Cation Binding Sites Research Articles

Conformational dynamics and putative substrate extrusion pathways ofthe N-glycosylated outer membrane factor CmeC from campylobacter jejuni.

The outer membrane factor CmeC of the efflux machinery CmeABC plays an important role in conferring antibiotic and bile resistance to Campylobacter jejuni. Curiously, the proteins in this machinery are N-glycosylated, with the glycans playing a key role in the effective function of the efflux system. Here we have employed atomistic equilibrium molecular dynamics simulations of CmeC in a representative model of the C. jejuni outer membrane to characterise the dynamics of the protein and its associated glycans. We show that the N-glycans are more conformationally labile than had previously been thought. The extracellular loops of CmeC visit the open and closed states freely suggesting the absence of a gating mechanism on this side, while the narrow periplasmic entrance remains tightly closed, regulated via coordination to solvated cations. We identify several cation binding sites on the interior surface of the protein. Additionally, we have used steered molecular dynamics simulations to elucidate translocation pathways for a bile acid, chenodeoxycholic acid, and a macrolide antibiotic, erythromycin. These, and additional equilibrium simulations suggest that the anionic bile acid utilizes multivalent cations to climb a ladder of acidic residues that line the interior surface of the protein. In combination, these results further our understanding of the dynamics of outer membrane factors and allow insight into the translocation pathways of antibiotics and other known substrates in this region of the efflux machinery.

Simultaneous visual and spectroscopic multi-analyte detection of Al3+ and AsO2− using simple salicylidene based D-π-A chromophore

The salicylidene-isoniazide Schiff base D-π-A chromophore ( L1 ) exhibits chromogenic and fluorogenic dual responsive behaviour with multiple ICT-PET-ESIPT interplaying sensing mechanism and provides diverse optical signals promising to selectively and simultaneously track multiple analytes in water rich media. • Salicylidene-isoniazide Schiff base ( L1) exhibited naked-eye detectable distinctive colour changes for Al 3+ and AsO 2 - • It displayed turn-on type distinct fluorogenic response for both Al 3+ and AsO 2 - • Multiple ICT-PET-ESIPT interplaying mechanism provided diverse optical signals to simultaneously track multiple analytes. • Applied for fluorescence imaging of Al 3+ and AsO 2 - in living cells. The present communication reports the efficacy of a salicylidene-isoniazide Schiff base based donor-π-acceptor (D-π-A) chemosensor ( L1 ) for chromogenic and fluorogenic dual mode sensing of Al 3+ and AsO 2 - in water rich semi-aqueous solution. L1 offers multiple benefits of simplicity, ease of synthesis, inexpensive, suitable binding sites for cations and anions, high sensitivity, noteworthy colourimetric response, naked-eye detection, selectivity, and good detection limits of 1.89 μM for Al 3+ and 2.2 μM for AsO 2 - . It shows selective turn-on fluorescence response for both Al 3+ and AsO 2 - , attributed to CHEF due to the combined effect of arrest of PET, inhibition of -CH=N- geometrical isomerization, prevention of ESIPT process and increase in conformational rigidity. Owing to specific interaction between Mo(VI) and arsenite, L1 -Mo(VI) ensemble system is successfully utilized for colourimetric detection of arsenite through displacement approach. The binding modes of the receptor L1 to these analytes are established through Job’s plot and 1 H-NMR spectroscopic analysis. The reversible off-on-off fluorescence switching behaviour of L1 upon binding to Al 3+ and AsO 2 - are applied to construct combinational OR, AND, and NOT molecular logic gates. Fluorescence turn-on sensing, cell permeability and low toxicity of the chemosensor allows monitoring of these analytes in living cells.

