Cation Binding Sites Research Articles

Long-Range Effects of Na(+) Binding in Na,K-ATPase Reported by ATP.

This paper addresses the question of long-range interactions between the intramembranous cation binding sites and the cytoplasmic nucleotide binding site of the ubiquitous ion-transporting Na,K-ATPase using (13)C cross-polarization magic-angle spinning (CP-MAS) solid-state nuclear magnetic resonance. High-affinity ATP binding is induced by the presence of Na(+) as well as of Na-like substances such as Tris(+), and these ions are equally efficient promoters of nucleotide binding. CP-MAS analysis of bound ATP with Na,K-ATPase purified from pig kidney membranes reveals subtle differences in the nucleotide interactions within the nucleotide site depending on whether Na(+) or Tris(+) is used to induce binding. Differences in chemical shifts for ATP atoms C1' and C5' observed in the presence of Na(+) or Tris(+) suggest alterations in the residues surrounding the bound nucleotide, hydrogen bonding, and/or conformation of the ribose ring. This is taken as evidence of a long-distance communication between the Na(+)-filled ion sites in the membrane interior and the nucleotide binding site in the cytoplasmic domain and reflects the first conformational change ultimately leading to phosphorylation of the enzyme. Stopped-flow fluorescence measurements with the nucleotide analogue eosin show that the dissociation rate constant for eosin is larger in Tris(+) than in Na(+), giving kinetic evidence of the difference in structural effects of Na(+) and Tris(+). According to the recent crystal structure of the E1·AlF4(-)·ADP·3Na(+) form, the coupling between the ion binding sites and the nucleotide side is mediated by, among others, the M5 helix.

NhaA: A Unique Structural Fold of Secondary Active Transporters

AbstractThe Na+/H+ antiporters transport sodium (or several other monovalent cations) in exchange for H+ across lipid bilayers in all kingdoms of life. They are critical in the pH homeostasis of cytoplasm and/or organelles. In humans, these proteins are associated with the pathophysiology of various diseases. Yet, the most extensively studied Na+/H+ antiporter is Ec‐NhaA, the main Na+/H+ antiporter of Escherichia coli. The crystal structure of an inactive, down‐regulated Ec‐NhaA, determined at acidic pH, has provided the first structural insights into the antiport mechanism and pH regulation of an Na+/H+ antiporter. It reveals a unique structural fold (called the NhaA fold) in which helical transmembrane segments (TMs) are organized in inverted‐topology repeats, including two antiparallel non‐helical extended chain regions that cross each other, forming a delicate electrostatic balance in the middle of the membrane. The NhaA fold contributes to the cation binding site and facilitates rapid conformational changes of Ec‐NhaA. The NhaA fold has now been recognized to be shared by four Na+/H+ antiporters (bacterial and archaeal) and two Na+ symporters. Thus far, no crystal structure of any of the human Na+/H+ antiporters has been determined. Nevertheless, the Ec‐NhaA crystal structure has enabled the structural modeling of NHE1, NHE9, and NHA2, which are involved in human pathophysiology. Future elucidation of structure‐function relationships of eukaryotic and prokaryotic Na+/H+ antiporters are likely to provide insights into the human pathophysiology. Here, we will focus on the NhaA structural fold which underpins the antiporter functionality of Ec‐NhaA and the secondary active transporters.

Preparation of 3-benzyloxy-2-pyridinone functional linkers: tools for the synthesis of 3,2-hydroxypyridinone (HOPO) and HOPO/hydroxamic acid chelators

