Absence Of Mg2 Research Articles

Mechanistic Insights into Substrate Recognition of Human Nucleoside Diphosphate Kinase C Based on Nucleotide-Induced Structural Changes.

Nucleoside diphosphate kinases (NDPKs) are encoded by nme genes and exist in various isoforms. Based on interactions with other proteins, they are involved in signal transduction, development and pathological processes such as tumorigenesis, metastasis and heart failure. In this study, we report a 1.25 Å resolution structure of human homohexameric NDPK-C bound to ADP and describe the yet unknown complexes formed with GDP, UDP and cAMP, all obtained at a high resolution via X-ray crystallography. Each nucleotide represents a distinct group of mono- or diphosphate purine or pyrimidine bases. We analyzed different NDPK-C nucleotide complexes in the presence and absence of Mg2+ and explain how this ion plays an essential role in NDPKs' phosphotransferase activity. By analyzing a nucleotide-depleted NDPK-C structure, we detected conformational changes upon substrate binding and identify flexible regions in the substrate binding site. A comparison of NDPK-C with other human isoforms revealed a strong similarity in the overall composition with regard to the 3D structure, but significant differences in the charge and hydrophobicity of the isoforms' surfaces. This may play a role in isoform-specific NDPK interactions with ligands and/or important complex partners like other NDPK isoforms, as well as monomeric and heterotrimeric G proteins. Considering the recently discovered role of NDPK-C in different pathologies, these high-resolution structures thus might provide a basis for interaction studies with other proteins or small ligands, like activators or inhibitors.

A metal mediated specific poly(cyclotriphosphazene-co-curcumin) fluorescent micro-probe for tetracycline sensing

The extensive use of tetracycline antibiotics (TCs) renders their widespread in the environment that may cause bacterial resistance. Thus, detection of residual TCs and understanding of their broader roles and impacts in the environment and soil ecology are is of critical significance. This paper describes the investigation of a Mg (II)-specific fluorescent micro-probe (PCC–Mg) based on cyclotriphosphazene (Cpz) and curcumin for classical TC•HCl sensing with a low detection limit of 29 nM and a fast response time of less than 10 s. The sensing mechanism is presumably attributed to specific Mg2+ binding with TCs since the fluorescence enhancement is not observed in the absence of Mg2+ and not influenced by the presence of several metal ions and anions. The binding of Mg2+ to curcumin/TC and the formation of TC-metal-curcumin ternary complex can be concluded with NMR and mass spectrometry. Moreover, for the purpose of easy applications, test strips are made to render visual detection of TC with noticeable and selective fluorescence enhancement. The formation of the ternary complex between Cur from the popular herb Curcuma longa and TC from Streptomyces spp. mediated by metal ions also hints a potential role of microbiolochemical-phytochemical interactions in maintaining biodiversity and equilibria in soil microecology.

Mg2+-dependent mechanism of environmental versatility in a multidrug efflux pump.

Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg2+, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg2+ and pH in modulating the dynamics of the periplasmic adaptor protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug efflux pump from Escherichia coli. In the absence of Mg2+, AcrA becomes increasingly plastic within acidic conditions, but when Mg2+ is bound this is ameliorated, resulting in domain specific organisation in neutral to weakly acidic regimes. We establish a unique histidine residue directs these structural dynamics and is essential for sustaining pump efflux activity across acidic, neutral, and alkaline conditions. Overall, we propose Mg2+ conserves the structural mobility of AcrA to ensure optimal AcrAB-TolC function within rapid changing environments commonly faced by the periplasm during bacterial infection and colonization. This work highlights that Mg2+ is an important mechanistic component in this pump class and possibly across other periplasmic lipoproteins.

