Cation Binding Sites Research Articles

Iridium Oxide Nanoparticles Supported on Polybenzimidazole-Wrapped Multi-Wall Carbon Nanotube As Efficient and Durable Electrocatalysts for Proton Exchange Membrane Water Electrolyzers

Water electrolysis technology has attracted much attention because it serves as the key part of a carbon-neutral energy storage system as follows: electricity generated from renewable resources such as sunlight, water power, and wind, is used to produce hydrogen by water electrolysis. Then, the hydrogen is supplied to fuel cell system for power generation, which only generates electricity and water. Proton exchange membrane water electrolyzers (PEMWEs) are currently the most promising candidate because polymer electrolyte membrane provides higher ionic conductivity, better load flexibility, and purer hydrogen than alkaline electrolyte solution, anion exchange membrane, and solid oxide electrolyte, etc. The key challenge for PEMWEs is to develop efficient anode electrocatalyst because of the high catalytic efficiency loss related with the oxygen evolution reaction (OER) on anode. In addition, the use of PEM requires the anode material durable under long-term anodic polarization in acid environment, which significantly limits the options of materials. At present stage, iridium oxide (IrO2­) is widely regarded as the most promising anode electrocatalyst for PEMWEs owing to its high activity for OER and chemical stability in strong acid environment. Considering the price and scarcity of Ir, however, it is highly desired to lower the amount of Ir in the anode. In view of this, many approaches have focused on enhancing the mass activity of Ir-based catalysts towards OER by designing novel nanostructures. Another effective way is to disperse IrO2 on electronic conductive support with large surface area. Unfortunately, the most common support material for electrocatalysts, carbon materials, are regarded as not appropriate for PEMWEs because it would encounter severe degradation at high oxidizing potential region. Instead, metal oxides with large surface area and resistance to acid are used. However, their low electrical conductivity limits the electrochemical performance for OER.In previous studies, our group wrapped pristine multi-wall carbon nanotubes (MWNTs) with polybenzimidazole (PBI). The thin layer of PBI (thickness<1 nm) provides abundant binding sites for cations. As a result, uniform Pt nanoparticles could be deposited homogeneously on the PBI-wrapped pristine MWNTs. The resultant composite catalyst exhibited ehnhanced electrocatalytic activity and extraordinary long-term durability even polarized at high potential region of 1.0-1.5 V vs. RHE because (1) the aggregation of Pt nanoparticles was significantly suppressed due to the constraint effect of polymer and (2) the highly crystallized graphitic surface of pristine MWNTs refrained the electro-oxidation of carbon. The results provide a new insight into carbon nanotubes as an excellent support material with high corrosion-resistance under anodic polarization in acid environment.In present work, a composite catalyst of pristine MWNT, PBI, and IrO2 prepared by similar strategy. For comparison, Ir was also deposited on carbon black (CB) and PBI-wrapped CB. The products were characterized by thermogravimetry (TG), X-ray diffraction (XRD) analysis, X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, and scanning transmission electron microscope (STEM). The electrochemical properties of the catalysts were investigated by half-cell tests.

Dual Sensing By Simple Heteroditopic Salt Receptors Containing an Anthraquinone Unit

We synthesized simple ion pair receptors consisting of a crown ether cation binding site and an anthraquinone supported thiourea anion binding domain and studied their anion, cation and salt binding properties using spectroscopic, spectrophotometric and electrochemical measurements in acetonitrille solution. Apart from carboxylate anions, which cause deprotonation all the anions tested were found to associate with receptors more strongly in the presence of sodium cations, whereas in the presence of potassium or ammonium cation the anion binding strength was greatly diminished. A homotopic anion receptor lacking a crown ether unit, was unable to bind sodium salt more strongly than tetrabutylammonium salts. Solution and solid state X-ray measurements revealed that strong sodium coordination with the cation binding domain is responsible for the salt binding enhancement. Electrochemical measurements showed that the addition of anions to the ditopic ion pair receptors pretreated with sodium cations resulted in greater changes in reduction potentials compared to the addition of anions to receptors in the absence of Na+. Karbarz, M.; Romanski, J. Inorg. Chem. 2016, 55, 3616-3623. Figure 1

Structural and functional characterization of the CAP domain of pathogen-related yeast 1 (Pry1) protein.

