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Herein, a set of non-homologous proteins (238) that could bind the cobalt(ii) cations was selected from all the available Protein Data Bank structures with Co2+ cations. The secondary structure motifs around the amino acid residues that most frequently bind the Co2+ cations (His, Asp, and Glu) as well as the amino acid contents of the inner and outer spheres of complexes were studied. The residues forming coordination bonds to Co2+ (from the inner spheres of the complexes) are overrepresented in the regions of random coil between two β strands, between a β strand and α helix, and in all types of β strands, except that situated between an α helix and β strand. The residues situated at a distance of less than 5 Å from the Co2+ cations, but unable to form coordination bond to them (from the outer spheres of the complexes), are overrepresented in the regions of coil between the β strand and α helix and between two β strands. The data obtained for the Co2+ binding sites was compared with the data obtained for the Mg2+ and Mn2+ binding sites. Although the preferable motifs of the secondary structure for Co2+ binding (beta strand-loop-beta strand and beta strand-loop-alpha helix) are the same as those for Mg2+ and Mn2+, there are some differences in the amino acid contents of the inner and outer spheres of these complexes. The Co2+ cations are preferably coordinated by a combination of His and Glu residues, whereas the Mn2+ and Mg2+ cations prefer a combination of His and Asp and just Asp residues, respectively. As a result, two computer algorithms were developed that could evaluate the possibility of Mg2+ and Mn2+ replacement by the Co2+ cations (chemres.bsmu.by). These algorithms should help to investigate the pathogenesis of cobalt intoxication occurring in patients with cobalt-containing artificial joints.

RNA molecules cannot fold in the absence of counterions. Experiments are typically performed in the presence of monovalent and divalent cations. How to treat the impact of a solution containing a mixture of both ion types on RNA folding has remained a challenging problem for decades. By exploiting the large concentration difference between divalent and monovalent ions used in experiments, we develop a theory based on the reference interaction site model (RISM), which allows us to treat divalent cations explicitly while keeping the implicit screening effect due to monovalent ions. Our theory captures both the inner shell and outer shell coordination of divalent cations to phosphate groups, which we demonstrate is crucial for an accurate calculation of RNA folding thermodynamics. The RISM theory for ion-phosphate interactions when combined with simulations based on a transferable coarse-grained model allows us to predict accurately the folding of several RNA molecules in a mixture containing monovalent and divalent ions. The calculated folding free energies and ion-preferential coefficients for RNA molecules (pseudoknots, a fragment of the rRNA, and the aptamer domain of the adenine riboswitch) are in excellent agreement with experiments over a wide range of monovalent and divalent ion concentrations. Because the theory is general, it can be readily used to investigate ion and sequence effects on DNA properties., Competing Interests: The authors declare no competing interest.

Endogenous ions play important roles in the function and pharmacology of G-protein coupled receptors (GPCRs). Historically the evidence for ionic modulation of GPCR function dates to 1973 with studies of opioid receptors, where it was demonstrated that physiologic concentrations of sodium allosterically attenuated agonist binding. This Na + -selective effect was distinct from effects of other monovalent and divalent cations, with the latter usually counteracting sodium's negative allosteric modulation of binding. Since then, numerous studies documenting the effects of mono- and divalent ions on GPCR function have been published. While ions can act selectively and nonselectively at many sites in different receptors, the discovery of the conserved sodium ion site in class A GPCR structures in 2012 revealed the unique nature of Na + site, which has emerged as a near-universal site for allosteric modulation of class A GPCR structure and function. In this review, we synthesize and highlight recent advances in the functional, biophysical, and structural characterization of ions bound to GPCRs. Taken together, these findings provide a molecular understanding of the unique roles of Na + and other ions as GPCR allosteric modulators. We will also discuss how this knowledge can be applied to the redesign of receptors and ligand probes for desired functional and pharmacological profiles. SIGNIFICANCE STATEMENT: The function and pharmacology of GPCRs strongly depend on the presence of mono and divalent ions in experimental assays and in living organisms. Recent insights into the molecular mechanism of this ion-dependent allosterism from structural, biophysical, biochemical, and computational studies provide quantitative understandings of the pharmacological effects of drugs in vitro and in vivo and open new avenues for the rational design of chemical probes and drug candidates with improved properties., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

RNAs are involved in myriad cellular events. In general, RNA function is affected by cellular conditions. For instance, molecular crowding promotes RNA folding through compaction of the RNA. Metabolites generally destabilize RNA secondary structure, which improves RNA folding cooperativity and increases the fraction of functional RNA. Our recent studies demonstrate that cellular concentrations of amino acid-chelated magnesium (aaCM) stimulate RNA folding and catalysis while protecting RNAs from magnesium ion-induced degradation. However, effects of other cellular magnesium ion chelators on RNA function have not been tested. Herein, we report that nucleotide diphosphate-chelated magnesium, which is of intermediate strength, promotes RNA catalysis much like aaCM. Nucleotides are some of the major metabolites in cells and have one to three phosphates, which have increasingly tight binding of magnesium. On the basis of binding calculations, ∼85% ATP, ∼40% ADP, and only 5% AMP are estimated to possess a magnesium ion under cellular conditions of 0.50 mM Mg 2+ free . We tested the self-cleaving activity of the hammerhead ribozyme in the presence of these chelated magnesium species. Our results indicate that NTP-chelated magnesium and NMP-chelated magnesium do not appreciably stimulate RNA catalysis, whereas NDP-chelated magnesium promotes RNA catalysis up to 6.5-fold. Inspired by NDP, we observed similar stimulatory effects for several other Mg 2+ diphosphate-containing metabolites, including UDP-GlcNAC and UDP-Glc; in addition, we found similar effects for a DNAzyme. Thus, rate stimulatory effects are general with respect to the diphosphate and nucleic acid enzyme. These results implicate magnesium-chelated diphosphate metabolites as general facilitators of RNA function inside cells.

TRPM2 is critically involved in diverse physiological processes including core temperature sensing, apoptosis, and immune response. TRPM2's activation by Ca 2+ and ADP ribose (ADPR), an NAD + -metabolite produced under oxidative stress and neurodegenerative conditions, suggests a role in neurological disorders. We provide a central concept between triple-site ligand binding and the channel gating of human TRPM2. We show consecutive structural rearrangements and channel activation of TRPM2 induced by binding of ADPR in two indispensable locations, and the binding of Ca 2+ in the transmembrane domain. The 8-Br-cADPR-an antagonist of cADPR-binds only to the MHR1/2 domain and inhibits TRPM2 by stabilizing the channel in an apo-like conformation. We conclude that MHR1/2 acts as a orthostatic ligand-binding site for TRPM2. The NUDT9-H domain binds to a second ADPR to assist channel activation in vertebrates, but not necessary in invertebrates. Our work provides insights into the gating mechanism of human TRPM2 and its pharmacology., Competing Interests: YH, BR, WL, JD No competing interests declared, (© 2019, Huang et al.)

Endothelial cells (ECs) constitute the innermost layer that lines all blood vessels from the larger arteries and veins to the smallest capillaries, including the lymphatic vessels. Despite the histological classification of endothelium of a simple epithelium and its homogeneous morphological appearance throughout the vascular system, ECs, instead, are extremely heterogeneous both structurally and functionally. The different arrangement of cell junctions between ECs and the local organization of the basal membrane generate different type of endothelium with different permeability features and functions. Continuous, fenestrated and discontinuous endothelia are distributed based on the specific function carried out by the organs. It is thought that a large number ECs functions and their responses to extracellular cues depend on changes in intracellular concentrations of calcium ion ([Ca 2+ ] i ). The extremely complex calcium machinery includes plasma membrane bound channels as well as intracellular receptors distributed in distinct cytosolic compartments that act jointly to maintain a physiological [Ca 2+ ] i , which is crucial for triggering many cellular mechanisms. Here, we first survey the overall notions related to intracellular Ca 2+ mobilization and later highlight the involvement of this second messenger in crucial ECs functions with the aim at stimulating further investigation that link Ca 2+ mobilization to ECs in health and disease.