Mg2+-promoted high-efficiency DNA conjugation on polydopamine surfaces for aptamer-based ochratoxin A detection

BackgroundSurface immobilization of DNA is the foundation of a broad range of applications in biosensing and specific DNA extraction. Polydopamine (PDA) coatings can serve as intermediate layers to immobilize amino- or thiol-labelled molecules, including DNA, onto various materials through Michael addition and/or Schiff base reactions. However, the conjugation efficiency is limited by electrostatic repulsion between negatively charged DNA and PDA. Recently, it has been reported that polyvalent metal ions (such as Mg2+ and Ca2+) can mediate the adsorption of DNA on PDA surfaces. Inspired by this, in this work we aimed to exploit polyvalent metal ions to facilitate the conjugation of DNA on PDA. ResultsMg2+ was used to promote the conjugation of amino-terminated DNA complementary to ochratoxin A (OTA) aptamer (cDNA-NH2) on PDA-coated magnetic nanoparticles (Fe3O4@PDA). After the reaction, the unlinked cDNA-NH2 adsorbed on Fe3O4@PDA mediated by Mg2+ was removed with EDTA. In the presence of 20 mM Mg2+, the amount of covalently linked cDNA-NH2 increased approximately 11-fold compared to that in the absence of Mg2+. The resulting Fe3O4@PDA@cDNA conjugates exhibited superior hybridization capacity towards OTA aptamers, minimal nonspecific adsorption, and excellent chemical stability. The conjugates combined with fluorophore-labelled aptamers were employed for OTA detection, achieving a limit of detection (LOD) of 2.77 ng mL−1. To demonstrate versatility, this conjugation method was extended to Ca2+-promoted conjugation of cDNA-NH2 on Fe3O4@PDA nanoparticles and Mg2+-promoted conjugation of cDNA-NH2 on PDA-coated 96-well plates. SignificanceThe conjugation efficiency of DNA on PDA was significantly improved with the assistance of polyvalent metal ions (Mg2+ and Ca2+), providing a facile and efficient method for DNA immobilization. Due to the substrate-independent adhesion property of PDA, this method demonstrates versatility in DNA surface modification and holds great potential for applications in target extraction, biosensing, and other fields.

Structure and properties of graphullerene: a semiconducting two-dimensional C60 crystal

Graphullerene is a recently discovered, two-dimensional allotrope of carbon formed from C60 molecules. It has been synthesized in the form (C60Mg4)n and subsequently transformed into (C60)n by removal of the Mg atoms. Ab initio calculations are employed to examine the structure and properties of this material. Structurally, graphullerene is composed of strained C60 molecules. Each of these molecules is connected to six neighbors in a hexagonal network with a total of eight chemical bonds. We find this structure to be meta-stable, owing to the strain produced by the covalent bonding of the molecules. However, the inclusion of Mg atoms transforms the cohesion energy from negative to positive values by forming additional C-Mg bonds, creating an energetically stable material. In the absence of Mg, this allotrope is a pure carbon semiconductor with an indirect band gap. Phonon spectrum calculations confirm the dynamical stability of the material and yield its in- and out-of-plane sound velocities. In addition, symmetry breaking of the C60 molecules results in a distribution of bond lengths and creates vibrational modes that serve as a signature of graphullerene.

Cryo-EM structures of human magnesium channel MRS2 reveal gating and regulatory mechanisms

Magnesium ions (Mg2+) play an essential role in cellular physiology. In mitochondria, protein and ATP synthesis and various metabolic pathways are directly regulated by Mg2+. MRS2, a magnesium channel located in the inner mitochondrial membrane, mediates the influx of Mg2+ into the mitochondrial matrix and regulates Mg2+ homeostasis. Knockdown of MRS2 in human cells leads to reduced uptake of Mg2+ into mitochondria and disruption of the mitochondrial metabolism. Despite the importance of MRS2, the Mg2+ translocation and regulation mechanisms of MRS2 are still unclear. Here, using cryo-EM we report the structures of human MRS2 in the presence and absence of Mg2+ at 2.8 Å and 3.3 Å, respectively. From the homo-pentameric structures, we identify R332 and M336 as major gating residues, which are then tested using mutagenesis and two cellular divalent ion uptake assays. A network of hydrogen bonds is found connecting the gating residue R332 to the soluble domain, potentially regulating the gate. Two Mg2+-binding sites are identified in the MRS2 soluble domain, distinct from the two sites previously reported in CorA, a homolog of MRS2 in prokaryotes. Altogether, this study provides the molecular basis for understanding the Mg2+ translocation and regulatory mechanisms of MRS2.