The mechanism of extracellular polymeric substances in the formation of activated sludge flocs

In this study, activated sludge (AS) presented high aggregation ability of 56.9% to form flocs. To elucidate the effects of extracellular polymeric substances (EPS) on the AS flocs formation, four EPS fractions, including soluble EPS (S-EPS), loosely bound EPS (LB-EPS), ionic tightly bound EPS (ionic TB-EPS) and hydrophobic tightly bound EPS (hydrophobic TB-EPS), were extracted and characterized. The ionic TB-EPS were the largest fraction, accounting for 51.3% of the total EPS. By extracting EPS fractions, it was shown that ionic TB-EPS followed by hydrophobic TB-EPS played positive contribution to the formation of AS flocs, while both S-EPS and LB-EPS had negative effects. Furthermore, chemical reagents (Urea, SDS and EDTA) assays, zeta potential and spectroscopic assays were conducted to explore the role of hydrogen bonds, electrostatic interaction and hydrophobic interaction in the formation of AS flocs. The result showed that ionic TB-EPS contained sufficient cation-binding sites, such as -OH and CO-O- groups, and promoted AS aggregation through ion bridging. Hydrophobic TB-EPS contained hydrophobic groups and promoted AS aggregation through hydrophobic action. A high proportion of uronic acids resulted in high surface negative charge, which might be the reason that LB-EPS and S-EPS inhibited AS aggregation through electrostatic repulsion. From the perspective of molecule structure, the proteins in ionic TB-EPS had a loose structure, which fully exposed the inner cation-binding sites. This might be the critical reason why ion bridging acted as the main force for the formation of AS flocs. This study provided a new sight for the extraction of EPS fractions and expanded the understanding of EPS in the formation of AS flocs.

Conformational dynamics and putative substrate extrusion pathways of the N-glycosylated outer membrane factor CmeC from Campylobacter jejuni.

The outer membrane factor CmeC of the efflux machinery CmeABC plays an important role in conferring antibiotic and bile resistance to Campylobacter jejuni. Curiously, the protein is N-glycosylated, with the glycans playing a key role in the effective function of this system. In this work we have employed atomistic equilibrium molecular dynamics simulations of CmeC in a representative model of the C. jejuni outer membrane to characterise the dynamics of the protein and its associated glycans. We show that the glycans are more conformationally labile than had previously been thought. The extracellular loops of CmeC visit the open and closed states freely suggesting the absence of a gating mechanism on this side, while the narrow periplasmic entrance remains tightly closed, regulated via coordination to solvated cations. We identify several cation binding sites on the interior surface of the protein. Additionally, we used steered molecular dynamics simulations to elucidate translocation pathways for a bile acid and a macrolide antibiotic. These, and additional equilibrium simulations suggest that the anionic bile acid utilises multivalent cations to climb the ladder of acidic residues that line the interior surface of the protein.

New insights into the pathogenicity of TMEM165 variants using structural modeling based on AlphaFold 2 predictions

TMEM165 is a Golgi protein playing a crucial role in Mn2+ transport, and whose mutations in patients are known to cause Congenital Disorders of Glycosylation. Some of those mutations affect the highly-conserved consensus motifs E-φ-G-D-[KR]-[TS] characterizing the CaCA2/UPF0016 family, presumably important for the transport of Mn2+ which is essential for the function of many Golgi glycosylation enzymes. Others, like the G>R304 mutation, are far away from these motifs in the sequence. Until recently, the classical membrane protein topology prediction methods were unable to provide a clear picture of the organization of TMEM165 inside the cell membrane, or to explain in a convincing manner the impact of patient and experimentally-generated mutations on the transporter function of TMEM165. In this study, AlphaFold 2 was used to build a TMEM165 model that was then refined by molecular dynamics simulation with membrane lipids and water. This model provides a realistic picture of the 3D protein scaffold formed from a two-fold repeat of three transmembrane helices/domains where the consensus motifs face each other to form a putative acidic cation-binding site at the cytosolic side of the protein. It sheds new light on the impact of mutations on the transporter function of TMEM165, found in patients and studied experimentally in vitro, formerly and within this study. More particularly and very interestingly, this model explains the impact of the G>R304 mutation on TMEM165’s function. These findings provide great confidence in the predicted TMEM165 model whose structural features are discussed in the study and compared to other structural and functional TMEM165 homologs from the CaCA2/UPF0016 family and the LysE superfamily.