In contrast to 2,3-dihydroxypyridine, the 3-benzyloxy protected derivative, 2, undergoes facile alkylation at ambient temperatures with a variety of functionalized alkyl halides in good yields. This alkylation has been used to prepare a number of linkers that permit the attachment of 3,2-HOPO moieties onto various scaffolds using a wide range of coupling methods. The Mitsunobu reaction of 2 with representative alcohols was found to be of limited value due to competing O-alkylation that led to product mixtures. The phthalimide 3j can be converted in two steps to HOPO isocyanate 6 in excellent yields. Isocyanate 6 can be coupled to amines at room temperature or to alcohols in refluxing dichloroethane to obtain the corresponding urea or carbamate linked ligand systems. The coupling of isocyanate 6 with TREN followed by deprotection gave the tris-HOPO 10, an interesting target as it has both cationic and anionic binding sites. The HOPO hydroxylamine linker 11 was shown to be especially valuable as its coupling with carboxylic acids proceeds with the concomitant generation of an additional hydroxamate ligand moiety in the framework. The utility of this linker was shown by the preparation of two mixed HOPO-hydroxamate chelators, 16 and 19, based on the structure of desferrioxamine, a well-known trihydroxamate siderophore.

Homogentisate phytyltransferase from the unicellular green alga Chlamydomonas reinhardtii

Homogentisate phytyltransferase (HPT) (EC 2.5.1.-) catalyzes the first committed step of tocopherol biosynthesis in all photosynthetic organisms. This paper presents the molecular characterization and expression analysis of HPT1 gene, and a study on the accumulation of tocopherols under different environmental conditions in the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HPT1 protein conserves all the prenylphosphate- and divalent cation-binding sites that are found in polyprenyltransferases and all the amino acids that are essential for its catalytic activity. Its hydrophobicity profile confirms that HPT is a membrane-bound protein. Chlamydomonas genomic DNA analysis suggests that HPT is encoded by a single gene, HPT1, whose promoter region contains multiple motifs related to regulation by jasmonate, abscisic acid, low temperature and light, and an ATCTA motif presents in genes involved in tocopherol biosynthesis and some photosynthesis-related genes. Expression analysis revealed that HPT1 is strongly regulated by dark and low-temperature. Under the same treatments, α-tocopherol increased in cultures exposed to darkness or heat, whereas γ-tocopherol did it in low temperature. The regulatory expression pattern of HPT1 and the changes of tocopherol abundance support the idea that different tocopherols play specific functions, and suggest a role for γ-tocopherol in the adaptation to growth under low-temperature.

Sequential substitution of K(+) bound to Na(+),K(+)-ATPase visualized by X-ray crystallography.

Na+,K+-ATPase transfers three Na+ from the cytoplasm into the extracellular medium and two K+ in the opposite direction per ATP hydrolysed. The binding and release of Na+ and K+ are all thought to occur sequentially. Here we demonstrate by X-ray crystallography of the ATPase in E2·MgF42−·2K+, a state analogous to E2·Pi·2K+, combined with isotopic measurements, that the substitution of the two K+ with congeners in the extracellular medium indeed occurs at different rates, substantially faster at site II. An analysis of thermal movements of protein atoms in the crystal shows that the M3–M4E helix pair opens and closes the ion pathway leading to the extracellular medium, allowing K+ at site II to be substituted first. Taken together, these results indicate that site I K+ is the first cation to bind to the empty cation-binding sites after releasing three Na+.

ChemInform Abstract: Coordination Possibility of Uracil and Applications of Some of Its Complexes: A Review

AbstractReview: 41 refs.

Inter-domain electron transfer in cellobiose dehydrogenase: modulation by pH and divalent cations.

The flavocytochrome cellobiose dehydrogenase (CDH) is secreted by wood-decomposing fungi, and is the only known extracellular enzyme with the characteristics of an electron transfer protein. Its proposed function is reduction of lytic polysaccharide mono-oxygenase for subsequent cellulose depolymerization. Electrons are transferred from FADH2 in the catalytic flavodehydrogenase domain of CDH to haem b in a mobile cytochrome domain, which acts as a mediator and transfers electrons towards the active site of lytic polysaccharide mono-oxygenase to activate oxygen. This vital role of the cytochrome domain is little understood, e.g. why do CDHs exhibit different pH optima and rates for inter-domain electron transfer (IET)? This study uses kinetic techniques and docking to assess the interaction of both domains and the resulting IET with regard to pH and ions. The results show that the reported elimination of IET at neutral or alkaline pH is caused by electrostatic repulsion, which prevents adoption of the closed conformation of CDH. Divalent alkali earth metal cations are shown to exert a bridging effect between the domains at concentrations of > 3 mm, thereby neutralizing electrostatic repulsion and increasing IET rates. The necessary high ion concentration, together with the docking results, show that this effect is not caused by specific cation binding sites, but by various clusters of Asp, Glu, Asn, Gln and the haem b propionate group at the domain interface. The results show that a closed conformation of both CDH domains is necessary for IET, but the closed conformation also increases the FAD reduction rate by an electron pulling effect.