The production, crystal structure, and functional characterization of the C-terminal cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domain of pathogen-related yeast protein-1 (Pry1) from Saccharomyces cerevisiae is presented. The CAP domain of Pry1 (Pry1CAP) is functional in vivo as its expression restores cholesterol export to yeast mutants lacking endogenous Pry1 and Pry2. Recombinant Pry1CAP forms dimers in solution, is sufficient for in vitro cholesterol binding, and has comparable binding properties as full-length Pry1. Two crystal structures of Pry1CAP are reported, one with Mg2+ coordinated to the conserved CAP tetrad (His208, Glu215, Glu233 and His250) in spacegroup I41 and the other without divalent cations in spacegroup P6122. The latter structure contains four 1,4-dioxane molecules from the crystallization solution, one of which sits in the cholesterol binding site. Both structures reveal that the divalent cation and cholesterol binding sites are connected upon dimerization, providing a structural basis for the observed Mg2+-dependent sterol binding by Pry1.

Crystal structure of the epithelial calcium channel TRPV6.

SummaryPrecise regulation of calcium homeostasis is essential for many physiological functions. The Ca2+-selective TRP channels TRPV5 and TRPV6 play vital roles in calcium homeostasis as Ca2+ uptake channels in epithelial tissues. Detailed structural bases for their assembly and Ca2+ permeation remain obscure. Here, we report the crystal structure of rat TRPV6 at 3.25 Å resolution. The overall architecture of TRPV6 reveals shared and unique features compared to other TRP channels. Intracellular domains engage in extensive interactions to form an intracellular “skirt” involved in allosteric modulation. In the K+ channel-like transmembrane domain, Ca2+ selectivity is determined by direct coordination of Ca2+ by a ring of aspartate side chains in the selectivity filter. Based on crystallographically identified cation binding sites at the pore axis and extracellular vestibule, we propose a Ca2+ permeation mechanism. Our results provide a structural foundation to understand the regulation of epithelial Ca2+ uptake and its role in pathophysiology.

Cations Stiffen Actin Filaments by Adhering a Key Structural Element to Adjacent Subunits.

Ions regulate the assembly and mechanical properties of actin filaments. Recent work using structural bioinformatics and site-specific mutagenesis favors the existence of two discrete and specific divalent cation binding sites on actin filaments, positioned in the long axis between actin subunits. Cation binding at one site drives polymerization, while the other modulates filament stiffness and plays a role in filament severing by the regulatory protein, cofilin. Existing structural methods have not been able to resolve filament-associated cations, and so in this work we turn to molecular dynamics simulations to suggest a candidate binding pocket geometry for each site and to elucidate the mechanism by which occupancy of the “stiffness site” affects filament mechanical properties. Incorporating a magnesium ion in the “polymerization site” does not seem to require any large-scale change to an actin subunit’s conformation. Binding of a magnesium ion in the “stiffness site” adheres the actin DNase-binding loop (D-loop) to its long-axis neighbor, which increases the filament torsional stiffness and bending persistence length. Our analysis shows that bound D-loops occupy a smaller region of accessible conformational space. Cation occupancy buries key conserved residues of the D-loop, restricting accessibility to regulatory proteins and enzymes that target these amino acids.

SLO2 Channels Are Inhibited by All Divalent Cations That Activate SLO1 K+ Channels

Two members of the family of high conductance K(+)channels SLO1 and SLO2 are both activated by intracellular cations. However, SLO1 is activated by Ca(2+)and other divalent cations, while SLO2 (Slack or SLO2.2 from rat) is activated by Na(+) Curiously though, we found that SLO2.2 is inhibited by all divalent cations that activate SLO1, with Zn(2+)being the most effective inhibitor with an IC50of ∼8 μmin contrast to Mg(2+), the least effective, with an IC50of ∼ 1.5 mm Our results suggest that divalent cations are not SLO2 pore blockers, but rather inhibit channel activity by an allosteric modification of channel gating. By site-directed mutagenesis we show that a histidine residue (His-347) downstream of S6 reduces inhibition by divalent cations. An analogous His residue present in some CNG channels is an inhibitory cation binding site. To investigate whether inhibition by divalent cations is conserved in an invertebrate SLO2 channel we cloned the SLO2 channel fromDrosophila(dSLO2) and compared its properties to those of rat SLO2.2. We found that, like rat SLO2.2, dSLO2 was also activated by Na(+)and inhibited by divalent cations. Inhibition of SLO2 channels in mammals andDrosophilaby divalent cations that have second messenger functions may reflect the physiological regulation of these channels by one or more of these ions.

