zur proteins, it regulates the transcription of the znuABC genes, which encode ABC‐type zinc uptake systems located at the inner membrane. Uniquely, however, SynZur also activates transcription of a bacterial metallothionein ( bmtA ). The latter small cysteine‐rich proteins have a high capacity to bind up to four zinc ions and are normally regulated by repressors of the SmtB family, in response to zinc excess. The ability to upregulate production of a zinc storage protein upon encountering ‘luxury’ zinc appears to enable Synechococcus sp. WH8102 and related oligotrophic strains to populate ecological niches that are poor in inorganic nutrients but may experience episodic rises in zinc levels.

CYP106A2 (cytochrome P450meg) is a bacterial enzyme originally isolated from B. megaterium, and has been shown to hydroxylate a wide variety of substrates, including steroids. The regio- and stereochemistry of CYP106A2 hydroxylation has been shown to be dependent on a variety of factors, and hydroxylation often occurs at more than one site and/or with lack of stereospecificity for some substrates. Comprehensive backbone 15N, 1H and 13C resonance assignments based on multidimensional nuclear magnetic resonance (NMR) experiments performed with uniform and selective isotopically labeled CYP106A2 samples are reported herein, and broadening and splitting of resonances assigned to regions of the enzyme shown to be affected by substrate binding in other P450 enzymes indicate that substrate binding does not reduce structural heterogeneity as has been observed previously in P450 enzymes CYP101A1 and MycG. Paramagnetic relaxation enhancement (PRE) due to proximity between substrate protons and the heme iron were measured for three different substrates, and the relatively uniform nature of the PREs support the proposal that multiple substrate binding modes are occupied at saturating substrate concentrations.

In marine systems, the availability of inorganic phosphate can limit primary production leading to bacterial and phytoplankton utilization of the plethora of organic forms available. Among these are phospholipids that form the lipid bilayer of all cells as well as released extracellular vesicles. However, information on phospholipid degradation is almost nonexistent despite their relevance for biogeochemical cycling. Here, we identify complete catabolic pathways for the degradation of the common phospholipid headgroups phosphocholine (PC) and phosphorylethanolamine (PE) in marine bacteria. Using Phaeobacter sp. MED193 as a model, we provide genetic and biochemical evidence that extracellular hydrolysis of phospholipids liberates the nitrogen-containing substrates ethanolamine and choline. Transporters for ethanolamine (EtoX) and choline (BetT) are ubiquitous and highly expressed in the global ocean throughout the water column, highlighting the importance of phospholipid and especially PE catabolism in situ. Thus, catabolic activation of the ethanolamine and choline degradation pathways, subsequent to phospholipid metabolism, specifically links, and hence unites, the phosphorus, nitrogen, and carbon cycles.

Serum albumin-Co2+ interactions are of clinical importance. They play a role in mediating the physiological effects associated with cobalt toxicity and are central to the albumin cobalt binding (ACB) assay for diagnosis of myocardial ischemia. To further understand these processes, a deeper understanding of albumin-Co2+ interactions is required. Here, we present the first crystallographic structures of human serum albumin (HSA; three structures) and equine serum albumin (ESA; one structure) in complex with Co2+. Amongst a total of sixteen sites bearing a cobalt ion across the structures, two locations were prominent, and they relate to metal-binding sites A and B. Site-directed mutagenesis and isothermal titration calorimetry (ITC) were employed to characterise sites on HSA. The results indicate that His9 and His67 contribute to the primary (putatively corresponding to site B) and secondary Co2+-binding sites (site A), respectively. The presence of additional multiple weak-affinity Co2+ binding sites on HSA was also supported by ITC studies. Furthermore, addition of 5 molar equivalents of the non-esterified fatty acid palmitate (C16:0) reduced the Co2+-binding affinity at both sites A and B. The presence of bound myristate (C14:0) in the HSA crystal structures provided insight into the fatty acid-mediated structural changes that diminish the affinity of the protein toward Co2+. Together, these data provide further support for the idea that ischemia-modified albumin corresponds to albumin with excessive fatty-acid loading. Collectively, our findings provide a comprehensive understanding of the molecular underpinnings governing Co2+ binding to serum albumin.

The role of the extracellular medium in influencing metal uptake into cells has not been described quantitatively. In a chemically-defined model system containing albumin, zinc influx into endothelial cells correlates with the extracellular free zinc concentration. Allosteric inhibition of zinc-binding to albumin by free fatty acids increased zinc flux.