The role of monovalent cations in the ATPase reaction of DNA gyrase.

Four new crystal structures of the ATPase domain of the GyrB subunit of Escherichia coli DNA gyrase have been determined. One of these, solved in the presence of K(+), is the highest resolution structure reported so far for this domain and, in conjunction with the three other structures, reveals new insights into the function of this domain. Evidence is provided for the existence of two monovalent cation-binding sites: site 1, which preferentially binds a K(+) ion that interacts directly with the α-phosphate of ATP, and site 2, which preferentially binds an Na(+) ion and the functional significance of which is not clear. The crystallographic data are corroborated by ATPase data, and the structures are compared with those of homologues to investigate the broader conservation of these sites.

Efficient Deep-Blue Emittier and Molecular-Scale Memory Device Based on Dipyridyl–Phenylimidazole–Terpyridine Assembly

In this paper we have utilized a heteroditopic polypyridyl–imidazole system 4′-[4-(4,5-dipyridin-2-yl-1H-imidazol-2-yl)-phenyl]-[2,2′;6′,2″]terpyridine (tpy-Hbzim-dipy), wherein the terpyridine moiety has been utilized as the cation binding site and the imidazole motif as the anion binding site. This system is an efficient deep-blue emitter in chloroform with Commission Internationale de L’Eclairage (CIE) coordinates of 0.14 and 0.06. On the basis of the response profiles in terms of absorption or emission intensity and wavelength toward selected anions and cations, we developed a molecular system which can mimic sequential Boolean logic functions capable of integrating into a “Writing–Reading–Erasing–Reading” combinational circuit. In this work, we also examined the effect of selective anions and cations on the optical properties of the receptor by means of computational (DFT and TD-DFT) studies.

A novel human mutation in the SLC9A1 gene results in abolition of Na+/H+ exchanger activity.

The SLC9A1 gene, the Na+/H+ exchanger isoform 1 is the principal plasma membrane Na+/H+ exchanger of mammalian cells and functions by exchanging one intracellular proton for one extracellular sodium. The human protein is 815 amino acids in length. Five hundred N-terminal amino acids make up the transport domain of the protein and are believed to form 12 transmembrane segments. Recently, a genetic mutation of the Na+/H+ exchanger isoform 1, N266H, was discovered in a human patient through exome sequencing. We examined the effect of this mutation on expression, targeting and activity of the Na+/H+ exchanger. Mutant N266H protein was expressed in AP-1 cells, which lack their endogenous Na+/H+ exchanger protein. Targeting of the mutant protein to the cell surface was normal and expression levels were only slightly reduced relative to the wild type protein. However, the N266H mutant protein had no detectable Na+/H+ exchanger activity. A histidine residue at this location may disrupt the cation binding site or the pore of the Na+/H+ exchanger protein.

