Advances in the molecular understanding of biological zinc transport

Between 5 and 10% of all proteins of a given organism are estimated to require zinc for function, and hence zinc is essential for almost any given metabolic process. It is therefore of great interest to understand major players and mechanisms that ensure the tight and correct control of zinc distribution and speciation in organisms and their individual cells. Significant progress has been made in recent years regarding 3-dimensional structures and modes of action of zinc sensor proteins, membrane-bound zinc transporters for cellular and sub-cellular uptake and efflux, as well as intracellular binding proteins. This feature article highlights advances in structures, zinc-binding sites and thermodynamics of proteins that are involved in zinc homeostasis and trafficking, including developments in understanding the metal selectivity of proteins

The reduced Co2+-binding ability of ischaemia-modified albumin is unlikely to be because of oxidative modification of the N-terminus

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The type 4 metallothionein from Brassica napus seeds folds in a metal-dependent fashion and favours zinc over other metals

The problem of handling zinc in the cell is of great importance because zinc is an indis-pensable micronutrient involved in most physiological processes in all living organisms. Moreover, our understanding of mechanisms governing the discrimination between mi-cronutrients and toxic metals on the level of individual proteins to the whole-organism level is incomplete. Metallothioneins are able to bind heavy metal ions, and roles in zinc homeostasis have been proposed. Here, we have studied the in vitro and in vivo metal-binding abilities of Brassica napus type 4 metallothionein (BnMT4) and its expression in germinating seeds in response to metal treatment. Our studies on the regulation of MT4 expression by metals at early stages of ontogenic development revealed for the first time that the mRNA levels of BnMT4 were elevated in response to cadmium and zinc. Given this unexpected metalloregulation, and the dramatic differences in protein folding as de-tected by 1H NMR spectroscopy, we suggest that the BnMT4 protein may not only have a role in zinc homeostasis in early ontogenesis, but also the potential to discriminate be-tween zinc and cadmium, perhaps via differential recognition of Cd- and Zn-complexes by cellular components involved in protein turnover

Earthworm Lumbricus rubellus MT-2 : metal binding and protein folding of a true Cadmium-MT

Earthworms express, as most animals, metallothioneins (MTs)—small, cysteine-rich proteins that bind d10 metal ions (Zn(II), Cd(II), or Cu(I)) in clusters. Three MT homologues are known for Lumbricus rubellus, the common red earthworm, one of which, wMT-2, is strongly induced by exposure of worms to cadmium. This study concerns composition, metal binding affinity and metal-dependent protein folding of wMT-2 expressed recombinantly and purified in the presence of Cd(II) and Zn(II). Crucially, whilst a single Cd7wMT-2 species was isolated from wMT-2-expressing E. coli cultures supplemented with Cd(II), expressions in the presence of Zn(II) yielded mixtures. The average affinities of wMT-2 determined for either Cd(II) or Zn(II) are both within normal ranges for MTs; hence, differential behaviour cannot be explained on the basis of overall affinity. Therefore, the protein folding properties of Cd- and Zn-wMT-2 were compared by 1H NMR spectroscopy. This comparison revealed that the protein fold is better defined in the presence of cadmium than in the presence of zinc. These differences in folding and dynamics may be at the root of the differential behaviour of the cadmium- and zinc-bound protein in vitro, and may ultimately also help in distinguishing zinc and cadmium in the earthworm in vivo

Differential reactivity of closely related zinc(II)-binding metallothioneins from the plant Arabidopsis thaliana