Formation and transformation of Fe(III)- and Ca-precipitates in aqueous solutions and effects on phosphate retention over time

Exfiltration of anoxic phosphate-rich groundwater into surface water leads to the oxidation of dissolved Fe(II) and the formation of Fe(III)-precipitates that can retain phosphate (PO4) and thereby attenuate eutrophication. Fresh Fe(III)-precipitates transform into more stable phases over time, and retained PO4 may be released again. In parallel, CO2 outgassing can promote the formation of Ca-phosphates or -carbonates that also sequester PO4. In laboratory experiments, we studied the effects of Mg, Ca, silicate (SiO4) and PO4 on these processes. Fresh Fe(III)-precipitates were formed in bicarbonate-buffered aqueous solutions at pH ∼ 7.0 via the oxidation of 0.5 mM Fe(II) in the presence of 0.15 or 0.025 mM PO4, at Mg or Ca concentrations of 0, 0.4, 1.2 or 4 mM and in the absence or presence of 0.5 mM SiO4. After CO2 outgassing, the suspensions were allowed to age for 100 d at pH ∼ 8. Changes in the composition and structure of Fe(III)- and Ca-precipitates over time were probed with spectroscopic and microscopic techniques and were linked to variations in the retention of PO4. The oxidation of Fe(II) led to effective PO4 removal via the formation of Fe(III)-precipitates that consisted of amorphous (Ca-)Fe(III)-phosphate ((Ca)FeP), ferrihydrite (Fh) and, in SiO4-free treatments, lepidocrocite (Lp). During aging, FeP and Fh that had formed in the absence of Mg, Ca and SiO4 rapidly and nearly completely transformed into Lp. Via effects on molecular- and nanoscale precipitate structure, Mg slowed down FeP transformation into Fh, stabilized Fh, and decreased the crystallinity of Lp (in SiO4-free suspensions), Ca stabilized CaFeP against transformation into Fh, and SiO4 stabilized Fh and (Ca)FeP. Core/shell CaFeP/Fh particles formed in electrolytes that contained Ca and SiO4 hardly transformed within 100 d. Calcite only formed at low dissolved PO4 concentrations and, by incorporation of PO4, contributed to PO4 retention. Higher levels of dissolved PO4 inhibited calcite formation but could induce Ca-phosphate precipitation. Differences in precipitate formation and transformation pathways and kinetics were reflected in the extents of PO4 release over the 100-d aging period, ranging from rapid release of 77% of the total PO4 in the treatment without Mg, Ca and SiO4 at 0.15 mM total PO4 to slow release of only 0.1% of the total PO4 at initial concentrations of 4 mM Ca, 0.5 mM SiO4, and 0.025 mM PO4. In summary, this study reveals the conditions and the extents and timescales over which Fe(III)- and Ca-precipitates form and transform and how these processes affect PO4 immobilization in near-neutral natural waters. The detailed new insights into the coupling between Fe(III)- and Ca-precipitate formation and into the interdependent effects of Mg, Ca, SiO4 and PO4 are not only relevant with respect to PO4 but also with respect to the cycling of trace elements in natural and engineered systems.

Facile Metal Release from Pore-Lining Phases Enables Unique Carbonate Zonation in a Basalt Carbon Mineralization Demonstration.

Carbon-negative strategies such as geologic carbon sequestration in continental flood basalts offers a promising route to the removal of greenhouse gases, such as CO2, via safe and permanent storage as stable carbonates. This potential has been successfully demonstrated at a field scale at the Wallula Basalt Carbon Storage Pilot Project where supercritical CO2 was injected into the Columbia River Basalt Group (CRBG). Here, we analyze recovered post-injection sidewall core cross-sections containing carbonate nodules using μ-XRF chemical mapping techniques that revealed compositional zonation within the nodules. The unique nature of the subsurface anthropogenic carbonates is highlighted by the near absence of Mg in an ankerite-like composition. Furthermore, a comparison between pre- and post-injection sidewall cores along with an in-depth chemical mapping of basalt pore lining cements provides a better understanding into the source and fate of critical cationic species involved in the precipitation of carbon mineralization products. Collectively, these results provide crucial insights into carbonate growth mechanisms under a time-dependent pore fluid composition. As such, these findings will enable parameterization of predictive models for future CO2 sequestration efforts in reactive reservoirs around the world.