A functional correlate of severity in alternating hemiplegia of childhood

ObjectiveMutations in ATP1A3, the gene that encodes the α3 subunit of the Na+/K+ ATPase, are the primary cause of alternating hemiplegia of childhood (AHC). Correlations between different mutations and AHC severity were recently reported, with E815K identified in severe and D801N and G947R in milder cases. This study aims to explore the molecular pathological mechanisms in AHC and to identify functional correlates for mutations associated with different levels of disease severity. MethodsHuman wild type ATP1A3, and E815K, D801N and G947R mutants were expressed in Xenopus laevis oocytes and Na+/K+ ATPase function measured. Structural homology models of the human α3 subunit containing AHC mutations were created. ResultsThe AHC mutations examined all showed similar levels of reduction in forward cycling. Wild type forward cycling was reduced by coexpression with any mutant, indicating dominant negative interactions. Proton transport was measured and found to be selectively impaired only in E815K. Homology modeling showed that D801 and G947 lie within or near known cation binding sites while E815 is more distal. Despite its effect on proton transport, E815K was also distant from the proposed proton transport route. InterpretationLoss of forward cycling and dominant negativity are common and likely necessary pathomechanisms for AHC. In addition, loss of proton transport correlated with severity of AHC. D801N and G947R are likely to directly disrupt normal Na+/K+ binding while E815K may disrupt forward cycling and proton transport via allosteric mechanisms yet to be elucidated.

Structures and characterization of digoxin- and bufalin-bound Na+,K+-ATPase compared with the ouabain-bound complex

Cardiotonic steroids (CTSs) are specific and potent inhibitors of the Na(+),K(+)-ATPase, with highest affinity to the phosphoenzyme (E2P) forms. CTSs are comprised of a steroid core, which can be glycosylated, and a varying number of substituents, including a five- or six-membered lactone. These functionalities have specific influence on the binding properties. We report crystal structures of the Na(+),K(+)-ATPase in the E2P form in complex with bufalin (a nonglycosylated CTS with a six-membered lactone) and digoxin (a trisaccharide-conjugated CTS with a five-membered lactone) and compare their characteristics and binding kinetics with the previously described E2P-ouabain complex to derive specific details and the general mechanism of CTS binding and inhibition. CTSs block the extracellular cation exchange pathway, and cation-binding sites I and II are differently occupied: A single Mg(2+) is bound in site II of the digoxin and ouabain complexes, whereas both sites are occupied by K(+) in the E2P-bufalin complex. In all complexes, αM4 adopts a wound form, characteristic for the E2P state and favorable for high-affinity CTS binding. We conclude that the occupants of the cation-binding site and the type of the lactone substituent determine the arrangement of αM4 and hypothesize that winding/unwinding of αM4 represents a trigger for high-affinity CTS binding. We find that the level of glycosylation affects the depth of CTS binding and that the steroid core substituents fine tune the configuration of transmembrane helices αM1-2.

Cationic nanocarriers induce cell necrosis through impairment of Na(+)/K(+)-ATPase and cause subsequent inflammatory response.

Nanocarriers with positive surface charges are known for their toxicity which has limited their clinical applications. The mechanism underlying their toxicity, such as the induction of inflammatory response, remains largely unknown. In the present study we found that injection of cationic nanocarriers, including cationic liposomes, PEI, and chitosan, led to the rapid appearance of necrotic cells. Cell necrosis induced by cationic nanocarriers is dependent on their positive surface charges, but does not require RIP1 and Mlkl. Instead, intracellular Na+ overload was found to accompany the cell death. Depletion of Na+ in culture medium or pretreatment of cells with the Na+/K+-ATPase cation-binding site inhibitor ouabain, protected cells from cell necrosis. Moreover, treatment with cationic nanocarriers inhibited Na+/K+-ATPase activity both in vitro and in vivo. The computational simulation showed that cationic carriers could interact with cation-binding site of Na+/K+-ATPase. Mice pretreated with a small dose of ouabain showed improved survival after injection of a lethal dose of cationic nanocarriers. Further analyses suggest that cell necrosis induced by cationic nanocarriers and the resulting leakage of mitochondrial DNA could trigger severe inflammation in vivo, which is mediated by a pathway involving TLR9 and MyD88 signaling. Taken together, our results reveal a novel mechanism whereby cationic nanocarriers induce acute cell necrosis through the interaction with Na+/K+-ATPase, with the subsequent exposure of mitochondrial damage-associated molecular patterns as a key event that mediates the inflammatory responses. Our study has important implications for evaluating the biocompatibility of nanocarriers and designing better and safer ones for drug delivery.