Allosteric interactions between the voltage-sensing domain (VSD), the Ca 2+ -binding sites, and the pore domain govern the mammalian Ca 2+ - and voltage-activated K + (BK) channel opening. However, the functional relevance of the crosstalk between the Ca 2+ - and voltage-sensing mechanisms on BK channel gating is still debated. We examined the energetic interaction between Ca 2+ binding and VSD activation by investigating the effects of internal Ca 2+ on BK channel gating currents. Our results indicate that Ca 2+ sensor occupancy has a strong impact on VSD activation through a coordinated interaction mechanism in which Ca 2+ binding to a single α-subunit affects all VSDs equally. Moreover, the two distinct high-affinity Ca 2+ -binding sites contained in the C-terminus domains, RCK1 and RCK2, contribute equally to decrease the free energy necessary to activate the VSD. We conclude that voltage-dependent gating and pore opening in BK channels is modulated to a great extent by the interaction between Ca 2+ sensors and VSDs., Competing Interests: YL, WC, KC, OA, RL No competing interests declared, (© 2019, Lorenzo-Ceballos et al.)

The endoplasmic reticulum ( ER ) imports ATP and uses energy from ATP hydrolysis for protein folding and trafficking. However, little is known about how this vital ATP transport occurs across the ER membrane. Here, using three commonly used cell lines (CHO, INS1 and HeLa), we report that ATP enters the ER lumen through a cytosolic Ca 2+ -antagonized mechanism, or CaATiER ( Ca 2+ - A ntagonized T ransport i nto ER ). Significantly, we show that mitochondria supply ATP to the ER and a SERCA-dependent Ca 2+ gradient across the ER membrane is necessary for ATP transport into the ER, through SLC35B1/AXER. We propose that under physiological conditions, increases in cytosolic Ca 2+ inhibit ATP import into the ER lumen to limit ER ATP consumption. Furthermore, the ATP level in the ER is readily depleted by oxidative phosphorylation ( OxPhos ) inhibitors and that ER protein misfolding increases ATP uptake from mitochondria into the ER. These findings suggest that ATP usage in the ER may increase mitochondrial OxPhos while decreasing glycolysis, i.e. an ' anti-Warburg ' effect., Competing Interests: JY, HB, SB, MS, AM, RM, RK No competing interests declared, (© 2019, Yong et al.)

Artificial counterparts of conical-shaped transmembrane protein channels are of interest in biomedical sciences for biomolecule detection and selective ion permeation based on ionic size and/or charge differences. However, industrial-scale applications such as seawater desalination, separation of mono- from divalent cations, and treatment of highly-saline industrial waste effluents are still big challenges for such biomimetic channels. A simple monomer seeding experimental approach is used to grow ionically conductive biomimetic charged nanocone pores at the surface of an acid-functionalized membrane. These readily scalable nanocone membranes enable ultra-fast cation permeation (Na + =8.4× vs. Mg 2+ =1.4×) and high ion charge selectivity (Na + /Mg 2+ =6×) compared to the commercial state-of-the-art permselective membrane (CSO, Selemion, Japan) owing to negligible surface resistance and positively charged conical pore walls., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Multiple sclerosis (MS) has been shown to feature oxidative damage, which can be modelled using the cuprizone model of demyelinating disease. Oxidative damage can occur as a result of excessive influx of calcium ions (Ca 2+ ) and oligodendroglia are particularly vulnerable. However, the effects of limiting excess Ca 2+ influx on oxidative damage, oligodendroglia and myelin structure are unknown., Objective: This study investigated the effects of limiting excess Ca 2+ flux on oxidative damage and associated changes in oligodendroglial densities and Node of Ranvier structure in the cuprizone model., Methods: The effects of three weeks of cuprizone administration and of treatment with a combination of three ion channel inhibitors (Lomerizine, Brilliant Blue G (BBG) and YM872), were semi-quantified immunohistochemically. Outcomes assessed were protein nitration (3-nitrotyrosine (3NT)) oxidative damage to DNA (8-hydroxy deoxyguanosine (8OHDG)), advanced glycation end-products (carboxymethyl lysine (CML)), immunoreactivity of microglia (Iba1) and astrocytes (glial acidic fibrillary protein (GFAP)), densities of oligodendrocyte precursor cells (OPCs) (platelet derived growth factor alpha receptor (PDGFαR) with olig2) and oligodendrocytes (olig2 and CC1), and structural elements of the Node of Ranvier (contactin associated protein (Caspr))., Results: The administration of cuprizone resulted in increased protein nitration, DNA damage, and astrocyte and microglial immunoreactivity, a decrease in the density of oligodendrocytes and OPCs, together with altered structure of the Node of Ranvier and reduced myelin basic protein immunoreactivity. Treatment with the ion channel inhibitor combination significantly lowered protein nitration, increased the density of OPCs and reduced the number of atypical Node of Ranvier complexes; other outcomes were unaffected., Conclusion: Our findings suggest that excess Ca 2+ influx contributes to protein nitration, and associated changes to OPC densities and Node of Ranvier structure in demyelinating disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Parkinson's disease is the second most common neurodegenerative disorder worldwide. Neurodegeneration in this pathology is characterized by the loss of dopaminergic neurons in the substantia nigra, coupled with cytoplasmic inclusions known as Lewy bodies containing α-synuclein. The brain is an organ that concentrates metal ions, and there is emerging evidence that a break-down in metal homeostasis may be a critical factor in a variety of neurodegenerative diseases. α-synuclein has emerged as an important metal-binding protein in the brain, whereas these interactions play an important role in its aggregation and might represent a link between protein aggregation, oxidative damage, and neuronal cell loss. Additionally, α-synuclein undergoes several post-translational modifications that regulate its structure and physiological function, and may be linked to the aggregation and/or oligomer formation. This review is focused on the interaction of this protein with physiologically relevant metal ions, highlighting the cases where metal-AS interactions profile as key modulators for its structural, aggregation, and membrane-binding properties. The impact of α-synuclein phosphorylation and N-terminal acetylation in the metal-binding properties of the protein are also discussed, underscoring a potential interplay between PTMs and metal ion binding in regulating α-synuclein physiological functions and its role in pathology. This article is part of the Special Issue "Synuclein"., (© 2019 International Society for Neurochemistry.)

The assembly and disassembly of the FtsZ ring drives the division of bacteria cells, including Streptococcus pneumoniae, which causes pneumonia and meningitis. In contrast to FtsZ from other bacterial species, Streptococcus pneumoniae (Spn) FtsZ contains two tryptophan residues. Here, we demonstrate that the assembly and disassembly of Streptococcus pneumoniae FtsZ (SpnFtsZ) monomers can be monitored by the intrinsic tryptophan fluorescence of FtsZ. We found that the assembly of SpnFtsZ is closely associated with its GTPase activity. Guanosine 5'-[β,γ-imido]triphosphate, a nonhydrolyzable analog of GTP, stabilized the FtsZ filaments without inducing their bundling. Using intrinsic tryptophan fluorescence, light scattering, and electron microscopy, we could differentiate the effects of divalent calcium and magnesium on the assembly of FtsZ. Though Mg 2+ increased the stability of the FtsZ filaments, it could not prevent the disassembly of the filaments under conditions where GTP was limiting. Thus, our results indicate that Mg 2+ primarily enhances the longitudinal assembly of FtsZ. Low concentrations of Ca 2+ strongly promoted the bundling of FtsZ filaments and inhibited the disassembly of the filaments, suggesting that low concentrations of Ca 2+ enhance the lateral interactions between the FtsZ filaments. Interestingly, Ca 2+ delayed the nucleation process of FtsZ assembly, indicating that Ca 2+ exerts paradoxical effects on the assembly of FtsZ. However, higher concentrations of Ca 2+ did not enhance the bundling of FtsZ filaments. In addition, Ca 2+ altered the secondary structure of FtsZ and increased the fluorescence of the FtsZ-1-anilinonaphthalene-8-sulfonic acid complex, indicating that Ca 2+ induces conformational changes in FtsZ. The study provides an interesting insight into the assembly of SpnFtsZ and its regulation by divalent cations., (© 2019 Federation of European Biochemical Societies.)

