Anion Binding Site Research Articles

Mapping the binding site of C1-inhibitor for polyanion cofactors

The serpin, C1-inhibitor (also known as SERPING1), plays a vital anti-inflammatory role in the body by controlling pro-inflammatory pathways such as complement and coagulation. The inhibitor’s action is enhanced in the presence of polyanionic cofactors, such as heparin and polyphosphate, by increasing the rate of association with key enzymes such as C1s of the classical pathway of complement. The cofactor binding site of the serpin has never been mapped. Here we show that residues Lys284, Lys285 and Arg287 of C1-inhibitor play key roles in binding heparin and delivering the rate enhancement seen in the presence of polyanions and thus most likely represent the key cofactor binding residues for the serpin. We also show that simultaneous binding of the anion binding site of C1s by the polyanion is required to deliver the rate enhancement. Finally, we have shown that it is unlikely that the two positively charged zones of C1-inhibitor and C1s interact in the encounter complex between molecules as ablation of the charged zones did not in itself deliver a rate enhancement as might have been expected if the zones interacted. These insights provide crucial information as to the mechanism of action of this key serpin in the presence and absence of cofactor molecules.

The second-order NLO property of a photoswitchable heteroditpioc ion-pair receptor based on 2-pyridyl acylhydrazone linking with 2,6-pyridine bisamide: The impacts of metal cations and anions

A photoswitchable heteroditpioc ion-pair receptor E-1 and its isomeride Z-1 (without the anion binding site), that are based on the 2-pyridyl acylhydrazone linking 2,6-pyridine bisamide, have brought our attention to systematically explore the second-order nonlinear optical (NLO) properties by the density functional theory (DFT). In this work, we mainly studied the influences of metal cations (M = Na+, K+, Mg2+, Ca2+, Hg2+ and Pb2+), anions (X = Cl−, Br− and I−) and ion-pair (NaCl, NaBr and NaI) on NLO responses for the receptor. In addition, the impacts of isomerization and poto-switching processes on NLO response for these systems also have been discussed detailedly. The results show that the isomerization process does not effectively adjust the NLO properties for our studied systems. But the poto-switching process that was triggered by light to capure or release ions plays an important role in improving the NLO properties. The receptors E-1 and Z-1 are excellent candidates to effectively detect metal cation Pb2+, because the first hyperpolarizability (βtot) values of E∗Pb2+ and Z∗Pb2+ increased by 13 times and 20 times relative to that of receptors E-1 (188.06 a.u.) and Z-1 (270.21 a.u.), respectively. In addition, the receptor E-1 has the possibility to detect anion I− due to the larger βtot values compared with other anion-complexes. However, the changes of NLO responses for ion-pair complexes are not obvious compared with corresponding anion-complexes. We are looking forward to the research would be beneficial for further theoretical and experimental studies on recognizing metal cations and anions based on large second-order NLO difference.

Colorimetric molecular receptors for the sensing of acetate, fluoride and mercury based on Schiff’s bases

The real-time monitoring of biologically relevant anions and mercuric ion concentration in aqueous and physiological media and its quantitative determination across a large concentration range are of vital importance in environmental, health-care issues, industrial and scientific fields. Anion-like fluoride and acetate play an important role in a wide range of chemical and biological systems. This study reports the synthesis of a series of new molecular receptors based on Schiff’s bases by adopting a green strategy. The adopted procedures were simple and less complicated. A new simple colorimetric receptor based on salicylaldehyde Schiff’s bases sharing –OH and C=N moieties as anion binding sites have been successfully synthesized through one-step synthesis, and the sensing studies revealed that the receptors synthesized were found to be an elegant solution to the colorimetric sensing of the anions, especially acetate and fluoride.

Donepezil Inhibits Acetylcholinesterase via Multiple Binding Modes at Room Temperature.