Photoinduced conformational changes in DNA by poly(vinyl alcohol) carrying a malachite green moiety for protecting DNA against attack by nuclease.

Light is a highly advantageous means of specific cell targeting. Though targeted gene delivery is an important characteristic of an ideal delivery vehicle, there has been little effort to develop a photoresponsive vector. Among nonviral vectors, cationic substances interact effectively with negatively charged DNA. With this property in mind, we designed copolymers of poly(vinyl alcohol) carrying a malachite green moiety (PVAMG) with different molecular weights. Though PVAMG has no affinity for DNA in the absence of light, it undergoes photoionization in the presence of light to afford cationic DNA binding sites. The DNA-PVAMG complex was investigated with respect to DNA conformational changes and its protective nature, which are important properties for nonviral vectors. PVAMG irradiation promoted DNA conformational transitions from coils to partial globules to compacted globules. The complex had a protective effect against DNase I after PVAMG irradiation, while DNA was degraded under dark conditions. The effect on DNA transition and the protective nature were sensitive to the molecular weight of PVAMG. The data regarding binding constants and binding mode provided insight into the structure of the DNA-PVAMG complex. To withstand DNase I attacks, complexation results in the compaction of DNA, which is further covered with PVAMG.

Phosphate binding by a novel Zn(II) complex featuring a trans-1,2-diaminocyclohexane ligand. Effective anion recognition in water.

In this work we have investigated the binding properties of a new synthetic receptor for phosphate anions that combines metal ion coordination with electrostatic and H-bonding interactions. The described receptor is obtained by assembling an iminodiacetic (IDA) fragment, as a Zn(II) binding site, with a polyamine macrocyclic portion containing two trans-1,2-diaminocyclohexane (DAC) units and a pyrrole ring, as a cationic binding site, into an adaptive structure appropriately spanning the length of di- and tridentate phosphates. Potentiometric measurements together with (1)H and (31)P NMR investigation showed that, in a wide pH range including values of physiological interest, the Zn(II) complex of the receptor binds di- and triphosphates, such as ADP, ATP, pyrophosphate (PP) and triphosphate (TP), far better than monophosphate (MP), and that TP is poorly bound by methyliminodiacetate (MIDA) as a model for the Zn(II) binding site. Besides the excellent selectivity over other phosphates, the affinity for TP is the largest reported to date for Zn(II) complexes in water.

Site-Specific Cation Release Drives Actin Filament Severing by Vertebrate Cofilin

Actin polymerization powers the directed motility of eukaryotic cells. Sustained motility requires rapid filament turnover and subunit recycling. The essential regulatory protein cofilin severs actin filaments, which accelerates network remodeling by increasing the concentration of filament ends available for elongation and subunit exchange. Although cofilin effects on actin filament assembly dynamics have been extensively studied, the molecular mechanism of cofilin-induced filament severing is not understood. Here we demonstrate that actin filament severing by vertebrate cofilin is driven by the linked dissociation of a single cation per bound cofilin, which alters filament mechanical properties. Vertebrate cofilin only weakly severs Saccharomyces cerevisiae actin filaments lacking this “stiffness cation” unless a stiffness cation-binding site is engineered into the actin molecule. Vertebrate cofilin rescues the viability of a Saccharomyces cerevisiae cofilin deletion mutant only when the stiffness cation site is simultaneously introduced into actin, demonstrating that filament severing is the essential function of cofilin in cells. The work presented here reveals that site-specific interactions with cations serve a key regulatory function in actin filament fragmentation and dynamics.

Extraction and complexation of alkali and alkaline earth metal cations by lower-rim calix[4]arene diethylene glycol amide derivatives

The metal-ion extraction abilities of calix[4]arene derivatives1and2are largely dependent on the type of the amide group forming the cation-binding site. Tertiary-amide derivative2was shown to be an excellent extractant for most alkali and alkaline earth cations.