The Glutamate Receptor Ionotropic NMDA-Associated Protein 1 (GRINA) belongs to the Lifeguard family and is involved in calcium homeostasis, which governs key processes, such as cell survival or the release of neurotransmitters. GRINA is mainly associated with membranes of the endoplasmic reticulum, Golgi, endosome, and the cell surface, but its presence in the nucleus has not been explained yet. Here we dissect, with the help of different software tools, the potential roles of GRINA in the cell and how they may be altered in diseases, such as schizophrenia or celiac disease. We describe for the first time that the cytoplasmic N-terminal half of GRINA (which spans a Proline-rich domain) contains a potential DNA-binding sequence, in addition to cleavage target sites and probable PY-nuclear localization sequences, that may enable it to be released from the rest of the protein and enter the nucleus under suitable conditions, where it could participate in the transcription, alternative splicing, and mRNA export of a subset of genes likely involved in lipid and sterol synthesis, ribosome biogenesis, or cell cycle progression. To support these findings, we include additional evidence based on an exhaustive review of the literature and our preliminary data of the protein-protein interaction network of GRINA., Competing Interests: The authors declare no conflict of interest.

Many biological processes are based on molecular recognition between highly charged molecules such as nucleic acids, inorganic ions, charged amino acids, etc. For such cases, it has been demonstrated that molecular simulations with fixed partial charges often fail to achieve experimental accuracy. Although incorporation of more advanced electrostatic models (such as multipoles, mutual polarization, etc.) can significantly improve simulation accuracy, it increases computational expense by a factor of 5-20×. Indirect free energy (IFE) methods can mitigate this cost by modeling intermediate states at fixed-charge resolution. For example, an efficient "reference" model such as a pairwise Amber, CHARMM, or OPLS-AA force field can be used to derive an initial estimate, followed by thermodynamic corrections to a more advanced "target" potential such as the polarizable AMOEBA model. Unfortunately, all currently described IFE methods encounter difficulties reweighting more than ∼50 atoms between resolutions due to extensive scaling of both the magnitude of the thermodynamic corrections and their statistical uncertainty. We present an approach called "simultaneous bookending" (SB) that is fundamentally different from existing IFE methods based on a tunable sampling approximation, which permits scaling to thousands of atoms. SB is demonstrated on the relative binding affinity of Mg 2+ /Ca 2+ to a set of metalloproteins with up to 2972 atoms, finding no statistically significant difference between direct AMOEBA results and those from correcting Amber to AMOEBA. The ability to change the resolution of thousands of atoms during reweighting suggests the approach may be applicable in the future to protein-protein binding affinities or nucleic acid thermodynamics.

Phytic acid (PA) is a natural-occurring antioxidant, which plays an important role in many biological processes. PA is recognized as a potent inhibitor of lipid peroxidation because of its high affinity to multivalent cations, and it can play a role in osteogenic processes. However, its powerful chelating capacity is controversial because it can lead to a severe reduction of mineral availability in the organism. For this reason, compounds with beneficial biological properties of PA, but a modular ion binding capacity, are of high interest. In this work, we report the synthesis and physicochemical characterization of two hydroxylic derivatives of PA, named glycerylphytates (GPhy), through a condensation reaction of PA with glycerol (G). Both derivatives present antioxidant properties, measured by ferrozine/FeCl 2 method and chelating activity with calcium ions depending on the content of glyceryl groups incorporated. Besides, the hydroxylic modification not only modulates the ion binding affinity of derivatives but also improves their cytocompatibility in human bone marrow mesenchymal cells (MSCs). Furthermore, GPhy derivatives display osteogenic properties, confirmed by COL1A and ALPL expression depending on composition. These positive features convert GPhy compounds into potent alternatives for those skeletal diseases treatments where PA is tentatively applied.

Aging is an important risk factor for cardiac dysfunction. Heart during aging exhibits a depressed mechanical activity, at least, through mitochondria-originated increases in ROS. Previously, we also have shown a close relationship between increased ROS and cellular intracellular free Zn 2+ ([Zn 2+ ] i ) in cardiomyocytes under pathological conditions as well as the contribution of some re-expressed levels of Zn 2+ -transporters for redistribution of [Zn 2+ ] i among suborganelles. Therefore, we first examined the cellular (total) [Zn 2+ ] and then determined the protein expression levels of Zn 2+ -transporters in freshly isolated ventricular cardiomyocytes from 24-month rat heart compared to those of 6-month rats. The [Zn 2+ ] i in the aged-cardiomyocytes was increased, at most, due to increased ZIP7 and ZnT8 with decreased levels of ZIP8 and ZnT7. To examine redistribution of the cellular [Zn 2+ ] i among suborganelles, such as Sarco/endoplasmic reticulum, S(E)R, and mitochondria ([Zn 2+ ] SER and [Zn 2+ ] Mit ), a cell model (with galactose) to mimic the aged-cell in rat ventricular cell line H9c2 was used and demonstrated that there were significant increases in [Zn 2+ ] Mit with decreases in [Zn 2+ ] SER . In addition, the re-distribution of these Zn 2+ -transporters were markedly changed in mitochondria (increases in ZnT7 and ZnT8 with no changes in ZIP7 and ZIP8) and S(E)R (increase in ZIP7 and decrease in ZnT7 with no changes in both ZIP8 and ZnT8) both of them isolated from freshly isolated ventricular cardiomyocytes from aged-rats. Furthermore, we demonstrated that cellular levels of ROS, both total and mitochondrial lysine acetylation (K-Acetylation), and protein-thiol oxidation were significantly high in aged-cardiomyocytes from 24-month old rats. Using a mitochondrial-targeting antioxidant, MitoTEMPO (1 µM, 5-h incubation), we provided an important data associated with the role of mitochondrial-ROS production in the [Zn 2+ ] i -dyshomeostasis of the ventricular cardiomyocytes from 24-month old rats. Overall, our present data, for the first time, demonstrated that a direct mitochondria-targeting antioxidant treatment can be a new therapeutic strategy during aging in the heart through a well-controlled [Zn 2+ ] distribution among cytosol and suborganelles with altered expression levels of the Zn 2+ -transporters.

Saccharomyces cerevisiae has high level of inorganic polyphosphate and a multicomponent system of its metabolism, including polyphosphatases Ppx1, Ppn1, Ddp1, and Ppn2. The aim of the study was to construct the yeast strain overexpressing Ppn2 and to compare the properties of Ppn2, Ppx1, Ppn1, and Ddp1 purified from overexpressing strains of S. cerevisiae. We overexpressed Ppn2 in S. cerevisiae under a strong constitutive promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase-encoding gene and suggested biochemical criteria for distinguishing among yeast polyphosphatases, which is important for their identification and understanding of their functions. Ppn2, Ppn1, and Ddp1 had endopolyphosphatase activities, whereas Ppx1 did not. Ppx1 and Ppn1 exhibited high and Ddp1 and Ppn2 low exopolyphosphatase activity: 240, 500, 0.05 and 0.1 U/mg protein, respectively. The enzymes had distinct patterns of exopolyphosphatase activities stimulation by divalent metal ions. Ppn2, Ppn1 and Ddp1 displayed endopolyphosphatase activity in the presence of 1 mM Mg 2+ . The endopolyphosphatase activities of Ppn2 and Ppn1 were induced by 0.01 mM of Co 2+ or Zn 2+ , whereas that of Ddp1 required 0.1 mM of these cations. The endopolyphosphatase activity of Ppn1 was inhibited by 0.01 mg mL -1 of heparin, while endopolphosphatase activity of Ppn2 was weakly sensitive to 0.25 mg mL -1 of heparin. The Ppx1 and Ppn1 activity with guanosine tetraphosphate was nearly 80% of activity with long-chain polyphosphates. The Ppn1 hydrolyzed dATP, while Ppx1 did not. The differences in the mode of polyphosphate hydrolysis, substrate specificity, metal ion dependence and cell localization suggest distinct roles of these enzymes in yeast., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

In this review, we explore the roles of divalent metal ions in structure and function within the extracellular matrix (ECM), specifically, their interaction with glycosaminoglycans (GAGs) during tumor progression. Metals and GAGs have been individually associated with physiological and pathological processes, however, their combined activities in regulating cell behavior and ECM remodeling have not been fully explored to date. During tumor progression, divalent metals and GAGs participate in central processes, such as cell migration and angiogenesis, either by modulating cell surface molecules, as well as soluble signaling factors. In addition, studies on metals and polysaccharides interactions have been of great value, as they provide structural information that can be correlated with function. Finally, we believe that understanding how metals are regulated in physiological and pathological conditions is paramount for the development of new treatment strategies, as well as diagnostic and exploratory tools., (© 2019 Federation of European Biochemical Societies.)

WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus. WalKR regulates peptidoglycan synthesis, but this function alone does not explain its essentiality. Here, to further understand WalKR function, we investigate a suppressor mutant that arose when WalKR activity was impaired; a histidine to tyrosine substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PAS CYT ) domain of the histidine kinase WalK. Introducing the WalK H271Y mutation into wild-type S. aureus activates the WalKR regulon. Structural analyses of the WalK PAS CYT domain reveal a metal-binding site, in which a zinc ion (Zn 2+ ) is tetrahedrally-coordinated by four amino acids including H271. The WalK H271Y mutation abrogates metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn 2+ -binding negatively regulates WalKR. Promoter-reporter experiments using S. aureus confirm Zn 2+ sensing by this system. Identification of a metal ligand recognized by the WalKR system broadens our understanding of this critical S. aureus regulon.

Oxidative aggregation of γ-crystallins induced by copper in aged lens increases the lens opacity and causes cataract formation. Therefore, chelation of free Cu 2+ by small molecules can inhibit metal-mediated aggregation of γ-crystallin. In this work, the inhibition potency of several naturally occurring flavonoid compounds was studied against aggregation of human γD-crystallin (HGD) mediated by copper ions. Among them, rutin demonstrated ~20% inhibition of HGD aggregation induced by Cu 2+ through its metal chelation ability. Not only that, the chaperone activity of lens chaperone, human αA-crystallin (HAA) was found to be enhanced in the presence of rutin. Subsequently, the molecular interactions between HAA and rutin were investigated using fluorescence and CD spectroscopy to understand the molecular basis of the chaperone activity enhancement by rutin. Quenching of HAA fluorescence by rutin with a quenching constant in the order of ~10 5  M -1 depicts a complexation between them. Entropy driven process of complexation between HAA and rutin suggests significant involvement of hydrophobic interactions. Fluorescence resonance energy transfer between protein and ligand can occur at a distance of 2.73 nm. Synchronous fluorescence and circular dichroism spectroscopy revealed that protein-ligand interaction does not cause any notable conformational changes in HAA. Experimental observations have been well substantiated by docking., (Copyright © 2019 Elsevier B.V. All rights reserved.)

P2X3 receptor channels expressed in sensory neurons are activated by extracellular ATP and serve important roles in nociception and sensory hypersensitization, making them attractive therapeutic targets. Although several P2X3 structures are known, it is unclear how physiologically abundant Ca 2+ -ATP and Mg 2+ -ATP activate the receptor, or how divalent cations regulate channel function. We used structural, computational and functional approaches to show that a crucial acidic chamber near the nucleotide-binding pocket in human P2X3 receptors accommodates divalent ions in two distinct modes in the absence and presence of nucleotide. The unusual engagement between the receptor, divalent ion and the γ-phosphate of ATP enables channel activation by ATP-divalent complex, cooperatively stabilizes the nucleotide on the receptor to slow ATP unbinding and recovery from desensitization, a key mechanism for limiting channel activity. These findings reveal how P2X3 receptors recognize and are activated by divalent-bound ATP, aiding future physiological investigations and drug development., Competing Interests: ML, YW, RB, FM, SS, MH No competing interests declared, JF, KS Reviewing editor, eLife

Senescence Marker Protein (SMP30) is a metalloenzyme that shows lactonase activity in the ascorbic acid (AA) biosynthesis pathway in non-primate mammals such as a mouse. However, AA biosynthesis does not occur in the primates including humans. Several studies have shown the role of SMP30 in maintaining calcium homeostasis in mammals. In addition, it is also reported to have promiscuous enzyme activity with an organophosphate (OP) substrate. Hence, this study aims to recombinantly express and purify the SMP30 proteins from both mouse and human, and to study their structural alterations and functional deviations in the presence of different divalent metals. For this, mouse SMP30 (MoSMP30) as well as human SMP30 (HuSMP30) were cloned in the bacterial expression vector. Proteins were overexpressed and purified from soluble fractions as well as from inclusion bodies as these proteins were expressed largely in insoluble fractions. The purified proteins were used to study the folding conformations in the presence of different divalent cations (Ca2+, Co2+, Mg2+, and Zn2+) with the help of circular dichroism (CD) spectroscopy. It was observed that both MoSMP30 and HuSMP30 acquired native folding conformations. To study the metal-binding affinity, dissociation constant (Kd values) were calculated from UV-VIS titration curve, which showed the highest affinity of MoSMP30 with Zn2+. However, HuSMP30 showed the highest affinity with Ca2+, suggesting the importance of HuSMP30 in maintaining calcium homeostasis. Enzyme kinetics were performed with γ-Thiobutyrolactone and Demeton-S in the presence of different divalent cations. Interestingly, both the proteins showed lactonase activity in the presence of Ca2+. In addition, MoSMP30 and HuSMP30 also showed lactonase activity in the presence of Co2+ and Zn2+ respectively. Moreover, both the proteins showed OP hydrolase activities in the presence of Ca2+ as well as Zn2+, suggesting the metal-dependent promiscuous nature of SMP30., Competing Interests: The authors have declared that no competing interests exist.

Accumulation of cadmium ions may result in adverse effects on plant due to the oxidative stress via destructions of antioxidants and antioxidant enzymes. As the core component of the glutathione antioxidant system, glutathione S-transferases (GSTs) have been reported as biomarkers for evaluating the metal-induced oxidative damage to plants, but the potential toxicity and underlying toxic molecular mechanisms remain unknown. This article investigated the molecular interactions of cadmium ions with Arabidopsis thaliana glutathione S-transferase phi8 (AtGSTF8) by multi-spectroscopic techniques and enzyme activity measurements. The intrinsic fluorescence of AtGSTF8 was quenched statically upon the addition of cadmium ions accompanied with the complex formation and structural and conformational alterations from multiple spectroscopic measurements, resulting in deconstructed protein skeleton and microenvironmental alterations around the Tyr and Trp residues. A single binding site was predicted for AtGSTF8 towards cadmium ions and the van der Walls interactions and hydrogen bonds are the major driving forces of the interaction. In addition, the transferase activity changes of AtGSTF8 upon the addition of cadmium ions have been observed. The implementation of this work helps to clarify the mechanism of oxidative damage and antioxidant enzymes response induced by heavy metal accumulation in plant at molecular level., (Copyright © 2019. Published by Elsevier B.V.)

