Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9.

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.

Ion channels in innate and adaptive immunity.

Ion channels and transporters mediate the transport of charged ions across hydrophobic lipid membranes. In immune cells, divalent cations such as calcium, magnesium, and zinc have important roles as second messengers to regulate intracellular signaling pathways. By contrast, monovalent cations such as sodium and potassium mainly regulate the membrane potential, which indirectly controls the influx of calcium and immune cell signaling. Studies investigating human patients with mutations in ion channels and transporters, analysis of gene-targeted mice, or pharmacological experiments with ion channel inhibitors have revealed important roles of ionic signals in lymphocyte development and in innate and adaptive immune responses. We here review the mechanisms underlying the function of ion channels and transporters in lymphocytes and innate immune cells and discuss their roles in lymphocyte development, adaptive and innate immune responses, and autoimmunity, as well as recent efforts to develop pharmacological inhibitors of ion channels for immunomodulatory therapy.

Magnesium sensing via LFA-1 regulates CD8+ T cell effector function.

The relevance of extracellular magnesium in cellular immunity remains largely unknown. Here, we show that the co-stimulatory cell-surface molecule LFA-1 requires magnesium to adopt its active conformation on CD8+ T cells, thereby augmenting calcium flux, signal transduction, metabolic reprogramming, immune synapse formation, and, as a consequence, specific cytotoxicity. Accordingly, magnesium-sufficiency sensed via LFA-1 translated to the superior performance of pathogen- and tumor-specific T cells, enhanced effectiveness of bi-specific T cell engaging antibodies, and improved CAR T cell function. Clinically, low serum magnesium levels were associated with more rapid disease progression and shorter overall survival in CAR T cell and immune checkpoint antibody-treated patients. LFA-1 thus directly incorporates information on the composition of the microenvironment as a determinant of outside-in signaling activity. These findings conceptually link co-stimulation and nutrient sensing and point to the magnesium-LFA-1 axis as a therapeutically amenable biologic system.

Lactate Elicits ER-Mitochondrial Mg2+ Dynamics to Integrate Cellular Metabolism.

Mg2+ is the most abundant divalent cation in metazoans and an essential cofactor for ATP, nucleic acids, and countless metabolic enzymes. To understand how the spatio-temporal dynamics of intracellular Mg2+ (iMg2+) are integrated into cellular signaling, we implemented a comprehensive screen to discover regulators of iMg2+ dynamics. Lactate emerged as an activator of rapid release of Mg2+ from endoplasmic reticulum (ER) stores, which facilitates mitochondrial Mg2+ (mMg2+) uptake in multiple cell types. We demonstrate that this process is remarkably temperature sensitive and mediated through intracellular but not extracellular signals. The ER-mitochondrial Mg2+ dynamics is selectively stimulated by L-lactate. Further, we show that lactate-mediated mMg2+ entry is facilitated by Mrs2, and point mutations in the intermembrane space loop limits mMg2+ uptake. Intriguingly, suppression of mMg2+ surge alleviates inflammation-induced multi-organ failure. Together, these findings reveal that lactate mobilizes iMg2+ and links the mMg2+ transport machinery with major metabolic feedback circuits and mitochondrial bioenergetics.

Magnesium Flux Modulates Ribosomes to Increase Bacterial Survival.

Bacteria exhibit cell-to-cell variability in their resilience to stress, for example, following antibiotic exposure. Higher resilience is typically ascribed to "dormant" non-growing cellular states. Here, by measuring membrane potential dynamics of Bacillus subtilis cells, we show that actively growing bacteria can cope with ribosome-targeting antibiotics through an alternative mechanism based on ion flux modulation. Specifically, we observed two types of cellular behavior: growth-defective cells exhibited a mathematically predicted transient increase in membrane potential (hyperpolarization), followed by cell death, whereas growing cells lacked hyperpolarization events and showed elevated survival. Using structural perturbations of the ribosome and proteomic analysis, we uncovered that stress resilience arises from magnesium influx, which prevents hyperpolarization. Thus, ion flux modulation provides a distinct mechanism to cope with ribosomal stress. These results suggest new approaches to increase the effectiveness of ribosome-targeting antibiotics and reveal an intriguing connection between ribosomes and the membrane potential, two fundamental properties of cells.

ERMA (TMEM94) is a P-type ATPase transporter for Mg2+ uptake in the endoplasmic reticulum.

