Bound Form Research Articles

Role of the redox state of the Pirin-bound cofactor on interaction with the master regulators of inflammation and other pathways.

Persistent cellular stress induced perpetuation and uncontrolled amplification of inflammatory response results in a shift from tissue repair toward collateral damage, significant alterations of tissue functions, and derangements of homeostasis which in turn can lead to a large number of acute and chronic pathological conditions, such as chronic heart failure, atherosclerosis, myocardial infarction, neurodegenerative diseases, diabetes, rheumatoid arthritis, and cancer. Keeping the vital role of balanced inflammation in maintaining tissue integrity in mind, the way to combating inflammatory diseases may be through identification and characterization of mediators of inflammation that can be targeted without hampering normal body function. Pirin (PIR) is a non-heme iron containing protein having two different conformations depending on the oxidation state of the iron. Through exploration of the Pirin interactome and using molecular docking approaches, we identified that the Fe2+-bound Pirin directly interacts with BCL3, NFKBIA, NFIX and SMAD9 with more resemblance to the native binding pose and higher affinity than the Fe3+-bound form. In addition, Pirin appears to have a function in the regulation of inflammation, the transition between the canonical and non-canonical NF-κB pathways, and the remodeling of the actin cytoskeleton. Moreover, Pirin signaling appears to have a critical role in tumor invasion and metastasis, as well as metabolic and neuro-pathological complications. There are regulatory variants in PIR that can influence expression of not only PIR but also other genes, including VEGFD and ACE2. Disparity exists between South Asian and European populations in the frequencies of variant alleles at some of these regulatory loci that may lead to differential occurrence of Pirin-mediated pathogenic conditions.

Buckwheat (Fagopyrum esculentum) Hulls Are a Rich Source of Fermentable Dietary Fibre and Bioactive Phytochemicals.

Pseudo-cereals such as buckwheat (Fagopyrum esculentum) are valid candidates to promote diet biodiversity and nutrition security in an era of global climate change. Buckwheat hulls (BHs) are currently an unexplored source of dietary fibre and bioactive phytochemicals. This study assessed the effects of several bioprocessing treatments (using enzymes, yeast, and combinations of both) on BHs' nutrient and phytochemical content, their digestion and metabolism in vitro (using a gastrointestinal digestion model and mixed microbiota from human faeces). The metabolites were measured using targeted LC-MS/MS and GC analysis and 16S rRNA gene sequencing was used to detect the impact on microbiota composition. BHs are rich in insoluble fibre (31.09 ± 0.22% as non-starch polysaccharides), protocatechuic acid (390.71 ± 31.72 mg/kg), and syringaresinol (125.60 ± 6.76 mg/kg). The bioprocessing treatments significantly increased the extractability of gallic acid, vanillic acid, p-hydroxybenzoic acid, syringic acid, vanillin, syringaldehyde, p-coumaric acid, ferulic acid, caffeic acid, and syringaresinol in the alkaline-labile bound form, suggesting the bioaccessibility of these phytochemicals to the colon. Furthermore, one of the treatments, EC_2 treatment, increased significantly the in vitro upper gastrointestinal release of bioactive phytochemicals, especially for protocatechuic acid (p < 0.01). The BH fibre was fermentable, promoting the formation mainly of propionate and, to a lesser extent, butyrate formation. The EM_1 and EC_2 treatments effectively increased the content of insoluble fibre but had no effect on dietary fibre fermentation (p > 0.05). These findings promote the use of buckwheat hulls as a source of dietary fibre and phytochemicals to help meet dietary recommendations and needs.

Angular-Momentum Transfer Mediated by a Vibronic-Bound-State.

The notion that phonons can carry pseudo-angular momentum has many major consequences, including topologically protected phonon chirality, Berry curvature of phonon band structure, and the phonon Hall effect. When a phonon is resonantly coupled to an orbital state split by its crystal field environment, a so-called vibronic bound state forms. Here, a vibronic bound state is observed in NaYbSe2 , a quantum spin liquid candidate. In addition, field and polarization dependent Raman microscopy is used to probe an angular momentum transfer of ΔJz = ±ℏ between phonons and the crystalline electric field mediated by the vibronic bound stat. This angular momentum transfer between electronic and lattice subsystems provides new pathways for selective optical addressability of phononic angular momentum via electronic ancillary states.