Semicarbazone and thiosemicarbazone appended 4-diethylamino-2‑hydroxybenzyl compounds as highly selective bifunctional chemosensors: An experimental and computational approach

Three bifunctional chemosensors, 2-(4-(diethylamino)-2-hydroxybenzylidene)hydrazinecarboxamide (S1), 2-(4-(diethylamino)-2-hydroxybenzylidene)hydrazinecarbothioamide (S2) and 2-(4-(diethylamino)-2-hydroxybenzylidene)-N-methylhydrazinecarbothioamide (S3) have been designed and synthesized for recognition of inorganic cations and anions in solution at room temperature. The structure of the receptor S2 was confirmed by single crystal X-ray diffraction technique, which crystallizes in the monoclinic P21/n space group. The selectivity and sensitivity of cations (Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+ and Pb2+) and anions (F−, Cl−, Br−, I−, HSO4− and H2PO4−) were investigated using colorimetric, UV–vis absorption, emission and 1H NMR studies. The receptors (S1–S3) with ONO/ONS donors allow bare-face recognition of Co2+, Ni2+ and Cu2+ cations, and F− anion in a mixture of solvent, CH3CN:CH3OH (8:2, v/v). The receptor S3 possesses high sensitivity and selectivity in comparison with the receptors S1 and S2. The binding constant and limit of detection of the receptors with sensed cations and anion showed high sensitivity and selectivity towards Co2+ cation with order of magnitude 6, greater than the other sensed cations. In addition, the receptors S3 also shows 6 order of magnitude for Cu2+ cation and F− anion. The optimized geometry determined by DFT studies shows that the receptors in its conjugated state provide binding sites for cations and F− anion. The HOMO-LUMO energy gap between the receptors and the Co2+ cation was found to be the least among the studied receptors-cations binding interaction, possibly due to charge transfer effect.

Tris(pyridin‐2‐ylmethyl)amine‐Based Ion Pair Receptors for Selective Lithium Salt Recognition

AbstractTripodal ion pair receptors 1 and 2 consisting of tris(pyridine‐2‐ylmethyl)amine as a cation binding site linked with nitrophenyl urea or amidonitroindole groups as anion binding motifs have been synthesized. 1H NMR spectroscopic analyses carried out in 10 % DMSO in acetonitrile revealed that both ion pair receptors 1 and 2 are able to bind the LiCl ion pair with high selectivity over NaCl, KCl, RbCl, and CsCl. 1H NMR spectroscopic analyses in combination with molecular mechanics calculations proved that the lithium cation is bound to the tris(pyridin‐2‐ylmethyl)amine subunit while the chloride anion interacts with the NHs of urea or amido indole groups via hydrogen bonds. The addition of fluoride to the lithium cation complexes of receptors 1 and 2 leads to the release of the lithium cation from the receptors whereas chloride and bromide are co‐bound with lithium forming LiCl and LiBr ion pair complexes, respectively. The affinity of receptor 1 for the lithium cation was found to be improved by 5.0‐fold when the counter anion of Cl− is co‐bound within the receptor.

Enhancing the affinity and adsorption efficiency of Glochidion ericarpum Champ leave extract to cotton for colouristic and functional properties integrating trimethyl chitosan and ultrasonic technique

Recently, sustainable fabrication of hygiene-related textiles combining bioactive substances and cleaner production technique has been increasingly emphasized. This research introduces an efficient ultrasonic-assisted electrostatic co-deposition strategy for colouristic, antibacterial and ultraviolet (UV) protective cotton fabric with trimethyl chitosan (TMC) – a chitosan derivative, and Glochidion ericarpum Champ leave extract (GECLE). Results reveal that TMC significantly promotes the adsorption of GECLE to cotton by creating additional cationic binding sites. Based on Pesudo first-/second-order kinetic models, chemisorption is verified as the dominant mechanism triggered by the electrostatic interaction between TMC and GECLE. Indicated by adsorption rate constant k, the efficiency under ultrasound is enhanced by 13.5 % (60 °C) and 23.8 % (80 °C). Correspondingly, the half adsorption time (t1/2) is shortened by 29.5 % (60 °C) and 37.9 % (80 °C), which approximately saves 16.66 × 103 kJ (60 °C) and 31.31 × 103 kJ (80 °C) of energy. Through mathematical modelling, the factors that influenced the colour depth occured in the following order of importance: Concentration > pH > Temperature. Under optimal condition (GECLE concentration 9.36 g/L, pH 1.63 and temperature 61.70 °C), a theoretical maximum K/S value of 10.25 is achieved. Integrating the analyses above, a triple interactive scheme among TMC, GECLE and cotton fibre is proposed. GECLE (10 g/L) treated cotton deactivates up to 95% of Escherichia coli with 80% retention after 5 repeated launderings, and also provides an ‘Excellent’ rate of UV shielding performance. In general, the ultrasonic-assisted eco-dyeing and finishing process for cotton is explored in depth from practical and theoretical perspectives, which pushes forward the development of sustainable textile industry.