Combined resistant dextrin and low-dose Mg oxide administration increases short-chain fatty acid and lactic acid production by gut microbiota

The consumption of resistant dextrin improves constipation, while its fermentation and degradation by the intestinal microbiota produce short-chain fatty acids (SCFA) and lactic acid, which have beneficial effects on host metabolism and immunity. Mg oxide (MgO) is an important mineral that is used to treat constipation. Therefore, resistant dextrin and MgO are often administered together to improve constipation. However, limited information is available regarding the effect of this combination on SCFA and lactic acid production. Crl:CD1(ICR) mice were fed a Mg-free diet with 5% resistant dextrin, followed by oral administration of MgO. We collected the cecum contents and measured SCFA and lactic acid levels. Additionally, the human subjects received resistant dextrin and Mg supplements as part of their habitual diet. The results of this study demonstrate that intestinal microbiota cannot promote SCFA and lactic acid production in the absence of Mg. In a mouse model, low doses of MgO promoted the production of SCFA and lactic acid, whereas high doses decreased their production. In humans, the combined consumption of resistant dextrin and Mg supplements increased the production of SCFA and lactic acid. The production of SCFA and lactic acid from dietary fiber may be augmented by the presence of MgO.

Conformation-specific Synthetic Antibodies Discriminate Multiple Functional States of the Ion Channel CorA

CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg2+, and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg2+. Two sABs from these selections, C12 and C18, showed different degrees of Mg2+-sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg2+-depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg2+-depleted conditions. We used X-ray crystallography to determine a structure at 2.0 Å resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg2+] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA.

TRPM7 is Involved in the Regulation of Proliferation, Migration and Osteogenic Differentiation of Human Dental Follicle Cells.

Dental follicle cells (DFCs) are promising candidates for tissue engineering. However, the molecular mechanisms that regulate the biological characteristics of DFCs are still unclear. Transient receptor potential melastatin 7 (TRPM7) is a Ca2+- and Mg2+-permeable cation channel. The aim of this study was to determine the impact of TRPM7 on the proliferation, migration and osteogenic differentiation of DFCs. PCR, Western blotting, Immunocytochemical staining and Patch clamp methods were used to identify the gene and protein expression of TRPM7 in DFCs. DFCs were infected with lentiviruses that expressed either TRPM7 specific shRNA or scrambled non-effective shRNA to investigate its functional role. Cell proliferation and migration were assessed using Cell Counting Kit-8 assays and transwell cell culture chambers separately. Cell osteogenic differentiation were determined by ALP assay kit and Alizarin Red staining. Gene and protein expression of TRPM7 were detected in DFCs, but not of TRPM6, which is a closely related channel with similar function. In the absence of Mg2+, typical whole cell TRPM7-like currents were recorded by patch clamp. These were inhibited by low concentrations of 2-APB, but activated by high concentrations of 2-APB. Functional studies demonstrated that suppression of TRPM7 expression inhibited the proliferation and migration of DFCs, and promoted their osteogenic differentiation. Furthermore, Mg2+ deficiency mimicked the effects of TRPM7 knockdown in terms of osteogenic differentiation of DFCs. These results demonstrate that TRPM7 is involved in regulating the proliferation, migration and osteogenic differentiation of DFCs.

Divergence between Hemichannel and Gap Junction Permeabilities of Connexin 30 and 26.