A physiologically based pharmacokinetic model of diethyl phthalates in humans

Phthalates are a family of industrial and consumer product chemicals, among which diethyl phthalate (DEP) has been widely used. DEP is metabolized into the active metabolite monoethyl phthalate (MEP) and exposure to DEP may induce male reproductive toxicity, developmental toxicity and hepatotoxicity. To better assess the toxicity of DEP and MEP, it is important to understand and predict their internal concentrations, especially in reproductive organs. Here we present a human physiologically based pharmacokinetic (PBPK) model of DEP. Implemented in R, the PBPK model consists of seven tissue compartments, including blood, gut, liver, fat, skin, gonad, and rest of body (RB). In the blood both DEP and MEP partition into free and bound forms, and tissue distribution is considered as blood flow-limited. DEP is metabolized in the gut and liver into MEP which is further glucuronidated and cleared through the urine. The chemical-specific parameters of the model were predicted in silico or estimated based on published human urinary MEP data after exposure to DEP in the air at 250 or 300 μg/m3 for 3 or 6 h through inhalation and dermal absorption. Sensitivity analysis identified important parameters including partition coefficients of DEP for fat, RB, and skin compartments, and the rate constants for glucuronidation of MEP and urinary excretion, with regard to Cmax, area under the curve (AUC), and clearance half-lives of DEP and MEP. A subset of the sensitive parameters was then included in hierarchical population Bayesian Markov chain Monte Carlo (MCMC) simulations to characterize the uncertainty and variability of these parameters. The model is consistent with the notion that dermal absorption represents a significant route of exposure to DEP in ambient air and clothing can be an effective barrier. The developed human PBPK model can be utilized upon further refinement as a quantitative tool for DEP risk assessment.

Comparison of fat determination methods and analysis of fatty acids in tea

Comparative analysis of two method for determining fat and analysis of fatty acid content in tea samples. The content of freefatand total fat in tea was determined by Soxhlet extraction method and acid hydrolysis method, and the content of fatty acids were determined by gas chromatography. The composition and content of fatty acids in 21 tea samples from 5 regions were analyzed. The freefat content of tea determined by Soxhlet extraction method was significantly lower than that determined by acid hydrolysis method. The totalfat content in tea determined by acid hydrolysis method was consistent with the total amount of fatty acids determined by gas chromatography, and their content conformed to the logical relationshipsimultaneously. The totalfat content in tea ranged from 0.6 to 4.1 g/100 g, which in green tea, white tea, yellow tea, and black tea were 2.2, 1.8, 1.6 and 0.6 g/100 g, respectively. The content of free fat in tea was less than 58%, with 42%-80% of the fat existing in a bound form. The fatty acids in tea were mainly unsaturated fatty acids, accounting for 67.52%-99.03% of the total fatty acids. There were differences in the composition of fatty acids in different types of tea, with the proportion of unsaturated fatty acids in yellow tea accounting for 98.84% of the total fatty acids, which was significantly higher than that of green tea, white tea, and black tea. The fatty acids with high content in green tea(except Tang chi xiaolan tea, Bawangjian green tea and Liuxi yuye tea)were α-linoleic acid, linoleic acid, and palmitic acid. Theacid hydrolysis method is more suitable for the determination of fat in tea samples. The composition and content of fat and fatty acids in tea vary depending onfactors such as the type of tea and the degree of fermentation.

Concentration of Phenolic Compounds and Phenolic Acids of Various Spelt Cultivars in Response to Growing Years