Second Coordination Sphere Effects on the Mechanistic Pathways for Dioxygen Activation by a Ferritin: Involvement of a Tyr Radical and the Identification of a Cation Binding Site.

Ferritins are ubiquitous diiron enzymes involved in iron(II) detoxification and oxidative stress responses and can act as metabolic iron stores. The overall reaction mechanisms of ferritin enzymes are still unclear, particularly concerning the role of the conserved, near catalytic center Tyr residue. Thus, we carried out a computational study of a ferritin using a large cluster model of well over 300 atoms including its first‐ and second‐coordination sphere. The calculations reveal important insight into the structure and reactivity of ferritins. Specifically, the active site Tyr residue delivers a proton and electron in the catalytic cycle prior to iron(II) oxidation. In addition, the calculations highlight a likely cation binding site at Asp65, which through long‐range electrostatic interactions, influences the electronic configuration and charge distributions of the metal center. The results are consistent with experimental observations but reveal novel detail of early mechanistic steps that lead to an unusual mixed‐valent iron(III)‐iron(II) center.

Molecular Basis for the Cation Selectivity of Salmonella typhimurium Melibiose Permease

Cation selectivity and coupling are important attributes of cation-coupled symporters. Salmonella typhimurium melibiose permease (MelBSt) catalyzes the co-transport of galactosides with cations (H+, Li+, or Na+). 3-D crystal structures of MelBSt have revealed the molecular recognition for sugar substrates, but the cation binding and coupling mechanisms have not been defined to atomic levels. In its human homolog MFSD2A, a lethal mutation was mapped at its Na+-binding pocket; however, none of the structures in this subfamily resolved its cation binding. In this study, molecular dynamics simulations reveal the binding interactions of Na+ and Li+ with MelBSt. Interestingly, Thr121, the lethal mutation position in MFSD2A, forms stable interaction with Na+ but is at a distance from Li+. Most mutations among 11 single-site Thr121 mutants of MelBSt exhibited little effects on the galactoside binding, but largely altered the cation selectivity with severe inhibitions on Na+ binding. Few mutants (Pro and Ala) completely lost the Na+ binding and Na+-coupled transport, but their Li+ or H+ modes of activity were largely retained. It can be concluded that Thr121 is necessary for Na+ binding, but not required for the binding of H+ or Li+, so a subset of the Na+-binding pocket is enough for Li+ binding. In addition, the protein stability for some mutants can be only retained in the presence of Li+, but not by Na+ due to the lack of affinity. This finding, together with other identified thermostable mutants, supports that the charge balance of the cation-binding site plays an important role in MelBSt protein stability.

Cryoelectron microscopy of Na+,K+-ATPase in the two E2P states with and without cardiotonic steroids

Cryoelectron microscopy (cryo-EM) was applied to Na+,K+-ATPase (NKA) to determine the structures of two E2P states, one (E2PATP) formed by ATP and Mg2+ in the forward reaction, and the other (E2PPi) formed by inorganic phosphate (Pi) and Mg2+ in the backward reaction, with and without ouabain or istaroxime, representatives of classical and new-generation cardiotonic steroids (CTSs). These two E2P states exhibit different biochemical properties. In particular, K+-sensitive acceleration of the dephosphorylation reaction is not observed with E2PPi, attributed to the presence of a Mg2+ ion in the transmembrane cation binding sites. The cryo-EM structures of NKA demonstrate that the two E2P structures are nearly identical but Mg2+ in the transmembrane binding cavity is identified only in E2PPi, corroborating the idea that it should be denoted as E2PPi·Mg2+. We can now explain why the absence of transmembrane Mg2+ in E2PATP confers the K+ sensitivity in dephosphorylation. In addition, we show that ATP bridges the actuator (A) and nucleotide binding (N) domains, stabilizing the E2PATP state; CTS binding causes hardly any changes in the structure of NKA, both in E2PATP and E2PPi·Mg2+, indicating that the binding mechanism is conformational selection; and istaroxime binds to NKA, extending its aminoalkyloxime group deep into the cation binding site. This orientation is upside down compared to that of classical CTSs with respect to the steroid ring. Notably, mobile parts of NKA are resolved substantially better in the electron microscopy (EM) maps than in previous X-ray structures, including sugars sticking out from the β-subunit and many phospholipid molecules.