Dual Sensing by Simple Heteroditopic Salt Receptors Containing an Anthraquinone Unit.

We synthesized simple ion pair receptors consisting of a crown ether cation binding site and an anthraquinone-supported thiourea anion binding domain and studied their anion-, cation-, and salt-binding properties using spectroscopic, spectrophotometric, and electrochemical measurements in acetonitrile solution. Apart from carboxylate anions, which cause deprotonation, all the anions tested were found to associate with receptor 1 more strongly in the presence of sodium cations, whereas in the presence of potassium or ammonium cation the anion binding strength was greatly diminished. A homotopic anion receptor 3, lacking a crown ether unit, was unable to bind sodium salt more strongly than tetrabutylammonium salts. Solution and solid-state X-ray measurements revealed that strong sodium coordination with the cation-binding domain is responsible for the salt-binding enhancement. Electrochemical measurements showed that the addition of anions to the receptor 1 pretreated with sodium cations resulted in greater changes in reduction potentials compared to the addition of anions to receptor 1 in the absence of Na(+).

Relationships among pH, aluminum solubility and aluminum complexation with organic matter in acid forest soils of the Northeastern United States

We investigated the interactions among pH, Al solubility and Al–soil organic matter (SOM) complexation to test the hypothesis that competition between Al and H ions for cation binding sites on soil organic matter (SOM) determines soil pH and Al solubility in acidic forest soils in the northeastern U.S. Samples from five soil horizons in 39 forested watersheds across the northeastern U.S. were used with two Al complexation models to test the hypothesis. Our results indicated that Al solubility increases with soil depth when pH is less than 4.5 and the ratio of the fractions of organically bound Al and H(NAl/NH) or the ratio of organically bound Al to total organic carbon (Alorg/C) remains roughly constant within each horizon. Both the NAl/NH and the Alorg/C ratios are effective measures of the ratio of sites bound to Al and H in Al–SOM complexation models. The log-linear relationship pAlKCl-p(Alorg/C)=0.75×pHKCl-1.56 (r=0.86,P<.0001) can be used to describe Al–pH relationships in Oie, Oa, Bh and Bs1 horizon samples from across the region. The competition between H+ and Al3+ for binding to SOM affects soil pH and Al solubility to a greater degree in Oa and Bs1 horizons than in Oie and E horizons. Our data are consistent with the conceptual model that portrays the Al–pH relationship in acid forest soils as a balance between organic acidity and alkalinity produced through the weathering of aluminosilicate minerals.

An Unusual Cation-Binding Site and Distinct Domain-Domain Interactions Distinguish Class II Enolpyruvylshikimate-3-phosphate Synthases.

Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes a critical step in the biosynthesis of a number of aromatic metabolites. An essential prokaryotic enzyme and the molecular target of the herbicide glyphosate, EPSPSs are the subject of both pharmaceutical and commercial interest. Two EPSPS classes that exhibit low sequence homology, differing substrate/glyphosate affinities, and distinct cation activation properties have previously been described. Here, we report structural studies of the monovalent cation-binding class II Coxiella burnetii EPSPS (cbEPSPS). Three cbEPSPS crystal structures reveal that the enzyme undergoes substantial conformational changes that alter the electrostatic potential of the active site. A complex with shikimate-3-phosphate, inorganic phosphate (Pi), and K(+) reveals that ligand induced domain closure produces an unusual cation-binding site bordered on three sides by the N-terminal domain, C-terminal domain, and the product Pi. A crystal structure of the class I Vibrio cholerae EPSPS (vcEPSPS) clarifies the basis of differential class I and class II cation responsiveness, showing that in class I EPSPSs a lysine side chain occupies the would-be cation-binding site. Finally, we identify distinct patterns of sequence conservation at the domain-domain interface and propose that the two EPSPS classes have evolved to differently optimize domain opening-closing dynamics.