The aims of this study were to evaluate and compare the radical scavenging activities (DPPH), phenolic concentrations and concentrations of selected phenolic acids (PAs) of spelt cultivars and breeding lines with common wheat in a three-year controlled field experiment under conditions of organic farming. No significant variations were observed in the total and free DPPH of Altgold, Ebners Rotkorn, Ostro and PN-1-36 with common wheat. The total DPPH ranged from 52.13% to the lowest value of 44.01% in Franckenkorn. Total, free and bound phenolic concentrations were the highest for common wheat (1902.55 µg FAE g−1 DM of total), while all spelt cultivars achieved significantly lower values (from 1434.94 µg FAE g−1 DM in Franckenkorn to 1650.22 µg FAE g−1 DM in Ebners Rotkorn). Bound phenolic compounds represented 86.3% of the total ones. An extremely dry and warm ripening period had a negative impact on the synthesis of phenolic compounds. The highest concentration of total PAs was observed in spelt Ebners Rotkorn (681.75 µg g−1 DM) and the lowest in common wheat (396.05 µg g−1 DM). The total share of free and bound PAs was 5.7% and 74.8%, respectively. The extremely dry and very warm grain filling period had a more evident negative impact on the concentration of free PAs compared to bound forms. The dominant free PA was ferulic (70.48%), followed by syringic (9.30%), p-HBA (5.59%), sinapic acid (5.40%), salicylic (4.18%), p-coumaric acid (3.22%) and caffeic acid (1.93%). Spelt cultivar Ebners Rotkorn was distinguished by the highest concentration of free and bound forms of PAs.

Metabolomic and transcriptomic integrated analysis revealed the decrease of monoterpenes accumulation in table grapes during long time low temperature storage

Low temperature is the commonly used technique for maintaining the quality of table grapes during postharvest storage. However, this technique could strongly affect the aromatic flavor of fruit. Monoterpenes are the key compounds contributing to the Muscat aromas of grapes. The detailed information and molecular mechanisms underlying the changes in monoterpenes during postharvest low temperature storage have not been thoroughly characterized. In this study, the effects of low temperature storage on the free and bound monoterpene profiles in four cultivars of table grape were determined at both the transcriptomic and metabolomic levels. A total of 27 compounds in both free and bound forms were identified in the four cultivars and showed quantitative differences between the cultivars. Hierarchical cluster and principal component analysis indicated that the free and bound monoterpene profiles were remarkably affected by the low temperature storage. The monoterpenes in the same biosynthesis pathway were clustered together and showed similar evolution trends during low temperature storage. And the content of most of free monoterpenes underwent a rapid decline during low-temperature storage at a certain stage, but the time was different in 4 grape cultivars. Transcriptomic analysis revealed that the expression of DXS, HDR, GPPS and TPS genes involved in the monoterpene synthesis pathway were consistent with the changes in the accumulation of monoterpene compounds. While the expression of HMGS, HMGR genes in MVA pathway and branch genes GGPPS and FPPS were negatively correlated with the accumulation of monoterpenes. The findings provide new insights into the underlying mechanisms of the berry aroma flavor change during low temperature storage.

Influence of sprouting on phenolic acids, carotenoids, and antioxidant activity of millet varieties

The kernels of two millet varieties (foxtail and red proso) were germinated for 24, 48, and 72 h at 30 °C. Changes in free and bound forms of phenolic acids, lutein/zeaxanthin, and antioxidant activity were evaluated in wholegrain flours from the germinated seeds. Lutein/zeaxanthin increased from 4.08 to 6.65 μg/g in foxtail and from 1.62 to 4.85 μg/g in red proso millets. The two millets significantly scavenged ABTS free radicals, increasing from 351.47 (0 h) to 521.58 μg AAE/g (72 h) in foxtail and from 288.12 (0 h) to 692.46 μg AAE/g (72 h) in red proso. A similar trend was observed in the millets regarding the DPPH assay, in which DPPH free radicals were scavenged from 140.69 (0 h) to 314.25 μg AAE/g (72 h) and from 290.59 to 432.05 μg AAE/g, respectively. Free ferulic acid content was higher in germinated flour for 24 h (1.57 mg/kg dry weight basis) than in ungerminated grains (0.05 mg/kg). The concentrations of bound caffeic acid increased with germination time, from 12.78 mg/kg (0 h) to 31.2 mg/kg in germinated flour for 24 h. The foxtail millet had significant changes in phenolic acid content during germination. This study may provide insights into utilizing different millet varieties and their sprouted flours in food industry.

Free Energy Landscapes of the Tautomeric Interconversion of Pyridoxal 5'-Phosphate Aldimines at the Active Site of Ornithine Decarboxylase in Aqueous Media.