Moss biomonitoring using lead isotope ratios requires careful attention: Evaluation of transboundary pollutants in Japan

Transboundary atmospheric pollution has become a serious threat to ecosystems and human health at the global scale. Lead isotope ratios in mosses are used to evaluate the influence of transboundary atmospheric pollutants owing to the traceability of the origin of these pollutants. However, uncertainties for these ratios remain because uptake of trace metals by mosses depends on the surrounding environment and their absorption processes. This study examined how these factors affect lead isotope ratios in mosses and discussed their usefulness as indicators of transboundary pollutants. Moss samples were collected from thirteen sites across Japan. On the continental side, the moss samples in areas with a high amount of winter precipitation exhibited lead isotope ratios similar to those of transboundary pollutants. These results can be explained by enhanced influence of transboundary pollutants on the mosses through winter precipitation bias, given that transboundary pollutants are primarily carried to Japan during winter and that mosses efficiently take up pollutants through wet deposition. The moss samples on Pb-enriched soil absorbed a large quantity of soil-derived Pb, which was reflected in their Pb isotope ratios. Furthermore, the Pb uptake by mosses can be inhibited by other trace metals that compete with Pb for cation binding sites at the cell wall. This inhibition can alter the contribution of atmospheric Pb deposition to the total Pb content in mosses, thereby affecting the Pb isotope ratios. In conclusion, these results validate the usefulness of Pb isotope ratios in mosses as indicators of transboundary pollutants; however, they also imply the need for meticulous interpretation of moss biomonitoring results. The enhanced influence of transboundary pollutants on mosses due to the winter precipitation bias emphasizes the importance of moss biomonitoring for evaluating ecosystem integrity, as it gives insights on actual changes in the Pb cycling in an ecosystem through Pb accumulation by mosses.

Mechanisms underlying TARP modulation of the GluA1/2-\u03b38 AMPA receptor

AMPA-type glutamate receptors (AMPARs) mediate rapid signal transmission at excitatory synapses in the brain. Glutamate binding to the receptor’s ligand-binding domains (LBDs) leads to ion channel activation and desensitization. Gating kinetics shape synaptic transmission and are strongly modulated by transmembrane AMPAR regulatory proteins (TARPs) through currently incompletely resolved mechanisms. Here, electron cryo-microscopy structures of the GluA1/2 TARP-γ8 complex, in both open and desensitized states (at 3.5 Å), reveal state-selective engagement of the LBDs by the large TARP-γ8 loop (‘β1’), elucidating how this TARP stabilizes specific gating states. We further show how TARPs alter channel rectification, by interacting with the pore helix of the selectivity filter. Lastly, we reveal that the Q/R-editing site couples the channel constriction at the filter entrance to the gate, and forms the major cation binding site in the conduction path. Our results provide a mechanistic framework of how TARPs modulate AMPAR gating and conductance.