Intracellular Mg2+ (iMg2+) is bound with phosphometabolites, nucleic acids, and proteins in eukaryotes. Little is known about the intracellular compartmentalization and molecular details of Mg2+ transport into/from cellular organelles such as the endoplasmic reticulum (ER). We found that the ER is a major iMg2+ compartment refilled by a largely uncharacterized ER-localized protein, TMEM94. Conventional and AlphaFold2 predictions suggest that ERMA (TMEM94) is a multi-pass transmembrane protein with large cytosolic headpiece actuator, nucleotide, and phosphorylation domains, analogous to P-type ATPases. However, ERMA uniquely combines a P-type ATPase domain and a GMN motif for ERMg2+ uptake. Experiments reveal that a tyrosine residue is crucial for Mg2+ binding and activity in a mechanism conserved in both prokaryotic (mgtB and mgtA) and eukaryotic Mg2+ ATPases. Cardiac dysfunction by haploinsufficiency, abnormal Ca2+ cycling in mouse Erma+/- cardiomyocytes, and ERMA mRNA silencing in human iPSC-cardiomyocytes collectively define ERMA as an essential component of ERMg2+ uptake in eukaryotes.

Structural basis of transcription: RNA polymerase II substrate binding and metal coordination using a free-electron laser.

Catalysis and translocation of multisubunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near-atomic resolution and precise arrangement of key active site components have been elusive. Here, we present the free-electron laser (FEL) structures of a matched ATP-bound Pol II and the hyperactive Rpb1 T834P bridge helix (BH) mutant at the highest resolution to date. The radiation-damage-free FEL structures reveal the full active site interaction network, including the trigger loop (TL) in the closed conformation, bonafide occupancy of both site A and B Mg2+, and, more importantly, a putative third (site C) Mg2+ analogous to that described for some DNA polymerases but not observed previously for cellular RNA polymerases. Molecular dynamics (MD) simulations of the structures indicate that the third Mg2+ is coordinated and stabilized at its observed position. TL residues provide half of the substrate binding pocket while multiple TL/BH interactions induce conformational changes that could allow translocation upon substrate hydrolysis. Consistent with TL/BH communication, a FEL structure and MD simulations of the T834P mutant reveal rearrangement of some active site interactions supporting potential plasticity in active site function and long-distance effects on both the width of the central channel and TL conformation, likely underlying its increased elongation rate at the expense of fidelity.

High-level succinic acid production by overexpressing a magnesium transporter in Mannheimia succiniciproducens.

Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens. Although the importance of magnesium (Mg2+) ion on SA production has been evident from our previous studies, the role of Mg2+ ion remains largely unexplored. In this study, we investigated the impact of Mg2+ ion on SA production and developed a hyper-SA producing strain of M. succiniciproducens by reconstructing the Mg2+ ion transport system. To achieve this, optimal alkaline neutralizer comprising Mg2+ ion was developed and the physiological effect of Mg2+ ion was analyzed. Subsequently, the Mg2+ ion transport system was reconstructed by introducing an efficient Mg2+ ion transporter from Salmonella enterica. A high-inoculum fed-batch fermentation of the final engineered strain produced 152.23 ± 0.99 g/L of SA, with a maximum productivity of 39.64 ± 0.69 g/L/h. These findings highlight the importance of Mg2+ ions and transportation system optimization in succinic acid production by M. succiniciproducens.

Magnesium ions mitigate metastable states in the regulatory landscape of mRNA elements.

Residing in the 5' untranslated region of the mRNA, the 2'-deoxyguanosine (2'-dG) riboswitch mRNA element adopts an alternative structure upon binding of the 2'-dG molecule, which terminates transcription. RNA conformations are generally strongly affected by positively charged metal ions (especially Mg2+). We have quantitatively explored the combined effect of ligand (2'-dG) and Mg2+ binding on the energy landscape of the aptamer domain of the 2'-dG riboswitch with both explicit solvent all-atom molecular dynamics simulations (99 µsec aggregate sampling for the study) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments. We show that both ligand and Mg2+ are required for the stabilization of the aptamer domain; however, the two factors act with different modalities. The addition of Mg2+ remodels the energy landscape and reduces its frustration by the formation of additional contacts. In contrast, the binding of 2'-dG eliminates the metastable states by nucleating a compact core for the aptamer domain. Mg2+ ions and ligand binding are required to stabilize the least stable helix, P1 (which needs to unfold to activate the transcription platform), and the riboswitch core formed by the backbone of the P2 and P3 helices. Mg2+ and ligand also facilitate a more compact structure in the three-way junction region.

PRL-1/2 phosphatases control TRPM7 magnesium-dependent function to regulate cellular bioenergetics.