The pyridoxal 5'-phosphate (PLP) acts as a coenzyme for a large number of biochemical reactions. It exists in mainly two bound forms at the active site of the concerned enzyme: the internal aldimine, in which the PLP is bound with the ϵ-amino group of lysine at the active site, and the external aldimine, where the PLP is bound to the substrate amino acid. Both the internal and external aldimines have Schiff base linkage (N-H-O) and can exist in two tautomeric structures of ketoenamine and enolimine forms. In this work, we have investigated the free energy landscape for the tautomeric proton transfer in the internal and external aldimines at the active site of the ornithine decarboxylase enzyme in an aqueous medium. We performed hybrid quantum-classical metadynamics and force field-based molecular dynamics simulations, which revealed that the ketoenamine tautomer is more stable than the enolimine form. The QM/MM metadynamics calculations show that the free energy difference between the ketoenamine and enolimine forms for the internal aldimine is 3.9 kcal/mol, and it is found to be 5.8 kcal/mol for the external aldimine, with the ketoenamine form being more stable in both cases. The results are further supported by calculations of the binding free energies from classical simulations and static quantum chemical calculations in different environments. We have also analyzed the configurational structure of the microenvironment at the active site in order to have better insights into the interactions of the active site residues with the PLP in its two tautomeric forms.

Biomedical engineering approaches for the delivery of JAGGED1 as a potential tissue regenerative therapy.

JAG1 is a ligand that activates the NOTCH signaling pathway which plays a crucial role in determining cell fate behavior through cell-to-cell signaling. JAG1-NOTCH signaling is required for mesenchymal stem cell (MSC) differentiation into cardiomyocytes and cranial neural crest (CNC) cells differentiation into osteoblasts, making it a regenerative candidate for clinical therapy to treat craniofacial bone loss and myocardial infarction. However, delivery of soluble JAG1 has been found to inhibit NOTCH signaling due to the requirement of JAG1 presentation in a bound form. For JAG1-NOTCH signaling to occur, JAG1 must be immobilized within a scaffold and the correct orientation between the NOTCH receptor and JAG1 must be achieved. The lack of clinically translatable JAG1 delivery methods has driven the exploration of alternative immobilization approaches. This review discusses the role of JAG1 in disease, the clinical role of JAG1 as a treatment, and summarizes current approaches for JAG1 delivery. An in-depth review was conducted on literature that used both in vivo and in vitro delivery models and observed the canonical versus non-canonical NOTCH pathway activated by JAG1. Studies were then compared and evaluated based on delivery success, functional outcomes, and translatability. Delivering JAG1 to harness its ability to control cell fate has the potential to serve as a therapeutic for many diseases.

Bound form terpenes in sweet potatoes and their distribution in flesh and peel of different cultivars

SummarySix prevalent sweet potato cultivars, New Orleans, Beauregard, Japanese, Hannah, Garnet and Jewel, were selected to determine the distribution of terpenes in their flesh and peel. Free‐form terpenes were not detected in both flesh and peel in all the cultivars by using a solid phase microextraction (SPME) and gas chromatography–mass spectrometer (GC–MS) method. Major terpenes in the sweet potatoes were linalool and α‐terpineol and existed in bound form in flesh and peel. They were released as free‐form and determined after acid hydrolysis treatment. However, ultrasonication‐assisted acid hydrolysis decreased the levels of terpenes with increased treatment time because the broken‐down free‐form terpenes were volatile and rapidly evaporated or oxidised. For the selected cultivars, the Garnet, Jewel and Japanese cultivars had the greatest levels of linalool, while the Beauregard and New Orleans cultivars had the greatest level of α‐terpineol. Also, the concentration of linalool or α‐terpineol in the peel of each cultivar was approximately two times greater than in its flesh. This study revealed the bound form terpenes and their distribution in sweet potatoes. The results explored the potential utilisation of sweet potato peel as a natural source of terpenes.

Sulfation Effects of Chondroitin Sulfate to Bind a Chemokine in Aqueous Medium: Conformational Heterogeneity and Dynamics from Molecular Simulation.