Specific Cation Binding Stiffens Actin Filaments by Adhering D-Loops to Adjacent Monomers

Ions play a key role in mediating interactions between cytoskeletal proteins, helping to govern the assembly process and mechanical properties of actin filaments, which are composed of negatively charged monomers. Recent work using structural bioinformatics and site-specific mutagenesis suggests the existence of two specific divalent cation binding sites, one of which participates in the actin polymerization process, while the other modulates actin filament stiffness and plays a role in cofilin-mediated filament severing. To date, structural methods have not been able to resolve cation-associated filaments, and so in this work we turn to molecular dynamics simulations to predict candidate binding pocket geometries and to elucidate the mechanism by which binding in the stiffness site effects a filament's mechanical properties. We find that little change is necessary to an actin monomer conformation in order to incorporate an additional magnesium ion in the “polymerization site,” positioned in the long axis between actin monomers. We furthermore show that binding of an additional magnesium ion in the “stiffness site” adheres the actin D-loop to the adjacent actin's target binding cleft, resulting in a decrease in torsional flexibility and an increase in filament persistence length, consistent with previous biochemical and biophysical studies. Given these candidate structures, our simulations allow us to dissect the mechanism for this stiffening by studying the conformational space accessible to the D-loop. We also compute the burial of certain key conserved residues in the D-loop with and without coordinated magnesium ions, and hence predict their accessibility to enzymes known to regulate actin filament dynamics by post-translational modification of these amino-acids.

Crystal Structure and Asymmetric Conformation of a K+ Channel RCK Domain

Regulator of conductance of K+ (RCK) domains govern K+ channel and transporter activity through binding of cytosolic nucleotides or ions, although the locations and ligand-selectivities of activation sites within these domains can vary. To gain further insight toward structures of activation sites and ligand-dependent conformational changes in RCK domains, we solved the crystal structure of the cytosolic RCK domain from a Ca2+-gated K+ channel cloned from Thermoplasma volcanium. Crystals were grown in the presence of Mg2+, an activator of the Thermoplasma K+ channel, and the structure was solved to 2.95 A resolution. The structure was found to contain two RCK domains per asymmetric unit, forming a homodimer. Interestingly, this homodimer was captured in an asymmetric state, and structural alignment of the chains with one another revealed a “hinge” that immediately followed the domain's N-terminal Rossmann fold region. This asymmetry suggested that the RCK dimer might either be in an intermediate, partially-liganded state, or (alternatively) that the asymmetric conformation represents the fully-liganded state. To determine the locations of divalent cation binding sites, the domain was co-crystallized with Ba2+. The Ba2+-bound structure yielded the same asymmetric conformation observed in the Mg2+ co-crystal, and the anomalous difference Fourier map revealed only one Ba2+ binding site, located at the RCK domain dimer interface and thus forming a metal bridge between adjacent subunits. Crystallization of the domain in a four-fold symmetrical “gating ring” form and small-angle X-ray scattering profiles obtained over a range of [Ca2+] are consistent with the idea that formation of the intersubunit metal bridge may drive a conformational change in the gating ring, which in turn may modulate channel gating.

Kinetics by X-Ray Crystallography: Sequential Substitution of K+ Bound To Na+, K+-ATPase