Support by Advantage West Midlands and the European Regional Development Fund (Birmingham Science City) is gratefully acknowledged.The dynamics of metal binding to and transfer from metalloproteins involved in metal homeostasis are important for understanding cellular distribution of metal ions. The dicotyledonous plant Arabidopsis thaliana has two type 4 seed-specific metallothionein homologues, MT4a and MT4b, with likely roles in zinc(II) homeostasis. These two metallothioneins are 84% identical, with full conservation of all metal-binding cysteine and histidine residues. Yet, differences in their spatial and temporal expression patterns suggested divergence in their biological roles. To investigate whether biological functions are reflected in molecular properties, we compare aspects of zinc(II)-binding dynamics of full-length MT4a and MT4b, namely the pH dependence of zinc(II) binding and protein folding, and zinc(II) transfer to the chelator EDTA. UV–Vis and NMR spectroscopies as well as native electrospray ionisation mass spectrometry consistently showed that transfer from Zn6MT4a is considerably faster than from Zn6MT4b, with pseudo-first-order rate constants for the fastest observed step of kobs = 2.8 × 10−4 s−1 (MT4b) and kobs = 7.5 × 10−4 s−1 (MT4a) (5 µM protein, 500 µM EDTA, 25 mM Tris buffer, pH 7.33, 298 K). 2D heteronuclear NMR experiments allowed locating the most labile zinc(II) ions in domain II for both proteins. 3D homology models suggest that reactivity of this domain is governed by the local environment around the mononuclear Cys2His2 site that is unique to type 4 MTs. Non-conservative amino acid substitutions in this region affect local electrostatics as well as whole-domain dynamics, with both effects rendering zinc(II) ions bound to MT4a more reactive in metal transfer reactions. Therefore, domain II of MT4a is well suited to rapidly release its bound zinc(II) ions, in broad agreement with a previously suggested role of MT4a in zinc(II) transport and delivery to other proteins.Publisher PDFPeer reviewe

Biophysical characterization of a protein for structure comparison : methods for identifying insulin structural changes

Although protein structure has been studied for many decades it remains the case that we cannot state with confidence whether two samples have the same molecular structure, particularly in solution. The increasing number of biosimilar biopharmaceutical drugs that are being tested means this is not an academic exercise. In this work we consider how four well-established techniques: dynamic light scattering (DLS), circular dichroism (CD), nuclear magnetic resonance spectroscopy (NMR), and molecular modelling can be combined to provide information about the supposedly well-understood protein insulin. A goal of this work was to establish a systematic means of detecting differences between insulin samples as a function of pH, temperature, and the presence or absence of zinc, all of which are known to change the oligomerisation state and to affect molecular structure. We used the recently developed Secondary Structure Neural Network (SSNN) circular dichroism algorithm to facilitate analysis of the CD spectra

O2-independent demethylation of trimethylamine N-oxide by Tdm of Methylocella silvestris

Bacterial trimethylamine N-oxide (TMAO) demethylase, Tdm, carries out an unusual oxygen-independent demethylation reaction, resulting in the formation of dimethylamine and formaldehyde. In this study, sitedirected mutagenesis, homology modelling and metal analyses by inorganic mass spectrometry have been applied to gain insight into metal stoichiometry and underlying catalytic mechanism of Tdm of Methylocella silvestris BL2. Herein, we demonstrate that active Tdm has 1 molar equivalent of Zn2+ and 1 molar equivalent of non-heme Fe2+. We further investigated Zn2+ and Fe2+-binding sites through homology modelling and sitedirected mutagenesis and found that Zn2+ is coordinated by a 3-sulfur-1-O motif. An aspartate residue (D198) likely bridges Fe2+ and Zn2+ centres, either directly or indirectly via H-bonding through a neighbouring H2O molecule. H276 contributes to Fe2+ binding, mutation of which results in an inactive enzyme, and the loss of iron, but not zinc. Site-directed mutagenesis of Tdm also led to the identification of three hydrophobic aromatic residues likely involved in substrate coordination (F259, Y305, W321), potentially through a cation- interaction. Furthermore, a cross-over experiment using a substrate analogue gave direct evidence that a trimethylamine-alike intermediate was produced during the Tdm catalytic cycle, suggesting TMAO has a dual role of being both a substrate and an oxygen donor for formaldehyde formation. Together, our results provide novel insight into the role of Zn2+ and Fe2+ in the catalysis of TMAO demethylation by this unique oxygenindependent enzyme

Metal Ion-Coordinating Properties in Aqueous Solution of the Antivirally Active Nucleotide Analogue (S)-9-[3-Hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA). Quantification of Complex Isomeric Equilibria