The sulfation patterns and degree of sulfation of chondroitin sulfate (CS), an important class of glycosaminoglycans (GAG), and their interactions with chemokines are accountable for various diseases. To realize the underlying mechanism of such complex biological phenomena at a molecular level and their application in rational drug design, a study on conformations and dynamics of CSs is necessary. To explore this, in this study, we performed a series of atomistic molecular dynamics (MD) simulations with different sulfated variants of octadecasaccharide CS, like CS-C, CS-E, and CS-T, in their free forms and when bound to the protein chemokine CXCL8 dimer in an aqueous medium. The calculated binding free energy of CSs with the CXCL8 dimer is favorable, and the degree of sulfation favors the complexation process further with prominent hydrophobic and hydrogen-bonded interactions. We find that the recognition is associated with the configurational entropy loss of the CS molecules as calculated from the Gaussian mixture approach, which supports that the degree of sulfation regulates the process. Cluster analysis through the k-means algorithm and end-to-end distance measurement revealed that although the free CS molecules adopted linear conformations, the nonlinear conformations during binding with protein were noted. Adaptation of nonlinear forms in the bound forms is noteworthy for the less-sulfated CS-C and CS-E. Apart from favorable 4C1 conformations, the occasional appearance of skew-boat forms from the free-energy map of ring pucker for the GlcUA unit was observed, which remains unaffected by the sulfation. We find that during recognition, the average relaxation time of intra-CS and inter-CS-CXCL8 hydrogen bonds (HBs) is about a magnitude lesser than that of CS-water HBs, most prominent on the involvement of higher sulfated CS-T analogues. The translational motion of surrounded water molecules in CSs exhibited sublinear diffusion, and the degree of sublinearity increases around the heavily sulfated molecules due to the hindrance created by them as well as the presence of the chemokine and exhibited markedly slow heterogeneous diffusion.

Development and validation of a method based on liquid chromatography-mass spectrometry for comprehensive profiling of phenolic compounds in rice

The consumption of rice grains containing phytochemicals associated with antioxidant activity can potentially reduce the risk of chronic diseases. These phytochemicals, which include phenolic acids, anthoxanthins, and anthocyanins, are present in both free and bound forms in rice grains. However, there is limited documentation on the development of a method for determining the simultaneous amount of both free and bound phenolic compounds using sensitive analytical techniques. To address this gap, a study was conducted to develop and validate a liquid chromatography-mass spectrometric detection method for quantifying eighteen phenolic compounds in rice grains, both in free and bound forms. Paracetamol was used as the internal standard to minimize the effect of matrix interferences. The method was validated by measuring its working range, linearity, accuracy, precision, limits for detection and quantification, robustness, and expanded uncertainty. The method has a wide working range of 1.0 to 30.0 mg/kg for each phenolic compound and good linearity, with regression coefficients of calibration graphs greater than 0.999. Accuracy was determined using the recoveries after fortification at three concentration levels, which were in the range of 83.1 to 101.2%. Precision was expressed as a percentage of relative standard deviation (%RSD), which was below 7.62% for all determined phenolic compounds. The detection limits (LoD) and quantification limits (LoQ) ranged between 0.53 and 0.75 mg/kg and 0.54–0.77 mg/kg, respectively. The percentages of expanded uncertainties at the coverage factor of 2 (k = 2) were below 11.7%. The method was also evaluated for its robustness by obtaining recoveries at satisfactory levels for three different kinds of cereal; wheat, maize, and millet. The validated method is an accurate and reproducible approach for the simultaneous determination of phenolic compounds present at low levels in a complex matrix such as rice. The study also found that the phenolic profiles vary among rice genotypes, which can significantly impact human health by varying the nutraceutical properties of rice grains as health-promoting aspects.

Iron Load Toxicity in Medicine: From Molecular and Cellular Aspects to Clinical Implications.

Iron is essential for all organisms and cells. Diseases of iron imbalance affect billions of patients, including those with iron overload and other forms of iron toxicity. Excess iron load is an adverse prognostic factor for all diseases and can cause serious organ damage and fatalities following chronic red blood cell transfusions in patients of many conditions, including hemoglobinopathies, myelodyspasia, and hematopoietic stem cell transplantation. Similar toxicity of excess body iron load but at a slower rate of disease progression is found in idiopathic haemochromatosis patients. Excess iron deposition in different regions of the brain with suspected toxicity has been identified by MRI T2* and similar methods in many neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Based on its role as the major biological catalyst of free radical reactions and the Fenton reaction, iron has also been implicated in all diseases associated with free radical pathology and tissue damage. Furthermore, the recent discovery of ferroptosis, which is a cell death program based on free radical generation by iron and cell membrane lipid oxidation, sparked thousands of investigations and the association of iron with cardiac, kidney, liver, and many other diseases, including cancer and infections. The toxicity implications of iron in a labile, non-protein bound form and its complexes with dietary molecules such as vitamin C and drugs such as doxorubicin and other xenobiotic molecules in relation to carcinogenesis and other forms of toxicity are also discussed. In each case and form of iron toxicity, the mechanistic insights, diagnostic criteria, and molecular interactions are essential for the design of new and effective therapeutic interventions and of future targeted therapeutic strategies. In particular, this approach has been successful for the treatment of most iron loading conditions and especially for the transition of thalassemia from a fatal to a chronic disease due to new therapeutic protocols resulting in the complete elimination of iron overload and of iron toxicity.