UVB-induced skin damage is attributable to reactive oxygen species, which are triggered by intracellular Ca 2+ signals. However, exactly how the reactive oxygen species are triggered by intracellular Ca 2+ upon UVB irradiation remains obscure. Here, we show that UVB induces Ca 2+ signals via sequential generation of the following Ca 2+ messengers: inositol 1,4,5-trisphosphate, nicotinic acid adenine dinucleotide phosphate, and cyclic ADP-ribose. UVB induced H 2 O 2 production through NADPH oxidase 4 activation, which is downstream to inositol 1,4,5-trisphosphate and nicotinic acid adenine dinucleotide phosphate. H 2 O 2 derived from NADPH oxidase 4 activated CD38 to produce cyclic ADP-ribose. UVB first evoked the pannexin channel to release ATP, which acts on P2X7 receptor to generate inositol 1,4,5-trisphosphate. Inhibitors of these messengers, as well as antioxidants, blocked UVB-induced Ca 2+ signals and IL-1β secretion in keratinocytes. Furthermore, ablation of CD38 and NADPH oxidase 4 protected against UVB-induced inflammation and IL-1β secretion in the murine epidermis. These results show that UVB induces IL-1β secretion through cross-talk between Ca 2+ and reactive oxygen species, providing insight towards potential targets against UVB-induced inflammation., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Ca 2+ is a potent signalling molecule that regulates many cellular processes. In cyanobacteria, Ca 2+ has been linked to cell growth, stress response and photosynthesis, and to the development of specialist heterocyst cells in certain nitrogen-fixing species. Despite this, the pathways of Ca 2+ signal transduction in cyanobacteria are poorly understood, and very few protein components are known. The current study describes a previously unreported Ca 2+ -binding protein which was called the Ca 2+ Sensor EF-hand (CSE), which is conserved in filamentous, nitrogen-fixing cyanobacteria. CSE is shown to bind Ca 2+ , which induces a conformational change in the protein structure. Poor growth of a strain of Anabaena sp. PCC 7120 overexpressing CSE was attributed to diminished photosynthetic performance. Transcriptomics, biophysics and proteomics analyses revealed modifications in the light-harvesting phycobilisome and photosynthetic reaction centre protein complexes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ca 2+ -transcription coupling controls gene expression patterns that define vascular smooth muscle cell (VSMC) phenotype. Although not well understood this allows normally contractile VSMCs to become proliferative following vessel injury, a process essential for repair but which also contributes to vascular remodelling, atherogenesis and restenosis. Here we show that the Ca 2+ /HCO 3 - -sensitive enzyme, soluble adenylyl cyclase (sAC), links Ca 2+ influx in human coronary artery smooth muscle cells (hCASMCs) to 3',5'-cyclic adenosine monophosphate (cAMP) generation and phosphorylation of the transcription factor Ca 2+ /cAMP response element binding protein (CREB). Store-operated Ca 2+ entry (SOCE) into hCASMCs expressing the FRET-based cAMP biosensor H187 induced a rise in cAMP that mirrored cytosolic [Ca 2+ ]. SOCE also activated the cAMP effector, protein kinase A (PKA), as determined by the PKA reporter, AKAR4-NES, and induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and CREB. Transmembrane adenylyl cyclase inhibition had no effect on the SOCE-induced rise in cAMP, while sAC inhibition abolished SOCE-generated cAMP and significantly reduced SOCE-induced VASP and CREB phosphorylation. This suggests that SOCE in hCASMCs activates sAC which in turn activates the cAMP/PKA/CREB axis. sAC, which is insensitive to G-protein modulation but responsive to Ca 2+ , pH and ATP, may therefore act as an overlooked regulatory node in vascular Ca 2+ -transcription coupling.

We have constructed a new coumarin based fluorescence probe BENZPYR with ICT character through condensation of N, N-diethylamino-3-acetyl coumarin with 2-hydrazinobenzothiazole. The absorbance and fluorescence spectral characteristics of BENZPYR revealed that the chemosensor can specifically detect for Cu 2+ ions over other different metal ions and the lowest limit of detection was found in nano molar range. The turn off sensor of BENZPYR is related to chelation enhanced quenching (CHEQ) and intramolecular charge transfer (ICT) processes were serve as excellent fluorescent detection of Cu 2+ ions in DMF medium. Fluorescence microscopy experiments revealed that probe BENZPYR may have application as a fluorophore to detect the Cu 2+ in living cells. The simulated DFT analysis of electronic and structural properties and also UV-vis absorption spectra are in well accordance with the experimental UV-vis absorption spectra., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Copper is an important element indispensable for human life and health. Many copper-determining probes have been created for exploring its functional behavior in various cell types but few of them contains both fluorescent and colorimetric characters. In the present study, we developed a set of copper probes by synthesizing several novel thiophene-based Schiff bases in order to make a suitable sensor for quantifying and imaging copper in living cells. We find that the ligand FS-1 has a splendid selectivity and affinity toward Cu 2+ among the common divalent metal ions. Living cell imaging show that FS-1 has a robust and repetitive fluorescence response in the presence of Cu 2+ only in the cytosolic space of Hepg2 cell and not in the other cells examined. These data suggest that we have developed a new copper probe that can be used as a Cu 2+ fluorescent and colorimetric sensor for in vivo and in vitro copper studies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ca 2+ -stimulated translocation of cytosolic phospholipase A 2 α (cPLA 2 α) to the Golgi induces arachidonic acid production, the rate-limiting step in pro-inflammatory eicosanoid synthesis. Structural insights into the cPLA 2 α preference for phosphatidylcholine (PC)-enriched membranes have remained elusive. Here, we report the structure of the cPLA 2 α C2-domain (at 2.2 Å resolution), which contains bound 1,2-dihexanoyl- sn -glycero-3-phosphocholine (DHPC) and Ca 2+ ions. Two Ca 2+ are complexed at previously reported locations in the lipid-free C2-domain. One of these Ca 2+ ions, along with a third Ca 2+ , bridges the C2-domain to the DHPC phosphate group, which also interacts with Asn65. Tyr96 plays a key role in lipid headgroup recognition via cation-π interaction with the PC trimethylammonium group. Mutagenesis analyses confirm that Tyr96 and Asn65 function in PC binding selectivity by the C2-domain and in the regulation of cPLA 2 α activity. The DHPC-binding mode of the cPLA 2 α C2-domain, which differs from phosphatidylserine or phosphatidylinositol 4,5-bisphosphate binding by other C2-domains, expands and deepens knowledge of the lipid-binding mechanisms mediated by C2-domains., Competing Interests: YH, YG, DS, NV, LM, DS, CC, DP, RB No competing interests declared, (© 2019, Hirano et al.)

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are considered noncompetitive inhibitors that structurally alter reverse transcriptase (RT) and dramatically decrease catalysis. In this report, biochemical analysis with various divalent cations was used to demonstrate that NNRTIs and divalent cation-dNTP complexes are mutually exclusive, inhibiting each other's binding to RT/primer/template (RT-P/T) complexes. The binding of catalytically competent divalent cation-dNTP complexes to RT-P/T was measured with Mg 2+ , Mn 2+ , Zn 2+ , Co 2+ , and Ni 2+ using Ca 2+ , a noncatalytic cation, for displacement. Binding strength order was Mn 2+ ≈ Zn 2+ ≫ Co 2+ > Mg 2+ ≈ Ni 2+ . Consistent with but not exclusive to mutually exclusive binding, primer extension assays showed that stronger divalent cation-dNTP complexes were more resistant to NNRTIs (efavirenz (EFV), rilpivirine (RPV), and nevirapine (NVP)). Filtration assays demonstrated that divalent cation-dNTP complexes inhibited the binding of 14 C-labeled EFV to RT-P/T with stronger binding complexes formed with Mn 2+ inhibiting more potently than those with Mg 2+ . Conversely, filter binding assays demonstrated that EFV inhibited 3 H-labeled dNTP binding to RT-P/T complexes with displacement of Mn 2+ -dNTP complexes requiring much greater concentrations of EFV than the more weakly bound Mg 2+ -dNTP complexes. EFV bound relatively weakly to the NNRTI resistant K103N RT; but, binding was modestly enhanced in the presence of P/T, and EFV was easily displaced by divalent cation-dNTP complexes. This suggests that K103N overcomes EFV inhibition mostly by binding more weakly to the drug and is in contrast to other reports that indicate K103N has little to no effect on drug or dNTP binding. Overall, this biochemical analysis supports recent biophysical analyses of NNRTI-RT interactions that indicate mutually exclusive binding.

Complex spike bursts (CSBs) represent a characteristic firing pattern of hippocampal pyramidal cells (PCs). In CA1PCs, CSBs are driven by regenerative dendritic plateau potentials, produced by correlated entorhinal cortical and CA3 inputs that simultaneously depolarize distal and proximal dendritic domains. However, in CA3PCs neither the generation mechanisms nor the computational role of CSBs are well elucidated. We show that CSBs are induced by dendritic Ca 2+ spikes in CA3PCs. Surprisingly, the ability of CA3PCs to produce CSBs is heterogeneous, with non-uniform synaptic input-output transformation rules triggering CSBs. The heterogeneity is partly related to the topographic position of CA3PCs; we identify two ion channel types, HCN and Kv2 channels, whose proximodistal activity gradients contribute to subregion-specific modulation of CSB propensity. Our results suggest that heterogeneous dendritic integrative properties, along with previously reported synaptic connectivity gradients, define functional subpopulations of CA3PCs that may support CA3 network computations underlying associative memory processes.