Na+, K+-ATPase is one of the most important members of the P-type ATPase family. It transports 3 Na+ from the cytoplasm into the extracellular medium and countertransports 2 K+ per ATP hydrolyzed in each reaction cycle, thereby establishing gradients for Na+ and K+ across the membrane. These ion gradients are used in many fundamental processes, notably, excitation of nerve cells. Na+, K+-ATPase is also regarded as a membrane receptor for cardiotonic steroids, such as ouabain and digoxin, which have been prescribed for treatment of heart failure for ∼200 years. Crystallographic studies of Ca2+-ATPase of sarcoplasmic reticulum have provided detailed information on the gating mechanism on the cytoplasmic side, but little on the gating on the other side. This is partly because H+ countertransported by Ca2+-ATPase is invisible to X-rays. In this regard, Na+, K+-ATPase has a fundamental advantage over Ca2+-ATPase, as it countertransports K+ and even its congeners of larger atomic numbers. Here, we carried out X-ray crystallography and isotopic measurements of the ATPase in a state analogous to E2·Pi·2K+ to visualize the substitution process of 2 bound K+ with congeners in the extracellular medium. Crystals were soaked in a buffer containing K+ congeners and anomalous diffraction data were collected. The electron density maps at various incubation times clearly show that the substitution of the 2 K+ with congeners occurs substantially faster at site II. An analysis of B-factors of protein atoms in the crystal shows that the M3-M4E helix pair opens and closes the ion pathway leading to the extracellular medium. These results indicate that site I K+ is the first cation to bind to the empty cation binding sites after releasing 3 Na+.

Base–acid-induced translational isomerism in a branched [4]rotaxane

A branched [4]rotaxane containing three switching arms with both secondary ammonium cation and aniline binding sites for threaded crown ethers was prepared. 1H NMR spectroscopy was used to show the translational isomerisation of the [4]rotaxane, and different absorption maxima and the oxidation potential for the [4]rotaxane possessing a 1,3,5-tris(4-aminophenyl)benzene centre were monitored by ultraviolet spectroscopy and electrochemical studies, respectively, as the [4]rotaxane was exposed to encircling dibenzo[24]crown-8 species.

Removal of hexavalent chromium from electroplating wastewaters using marine macroalga Pelvetia canaliculata as natural electron donor

This paper reports a treatment strategy for an electroplating wastewater containing high amounts of hexavalent and trivalent chromium and zinc, and residual iron. The brown macroalga Pelvetia canaliculata was used as a natural electron donor for the reduction of Cr(VI) to Cr(III) at acidic pH, and as a natural cation exchanger for zinc, iron and trivalent chromium sequestration. The strategy adopted for the wastewater treatment involves: (i) the reduction of Cr(VI) to Cr(III) using the macroalga as electron donor; (ii) trivalent chromium, zinc and iron precipitation at pH 8.5; and (iii) the removal of residual zinc ions (13mg/L) by cation exchange at pH 8.5, using the negatively charged functional groups present at the surface of P. canaliculata.The Cr(VI) reduction was evaluated as a function of the biomass and Cr(VI) concentration, pH and temperature. The reaction was promoted through biomass oxidation in acidic medium. The Cr(VI) reduction capacities of raw and protonated P. canaliculata were around 1.8 and 2.3mmol/g and the values for the Cr(III) uptake capacity of the oxidized biomass were 0.8 and 1.9mmol/g, respectively. The results suggest that the oxidation of the biomass during Cr(VI) reduction generates new negatively charged active sites for cation binding. The continuous treatment of the wastewater containing Cr(VI) was evaluated in a column packed with raw P. canaliculata, and a maximum Cr(VI) reduction capacity of around 2.1mmol/g was achieved. After the Cr(VI) removal step, 100% (below the detection limit) and 95% of the remaining trivalent chromium and zinc, respectively, can be eliminated by precipitation at pH 8.5.

Sodium-Proton (Na(+)/H(+)) Antiporters: Properties and Roles in Health and Disease.