Donepezil is a second generation acetylcholinesterase (AChE) inhibitor for treatment of Alzheimer's disease (AD). AChE is important for neurotransmission at neuromuscular junctions and cholinergic brain synapses by hydrolyzing acetylcholine into acetate and choline. In vitro data support that donepezil is a reversible, mixed competitive and noncompetitive inhibitor of AChE. The experimental fact then suggests a more complex binding mechanism beyond the molecular view in X-ray models resolved at cryogenic temperatures that show a unique binding mode of donepezil in the active site of the enzyme. Aiming at clarifying the mechanism behind that mixed competitive and noncompetitive nature of the inhibitor, we have applied molecular dynamics (MD) simulations and docking and free-energy calculations to investigate microscopic details and energetics of donepezil association for conditions of substrate-free and -bound states of the enzyme. Liquid-phase MD simulation at room temperature shows AChE transits between "open" and "closed" conformations to control accessibility to the active site and ligand binding. As shown by docking and free-energy calculations, association of donepezil involves its reversible axial displacement and reorientation in the active site of the enzyme, assisted by water molecules. Donepezil binds equally well the main-door anionic binding site PAS, the acyl pocket, and the catalytic site CAS by respectively adopting outward-inward-inward orientations regardless of substrate occupancy-the overall stability of that reaction process depends however on co-occupancy of the enzyme being preferential for its substrate-free state. All together, our findings support a physiologically relevant mechanism of AChE inhibition by donepezil involving multistable interactions modes at the molecular origin of the inhibitor's activity.

Phosphonium Boranes for the Selective Transport of Fluoride Anions across Artificial Phospholipid Membranes.

With the view of developing selective transmembrane anion transporters, a series of phosphonium boranes of general formula [p-RPh2 P(C6 H4 )BMes2 ]+ have been synthesized and evaluated. The results demonstrate that variation of the R group appended to the phosphorus atom informs the lipophilicity of these compounds, their Lewis acidity, as well as their transport activity. Anion transport experiments in POPC-based large unilamellar vesicles show that these main-group cations are highly selective for the fluoride anion, which is transported more than 20 times faster than the chloride anion. Finally, this work shows that the anion transport properties of these compounds are extremely sensitive to the steric crowding about the boron atom, pointing to the crucial involvement of the Group 13 element as the anion binding site.

Phosphonium Boranes for the Selective Transport of Fluoride Anions across Artificial Phospholipid Membranes

AbstractWith the view of developing selective transmembrane anion transporters, a series of phosphonium boranes of general formula [p‐RPh2P(C6H4)BMes2]+ have been synthesized and evaluated. The results demonstrate that variation of the R group appended to the phosphorus atom informs the lipophilicity of these compounds, their Lewis acidity, as well as their transport activity. Anion transport experiments in POPC‐based large unilamellar vesicles show that these main‐group cations are highly selective for the fluoride anion, which is transported more than 20 times faster than the chloride anion. Finally, this work shows that the anion transport properties of these compounds are extremely sensitive to the steric crowding about the boron atom, pointing to the crucial involvement of the Group 13 element as the anion binding site.

Casein kinase 1 dynamics underlie substrate selectivity and the PER2 circadian phosphoswitch.

Post-translational control of PERIOD stability by Casein Kinase 1δ and ε (CK1) plays a key regulatory role in metazoan circadian rhythms. Despite the deep evolutionary conservation of CK1 in eukaryotes, little is known about its regulation and the factors that influence substrate selectivity on functionally antagonistic sites in PERIOD that directly control circadian period. Here we describe a molecular switch involving a highly conserved anion binding site in CK1. This switch controls conformation of the kinase activation loop and determines which sites on mammalian PER2 are preferentially phosphorylated, thereby directly regulating PER2 stability. Integrated experimental and computational studies shed light on the allosteric linkage between two anion binding sites that dynamically regulate kinase activity. We show that period-altering kinase mutations from humans to Drosophila differentially modulate this activation loop switch to elicit predictable changes in PER2 stability, providing a foundation to understand and further manipulate CK1 regulation of circadian rhythms.