Acyclic nucleoside phosphonates are of medical relevance and deserve detailed chemical characterization. We focus here on ( S )‐9‐[3‐hydroxy‐2‐(phosphonomethoxy)propyl]adenine (HPMPA) and include for comparison 9‐[2‐(phosphonomethoxy)ethyl]adenine (PMEA), as well as the nucleobase‐free (phosphonomethoxy)ethane (PME) and ( R )‐hydroxy‐2‐(phosphonomethoxy)propane (HPMP). The acidity constants of H 3 (HPMPA) + were determined and compared with those of the related phosph(on)ate derivatives; they are also needed to understand the properties of the metal ion complexes. Given that in vivo nucleotides and their analogues participate in reactions typically as divalent metal ion (M 2+ ) complexes, the stability constants of the M(H;HPMPA) + and M(HPMPA) species with M 2+ = Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , and Cd 2+ were measured. Comparisons between the results for HPMPA 2- and the previous data for PMEA 2- , HPMP 2- and PME 2- revealed that for most M(HPMPA) complexes the enhanced stability (the enhancement relative to the stability of a simple phosphonate‐M 2+ coordination), can solely be explained by the formation of 5‐membered chelates involving the ether oxygen. These chelates occur in equilibrium with simple ′open′ phosphonate‐M 2+ species, the phosphonate group being the primary binding site. The only exceptions are the M(HPMPA) complexes of Ni 2+ , Cu 2+ , and Zn 2+ , which show an additional stability enhancement; in these instances not only the indicated 5‐membered chelates are formed, but M 2+ coordinates in addition to N3 of the adenine residue forming a 7‐membered chelate ring. This observation regarding N3 is important because it emphasizes the metal ion affinity of this site (which is often ignored). Note that in the DNA double helix N3 is exposed to the solvent in the minor groove. The stability data for the monoprotonated M(H;HPMPA) + complexes suggest that these carry H + at the phosphonate group whereas M 2+ is partly at the nucleobase and partly also at the phosphonate group. The ratios of these isomers depend on the metal ion involved, e.g., for Cu(H;HPMPA) the ratio of the isomers is about 1:1

Allosteric modulation of zinc speciation by fatty acids

Background: Serum albumin is the major protein component of blood plasma and is responsible for the circulatory transport of a range of small molecules that include fatty acids, hormones, metal ions and drugs. Studies examining the ligand-binding properties of albumin make up a large proportion of the literature. However, many of these studies do not address the fact that albumin carries multiple ligands (including metal ions) simultaneously in vivo. Thus the binding of a particular ligand may influence both the affinity and dynamics of albumin interactions with another. Scope of review: Here we review the Zn2 + and fatty acid transport properties of albumin and highlight an important interplay that exists between them. Also the impact of this dynamic interaction upon the distribution of plasma Zn2 +, its effect upon cellular Zn2 + uptake and its importance in the diagnosis of myocardial ischemia are considered. Major conclusions: We previously identified the major binding site for Zn2 + on albumin. Furthermore, we revealed that Zn2 +-binding at this site and fatty acid-binding at the FA2 site are interdependent. This suggests that the binding of fatty acids to albumin may serve as an allosteric switch to modulate Zn2 +-binding to albumin in blood plasma. General significance: Fatty acid levels in the blood are dynamic and chronic elevation of plasma fatty acid levels is associated with some metabolic disorders such as cardiovascular disease and diabetes. Since the binding of Zn2 + to albumin is important for the control of circulatory/cellular Zn2 + dynamics, this relationship is likely to have important physiological and pathological implications. This article is part of a Special Issue entitled Serum Albumin

The interplay between non-esterified fatty acids and plasma zinc and its influence on thrombotic risk in obesity and type 2 diabetes

This work is funded by the British Heart Foundation (grant numbers: FS/20/3/34956 and FS/19/69/34639), the China Scholarship Council and the Leverhulme Trust (grant number: RPG-2017-214).Thrombosis is a major comorbidity of obesity and type-2 diabetes mellitus (T2DM). Despite the development of numerous effective treatments and preventative strategies to address thrombotic disease in such individuals, the incidence of thrombotic complications remains high. This suggests that not all the pathophysiological mechanisms underlying these events have been identified or targeted. Non-esterified fatty acids (NEFAs) are increasingly regarded as a nexus between obesity, insulin resistance and vascular disease. Notably, plasma NEFA levels are consistently elevated in obesity and T2DM and may impact haemostasis in several ways. A potentially unrecognised route of NEFA-mediated thrombotic activity is their ability to disturb Zn2+ speciation in the plasma. Zn2+ is a potent regulator of coagulation and its availability in the plasma is monitored carefully through buffering by human serum albumin (HSA). The binding of long-chain NEFAs such as palmitate and stearate however trigger a conformational change in HSA that reduce its ability to bind Zn2+ thus increasing the ion’s availability to bind and activate coagulation proteins. NE-FA-mediated perturbation of HSA-Zn2+ binding is thus predicted to contribute to the prothrom-botic milieu in obesity and T2DM, representing a novel targetable disease mechanism in these disorders.Publisher PDFPeer reviewe