Cx30 has been proposed to play physiological functions in the kidney and cochlea, and this has often been associated with its hemichannel role (deafness mutants frequently affecting hemichannels more than gap junctions), implicated in ATP release. Here, we used heterologous expression systems (Xenopus oocytes and N2A cells) to describe the properties of Cx30 hemichannels, with the objective of better understanding their physiological functions. As previously observed, Cx30 hemichannels gated in response to transmembrane voltage (V0) and extracellular [Ca2+] (pK[Ca2+] of 1.9 μM in the absence of Mg++). They show minimal charge selectivity with respect to small ions (ratio of Na+: K+: Cl- of 1: 0.4: 0.6) and an MW cut-off for Alexa Dyes between 643 (Alex 488) and 820 Da (Alexa 594). However, while cations follow the expected drop in conductance with size (Na+ to TEA+ is 1: 0.3), anions showed an increase, with a ratio of Cl- to gluconate conductance of 1:1.4, suggesting favorable interactions between larger anions and the pore. This was further explored by comparing the permeabilities of both hemichannels and gap junctions to the natural anion (ATP), the release of which has been implicated in Ca++ signaling through hemichannels. We extended this analysis to two closely related connexins co-expressed in the cochlear, Cx26 and Cx30. Cx30 and 26 hemichannels displayed similar permeabilities to ATP, but surprisingly Cx26 gap junctions were six times more permeable than their hemichannels and four times more permeable than Cx30 gap junctions. This suggests a significant physiological difference in the functions of Cx26 and Cx30 gap junctions in organs where they are co-expressed, at least with regard to the distribution of energy resources of the cells. It also demonstrates that the permeability characteristics of hemichannels can significantly diverge from that of their gap junctions for some connexins but not others.

Conformational changes linked to ADP release from human cardiac myosin bound to actin-tropomyosin.

Following binding to the thin filament, β-cardiac myosin couples ATP-hydrolysis to conformational rearrangements in the myosin motor that drive myofilament sliding and cardiac ventricular contraction. However, key features of the cardiac-specific actin-myosin interaction remain uncertain, including the structural effect of ADP release from myosin, which is rate-limiting during force generation. In fact, ADP release slows under experimental load or in the intact heart due to the afterload, thereby adjusting cardiac muscle power output to meet physiological demands. To further elucidate the structural basis of this fundamental process, we used a combination of cryo-EM reconstruction methodologies to determine structures of the human cardiac actin-myosin-tropomyosin filament complex at better than 3.4 Å-resolution in the presence and in the absence of Mg2+·ADP. Focused refinements of the myosin motor head and its essential light chains in these reconstructions reveal that small changes in the nucleotide-binding site are coupled to significant rigid body movements of the myosin converter domain and a 16-degree lever arm swing. Our structures provide a mechanistic framework to understand the effect of ADP binding and release on human cardiac β-myosin, and offer insights into the force-sensing mechanism displayed by the cardiac myosin motor.

Molecular dynamics simulation study on Bacillus subtilis EngA: the presence of Mg2+ at the active-sites promotes the functionally important conformation

EngA, a GTPase contains two GTP binding domains [GD1, GD2], and the C-terminal KH domain shown to be involved in the later stages of ribosome maturation. Association of EngA to the ribosomal subunit in the intermediate stage of maturation is essential for complete ribosome maturation. However, this association was shown to be dependent on the nucleotide bound combinations. This nucleotide dependent association tendency is attributed to the conformational changes that occur among different nucleotide bound combinations. Therefore, to explore the conformational changes, all-atom molecular dynamics simulations for Bacillus subtilis EngA in different nucleotide bound combinations along with the presence or absence of Mg2+ in the active-sites were carried out. The presence of Mg2+ along with the bound nucleotide at the GD2 active-site dictates the GD2-Sw-II mobility, but the GD1-Sw-II mobility has not shown any nucleotide or Mg2+ dependent movement. However, the GD1-Sw-II secondary conformations are shown to be influenced by the GD2 nucleotide bound state. This allosteric connection between the GD2 active-site and the GD1-Sw-II is also observed through the dynamic network analysis. Further, the exploration of the GD1-KH interface interactions exhibited a more attractive tendency when GD1 is bound to GTP-Mg2+. In addition, the presence of Mg2+ stabilizes active-site water and also increases the distances between the α- and γ- phosphates of the bound GTP. Curiously, three water molecules in the GD1 active-site and only one water molecule in the GD2 active-site are stabilized. This indicates that the probability of GTP hydrolysis is more in GD1 compared to GD2. Communicated by Ramaswamy H. Sarma

Short-Range Cooperative Slow-down of Water Solvation Dynamics Around SO4 2--Mg2+ Ion Pairs.