Negatively Charged Amino Acids Near and in Transient Receptor Potential (TRP) Domain of TRPM4 Channel Are One Determinant of Its Ca2+ Sensitivity

Transient receptor potential (TRP) channel melastatin subfamily member 4 (TRPM4) is a broadly expressed nonselective monovalent cation channel. TRPM4 is activated by membrane depolarization and intracellular Ca(2+), which is essential for the activation. The Ca(2+) sensitivity is known to be regulated by calmodulin and membrane phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Although these regulators must play important roles in controlling TRPM4 activity, mutation analyses of the calmodulin-binding sites have suggested that Ca(2+) binds to TRPM4 directly. However, the intrinsic binding sites in TRPM4 remain to be elucidated. Here, by using patch clamp and molecular biological techniques, we show that there are at least two functionally different divalent cation-binding sites, and the negatively charged amino acids near and in the TRP domain in the C-terminal tail of TRPM4 (Asp-1049 and Glu-1062 of rat TRPM4) are required for maintaining the normal Ca(2+) sensitivity of one of the binding sites. Applications of Co(2+), Mn(2+), or Ni(2+) to the cytosolic side potentiated TRPM4 currents, increased the Ca(2+) sensitivity, but were unable to evoke TRPM4 currents without Ca(2+). Mutations of the acidic amino acids near and in the TRP domain, which are conserved in TRPM2, TRPM5, and TRPM8, deteriorated the Ca(2+) sensitivity in the presence of Co(2+) or PI(4,5)P2 but hardly affected the sensitivity to Co(2+) and PI(4,5)P2. These results suggest a novel role of the TRP domain in TRPM4 as a site responsible for maintaining the normal Ca(2+) sensitivity. These findings provide more insights into the molecular mechanisms of the regulation of TRPM4 by Ca(2+).

Vesicular Glutamate Transporters Use Flexible Anion and Cation Binding Sites for Efficient Accumulation of Neurotransmitter

Vesicular glutamate transporters (VGLUTs) accumulate the neurotransmitter glutamate in synaptic vesicles. Transport depends on a V-ATPase-dependent electrochemical proton gradient (ΔμH+) and requires chloride ions, but how chloride acts and how ionic and charge balance is maintained during transport is controversial. Using a reconstitution approach, we used an exogenous proton pump to drive VGLUT-mediated transport either in liposomes containing purified VGLUT1 or in synaptic vesicles fused with proton-pump-containing liposomes. Our data show that chloride stimulation can be induced at both sides of the membrane. Moreover, chloride competes with glutamate at high concentrations. In addition, VGLUT1 possesses a cation binding site capable of binding H+ or K+ ions, allowing for proton antiport or K+ / H+ exchange. We conclude that VGLUTs contain two anion binding sites and one cation binding site, allowing the transporter to adjust to the changing ionic conditions during vesicle filling without being dependent on other transporters or channels.

Structural basis for ion selectivity revealed by high-resolution crystal structure of Mg2+ channel MgtE.

Magnesium is the most abundant divalent cation in living cells and is crucial to several biological processes. MgtE is a Mg2+ channel distributed in all domains of life that contributes to the maintenance of cellular Mg2+ homeostasis. Here we report the high-resolution crystal structures of the transmembrane domain of MgtE, bound to Mg2+, Mn2+ and Ca2+. The high-resolution Mg2+-bound crystal structure clearly visualized the hydrated Mg2+ ion within its selectivity filter. Based on those structures and biochemical analyses, we propose a cation selectivity mechanism for MgtE in which the geometry of the hydration shell of the fully hydrated Mg2+ ion is recognized by the side-chain carboxylate groups in the selectivity filter. This is in contrast to the K+-selective filter of KcsA, which recognizes a dehydrated K+ ion. Our results further revealed a cation-binding site on the periplasmic side, which regulate channel opening and prevents conduction of near-cognate cations.