The speciation of essential metal ions in biological fluids, such as blood plasma and serum, is of fundamental importance to understand the homeostasis of these elements. The activity of metal ions such as Zn2+ in extra -cellular media is thought to affect their interaction with membrane-bound transporters, and thus is critical for their cellular uptake. Previous approaches to determine "free " Zn2+ (i.e. the hexaaquo ion) are based on separation by either chromatography or ultrafiltration, or on metallochromic dyes. However, both types of approach are prone to affect the relevant equilibria. These drawbacks can be circumvented with the electroanalytical technique AGNES (Absence of Gradients and Nernstian Equilibrium Stripping), since it can measure free zinc concentration without perturbing the sample speciation. Here, a Bovine Serum Albumin (BSA) + Zn synthetic mixture and Fetal Bovine Serum (FBS) are analyzed as proof of concept. Adsorption of BSA on the surface of the Hanging Mercury Drop Electrode (HMDE), despite the advantage of its renewal, is so intense that it blocks appropriate attainment of the required equilibrium, and only estimations of [Zn2+] can be derived. In contrast, a rotating disc electrode with a thin mercury film deposited on it (TMF-RDE) is advantageous because of its small volume and enhanced mass transfer. Protein adsorption can be prevented by covering the TMF-RDE with Nafion. A free Zn concentration [Zn2+] = 2.7 nmol L-1 was found at pH 7.0, total Zn 20 mu mu mol L(-1 )and BSA 600 mu mu mol L-1. A sample of FBS with fixed pH 7.2 (MOPS 0.08 mol L-1) yielded [Zn2+] = 0.25 nmol L-1. This methodology opens the way to free metal concentration determinations in biological fluids.

The study of chemical speciation and the refinement and expansion of omics-based methods are both consolidated and highly active research fields. Although well established, such fields are extremely dynamic and are driven by the emergence of new strategies and improvements in instrumentation. In the case of omics-based studies, subareas including lipidomics, proteomics, metallomics, metabolomics and foodomics have emerged. Here, speciomics is being proposed as an "umbrella" term, that incorporates all of these subareas, to capture studies where the evaluation of chemical species is carried out using omics approaches. This paper contextualizes both speciomics and the speciome, and reviews omics applications used for species identification through examination of proteins, metalloproteins, metabolites, and nucleic acids. In addition, some implications from such studies and a perspective for future development of this area are provided. SIGNIFICANCE: The synergic effect between chemical speciation and omics is highlighted in this work, demonstrating an emerging area of research with a multitude of possibilities in terms of applications and further developments. This work not only defines and contextualizes speciomics and individual speciomes, but also demonstrates with some examples the great potential of this new interdisciplinary area of research.

Marine cyanobacteria are critical players in global nutrient cycles that crucially depend on trace metals in metalloenzymes, including zinc for CO 2 fixation and phosphorus acquisition. How strains proliferating in the vast oligotrophic ocean gyres thrive at ultra-low zinc concentrations is currently unknown. Using Synechococcus sp. WH8102 as a model we show that its zinc-sensor protein Zur differs from all other known bacterial Zur proteins in overall structure and the location of its sensory zinc site. Uniquely, Synechococcus Zur activates metallothionein gene expression, which supports cellular zinc quotas spanning two orders of magnitude. Thus, a single zinc sensor facilitates growth across pico- to micromolar zinc concentrations with the bonus of banking this precious resource. The resultant ability to grow well at both ultra-low and excess zinc, together with overall lower zinc requirements, likely contribute to the broad ecological distribution of Synechococcus across the global oceans.