Is it possible to ”simply” predict the photoejection of a cation? Example of azacrown-substituted [(bpy)Re(CO)[formula omitted]L][formula omitted] complexes

In the context of designing efficient photocontrolled ion-release systems, we propose a ”simple” computational strategy able to predict the cation photoejection. Our strategy is based on TD-DFT calculations and the analysis of the charge transfer (CT) parameters of the excited states that can be populated for a given excitation wavelength, in the Franck–Condon region.With the help of CT descriptors (the variation of the electronic density, the so-called DCT index and the variation of natural population analysis NPA charges), we aim to identify the CT states presenting a depletion of electronic density on the complexation site after excitation. If the loss of electronic density is large enough in the cation binding site, the population of such a state should be the first step of the mechanism leading to the cation photorelease.We test this strategy on ReAZBAP+-Mn+ (Mn+ = Li+, Na+, Mg2+, Ca2＋, Ba2+) and ReAZAT+-Ba2+ systems, a series of complexes synthesized and studied by Moore and co-workers [Lewis et al. (2004)]. We show that our first-order approach that does not take into account the complex excited-state decay routes, is able to correctly predict the photorelease properties with a success rate of ca. 80 percent. This method should thus be considered as a valuable tool in the framework of the in silico design of light-controlled ion release.

A Novel Calix[4]Crown-Based 1,3,4-Oxadiazole as a Fluorescent Chemosensor for Copper(II) Ion Detection.

The synthesis and characterization of a novel florescent chemosensor 1 with two different types of cationic binding sites have been reported in this work, which is a calix[4]crown derivative in 1,3-alternate conformation bearing two 2-phenyl-5-(4-dimethylaminopyenyl)-1,3,4-oxadiazole units. The recognition behaviors of 1 in dichloromethane/acetonitrile solution to alkali metal ions (Na+ and K+), alkaline earth metal ions (Mg2+ and Ca2+), and transition metal ions (Co2+, Ni2+, Zn2+, Cd2+, Cu2+, Mn2+, and Ag+) have been investigated by UV-Vis and fluorescence spectra. The fluorescence of 1 might be quenched selectively by Cu2+ due to the photo-induced electron transfer mechanism, and the quenched emission from 1 could be partly revived by the addition of Ca2+ or Mg2+; thus, the receptor 1 might be worked as an on–off switchable fluorescent chemosensor triggered by metal ion exchange.

Gastric proton pump with two occluded K+ engineered with sodium pump-mimetic mutations

The gastric H+,K+-ATPase mediates electroneutral exchange of 1H+/1K+ per ATP hydrolysed across the membrane. Previous structural analysis of the K+-occluded E2-P transition state of H+,K+-ATPase showed a single bound K+ at cation-binding site II, in marked contrast to the two K+ ions occluded at sites I and II of the closely-related Na+,K+-ATPase which mediates electrogenic 3Na+/2K+ translocation across the membrane. The molecular basis of the different K+ stoichiometry between these K+-counter-transporting pumps is elusive. We show a series of crystal structures and a cryo-EM structure of H+,K+-ATPase mutants with changes in the vicinity of site I, based on the structure of the sodium pump. Our step-wise and tailored construction of the mutants finally gave a two-K+ bound H+,K+-ATPase, achieved by five mutations, including amino acids directly coordinating K+ (Lys791Ser, Glu820Asp), indirectly contributing to cation-binding site formation (Tyr340Asn, Glu936Val), and allosterically stabilizing K+-occluded conformation (Tyr799Trp). This quintuple mutant in the K+-occluded E2-P state unambiguously shows two separate densities at the cation-binding site in its 2.6 Å resolution cryo-EM structure. These results offer new insights into how two closely-related cation pumps specify the number of K+ accommodated at their cation-binding site.

Polyethyleneimine impregnated alginate capsule as a high capacity sorbent for the recovery of monovalent and trivalent gold

For the first time, a polyethyleneimine-impregnated alginate capsule (PEIIAC) with a high adsorption capacity is developed for the recovery of monovalent and trivalent gold from an acidic solution. The strategy results in a new type of adsorbent, polyethyleneimine impregnated alginate capsule (PEIIAC) with a core–shell structure having a large number of amine groups as cationic binding site, facilitating maximum uptake of anionic auric chloride. The maximum uptake of PEIIAC was 3078 and 929 mg/g for Au (III) and Au (I), respectively, are recordable compared to other reported adsorbents to date. The as-prepared material was executed to check the sorption efficacy for Au (III) and Au (I) in the pH range of 1–12. With an increment in pH, the uptake capacity for Au (III) increased, while the uptake capacity for Au (I) decreased. The FTIR, XRD, and XPS studies revealed that the gold adsorption mechanism includes ionic interactions and reduction, wherein the amine, hydroxyl, and carboxyl groups are involved. The capsule showed a higher adsorption efficiency than other reported sorbents, making the material applicable in acidic solutions for the recovery of Au (I) and Au (III).