The presence of ions affects the structure and dynamics of water on a multitude of length and time scales. In this context, pairs of Mg2+ and SO4 2- ions in water constitute a prototypical system for which conflicting pictures of hydration geometries and dynamics have been reported. Key issues are the molecular pair and solvation shell geometries, the spatial range of electric interactions, and their impact on solvation dynamics. Here, we introduce asymmetric SO4 2- stretching vibrations as new and most specific local probes of solvation dynamics that allow to access ion hydration dynamics at the dilute concentration (0.2 M) of a native electrolyte environment. Highly sensitive heterodyne 2D-IR spectroscopy in the fingerprint region of the SO4 2- ions around 1100 cm-1 reveals a specific slow-down of solvation dynamics for hydrated MgSO4 and for Na2SO4 in the presence of Mg2+ ions, which manifests as a retardation of spectral diffusion compared to aqueous Na2SO4 solutions in the absence of Mg2+ ions. Extensive molecular dynamics and density functional theory QM/MM simulations provide a microscopic view of the observed ultrafast dephasing and hydration dynamics. They suggest a molecular picture where the slow-down of hydration dynamics arises from the structural peculiarities of solvent-shared SO4 2--Mg2+ ion pairs.

Theoretical study of the heterometal complexation of Pr(III) with L-isoleucine in the presence/absence of Mg(II) in solution: 4f-4f transition spectra as probe

The Judd-Ofelt intensity Tλ (λ=2, 4, 6), oscillator strength (P) and other energy interaction parameters like Slater Condon (FK), Racah (EK), Lande (ξ4f), nephelauxetic ratio (β), bonding (b1/2), percent covalency (δ) were evaluated to study the mode of interaction of Pr(III) with L-isoleucine in the presence/ absence of Mg(II) in different aquated organic solvents (DMF, dioxane, methanol and acetonitrile) and also at pH levels 2, 4, 6, using the 4f-4f transition spectra as a probe. The variation in values of both intensity and energy interaction parameters could reveal the formation of a bond among Pr(III), L-isoleucine, and Mg(II). Multimetal complexation among Pr(III) and the ligands were found to be most favourable in DMF solvent and at pH 6.

Membrane destabilization and pore formation induced by the Synechocystis IM30 protein

The inner membrane-associated protein of 30 kDa (IM30) is essential in chloroplasts and cyanobacteria. The spatio-temporal cellular localization of the protein appears to be highly dynamic and triggered by internal as well as external stimuli, mainly light intensity. The soluble fraction of the protein is localized in the cyanobacterial cytoplasm or the chloroplast stroma, respectively. Additionally, the protein attaches to the thylakoid membrane as well as to the chloroplast inner envelope or the cyanobacterial cytoplasmic membrane, respectively, especially under conditions of membrane stress. IM30 is involved in thylakoid membrane biogenesis and/or maintenance, where it either stabilizes membranes and/or triggers membrane-fusion processes. These apparently contradicting functions have to be tightly controlled and separated spatiotemporally in chloroplasts and cyanobacteria. IM30’s fusogenic activity depends on Mg2+ binding to IM30; yet, it still is unclear how Mg2+-loaded IM30 interacts with membranes and promotes membrane fusion. Here, we show that the interaction of Mg2+ with IM30 results in increased binding of IM30 to native, as well as model, membranes. Via atomic force microscopy in liquid, IM30-induced bilayer defects were observed in solid-supported bilayers in the presence of Mg2+. These structures differ dramatically from the membrane-stabilizing carpet structures that were previously observed in the absence of Mg2+. Thus, Mg2+-induced alterations of the IM30 structure switch the IM30 activity from a membrane-stabilizing to a membrane-destabilizing function, a crucial step in membrane fusion.

Pharmacological Comparative Characterization of REL-1017 (Esmethadone-HCl) and Other NMDAR Channel Blockers in Human Heterodimeric N-Methyl-D-Aspartate Receptors.