Dual sensing of copper ion and chromium (VI) oxyanions by benzotriazole functionalized UiO-66 metal-organic framework in aqueous media

A new dual chemosensor UiO-66-NH-BT (BT=1-methylenebenzotriazole) based on the UiO-66 (University of Oslo) framework, containing benzotriazole functionalized dicarboxylate struts was synthesized and characterized. This isoreticular Metal-Organic Framework (MOF) was found to be a very selective and ultrasensitive for copper ion and chromium oxyanions in aqueous media. It showed a detection limit of 16.9 ppb (0.266 μM) for Cu2+ ion, 280 ppb (1.3 μM) for Cr2O72− and 47.7 ppb (0.411 μM) for CrO42− anions. The quenching constants (Ksv) for Cu2+, Cr2O72−, CrO42− was found to be 1.1x105 M−1, 3.9x103 M−1, and 6.7x103 M−1 respectively. The covalently bonded benzotriazole moiety with the UiO-66 framework not only produces an emission peak at 491 nm but also act as an intrinsic binding site for both cations and anions. The nature of the coordinative interaction between the analytes and the UiO-66-NH-BT has also been elaborated with the help of ICP and FTIR. This chemosensor also demonstrated a regenerative property without the loss in performance for five consecutive cycles.

Imidodiphosphonate Ligands for Enhanced Sensitization and Shielding of Visible and Near-Infrared Lanthanides.

The design of coordination sites around lanthanide ions has a strong impact on the sensitization of their luminescent signal. An imidodiphosphonate anionic binding site is attractive as it can be functionalized with "remote" sensitizer units, such as phenoxy moieties, namely, HtpOp, accompanied by an increased distance of the lanthanide from the ligand high-energy stretching vibrations which quench the luminescence signal, hence providing flexible shielding of the lanthanide. We report the formation and isolation of Ln(tpOp)3 complexes where Ln = Er, Gd, Tb, Dy, Eu, and Yb and the Y(tpOp)3 diamagnetic analogue. The complexes are formed from reaction of KtpOp and the corresponding LnCl3·6H2O salt either by titration and in situ formation or by mixing and isolation. All complexes are seven-coordinated by three tpOp ligand plus one ethanol molecule, except for Yb(tpOp)3 which has no solvent coordinated. Phosphorus NMR shows characteristic shifts to support the coordination of the lanthanide complexes. The complexes display visible and near-infrared luminescence with long lifetimes even for the near-infrared complexes which range from 3.3 μs for Nd(tpOp)3 to 20 μs for Yb(tpOp)3. The ligand shows more efficient sensitization than the imidodiphosphinate analogues for all lanthanide complexes with a notable quantum yield of the Tb(tpOp)3 complex at 45%. We attribute this to the properties of the remote sensitizer unit and its positioning further away from the lanthanide, eliminating quenching of high energy C-H vibrations from the ligand shell. Calculations of the ligand shielding support the photophysical properties of the complexes. These results suggest that these binding sites are promising in the further development of the lanthanide complexes in optoelectronic devices for telecommunications and new light emitting materials.

Role of Fine Structural Dynamics in Recognition of Histone H3 by HP1γ(CSD) Dimer and Ability of Force Fields to Describe Their Interaction Network.

Human heterochromatin protein 1 (HP1) is a key factor in heterochromatin formation and maintenance. Its chromo-shadow domain (CSD) homodimerizes, and the HP1 dimer acts as a hub, transiently interacting with diverse binding partner (BP) proteins. We analyze atomistic details of interactions of the HP1γ(CSD) dimer with one of its targets, the histone H3 N-terminal tail, using molecular dynamics (MD) simulations. The goal is to complement the available X-ray crystallography data and unravel potential dynamic effects in the molecular recognition. Our results suggest that HP1(CSD)-BP recognition involves structural dynamics of both partners, including structural communication between adjacent binding pockets that may fine-tune the sequence recognition. For example, HP1 Trp174 sidechain substates may help in distinguishing residues bound in the conserved HP1(CSD) ±2 hydrophobic pockets. Further, there is intricate competition between the binding of negatively charged HP1 C-terminal extension and solvent anions near the ±2 hydrophobic pockets, which is also influenced by the BP sequence. Phosphorylated H3 Y41 can interact with the same site. We also analyze the ability of several pair-additive force fields to describe the protein-protein interface. ff14SB and ff99SB-ILDN* provide the closest correspondence with the crystallographic model. The ff15ipq local dynamics are somewhat less consistent with details of the experimental structure, while larger perturbations of the interface commonly occur in CHARMM36m simulations. The balance of some interactions, mainly around the anion binding site, also depends on the ion parameters. Some differences between the simulated and experimental structures are attributable to crystal packing.

Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl−/H+ antiporter

The CLC family of proteins are involved in a variety of physiological processes to control cellular chloride concentration. Two distinct classes of CLC proteins, Cl- channels and Cl-/H+ antiporters, have been functionally and structurally investigated over the last several decades. Previous studies have suggested that the conformational heterogeneity of the critical glutamate residue, Gluex, could explain the transport cycle of CLC-type Cl-/H+ antiporters. However, the presence of multiple conformations (Up, Middle, and Down) of the Gluex has been suggested from combined structural snapshots of 2 different CLC antiporters: CLC-ec1 from Escherichia coli and cmCLC from a thermophilic red alga, Cyanidioschyzon merolae Thus, we aimed to investigate further the heterogeneity of Gluex-conformations in CLC-ec1, the most deeply studied CLC antiporter, at both functional and structural levels. Here, we show that the crystal structures of the Gluex mutant E148D and wild-type CLC-ec1 with varying anion concentrations suggest a structural intermediate, the "Midlow" conformation. We also found that an extra anion can be located above the external Cl--binding site in the E148D mutant when the anion concentration is high. Moreover, we observed that a carboxylate in solution can occupy either the external or central Cl--binding site in the ungated E148A mutant using an anomalously detectable short carboxylic acid, bromoacetate. These results lend credibility to the idea that the Gluex can take at least 3 distinct conformational states during the transport cycle of a single CLC antiporter.

New classes of carbazoles as potential multi-functional anti-Alzheimer’s agents

Multi-Target approach is particularly promising way to drug discovery against Alzheimer's disease. In the present study, we synthesized a series of compounds comprising the carbazole backbone linked to the benzyl piperazine, benzyl piperidine, pyridine, quinoline, or isoquinoline moiety through an aliphatic linker and evaluated as cholinesterase inhibitors. The synthesized compounds showed IC50 values of 0.11–36.5 µM and 0.02–98.6 µM against acetyl- and butyrylcholinesterase (AChE and BuChE), respectively. The ligand-protein docking simulations and kinetic studies revealed that compound 3s could bind effectively to the peripheral anionic binding site (PAS) and anionic site of the enzyme with mixed-type inhibition. Compound 3s was the most potent compound against AChE and BuChE and showed acceptable inhibition potency for self- and AChE-induced Aβ1-42 aggregation. Moreover, compound 3s could significantly protect PC12 cells against H2O2-induced toxicity. The results suggested that the compounds 3s could be considered as a promising multi-functional agent for further drug discovery development against Alzheimer's disease.

Cryo-EM structures and functional characterization of murine Slc26a9 reveal mechanism of uncoupled chloride transport.

The epithelial anion transporter SLC26A9 contributes to airway surface hydration and gastric acid production. Colocalizing with CFTR, SLC26A9 has been proposed as a target for the treatment of cystic fibrosis. To provide molecular details of its transport mechanism, we present cryo-EM structures and a functional characterization of murine Slc26a9. These structures define the general architecture of eukaryotic SLC26 family members and reveal an unusual mode of oligomerization which relies predominantly on the cytosolic STAS domain. Our data illustrates conformational transitions of Slc26a9, supporting a rapid alternate-access mechanism which mediates uncoupled chloride transport with negligible bicarbonate or sulfate permeability. The characterization of structure-guided mutants illuminates the properties of the ion transport path, including a selective anion binding site located in the center of a mobile module within the transmembrane domain. This study thus provides a structural foundation for the understanding of the entire SLC26 family and potentially facilitates their therapeutic exploitation.

Degradable dendritic nanogels as carriers for antimicrobial peptides

In the present study, we investigate degradable anionic dendritic nanogels (DNG) as carriers for antimicrobial peptides (AMPs). In such systems, the dendritic part contains carboxylic acid-based anionic binding sites for cationic AMPs, whereas linear poly(ethylene glycol) (PEG) chains form a shell for promotion of biological stealth. In order to clarify factors influencing membrane interactions of such systems, we here address effects of nanogel charge, cross-linking, and degradation on peptide loading/release, as well as consequences of these factors for lipid membrane interactions and antimicrobial effects. The DNGs were found to bind the AMPs LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) and DPK-060 (GKHKNKGKKNGKHNGWKWWW). For the smaller DPK-060 peptide, loading was found to increase with increasing nanogel charge density. For the larger LL-37, on the other hand, peptide loading was largely insensitive to nanogel charge density. In line with this, results on the secondary structure, as well as on the absence of stabilization from proteolytic degradation by the nanogels, show that the larger LL-37 is unable to enter into the interior of the nanogels. While 40–60% nanogel degradation occurred over 10 days, promoted at high ionic strength and lower cross-linking density/higher anionic charge content, peptide release at physiological ionic strength was substantially faster, and membrane destabilization not relying on nanogel degradation. Ellipsometry and liposome leakage experiments showed both free peptide and peptide/DNG complexes to cause membrane destabilization, indicated also by antimicrobial activities being comparable for nanogel-bound and free peptide. Finally, the DNGs were demonstrated to display low toxicity towards erythrocytes even at peptide concentrations of 100 µM.