Solution structure of the HOIL-1L NZF domain reveals a conformational switch regulating linear ubiquitin affinity

Attachment of polyubiquitin (poly-Ub) chains to proteins is a major posttranslational modification in eukaryotes. Linear ubiquitin chain assembly complex, consisting of HOIP (HOIL-1-interacting protein), HOIL-1L (heme-oxidized IRP2 Ub ligase 1), and SHARPIN (Shank-associated RH domain-interacting protein), specifically synthesizes "head-to-tail" poly-Ub chains, which are linkedviathe N-terminal methionine α-amino and C-terminal carboxylate of adjacent Ub units and are thus commonly called "linear" poly-Ub chains. Linear ubiquitin chain assembly complex-assembled linear poly-Ub chains play key roles in immune signaling and suppression of cell death and have been associated with immune diseases and cancer; HOIL-1L is one of the proteins known to selectively bind linear poly-Ubviaits Npl4 zinc finger (NZF) domain. Although the structure of the bound form of the HOIL-1L NZF domain with linear di-Ub is known, several aspects of the recognition specificity remain unexplained. Here, we show using NMR and orthogonal biophysical methods, how the NZF domain evolves from a free to the specific linear di-Ub-bound state while rejecting other potential Ub species after weak initial binding. The solution structure of the free NZF domain revealed changes in conformational stability upon linear Ub binding, and interactions between the NZF core and tail revealed conserved electrostatic contacts, which were sensitive to charge modulation at a reported phosphorylation site: threonine-207. Phosphomimetic mutations reduced linear Ub affinity by weakening the integrity of the linear di-Ub-bound conformation. The described molecular determinants of linear di-Ub binding provide insight into the dynamic aspects of the Ub code and the NZF domain's role in full-length HOIL-1L.

New Crystal Form of Human Neuropilin-1 b1 Fragment with Six Electrostatic Mutations Complexed with KDKPPR Peptide Ligand.

Neuropilin 1 (NRP1), a cell-surface co-receptor of a number of growth factors and other signaling molecules, has long been the focus of attention due to its association with the development and the progression of several types of cancer. For example, the KDKPPR peptide has recently been combined with a photosensitizer and a contrast agent to bind NRP1 for the detection and treatment by photodynamic therapy of glioblastoma, an aggressive brain cancer. The main therapeutic target is a pocket of the fragment b1 of NRP1 (NRP1-b1), in which vascular endothelial growth factors (VEGFs) bind. In the crystal packing of native human NRP1-b1, the VEGF-binding site is obstructed by a crystallographic symmetry neighbor protein, which prevents the binding of ligands. Six charged amino acids located at the protein surface were mutated to allow the protein to form a new crystal packing. The structure of the mutated fragment b1 complexed with the KDKPPR peptide was determined by X-ray crystallography. The variant crystallized in a new crystal form with the VEGF-binding cleft exposed to the solvent and, as expected, filled by the C-terminal moiety of the peptide. The atomic interactions were analyzed using new approaches based on a multipolar electron density model. Among other things, these methods indicated the role played by Asp320 and Glu348 in the electrostatic steering of the ligand in its binding site. Molecular dynamics simulations were carried out to further analyze the peptide binding and motion of the wild-type and mutant proteins. The simulations revealed that specific loops interacting with the peptide exhibited mobility in both the unbound and bound forms.