Phosphatases of regenerating liver (PRL-1, PRL-2, PRL-3; also known as PTP4A1, PTP4A2, PTP4A3, respectively) control intracellular magnesium levels by interacting with the CNNM magnesium transport regulators. Still, the exact mechanism governing magnesium transport by this protein complex is not well understood. Herein, we have developed a genetically encoded intracellular magnesium-specific reporter and demonstrate that the CNNM family inhibits the function of the TRPM7 magnesium channel. We show that the small GTPase ARL15 increases CNNM3/TRPM7 protein complex formation to reduce TRPM7 activity. Conversely, PRL-2 overexpression counteracts ARL15 binding to CNNM3 and enhances the function of TRPM7 by preventing the interaction between CNNM3 and TRPM7. Moreover, while TRPM7-induced cell signaling is promoted by PRL-1/2, it is reduced when CNNM3 is overexpressed. Lowering cellular magnesium levels reduces the interaction of CNNM3 with TRPM7 in a PRL-dependent manner, whereby knockdown of PRL-1/2 restores the protein complex formation. Cotargeting of TRPM7 and PRL-1/2 alters mitochondrial function and sensitizes cells to metabolic stress induced by magnesium depletion. These findings reveal the dynamic regulation of TRPM7 function in response to PRL-1/2 levels, to coordinate magnesium transport and reprogram cellular metabolism.

Photosynthetic plasticity aggravates the susceptibility of magnesium-deficient leaf to high light in rapeseed plants: the importance of Rubisco and mesophyll conductance.

Plants grown under low magnesium (Mg) soils are highly susceptible to encountering light intensities that exceed the capacity of photosynthesis (A), leading to a depression of photosynthetic efficiency and eventually to photooxidation (i.e., leaf chlorosis). Yet, it remains unclear which processes play a key role in limiting the photosynthetic energy utilization of Mg-deficient leaves, and whether the plasticity of A in acclimation to irradiance could have cross-talk with Mg, hence accelerating or mitigating the photodamage. We investigated the light acclimation responses of rapeseed (Brassica napus) grown under low- and adequate-Mg conditions. Magnesium deficiency considerably decreased rapeseed growth and leaf A, to a greater extent under high than under low light, which is associated with higher level of superoxide anion radical and more severe leaf chlorosis. This difference was mainly attributable to a greater depression in dark reaction under high light, with a higher Rubisco fallover and a more limited mesophyll conductance to CO2 (gm ). Plants grown under high irradiance enhanced the content and activity of Rubisco and gm to optimally utilize more light energy absorbed. However, Mg deficiency could not fulfill the need to activate the higher level of Rubisco and Rubisco activase in leaves of high-light-grown plants, leading to lower Rubisco activation and carboxylation rate. Additionally, Mg-deficient leaves under high light invested more carbon per leaf area to construct a compact leaf structure with smaller intercellular airspaces, lower surface area of chloroplast exposed to intercellular airspaces, and CO2 diffusion conductance through cytosol. These caused a more severe decrease in within-leaf CO2 diffusion rate and substrate availability. Taken together, plant plasticity helps to improve photosynthetic energy utilization under high light but aggravates the photooxidative damage once the Mg nutrition becomes insufficient.

A plasma membrane-localized transporter remobilizes aleurone layer magnesium for seed germination in rice.

The aleurone layer in cereal grains acts as a major reservoir of essential mineral nutrients, significantly influencing seed germination. However, the molecular mechanism underlying the redistribution of nutrients from the aleurone layer in the germinating seed is still not well understood. Here, in rice, we identified a plasma membrane (PM) localized magnesium transporter, MAGNESIUM RELEASE TRANSPORTER 3 (MGR3), is critical for seed germination. OsMGR3 is predominantly expressed in the aleurone layer cells of endosperm, facilitating magnesium remobilization during germination. Non-invasive Micro-test Technology assay data demonstrated that the loss-of-function of OsMGR3 restrained magnesium efflux from the aleurone layer. In the embryo/endosperm grafting experiment, we observed that the mutation of OsMGR3 in the aleurone layer suppressed the growth and differentiation of the embryo during germination. Furthermore, magnesium fluorescence imaging revealed the osmgr3 mutant seeds showed impaired exportation of aleurone layer-stored magnesium to the embryo, consequently delaying germination. Importantly, we discovered that disrupting OsMGR3 could inhibit pre-harvest sprouting without affecting rice yield and quality. Therefore, the magnesium efflux transporter OsMGR3 in the aleurone layer represents a promising genetic target for future agronomic trait improvement.