A perylenebisimide–tetraphenylethene dyad for sensing of phosphate anions

A new perylenebisimide–tetraphenylethene dyad (1) linked by imidazolium/amide anion binding sites at the imide position of the PBI core, has been designed and characterized. The recognition ability of 1 toward anions has been investigated in detail by UV–vis and emission spectroscopy. In CH3CN solution, this probe shows a specific fluorescence quenching response toward H2PO4− (Pi) and HP2O73− (PPi) over other test anions (F−, Cl−, Br−, I−, AcO− and HSO4−). The UV–vis titration, emission titration, 1H NMR titration, Tyndall and DLS experimental results reveal that the binding of Pi or PPi leads to an anion-induced aggregation of the probe with a fluorescence quenching. The probe has been successfully applied to the detection of Pi and PPi in a form of test paper. Furthermore, probe 1 still shows a good fluorescence response toward PPi even in competitive H2O–CH3CN mixtures which can discriminate PPi from Pi and other anions.

An Electrostatic Switch for Gating the Electromechanical Activity of SLC26A5 (prestin)

SLC26 comprises a family of multifunctional anion transporters that mediate passive or coupled transport of small anions. SLC26A5 (prestin) stands out by functioning as a voltage-driven mechanical actuator in sensory outer hair cells. The quasi-piezoelectric function of prestin depends on binding of intracellular anions, suggesting that the underlying dynamics may be derived from an ancestral anion transport mechanism. We previously identified the structural architecture of prestin as a 7 transmembrane domain inverted repeat architecture, a layout shared with SLC4 (e.g., AE1) and SLC23 transporters. To address the molecular mode of action of anions in electromechanical function, we conducted a glutamate scan of the putative anion binding site. While introduction of glutamate at most positions probed was disruptive for protein function, glutamate at a central position (S396E) was tolerated. Moreover, S396E rendered prestin anion-independent and insensitive to competitive anionic blockers. We conclude that S396E reveals location and coordination of substrate anions. Anion binding may enable the voltage-dependent conformational transition by perturbing a local hydrogen or electrostatic network. To gain insights into the conformational dynamics underlying electromechanical activity we performed cysteine accessibility scanning mutagenesis of transmembrane segments 3 and 10, which mainly form the binding site as defined by glutamate substitution. Accessibility was essentially restricted to intracellular reagents. However, a position within TM3 near the substrate binding site was accessible both from the inside and from the outside - contrary to the crystal structure of the bacterial homolog SLC26Dg predicting inaccessibility to extracellular solutes in the inside-open conformation. Two-sided accessibility thus indicates movement of the rigid TM bundle comprising TMs 3 and 10 towards the extracellular side, possibly in an elevator-like movement. We speculate that this partial movement constitutes the conformational transition underlying prestin's electromechanical activity.

CO2 Capture via Crystalline Hydrogen-Bonded Bicarbonate Dimers

Summary Limiting global temperature rises is increasingly dependent on the development of energy-efficient carbon-capture methods. Here, we report a simple CO2-separation cycle using an aqueous bis(iminoguanidine) (BIG) sorbent that reacts with CO2 and crystallizes into an insoluble bicarbonate salt. X-ray diffraction analysis of the bicarbonate crystals revealed “anti-electrostatic” hydrogen-bonded (HCO3−)2 dimers, stabilized by guanidinium cations and water. Mild heating of the crystals releases the CO2 and regenerates the BIG sorbent quantitatively, thereby closing the CO2-separation cycle. Experimental and computational investigations support a CO2-release mechanism consisting of surface-initiated low-barrier proton transfer from guanidinium groups to bicarbonate anions with the formation of carbonic acid dimers, followed by CO2 and H2O release in the rate-limiting step, with a measured activation energy of 102 ± 12 kJ/mol. The minimum energy required for sorbent regeneration is 151.5 kJ/mol CO2, which is 24% lower than the regeneration energy of monoethanolamine, a benchmark industrial sorbent.