The transmembranal 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 pH homeostasis of the cytoplasm and/or organelles. A particularly notable example is the SLC9 gene family, which encodes Na(+)/H(+) exchangers (NHEs) in many species from prokaryotes to eukaryotes. 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 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 transmembrane segments (TMs) are organized in inverted-topology repeats, including two antiparallel unfolded regions that cross each other, forming a delicate electrostatic balance in the middle of the membrane. This unique structural fold (The NhaA fold) contributes to the cation binding site and facilitates the rapid conformational changes expected for Ec-NhaA. The NhaA fold has now been recognized to be shared by four Na(+)/H(+) antiporters (bacterial and archaeal) and a Na(+) symporter. Remarkably, no crystal structure of any of the human Na(+)/H(+) antiporters exists. Nevertheless, the Ec-NhaA crystal structure has enabled the structural modeling of NHE1, NHE9, and NHA2, three human plasmalemmal proteins that are members of the SLC9 family that are involved in human pathophysiology. Moreover, as outlined in this review, developments in the field, including cellular and biophysical methods that enable ion levels and fluxes to be measured in intact cells as well as in knockout mice, have led to striking advances in the identification and characterization of plasma membrane NHEs and NHA.Very little is known about the endomembrane isoforms of NHE. These intracellular exchangers may serve a function in cation homeostasis and/or osmoregulation, and not in pH regulation as is the case for the plasmalemmal isoforms. This intriguing possibility should be borne in mind when designing future studies. Future progress towards gaining an understanding of the SLC9 gene family, including its structure-function relationships and regulatory mechanisms in health and in disease, is likely to include insights into the pathophysiology of multiple diseases.

MATE transport of the E. coli-derived genotoxin colibactin.

Various forms of cancer have been linked to the carcinogenic activities of microorganisms(1-3). The virulent gene island polyketide synthase (pks) produces the secondary metabolite colibactin, a genotoxic molecule(s) causing double-stranded DNA breaks(4) and enhanced colorectal cancer development(5,6). Colibactin biosynthesis involves a prodrug resistance strategy where an N-terminal prodrug scaffold (precolibactin) is assembled, transported into the periplasm and cleaved to release the mature product(7-10). Here, we show that ClbM, a multidrug and toxic compound extrusion (MATE) transporter, is a key component involved in colibactin activity and transport. Disruption of clbM attenuated pks+ E. coli-induced DNA damage in vitro and significantly decreased the DNA damage response in gnotobiotic Il10(-/-) mice. Colonization experiments performed in mice or zebrafish animal models indicate that clbM is not implicated in E. coli niche establishment. The X-ray structure of ClbM shows a structural motif common to the recently described MATE family. The 12-transmembrane ClbM is characterized as a cation-coupled antiporter, and residues important to the cation-binding site are identified. Our data identify ClbM as a precolibactin transporter and provide the first structure of a MATE transporter with a defined and specific biological function.

Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near -65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼ 15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor-based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure-function relationships of the light-gated pore.

Platelet Phosphatidylserine Exposure, Survival and Blood Coagulation in Mice Lacking TMEM16F

Scott Syndrome is a rare, moderately severe bleeding disorder caused by a defect in platelet, red cell, and lymphocyte phosphatidylserine exposure. The syndrome has been linked to mutations in TMEM16F, a Ca++-activated ion channel. A moderately severe bleeding disorder in German Shephard dogs, characterized by decreased platelet phosphatidylserine exposure, has also linked to mutations in TMEM16F.TMEM16F, is a member of a recently-identified family of calcium-activated chloride channels that are also called anoctamins. Members of the family apparently serve both as ion channels and phospholipid scrambling channels. A crystal structure of one member of the TMEM16 family shows a homo-dimeric structure of an integral membrane protein with each unit containing 10 transmembrane helices. A transmembrane hydrophilic cavity lies at the interface, and contains a cation binding site, and a slot with dimensions that could accommodate acyl chains.Tmem16f-/- mice were recently reported to be viable with a prolonged tail snip bleeding time but no spontaneous bleeding. We have developed an independent gene-targeted (gt) Tmem16f allele generated in C57BL/6 ES cells and find results that both confirm prior reports and contrast with them.JM8 ES cells were obtained from EUCOMM, and Tmem16f gene targeting in intron 1 was confirmed by PCR and sequencing. Genotyping of 120 Tmem16f+/gt intercross progeny identified no surviving Tmem16fgt/gt mice at weaning (p<0.001). However, +/gt intercrosses generated the expected Mendelian genotype ratios at both E10.5 and E17.5, with gt/gt embryos. Blinded pathological evaluation of E17.5 gt/gt pups indicated reduction of ossification and angular limb changes, consistent with 2 prior reports. Thus, our results confirm previously reported bone changes, but in contrast with prior reports, indicate that Tmem16f deficiency is lethal in the C57BL/6 genetic background.An F2 intercross of +/gt mice outcrossed one generation to 129x16vJ resulted in gt/gt mice surviving to weaning, though at only 30% of the expected Mendelian frequency. Progeny analysis points to a single autosomal dominant 129x1SvJ-associated genetic modifier. Preliminary genetic analysis of these mice appears to map this locus to the proximal region of chromosome 3. Further efforts to localize the responsible gene(s) are underway.Tail bleeding times for gt/gt were >10min, whereas littermate +/gt and +/+ mice bleeding ceased at 8 ± 1 min and 6 ± 0.8 min, respectively, each significantly different than gt/gt (p<0.05). Notably, platelets from +/gt mice exhibited a trend toward reduced PS exposure, detected with FITC-labelled lactadherin, in response to PAR4 agonist peptide, whereas gt/gt mice had significantly reduced PS exposure (p < 0.05). gt/gt platelets showed a trend toward reduced PS exposure in response to A23187, as well as prolonged platelet rich plasma clotting times, and less efficient lactadherin inhibition of platelet clotting time.Our data suggest the existence of a viability-determining genetic modifier of the TMEM16F deficiency phenotype in the 129x1SvJ mouse strain. Identification of the responsible gene may uncover a novel regulator of hemostatic function. The observation that heterozygous deficiency leads to a PS exposure and hemostatic phenotype also suggests the possibility that heterozygous TMEM16F mutations may influence hemostasis or thrombosis in humans. DisclosuresNo relevant conflicts of interest to declare.