Excessive Ca2+ currents via N-methyl-D-aspartate receptors (NMDARs) have been implicated in many disorders. Uncompetitive NMDAR channel blockers are an emerging class of drugs in clinical use for major depressive disorder (MDD) and other neuropsychiatric diseases. The pharmacological characterization of uncompetitive NMDAR blockers in clinical use may improve our understanding of NMDAR function in physiology and pathology. REL-1017 (esmethadone-HCl), a novel uncompetitive NMDAR channel blocker in Phase 3 trials for the treatment of MDD, was characterized together with dextromethorphan, memantine, (±)-ketamine, and MK-801 in cell lines over-expressing NMDAR subtypes using fluorometric imaging plate reader (FLIPR), automated patch-clamp, and manual patch-clamp electrophysiology. In the absence of Mg2+, NMDAR subtypes NR1-2D were most sensitive to low, sub-μM glutamate concentrations in FLIPR experiments. FLIPR Ca2+ determination demonstrated low μM affinity of REL-1017 at NMDARs with minimal subtype preference. In automated and manual patch-clamp electrophysiological experiments, REL-1017 exhibited preference for the NR1-2D NMDAR subtype in the presence of 1 mM Mg2+ and 1 μM L-glutamate. Tau off and trapping characteristics were similar for (±)-ketamine and REL-1017. Results of radioligand binding assays in rat cortical neurons correlated with the estimated affinities obtained in FLIPR assays and in automated and manual patch-clamp assays. In silico studies of NMDARs in closed and open conformation indicate that REL-1017 has a higher preference for docking and undocking the open-channel conformation compared to ketamine. In conclusion, the pharmacological characteristics of REL-1017 at NMDARs, including relatively low affinity at the NMDAR, NR1-2D subtype preference in the presence of 1 mM Mg2+, tau off and degree of trapping similar to (±)-ketamine, and preferential docking and undocking of the open NMDAR, could all be important variables for understanding the rapid-onset antidepressant effects of REL-1017 without psychotomimetic side effects.

Heterotropic roles of divalent cations in the establishment of allostery and affinity maturation of integrin αXβ2.

SUMMARYAllosteric activation and silencing of leukocyte β2-integrins transpire through cation-dependent structural changes, which mediate integrin biosynthesis and recycling, and are essential to designing leukocyte-specific drugs. Stepwise addition of Mg2+ reveals two mutually coupled events for the αXβ2 ligand-binding domain—the αX I-domain—corresponding to allostery establishment and affinity maturation. Electrostatic alterations in the Mg2+-binding site establish long-range couplings, leading to both pH− and Mg2+-occupancy-dependent biphasic stability change in the αX I-domain fold. The ligand-binding sensorgrams show composite affinity events for the αX I-domain accounting for the multiplicity of the αX I-domain conformational states existing in the solution. On cell surfaces, increasing Mg2+ concentration enhanced adhesiveness of αXβ2. This work highlights how intrinsically flexible pH− and cation-sensitive architecture endows a unique dynamic continuum to the αI-domain structure on the intact integrin, thereby revealing the importance of allostery establishment and affinity maturation in both extracellular and intracellular integrin events.

Self-construction of actin networks through phase separation–induced abLIM1 condensates

The abLIM1 is a nonerythroid actin-binding protein critical for stable plasma membrane-cortex interactions under mechanical tension. Its depletion by RNA interference results in sparse, poorly interconnected cortical actin networks and severe blebbing of migrating cells. Its isoforms, abLIM-L, abLIM-M, and abLIM-S, contain, respectively four, three, and no LIM domains, followed by a C terminus entirely homologous to erythroid cortex protein dematin. How abLIM1 functions, however, remains unclear. Here we show that abLIM1 is a liquid-liquid phase separation (LLPS)-dependent self-organizer of actin networks. Phase-separated condensates of abLIM-S-mimicking ΔLIM or the major isoform abLIM-M nucleated, flew along, and cross-linked together actin filaments (F-actin) to produce unique aster-like radial arrays and interconnected webs of F-actin bundles. Interestingly, ΔLIM condensates facilitated actin nucleation and network formation even in the absence of Mg2+. Our results suggest that abLIM1 functions as an LLPS-dependent actin nucleator and cross-linker and provide insights into how LLPS-induced condensates could self-construct intracellular architectures of high connectivity and plasticity.