New Insights into the Mechanism of NO3- Selectivity in the Human Kidney Chloride Channel ClC-Ka and the CLC Protein Family.

The mechanism of anion selectivity in the human kidney chloride channels ClC-Ka and ClC-Kb is unknown. However, it has been thought to be very similar to that of other channels and antiporters of the CLC protein family, and to rely on anions interacting with a conserved Ser residue (Sercen) at the center of three anion binding sites in the permeation pathway Scen. In both CLC channels and antiporters, mutations of Sercen alter the anion selectivity. Structurally, the side chain of Sercen of CLC channels and antiporters typically projects into the pore and coordinates the anion bound at Scen. To investigate the role of several residues in anion selectivity of ClC-Ka, we created mutations that resulted in amino acid substitutions in these residues. We also used electrophysiologic techniques to assess the properties of the mutants. Mutations in ClC-Ka that change Sercen to Gly, Pro, or Thr have only minor effects on anion selectivity, whereas the mutations in residues Y425A, F519A, and Y520A increase the NO3-/Cl- permeability ratio, with Y425A having a particularly strong effect. s ClC-Ka's mechanism of anion selectivity is largely independent of Sercen, and it is therefore unique in the CLC protein family. We identified the residue Y425 in ClC-Ka-and the corresponding residue (A417) in the chloride channel ClC-0-as residues that contribute to NO3- discrimination in these channels. This work provides important and timely insight into the relationship between structure and function for the kidney chloride channels ClC-Ka and ClC-Kb, and for CLC proteins in general.

Exploiting 1,4-naphthoquinone and 3-iodo-1,4-naphthoquinone motifs as anion binding sites by hydrogen or halogen-bonding interactions.

We describe here the utilization of 1,4-naphthoquinone and 3-iodo-1,4-naphthoquinone motifs as new anion binding sites by hydrogen- or halogen-bonding interactions, respectively. These binding sites have been integrated in bidentate ester based receptors. Emission experiments reveal that both receptors selectively recognize sulfate anions, which induced a remarkable increase of a new emission band attributed to the formation of π-stacking interactions between two 1,4-naphthoquinone units. Absorption spectroscopy and mass spectrometry indicate the disruption of the ester group of the 1,4-naphthoquinone based receptor in the presence of HP2O73-, H2PO4-, F-, AcO- and C6H5CO2- and in the halogenated receptor with HP2O73-, F- and AcO- anions, while the presence of sulfate anions showed the clasical complexation behaviour. The 1H-NMR experiment showed a slow exchange process of the receptors with their sulfate complexes. The binding mode of the receptors with sulfate has been studied by DFT calculations along with the Molecular Electrostatic Potential (MEP) surface computational tool that reveals those parts of the receptors which are more suitable for interacting with anions.

Effects of the H6R and D7H Mutations on the Heparin-Dependent Modulation of Zinc-Induced Aggregation of Amyloid β

Zinc ions and glycosaminoglycans (GAGs) are found in amyloid deposits and are known to modulate the β-amyloid peptide (Aβ) aggregation, which is thought to be a key event in the pathogenesis of Alzheimer's disease (AD). Correlation spectroscopy was used to study how the H6R and D7H mutations of the metal-binding domain (MBD) of Aβ42 affect the modulation of its zinc-induced aggregation by the model GAG heparin. The H6R mutation was shown to decrease and the D7H mutation to increase the Aβ42 propensity to aggregate in the presence of zinc ions. In addition, H6R diminished and D7H enhanced the modulating effect of heparin. The difference in the heparin-dependent modulation was associated with coordination of zinc ions within the MBDs of the mutant peptides. The findings indicate that anion-binding sites formed by complexes of zinc ions with the Aβ MBD play an essential role in the interaction of zinc-induced Aβ aggregates with heparin.