Streptococcal 5′-Nucleotidase A (S5nA), a Novel Streptococcus pyogenes Virulence Factor That Facilitates Immune Evasion

Streptococcus pyogenes is an important human pathogen that causes a wide range of diseases. Using bioinformatics analysis of the complete S. pyogenes strain SF370 genome, we have identified a novel S. pyogenes virulence factor, which we termed streptococcal 5'-nucleotidase A (S5nA). A recombinant form of S5nA hydrolyzed AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. Michaelis-Menten kinetics revealed a Km of 169 μm and a Vmax of 7550 nmol/mg/min for the substrate AMP. Furthermore, recombinant S5nA acted synergistically with S. pyogenes nuclease A to generate macrophage-toxic deoxyadenosine from DNA. The enzyme showed optimal activity between pH 5 and pH 6.5 and between 37 and 47 °C. Like other 5'-nucleotidases, S5nA requires divalent cations and was active in the presence of Mg(2+), Ca(2+), or Mn(2+). However, Zn(2+) inhibited the enzymatic activity. Structural modeling combined with mutational analysis revealed a highly conserved catalytic dyad as well as conserved substrate and cation-binding sites. Recombinant S5nA significantly increased the survival of the non-pathogenic bacterium Lactococcus lactis during a human whole blood killing assay in a dose-dependent manner, suggesting a role as an S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5'-nucleotidase activity and immune evasion properties.

Structural and Dynamic Basis for Low-Affinity, High-Selectivity Binding of L-Glutamine by the Glutamine Riboswitch.

Naturally occurring L-glutamine riboswitches occur in cyanobacteria and marine metagenomes, where they reside upstream of genes involved in nitrogen metabolism. By combining X-ray, NMR, and MD, we characterized an L-glutamine-dependent conformational transition in the Synechococcus elongatus glutamine riboswitch from tuning fork to L-shaped alignment of stem segments. This transition generates an open ligand-binding pocket with L-glutamine selectivity enforced by Mg(2+)-mediated intermolecular interactions. The transition also stabilizes the P1 helix through a long-range "linchpin" Watson-Crick G-C pair-capping interaction, while melting a short helix below P1 potentially capable of modulating downstream readout. NMR data establish that the ligand-free glutamine riboswitch in Mg(2+) solution exists in a slow equilibrium between flexible tuning fork and a minor conformation, similar, but not identical, to the L-shaped bound conformation. We propose that an open ligand-binding pocket combined with a high conformational penalty for forming the ligand-bound state provide mechanisms for reducing binding affinity while retaining high selectivity.