Fluctuations of formin binding in the generation of membrane patterns

Circular actin waves that propagate on the substrate-attached membrane of Dictyostelium cells separate two distinct membrane domains from each other: an inner territory rich in phosphatidyl-(3,4,5) trisphosphate (PIP3) and an external area decorated with the PIP3-degrading 3-phosphatase PTEN. During wave propagation, the inner territory increases at the expense of the external area. Beyond a size limit, the inner territory becomes unstable, breaking into an inner and an external domain. The sharp boundary between these domains is demarcated by the insertion of an actin wave. During the conversion of inner territory to external area, the state of the membrane fluctuates, as visualized by dynamic landscapes of formin B binding. Here we analyze the formin B fluctuations in relation to three markers of the membrane state: activated Ras, PIP3, and PTEN.

Crystal structures of Na+ ,K+ -ATPase reveal the mechanism that converts the K+ -bound form to Na+ -bound form and opens and closes the cytoplasmic gate.

Na+ ,K+ -ATPase (NKA) plays a pivotal role in establishing electrochemical gradients for Na+ and K+ across the cell membrane by alternating between the E1 (showing high affinity for Na+ and low affinity for K+ ) and E2 (low affinity to Na+ and high affinity to K+ ) forms. Presented here are two crystal structures of NKA in E1·Mg2+ and E1·3Na+ states at 2.9 and 2.8 Å resolution, respectively. These two E1 structures fill a gap in our description of the NKA reaction cycle based on the atomic structures. We describe how NKA converts the K+ -bound E2·2K+ form to an E1 (E1·Mg2+ ) form, which allows high-affinity Na+ binding, eventually closing the cytoplasmic gate (in E1 ~ P·ADP·3Na+ ) after binding three Na+ , while keeping the extracellular ion pathway sealed. We now understand previously unknown functional roles for several parts of NKA and that NKA uses even the lipid bilayer for gating the ion pathway.

Bacterioferritin nanocage structures uncover the biomineralization process in ferritins.

Iron is an essential element involved in various metabolic processes. The ferritin family of proteins forms nanocage assembly and is involved in iron oxidation, storage, and mineralization. Although several structures of human ferritins and bacterioferritins have been solved, there is still no complete structure that shows both the trapped Fe-biomineral cluster and the nanocage. Furthermore, whereas the mechanism of iron trafficking has been explained using various approaches, structural details on the biomineralization process (i.e. the formation of the mineral itself) are generally lacking. Here, we report the cryo-electron microscopy (cryo-EM) structures of apoform and biomineral bound form (holoforms) of the Streptomyces coelicolor bacterioferritin (ScBfr) nanocage and the subunit crystal structure. The holoforms show different stages of Fe-biomineral accumulation inside the nanocage, in which the connections exist in two of the fourfold channels of the nanocage between the C-terminal of the ScBfr monomers and the Fe-biomineral cluster. The mutation and truncation of the bacterioferritin residues involved in these connections significantly reduced the iron and phosphate binding in comparison with those of the wild type and together explain the underlying mechanism. Collectively, our results represent a prototype for the bacterioferritin nanocage, which reveals insight into its biomineralization and the potential channel for bacterioferritin-associated iron trafficking.

The battle for silver binding: How the interplay between the SilE, SilF, and SilB proteins contributes to the silver efflux pump mechanism

The resistance of Gram-negative bacteria to silver ions is mediated by a silver efflux pump, which mainly relies on a tripartite efflux complex SilCBA, a metallochaperone SilF and an intrinsically disordered protein SilE. However, the precise mechanism by which silver ions are extruded from the cell, and the different roles of SilB, SilF and SilE remain poorly understood. To address these questions, we employed Nuclear Magnetic Resonance and Mass Spectrometry to investigate the interplay between these proteins. We first solved the solution structures of SilF in its free and Ag+-bound forms and we demonstrated that SilB exhibits two silver binding sites in its N- and C-termini. Conversely to the homologous Cus system, we determined that SilF and SilB interact without the presence of silver ions and that the rate of silver dissociation is eight times faster when SilF is bound to SilB, indicating the formation of a SilF-Ag-SilB intermediate complex. Finally, we have shown that SilE does not bind to either SilF or SilB, regardless of the presence or absence of silver ions, further corroborating that it merely acts as a regulator that prevents the cell from being overloaded with silver. Collectively, we have provided further insight into protein interactions within the sil system that contribute to bacterial resistance to silver ions.