Bacterial metallothioneins are known for a limited range of phyla including cyanobacteria. We have characterised the BmtA from the marine cyanobacterium Synechococcus sp. WH8102 (SynBmtA). This strain inhabits the open ocean, one of the most nutrient-poor environments on Earth, with very low total and free Zn2+ concentrations. Therefore, the presence of a metallothionein, usually associated with zinc and cadmium tolerance, in this strain is intriguing. Previous transcriptomics work revealed that unprecedentedly, expression of SynBmtA is activated by the Synechococcus sp. WH8102 "zinc uptake regulator" (SynZur) at elevated [Zn2+]. SynBmtA binds four Zn2+ ions, and its first 37 residues adopt the zinc-finger fold characteristic of BmtAs. In contrast, sequence similarity to other BmtAs in the C-terminal stretch is low. This is expected to affect especially the most reactive site in zinc-transfer reactions. Indeed, chelators were unable to extract Zn2+ from SynBmtA, even in the presence of denaturant. This indicates an extremely stable protein fold, with no accessibility to any bound zinc ions in the folded protein. In addition, the zinc-binding affinity of SynBmtA exceeds those of any other metallothioneins. Apo-SynBmtA is capable of removing zinc from the sensory site of SynZur, providing one possible avenue of de-activating transcription of the synbmtA gene. All of these properties are consistent with a role in safely sequestering any excess zinc, to prevent toxic effects. The fact that this strain stores zinc in a metallothionein rather than employing an efflux pump implies that zinc is a valuable resource for Synechococcus sp. WH8102 and related strains.

The initiation, maintenance and regulation of blood coagulation is inexorably linked to the actions of Zn2+ in blood plasma. Zn2+ interacts with a variety of haemostatic proteins in the bloodstream including fibrinogen, histidine-rich glycoprotein (HRG) and high molecular weight kininogen (HMWK) to regulate haemostasis. The availability of Zn2+ to bind such proteins is controlled by human serum albumin (HSA), which binds 70–85% of plasma Zn2+ under basal conditions. HSA also binds and transports non-esterified fatty acids (NEFAs). Upon NEFA binding, there is a change in the structure of HSA which leads to a reduction in its affinity for Zn2+. This enables other plasma proteins to better compete for binding of Zn2+. In diseases where elevated plasma NEFA concentrations are a feature, such as obesity and diabetes, there is a concurrent increase in hypercoagulability. Evidence indicates that NEFA-induced perturbation of Zn2+-binding by HSA may contribute to the thrombotic complications frequently observed in these pathophysiological conditions. This review highlights potential interventions, both pharmaceutical and non-pharmaceutical that may be employed to combat this dysregulation. Lifestyle and dietary changes have been shown to reduce plasma NEFA concentrations. Furthermore, drugs that influence NEFA levels such as statins and fibrates may be useful in this context. In severely obese patients, more invasive therapies such as bariatric surgery may be useful. Finally, other potential treatments such as chelation therapies, use of cholesteryl transfer protein (CETP) inhibitors, lipase inhibitors, fatty acid inhibitors and other treatments are highlighted, which with additional research and appropriate clinical trials, could prove useful in the treatment and management of thrombotic disease through amelioration of plasma Zn2+ dysregulation in high-risk individuals.

Protonation equilibria for the parent compound of three highly potent antivirals have been studied by 1H NMR spectroscopy.

Zn2+ is an essential regulator of coagulation and is released from activated platelets. In plasma, the free Zn2+ concentration is fine-tuned through buffering by human serum albumin (HSA). Importantly, the ability of HSA to bind/buffer Zn2+ is compromised by co-transported non-esterified fatty acids (NEFAs). Given the role of Zn2+ in blood clot formation, we hypothesise that Zn2+ displacement from HSA by NEFAs in certain conditions (such as type 2 diabetes mellitus, T2DM) impacts on the cellular and protein arms of coagulation. To test this hypothesis, we assessed the extent to which increasing concentrations of a range of medium- and long-chain NEFAs reduced Zn2+-binding ability of HSA. Amongst the NEFAs tested, palmitate (16 : 0) and stearate (18 : 0) were the most effective at suppressing zinc-binding, whilst the mono-unsaturated palmitoleate (16 : 1c9) was markedly less effective. Assessment of platelet aggregation and fibrin clotting parameters in purified systems and in pooled plasma suggested that the HSA-mediated impact of the model NEFA myristate on zinc speciation intensified the effects of Zn2+ alone. The effects of elevated Zn2+ alone on fibrin clot density and fibre thickness in a purified protein system were mirrored in samples from T2DM patients, who have derranged NEFA metabolism. Crucially, T2DM individuals had increased total plasma NEFAs compared to controls, with the concentrations of key saturated (myristate, palmitate, stearate) and mono-unsaturated (oleate, cis-vaccenate) NEFAs positively correlating with clot density. Collectively, these data strongly support the concept that elevated NEFA levels contribute to altered coagulation in T2DM through dysregulation of plasma zinc speciation.