Involvement of Escherichia coli YbeX/CorC in ribosomal metabolism.

YbeX of Escherichia coli, a member of the CorC protein family, is encoded in the same operon with ribosome-associated proteins YbeY and YbeZ. Here, we report the involvement of YbeX in ribosomal metabolism. The ΔybeX cells accumulate distinct 16S rRNA degradation intermediates in the 30S particles and the 70S ribosomes. E. coli lacking ybeX has a lengthened lag phase upon outgrowth from the stationary phase. This growth phenotype is heterogeneous at the individual cell level and especially prominent under low extracellular magnesium levels. The ΔybeX strain is sensitive to elevated growth temperatures and to several ribosome-targeting antibiotics that have in common the ability to induce the cold shock response in E. coli. Although generally milder, the phenotypes of the ΔybeX mutant overlap with those caused by ybeY deletion. A genetic screen revealed partial compensation of the ΔybeX growth phenotype by the overexpression of YbeY. These findings indicate an interconnectedness among the ybeZYX operon genes, highlighting their roles in ribosomal assembly and/or degradation.

Cardiolipin deficiency leads to the destabilization of mitochondrial magnesium channel MRS2 in Barth syndrome.

Barth syndrome (BTHS) is a debilitating X-linked cardio-skeletal myopathy caused by loss-of-function mutations in TAFAZZIN, a cardiolipin (CL)-remodeling enzyme required for the maintenance of normal levels of CL species in mitochondrial membranes. At present, how perturbations in CL abundance and composition lead to many debilitating clinical presentations in BTHS patients have not been fully elucidated. Inspired by our recent findings that CL is essential for optimal mitochondrial calcium uptake, we measured the levels of other biologically important metal ions in BTHS mitochondria and found that in addition to calcium, magnesium levels are significantly reduced. Consistent with this observation, we report a decreased abundance of the mitochondrial magnesium influx channel MRS2 in multiple models of BTHS including yeast, murine myoblast, and BTHS patient cells and cardiac tissue. Mechanistically, we attribute reduced steady-state levels of MRS2 to its increased turnover in CL-deficient BTHS models. By expressing Mrs2 in well-characterized yeast mutants of the phospholipid biosynthetic pathways, we demonstrate a specific requirement of CL for Mrs2 abundance and assembly. Finally, we provide in vitro evidence for the direct binding of CL with human MRS2. Together, our study has identified a critical requirement of CL for MRS2 stability and suggests perturbation of mitochondrial magnesium homeostasis as a novel contributing factor to BTHS pathology.

Intrinsic Ribosome Destabilization Underlies Translation and Provides an Organism with a Strategy of Environmental Sensing.

Nascent polypeptides can modulate the polypeptide elongation speed on the ribosome. Here, we show that nascent chains can even destabilize the translating Escherichia coli ribosome from within. This phenomenon, termed intrinsic ribosome destabilization (IRD), occurs in response to a special amino acid sequence of the nascent chain, without involving the release or the recycling factors. Typically, a consecutive array of acidic residues and those intermitted by alternating prolines induce IRD. The ribosomal protein bL31, which bridges the two subunits, counteracts IRD, such that only strong destabilizing sequences abort translation in living cells. We found that MgtL, the leader peptide of a Mg2+ transporter (MgtA), contains a translation-aborting sequence, which sensitizes the ribosome to a decline in Mg2+ concentration and thereby triggers the MgtA-upregulating genetic scheme. Translation proceeds at an inherent risk of ribosomal destabilization, and nascent chain-ribosome complexes can function as a Mg2+ sensor by harnessing IRD.

Carbon-negative cement manufacturing from seawater-derived magnesium feedstocks.

This study describes and demonstrates key steps in a carbon-negative process for manufacturing cement from widely abundant seawater-derived magnesium (Mg) feedstocks. In contrast to conventional Portland cement, which starts with carbon-containing limestone as the source material, the proposed process uses membrane-free electrolyzers to facilitate the conversion of carbon-free magnesium ions (Mg2+) in seawater into magnesium hydroxide [Mg(OH)2] precursors for the production of Mg-based cement. After a low-temperature carbonation curing step converts Mg(OH)2 into magnesium carbonates through reaction with carbon dioxide (CO2), the resulting Mg-based binders can exhibit compressive strength comparable to that achieved by Portland cement after curing for only 2 days. Although the proposed "cement-from-seawater" process requires similar energy use per ton of cement as existing processes and is not currently suitable for use in conventional reinforced concrete, its potential to achieve a carbon-negative footprint makes it highly attractive to help decarbonize one of the most carbon-intensive industries.