Computational study for the electrophilic reactivity prediction of crown ethers

Crown ethers are kinds of macrocyclic compounds with high selectivity to metal cations, which can be used as ion sensors, ion analytical reagents and ionophores for biological applications. The chemical reactivity of 1,8-dihydroxyl-4,4,5,5-tetramethylbenzo-14-crown-4 had been investigated at the level of M062X/def2TZVP with Gauss16. The conformations were studied though the high-temperature annealing process with Gromacs. The applicability of Frontier molecular orbital theory, Conceptual density functional theory and Molecular electrostatic potential analysis for the reactivity prediction were discussed. The analysis results show that the methods reflecting the electronic softness property such as Fukui function, global softness, relative nucleophilicity, are suitable for the prediction of electrophilic substitution reaction sites. The electrophilic substitution reaction sites of different conformations are at the C25 or C26 atom, which has the largest HOMO orbital compositions. The oxygen atoms of the main ring and the hydroxyl groups are the binding sites of metal cations. The lowest electrostatic potential is −74.81 kcal/mol on the vdW surface of (1R, 8S) 1,8-dihydroxyl-4,4,5,5-tetramethylbenzo-14-crown-4 contributing to the lone pair electrons and π-electron cloud. The hydroxyl groups and the benzene ring can decrease the surface potential and increase the reactivity.

The accurate prediction and characterization of cancerlectin by a combined machine learning and GO analysis.

Cancerlectins, lectins linked to tumor progression, have become the focus of cancer therapy research for their carbohydrate-binding specificity. However, the specific characterization for cancerlectins involved in tumor progression is still unclear. By taking advantage of the g-gap tripeptide and tetrapeptide composition feature descriptors, we increased the accuracy of the classification model of cancerlectin and lectin to 98.54% and 95.38%, respectively. About 36 cancerlectin and 135 lectin features were selected for functional characterization by P/N feature ranking method, which particularly selects the features in positive samples. The specific protein domains of cancerlectins are found to be p-GalNAc-T, crystal and annexin by comparing with lectins through the exclusion method. Moreover, the combined GO analysis showed that the conserved cation binding sites of cancerlectin specific domains are covered by selected feature peptides, suggesting that the capability of cation binding, critical for enzyme activity and stability, could be the key characteristic of cancerlectins in tumor progression. These results will help to identify potential cancerlectin and provide clues for mechanism study of cancerlectin in tumor progression.

Geometry and water accessibility of the inhibitor binding site of Na+-pump: Pulse- and CW-EPR study

Spin labels based on cinobufagin, a specific inhibitor of the Na,K-ATPase, have proved valuable tools to characterize the binding site of cardiotonic steroids (CTSs), which also constitutes the extracellular cation pathway. Because existing literature suggests variations in the physiological responses caused by binding of different CTSs, we extended the original set of spin-labeled inhibitors to the more potent bufalin derivatives. Positioning of the spin labels within the Na,K-ATPase site was defined and visualized by molecular docking. Although the original cinobufagin labels exhibited lower affinity, continuous-wave electron paramagnetic resonance spectra of spin-labeled bufalins and cinobufagins revealed a high degree of pairwise similarity, implying that these two types of CTS bind in the same way. Further analysis of the spectral lineshapes of bound spin labels was performed with emphasis on their structure (PROXYL vs. TEMPO), as well as length and rigidity of the linkers. For comparable structures, the dynamic flexibility increased in parallel with linker length, with the longest linker placing the spin label at the entrance to the binding site. Temperature-related changes in spectral lineshapes indicate that six-membered nitroxide rings undergo boat-chair transitions, showing that the binding-site cross section can accommodate the accompanying changes in methyl-group orientation. D2O-electron spin echo envelope modulation in pulse-electron paramagnetic resonance measurements revealed high water accessibilities and similar polarity profiles for all bound spin labels, implying that the vestibule leading to steroid-binding site and cation-binding sites is relatively wide and water-filled.