Manganese (Mn 2+ ) is extruded from the cell by the zinc transporter 10 (ZnT10). Loss of ZnT10 expression caused by autosomal mutations in the ZnT10 gene leads to hypermanganesemia in multiple organs. Here, combining fluorescent monitoring of cation influx in HEK293-T cells expressing human ZnT10 with molecular modeling of ZnT10 cation selectivity, we show that ZnT10 is exploiting the transmembrane Ca 2+ inward gradient for active cellular exchange of Mn 2+ In analyzing ZnT10 activity we used the ability of Fura-2 to spectrally distinguish between Mn 2+ and Ca 2+ fluxes. We found that ( a ) application of Mn 2+ -containing Ca 2+ -free solution to ZnT10-expressing cells triggers an influx of Mn 2+ , ( b ) reintroduction of Ca 2+ leads to cellular Mn 2+ extrusion against an inward Mn 2+ gradient, and ( c ) the cellular transport of Mn 2+ by ZnT10 is coupled to a reciprocal movement of Ca 2+ Remarkably, replacing a single asparagine residue in ZnT10 (Asp-43) with threonine (ZnT10 N43T) converted the Mn 2+ /Ca 2+ exchange to an uncoupled channel mode, permeable to both Ca 2+ and Mn 2+ The findings in our study identify the first ion transporter that uses the Ca 2+ gradient for active counter-ion exchange. They highlight a remarkable versatility in metal selectivity and mode of transport controlled by the tetrahedral metal transport site of ZnT proteins., (© 2019 Levy et al.)

Protein phosphatase 5 (PP5), an important member of serine/threonine protein phosphatases, has been associated with diverse human cancers. Structural data demonstrate that two metal ions and two water molecules are located in the active site of PP5 and are involved in the dephosphorylations of the substrate. Recently, the catalytic reaction of PP5 has been studied by a density functional theory (DFT) method, and the role of the water molecules in regulating the substrate binding of PP5 was explored by our previous molecular dynamics (MD) study. These two studies enrich our understanding of the catalytic mechanism of PP5. However, the questions of how the metal ions regulate the substrate binding and the contribution of each metal ion to the catalysis remain not answered. To this end, the role of the catalytic Mn 2+ ions in regulating the binding of PP5 with its substrate was investigated through MD simulations. Results reveal that the different states of Mn 2+ ions can alter the structure of the active site and the conformations of the residues coordinating the Mn 2+ ions or the substrate. This leads to changes in the secondary structure and dynamical fluctuation of PP5, especially for that of helix αJ. With the position change of helix αJ, the volume of the substrate binding pocket is altered by the different states of Mn 2+ ions, indicating the regulatory role of Mn 2+ ions in PP5 activity. Furthermore, combined with the simulation data of our previous study, the sequential steps for PP5 binding of the substrate, the two active site water molecules, and the two Mn 2+ ions are proposed. This may help to develop new inhibitors to treat the PP5-related cancers., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Bacteria sense environmental chemicals using chemosensor proteins, most of which are present in the cytoplasmic membrane. Canonical chemoreceptors bind their specific ligands in their periplasmic domain, and the ligand binding creates a molecular stimulus that is transmitted into the cytoplasm, leading to various cellular responses, such as chemotaxis and specific gene expression. Vibrio cholerae , the causative agent of cholera, contains about 44 putative sensor proteins, which are homologous to methyl-accepting chemotaxis proteins involved in chemotaxis. Two of them, Mlp24 and Mlp37, have been identified as chemoreceptors that mediate chemotactic responses to various amino acids. Although most of the residues of Mlp37 involved in ligand binding are conserved in Mlp24, these chemoreceptors bind the same ligands with different affinities. Moreover, they have distinct cellular roles. Here we determined a series of ligand complex structures of the periplasmic domains of Mlp24 (Mlp24p). The structures revealed that Ca 2+ binds to the loop that forms the upper wall of the ligand-binding pocket. Ca 2+ does not bind to the corresponding loop of Mlp37, implying that the structural difference of the loop may cause the ligand affinity difference. Isothermal titration calorimetry (ITC) measurements indicated that Ca 2+ changes the ligand binding affinity of Mlp24p. Furthermore, Ca 2+ affected chemotactic behaviors to various amino acids mediated by Mlp24. Thus, Ca 2+ is suggested to serve as a cosignal for the primary signal mediated by Mlp24p, and V. cholerae fine-tunes its chemotactic behavior depending on the Ca 2+ concentration by modulating the ligand sensitivity of Mlp24. IMPORTANCE Mlp24 and Mlp37 are homologous chemoreceptors of Vibrio cholerae that bind various amino acids. Although most of the residues involved in ligand interaction are conserved, these chemoreceptors show different affinities for the same ligand and play different cellular roles. A series of ligand complex structures of the periplasmic region of Mlp24 (Mlp24p) and following ITC analysis revealed that Ca 2+ binds to the loop of Mlp24p and modulates the ligand binding affinity of Mlp24p. Moreover, Ca 2+ changes the chemotactic behaviors mediated by Mlp24. We propose that Ca 2+ acts as a cosignal that modulates the affinity of Mlp24 for the primary signal, thereby changing the chemotactic behavior of V. cholerae ., (Copyright © 2019 American Society for Microbiology.)

A broad range of biochemicals, from proteins to nucleic acids, function properly only when associated with a metal, usually a divalent cation. Not any divalent metal will do: these metals differ in their ionic radius, dissociation in water, ionization potential, and number of unpaired electrons in their outer shells, and so substituting one metal for another often changes substrate positioning, redox reactivities, and physiological performance, and thus may serve as a regulatory mechanism. For instance, exchanging manganese for magnesium in several chloroplast enzymes maintains plant carbon-nitrogen balance under rising atmospheric CO 2 concentrations. Here, we review this and a few other cases where association of proteins or nucleic acids with different metals control metabolism., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Novel Ca 2+ -independent C-type lectins, SPL-1 and SPL-2, were purified from the bivalve Saxidomus purpuratus. They are composed of dimers with either identical (SPL-2 composed of two B-chains) or distinct (SPL-1 composed of A- and B-chains) polypeptide chains, and show affinity for N-acetylglucosamine (GlcNAc)- and N-acetylgalactosamine (GalNAc)-containing carbohydrates, but not for glucose or galactose. A database search for sequence similarity suggested that they belong to the C-type lectin family. X-ray crystallographic analysis revealed definite structural similarities between their subunits and the carbohydrate-recognition domain (CRD) of the C-type lectin family. Nevertheless, these lectins (especially SPL-2) showed Ca 2+ -independent binding affinity for GlcNAc and GalNAc. The crystal structure of SPL-2/GalNAc complex revealed that bound GalNAc was mainly recognized via its acetamido group through stacking interactions with Tyr and His residues and hydrogen bonds with Asp and Asn residues, while widely known carbohydrate-recognition motifs among the C-type CRD (the QPD [Gln-Pro-Asp] and EPN [Glu-Pro-Asn] sequences) are not involved in the binding of the carbohydrate. Carbohydrate-binding specificities of individual A- and B-chains were examined by glycan array analysis using recombinant lectins produced from Escherichia coli cells, where both subunits preferably bound oligosaccharides having terminal GlcNAc or GalNAc with α-glycosidic linkages with slightly different specificities., (© 2019 The Protein Society.)

Risk for Atrial Fibrillation (AF), the most common human arrhythmia, has a major genetic component. The T-box transcription factor TBX5 influences human AF risk, and adult-specific Tbx5 -mutant mice demonstrate spontaneous AF. We report that TBX5 is critical for cellular Ca 2+ homeostasis, providing a molecular mechanism underlying the genetic implication of TBX5 in AF. We show that cardiomyocyte action potential (AP) abnormalities in Tbx5 -deficient atrial cardiomyocytes are caused by a decreased sarcoplasmic reticulum (SR) Ca 2+ ATPase (SERCA2)-mediated SR calcium uptake which was balanced by enhanced trans-sarcolemmal calcium fluxes (calcium current and sodium/calcium exchanger), providing mechanisms for triggered activity. The AP defects, cardiomyocyte ectopy, and AF caused by TBX5 deficiency were rescued by phospholamban removal, which normalized SERCA function. These results directly link transcriptional control of SERCA2 activity, depressed SR Ca 2+ sequestration, enhanced trans-sarcolemmal calcium fluxes, and AF, establishing a mechanism underlying the genetic basis for a Ca 2+ -dependent pathway for AF risk., Competing Interests: WD, BL, LT, KS, RN, FA, SM, SL, MG, YW, YL, HV, LS, MB, IM, CW No competing interests declared, (© 2019, Dai et al.)