Designing a Magnesium/Sodium Hybrid Battery Using Hierarchical Iron Selenide Architecture as Cathode Material and Modified Dual-Ion Salts in Ether as Electrolyte.

We developed a magnesium/sodium (Mg/Na) hybrid battery using a hierarchical disk-whisker FeSe2 architecture (HD-FeSe2) as the cathode material and a modified dual-ion electrolyte. The polarizable Se2- anion reduced the Mg2+ migration barrier, and the 3D configuration possessed a large surface area, which facilitated both Mg2+/Na+ cation diffusion and electron transport. The dual-ion salts with NaTFSI in ether reduced the Mg plating/stripping overvoltage in a symmetric cell. The hybrid battery exhibited an energy density of 260.9 Wh kg-1 and a power density of 600.8 W kg-1 at 0.2 A g-1. It showed a capacity retention of 154 mAh g-1 and a Coulombic efficiency of over 99.5% under 1.0 A g-1 after 800 long cycles. The battery also displayed outstanding temperature tolerance. The findings of 3D architecture as cathode material and hybrid electrolyte provide a pathway to design a highly reliable Mg/Na hybrid battery.

Unlocking a Fast Track for Magnesium Migration in Cu1.8S1-xSex via Anion-Tuning Chemistry.

Rechargeable magnesium batteries (rMBs) are promising candidates for next-generation batteries in which sulfides are widely used as cathode materials. The slow kinetics, low redox reversibility, and poor magnesium storage stability induced by the large Coulombic resistance and ionic polarization of Mg2+ ions have obstructed the development of high-performance rMBs. Herein, a Cu1.8S1-xSex cathode material with a two-dimensional sheet structure has been prepared by an anion-tuning strategy, achieving improved magnesium storage capacity and cycling stability. Element-specific synchrotron radiation analysis is evidence that selenium incorporation has indeed changed the chemical state of Cu species. Density functional theory calculations combined with kinetics analysis reveal that the anionic substitution endows the Cu1.8S1-xSex electrode with favorable charge-transfer kinetics and low ion diffusion barrier. The principal magnesium storage mechanisms and structural evolution process have been revealed in details based on a series of ex situ investigations. Our findings provide an effective heteroatom-tuning tactic of optimizing electrode structure toward advanced energy storage devices.

Self-Healable, High-Stability Anode for Rechargeable Magnesium Batteries Realized by Graphene-Confined Gallium Metal.

In this work, a self-healable, high-stability anode material for rechargeable magnesium batteries (RMBs) has been developed by introducing a core-shell structure of Ga confined by reduced graphene oxide (Ga@rGO). Via this Ga@rGO anode, a specific capacity of 150 mAh g-1 at a current of 0.5 A g-1 stable up to 1200 cycles at room temperature and a specific capacity of 100 mAh g-1 under an ultrahigh current of 1 A g-1 stable up to 700 cycles at a slightly elevated temperature of 40 °C have been achieved. Additionally, the ultrahigh rate, high-cycling stability, and long-cycle life of the anode are attributed to the stabilized structure; such a low-cost, simple, and environmentally friendly direct drop coating (DDC) method is developed to maximize the original state of the active materials. Remarkably, the self-healing ability of anodes is still presented under the ultrahigh charging current. This anode is promising for the development of high rate and high stability RMBs.

Stability of Plasmonic Mg-MgO Core-Shell Nanoparticles in Gas-Phase Oxidative Environments.

Magnesium is a recent addition to the plasmonic toolbox: nanomaterials that efficiently utilize photons' energy due to their ability to sustain localized surface plasmon resonances. Magnesium nanoparticles protected by a native oxide shell can efficiently absorb light across the solar spectrum, making them a promising photocatalytic material. However, their inherent reactivity toward oxidation may limit the number of reactions in which Mg-MgO can be used. Here, we investigate the stability of plasmonic Mg-MgO core-shell nanoplates under oxidative conditions. We demonstrate that the MgO shell stabilizes the metallic Mg core against oxidation in air at up to 400 °C. Furthermore, we show that the reactivity of Mg-MgO nanoplates with water vapor (3.5 vol % in N2) decreases with temperature, with no oxidation of the Mg core detected from 200 to 400 °C. This work unravels the potential of Mg-MgO nanoparticles for a broad range of catalytic transformations occurring in oxidative environments.