Pb 2+ exposure leads to diverse neurological disorders; however, the mechanism of Pb 2+ -induced neurotoxicity is not clearly understood. Here we demonstrate that Pb 2+ binds to EF-hands in apo-DREAM (downstream regulatory element antagonist modulator) with a lower equilibrium dissociation constant ( K d = 20 ± 2 nM) than Ca 2+ ( K d = 1 μM). Based on the Trp169 emission and CD spectra, we report that Pb 2+ association triggers changes in the protein secondary and tertiary structures that are analogous to those previously observed for Ca 2+ -bound protein. The hydrophobic cavity in the C-terminal domain of DREAM is solvent exposed in the presence of Pb 2+ as determined using a hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS). Pb 2+ association with DREAM also modulates interactions between DREAM and its intracellular partners as evident from the fact that Pb 2+ -bound DREAM associates with peptide-based model systems, presenilin-1 helix-9 "PS1HL9" K V 4.3(70-90) "site-2" and K V 4.3(2-22) "site 1". Namely, dissociation constants for Pb 2+ -bound DREAM interaction with PS1HL9 ( K d = 2.4 ± 0.1 μM), site-2 ( K d = 11.0 ± 0.5 μM) and site 1 ( K d = 5.0 ± 0.6 μM) are nearly identical to those observed for Ca 2+ bound DREAM. Isothermal titration calorimetry data reveal that Pb 2+ binds to two high-affinity sites in Ca 2+ bound DREAM with the overall apparent constant of 4.81 ± 0.06 μM and its binding to Ca 2+ bound DREAM is entropy-driven. Taking into account the structural and sequence similarity between DREAM and other neuronal calcium sensor (NCS) proteins, these results strongly indicate that DREAM and possibly other NCS proteins bind Pb 2+ with a higher affinity than that for Ca 2+ and Pb 2+ interactions with NCS proteins can contribute to Pb 2+ -induced neurotoxicity.

Mandalate racemase (MR) from Pseudomonas putida requires a divalent metal cation, usually Mg2+, to catalyse the interconversion of the enantiomers of mandelate. Although the active site Mg2+ may be replaced by Mn2+, Co2+, or Ni2+, substitution by these metal ions does not markedly (<10-fold) alter the kinetic parameters Kappm, kappcat, and (kcat/Km)app for the substrates (R)- and (S)-mandelate, and the alternative substrate (S)-trifluorolactate. Viscosity variation experiments with Mn2+-MR showed that the metal ion plays a role in the uniform binding of the transition states for enzyme-substrate association, the chemical step, and enzyme-product dissociation. Surprisingly, the competitive inhibition constants (Ki) for inhibition of each metalloenzyme variant by benzohydroxamate did not vary significantly with the identity of the metal ion unlike the marked variation of the stability constants (K1) observed for M2+·BzH complex formation in solution. A similar trend was observed for the inhibition of the metalloenzyme variants by F-, except for Mg2+-MR, which bound F- tighter than would be predicted based on the stability constants for formation of M2+·F- complexes in solution. Thus, the enzyme modifies the enatic state of the bound metal ion cofactor so that the apparent electrophilicity of Mg2+ is enhanced, while that of Ni2+ is attenuated, resulting in a levelling effect relative to the trends observed for the free metals in solution.

In most species, fertilization induces Ca 2+ transients in the egg. In mammals, the Ca 2+ rises are triggered by phospholipase Cζ (PLCζ) released from the sperm; IP 3 generated by PLCζ induces Ca 2+ release from the intracellular Ca 2+ store through IP 3 receptor, termed IP 3 -induced Ca 2+ release. Here, we developed new fluorescent IP 3 sensors (IRIS-2s) with the wider dynamic range and higher sensitivity (Kd = 0.047-1.7 μM) than that we developed previously. IRIS-2s employed green fluorescent protein and Halo-protein conjugated with the tetramethylrhodamine ligand as fluorescence resonance energy transfer (FRET) donor and acceptor, respectively. For simultaneous imaging of Ca 2+ and IP 3 , using IRIS-2s as the IP 3 sensor, we developed a new single fluorophore Ca 2+ sensor protein, DYC3.60. With IRIS-2s and DYC3.60, we found that, right after fertilization, IP 3 concentration ([IP 3 ]) starts to increase before the onset of the first Ca 2+ wave. [IP 3 ] stayed at the elevated level with small peaks followed after Ca 2+ spikes through Ca 2+ oscillations. We detected delays in the peak of [IP 3 ] compared to the peak of each Ca 2+ spike, suggesting that Ca 2+ -induced regenerative IP 3 production through PLC produces small [IP 3 ] rises to maintain [IP 3 ] over the basal level, which results in long lasting Ca 2+ oscillations in fertilized eggs.

CvsSR is a Ca 2+ -induced two-component system (TCS) in the plant pathogen Pseudomonas syringae pv. tomato DC3000. Here, we discovered that CvsSR is induced by Fe 3+ , Zn 2+ , and Cd 2+ However, only supplementation of Ca 2+ to medium resulted in rugose, opaque colonies in Δ cvsS and Δ cvsR strains. This phenotype corresponded to formation of calcium phosphate precipitation on the surface of Δ cvsS and Δ cvsR colonies. CvsSR regulated swarming motility in P. syringae pv. tomato in a Ca 2+ -dependent manner, but swarming behavior was not influenced by Fe 3+ , Zn 2+ , or Cd 2+ We hypothesized that reduced swarming displayed by Δ cvsS and Δ cvsR strains was due to precipitation of calcium phosphate on the surface of Δ cvsS and Δ cvsR cells grown on agar medium supplemented with Ca 2+ By reducing the initial pH or adding glucose to the medium, calcium precipitation was inhibited, and swarming was restored to Δ cvsS and Δ cvsR strains, suggesting that calcium precipitation influences swarming ability. Constitutive expression of a CvsSR-regulated carbonic anhydrase and a CvsSR-regulated putative sulfate major facilitator superfamily transporter in Δ cvsS and Δ cvsR strains inhibited formation of calcium precipitates and restored the ability of Δ cvsS and Δ cvsR bacteria to swarm. Lastly, we found that glucose inhibited Ca 2+ -based induction of CvsSR. Hence, CvsSR is a key regulator that controls calcium precipitation on the surface of bacterial cells. IMPORTANCE Bacteria are capable of precipitating and dissolving minerals. We previously reported the characterization of the two-component system CvsSR in the plant-pathogenic bacterium Pseudomonas syringae CvsSR responds to the presence of calcium and is important for causing disease. Here, we show that CvsSR controls the ability of the bacterium to prevent calcium phosphate precipitation on the surface of cells. We also identified a carbonic anhydrase and transporter that modulate formation of surface-associated calcium precipitates. Furthermore, our results demonstrate that the ability of the bacterium to swarm is controlled by the formation and dissolution of calcium precipitates on the surface of cells. Our study describes new mechanisms for microbially induced mineralization and provides insights into the role of mineral deposits on bacterial physiology. The discoveries may lead to new technological and environmental applications.

T regulatory (Treg) cells maintain immunological tolerance and organ homeostasis. Activated Treg cells differentiate into effector Treg subsets that acquire tissue-specific functions. Ca 2+ influx via Ca 2+ release-activated Ca 2+ (CRAC) channels formed by STIM and ORAI proteins is required for the thymic development of Treg cells, but its function in mature Treg cells remains unclear. Here we show that deletion of Stim1 and Stim2 genes in mature Treg cells abolishes Ca 2+ signaling and prevents their differentiation into follicular Treg and tissue-resident Treg cells. Transcriptional profiling of STIM1/STIM2-deficient Treg cells reveals that Ca 2+ signaling regulates transcription factors and signaling pathways that control the identity and effector differentiation of Treg cells. In the absence of STIM1/STIM2 in Treg cells, mice develop a broad spectrum of autoantibodies and fatal multiorgan inflammation. Our findings establish a critical role of CRAC channels in controlling lineage identity and effector functions of Treg cells.

A 15-state kinetic model for Na^{+} channel gating that describes the coupling between three activation sensors, a two-stage fast inactivation process, and slow inactivated states may be reduced to equations for a 6-state system by application of the method of multiple scales. By expressing the occupation probabilities for closed states and the open state in terms of activation and fast inactivation variables, and assuming that activation has a faster relaxation than inactivation and that the activation sensors are mutually independent, the kinetic equations may be further reduced to rate equations for activation, and coupled fast and slow inactivation that describe spike frequency adaptation, a repetitive bursting oscillation in the neural membrane, and a cardiac action potential with a plateau oscillation. The fast inactivation rate function is, in general, dependent on the activation variable m(t) but may be approximated by a voltage-dependent function, and the rate function for entry into the slow inactivated state is dependent on the fast inactivation variable.

Development of the nervous system relies on a balance between axon and dendrite growth and subsequent pruning and degeneration. The developmental degeneration of dorsal root ganglion (DRG) sensory axons has been well studied in part because it can be readily modeled by removing the trophic support by nerve growth factor (NGF) in vitro . We have recently reported that axonal fragmentation induced by NGF withdrawal is dependent on Ca 2+ , and here, we address the mechanism of Ca 2+ entry required for developmental axon degeneration of mouse embryonic DRG neurons. Our results show that the transient receptor potential vanilloid family member 1 (TRPV1) cation channel plays a critical role mediating Ca 2+ influx in DRG axons withdrawn from NGF. We further demonstrate that TRPV1 activation is dependent on reactive oxygen species (ROS) generation that is driven through protein kinase C (PKC) and NADPH oxidase (NOX)-dependent pathways that become active upon NGF withdrawal. These findings demonstrate novel mechanistic links between NGF deprivation, PKC activation, ROS generation, and TRPV1-dependent Ca 2+ influx in sensory axon degeneration.

Voltage-activated ion channels contain S1-S4 domains that sense membrane voltage and control opening of ion-selective pores, a mechanism that is crucial for electrical signaling. Related S1-S4 domains have been identified in voltage-sensitive phosphatases and voltage-activated proton channels, both of which lack associated pore domains. hTMEM266 is a protein of unknown function that is predicted to contain an S1-S4 domain, along with partially structured cytoplasmic termini. Here we show that hTMEM266 forms oligomers, undergoes both rapid (µs) and slow (ms) structural rearrangements in response to changes in voltage, and contains a Zn 2+ binding site that can regulate the slow conformational transition. Our results demonstrate that the S1-S4 domain in hTMEM266 is a functional voltage sensor, motivating future studies to identify cellular processes that may be regulated by the protein. The ability of hTMEM266 to respond to voltage on the µs timescale may be advantageous for designing new genetically encoded voltage indicators., Competing Interests: FP, SL, OS, EB, JS, TC, MB, GT No competing interests declared, KS Reviewing editor, eLife

Motilin is a gastrointestinal peptide hormone that stimulates gastrointestinal motility. Motilin is produced primarily in the duodenum and jejunum. Motilin receptors (MTLRs) are G protein-coupled receptors that may represent a clinically useful pharmacological target as they can be activated by erythromycin. The functions of motilin are highly species-dependent and remain poorly understood. As a functional motilin system is absent in rodents such as rats and mice, these species are not commonly used for basic studies. In this study, we examine the usefulness of human MTLR-overexpressing transgenic (hMTLR-Tg) mice by identifying the mechanisms of the gastric motor response to human motilin and erythromycin. The distribution of hMTLR was examined immunohistochemically in male wild-type (WT) and hMTLR-Tg mice. The contractile response of gastric strips was measured isometrically in an organ bath, while gastric emptying was determined using phenol red. hMTLR expression was abundant in the gastric smooth muscle layer. Interestingly, higher levels of hMTLR expression were observed in the myenteric plexus of hMTLR-Tg mice but not WT mice. hMTLR was not co-localized with vesicular acetylcholine transporter, a marker of cholinergic neurons in the myenteric plexus. Treatment with human motilin and erythromycin caused concentration-dependent contraction of gastric strips obtained from hMTLR-Tg mice but not from WT mice. The contractile response to human motilin and erythromycin in hMTLR-Tg mice was affected by neither atropine nor tetrodotoxin and was totally absent in Ca2+-free conditions. Furthermore, intraperitoneal injection of erythromycin significantly promoted gastric emptying in hMTLR-Tg mice but not in WT mice. Human motilin and erythromycin stimulate gastric smooth muscle contraction in hMTLR-Tg mice. This action is mediated by direct contraction of smooth muscle via the influx of extracellular Ca2+. Thus, hMTLR-Tg mice may be useful for the evaluation of MTLR agonists as gastric prokinetic agents., Competing Interests: The authors have declared that no competing interests exist.

The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of murine TMEM16F (mTMEM16F) that underlie its function as a lipid scramblase and an ion channel. The cryo-EM data of mTMEM16F in absence and presence of Ca 2+ define the ligand-free closed conformation of the protein and the structure of a Ca 2+ -bound intermediate. Both conformations resemble their counterparts of the scrambling-incompetent anion channel mTMEM16A, yet with distinct differences in the region of ion and lipid permeation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, both functions appear to be mediated by alternate protein conformations that are at equilibrium in the ligand-bound state., Competing Interests: CA, NL, VC, GO, RD, CP No competing interests declared, (© 2019, Alvadia et al.)

PrimPol is the most recently discovered human DNA polymerase/primase and plays an emerging role in nuclear and mitochondrial genomic maintenance. As a member of archaeo-eukaryotic primase superfamily enzymes, PrimPol possesses DNA polymerase and primase activities that are important for replication fork progression in vitro and in cellulo. The enzymatic activities of PrimPol are critically dependent on the nucleotidyl-transfer reaction to incorporate deoxyribonucleotides successively; however, our knowledge concerning the kinetic mechanism of the reaction remains incomplete. Using enzyme kinetic analyses and computer simulations, we dissected the mechanism by which PrimPol transfers a nucleotide to a primer-template DNA, which comprises DNA binding, conformational transition, nucleotide binding, phosphoester bond formation, and dissociation steps. We obtained the rate constants of the steps by steady-state and pre-steady-state kinetic analyses and simulations. Our data demonstrate that the rate-limiting step of PrimPol-catalyzed DNA elongation depends on the metal cofactor involved. In the presence of Mn 2+ , a conformational transition step from non-productive to productive PrimPol:DNA complexes limits the enzymatic turnover, whereas in the presence of Mg 2+ , the chemical step becomes rate limiting. As evidenced from our kinetic and simulation data, PrimPol maintains the same kinetic mechanism under either millimolar or physiological micromolar Mn 2+ concentration. Our study revealed the underlying mechanism by which PrimPol catalyzes nucleotide incorporation with two common metal cofactors and provides a kinetic basis for further understanding the regulatory mechanism of this functionally diverse primase-polymerase., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The binding of orthosteric ligands to integrins requires the presence of divalent metal cations bound to metal ion-binding sites located in the I domains of the integrin α and β subunits. In this study the influence of the type and concentration of divalent metal cation present was investigated on a single arginyl-glycinyl-aspartic acid (RGD) ligand across the αv integrin sub-family and single αv integrin (αvβ6) with different ligands. These relationships were determined using radioligand binding studies completed with [ 3 H] ligands and purified αv integrin protein preparations. The binding of [ 3 H]compound 1 to the RGD site on individual αv integrins demonstrated a unique profile in relation to the type and concentration of divalent metal cation present. The use of physiological concentrations of Mg 2+ and Ca 2+ in simulated lung fluid altered the αv integrin selectivity profile of [ 3 H]compound 1 in terms of affinity and the level of receptor occupancy. In addition, different RGD ligands for the αvβ6 integrin behaved differently under the same divalent metal cation conditions. In conclusion, this study demonstrates the need to determine the individual relationship between RGD ligands and the integrins they may engage in vivo, especially when determining selectivity profiles for potential RGD-mimetic small molecule therapeutics, with organ and disease state also considered., (Copyright © 2018 GlaxoSmithKline Plc. Published by Elsevier Masson SAS.. All rights reserved.)