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Magnesium is essential for cellular life, but how it is homeostatically controlled still remains poorly understood. Here, we report that members of CNNM family, which have been controversially implicated in both cellular Mg 2+ influx and efflux, selectively bind to the TRPM7 channel to stimulate divalent cation entry into cells. Coexpression of CNNMs with the channel markedly increased uptake of divalent cations, which is prevented by an inactivating mutation to the channel’s pore. Knockout (KO) of TRPM7 in cells or application of the TRPM7 channel inhibitor NS8593 also interfered with CNNM-stimulated divalent cation uptake. Conversely, KO of CNNM3 and CNNM4 in HEK-293 cells significantly reduced TRPM7-mediated divalent cation entry, without affecting TRPM7 protein expression or its cell surface levels. Furthermore, we found that cellular overexpression of phosphatases of regenerating liver (PRLs), known CNNMs binding partners, stimulated TRPM7-dependent divalent cation entry and that CNNMs were required for this activity. Whole-cell electrophysiological recordings demonstrated that deletion of CNNM3 and CNNM4 from HEK-293 cells interfered with heterologously expressed and native TRPM7 channel function. We conclude that CNNMs employ the TRPM7 channel to mediate divalent cation influx and that CNNMs also possess separate TRPM7-independent Mg 2+ efflux activities that contribute to CNNMs’ control of cellular Mg 2+ homeostasis.

RNA molecules cannot fold in the absence of counterions. Experiments are typically performed in the presence of monovalent and divalent cations. How to treat the impact of a solution containing a mixture of both ion types on RNA folding has remained a challenging problem for decades. By exploiting the large concentration difference between divalent and monovalent ions used in experiments, we develop a theory based on the reference interaction site model (RISM), which allows us to treat divalent cations explicitly while keeping the implicit screening effect due to monovalent ions. Our theory captures both the inner shell and outer shell coordination of divalent cations to phosphate groups, which we demonstrate is crucial for an accurate calculation of RNA folding thermodynamics. The RISM theory for ion–phosphate interactions when combined with simulations based on a transferable coarse-grained model allows us to predict accurately the folding of several RNA molecules in a mixture containing monovalent and divalent ions. The calculated folding free energies and ion-preferential coefficients for RNA molecules (pseudoknots, a fragment of the rRNA, and the aptamer domain of the adenine riboswitch) are in excellent agreement with experiments over a wide range of monovalent and divalent ion concentrations. Because the theory is general, it can be readily used to investigate ion and sequence effects on DNA properties.

Zn 2+ , Mg 2+ and Ca 2+ are essential divalent cations implicated in many metabolic processes and signalling pathways. An emerging new paradigm is that the organismal balance of these cations predominantly depends on a common gatekeeper, the channel-kinase TRPM7. Despite extensive electrophysiological studies and recent cryo-EM analysis, an open question is how the channel activity of TRPM7 is activated. Here, we performed site-directed mutagenesis of mouse TRPM7 in conjunction with patch-clamp assessment of whole-cell and single-channel activity and molecular dynamics (MD) simulations to show that the side chains of conserved N1097 form an inter-subunit Mg 2+ regulatory site located in the lower channel gate of TRPM7. Our results suggest that intracellular Mg 2+ binds to this site and stabilizes the TRPM7 channel in the closed state, whereas the removal of Mg 2+ favours the opening of TRPM7. Hence, our study identifies the structural underpinnings through which the TRPM7 channel is controlled by cytosolic Mg 2+ , representing a new structure–function relationship not yet explored among TRPM channels.

Efficient enzymatic conversion of crystalline polysaccharides is crucial for an economically and environmentally sustainable bioeconomy but remains unfavorably inefficient. We describe an enzyme that acts on the surface of crystalline chitin, where it introduces chain breaks and generates oxidized chain ends, thus promoting further degradation by chitinases. This enzymatic activity was discovered and further characterized by using mass spectrometry and chromatographic separation methods to detect oxidized products generated in the absence or presence of H₂¹⁸O or ¹⁸O₂. There are strong indications that similar enzymes exist that work on cellulose. Our findings not only demonstrate the existence of a hitherto unknown enzyme activity but also provide new avenues toward more efficient enzymatic conversion of biomass.

Precise regulation of calcium homeostasis is essential for many physiological functions. The Ca 2+ -selective transient receptor potential (TRP) channels TRPV5 and TRPV6 play vital roles in calcium homeostasis as Ca 2+ uptake channels in epithelial tissues. Detailed structural bases for their assembly and Ca 2+ permeation remain obscure. Here we report the crystal structure of rat TRPV6 at 3.25 Å resolution. The overall architecture of TRPV6 reveals shared and unique features compared with other TRP channels. Intracellular domains engage in extensive interactions to form an intracellular ‘skirt’ involved in allosteric modulation. In the K + channel-like transmembrane domain, Ca 2+ selectivity is determined by direct coordination of Ca 2+ by a ring of aspartate side chains in the selectivity filter. On the basis of crystallographically identified cation-binding sites at the pore axis and extracellular vestibule, we propose a Ca 2+ permeation mechanism. Our results provide a structural foundation for understanding the regulation of epithelial Ca 2+ uptake and its role in pathophysiology. The X-ray crystal structure of rat transient receptor potential channel TRPV6 at 3.25 Å resolution is reported, providing new insights into its assembly and calcium-selective permeation. TRPV6 Ca 2+ channel structure The transient receptor potential (TRP) channels are a superfamily of cation-permeable ion channels that act as transducers of sensory modalities, including temperature, taste, olfaction, vision, hearing and touch. TRPV5 and TRPV6 are TRP channels that are highly selective for Ca 2+ , and they play vital roles in calcium homeostasis. In this manuscript, the authors report the X-ray crystal structure of rat TRPV6 at 3.25 Å resolution. Although the overall architecture of TRPV6 is fairly similar to that of TRPV1, this new structure reveals that exceptionally high Ca 2+ selectivity is achieved via the direct coordination of Ca 2+ by a ring of aspartate side chains in the selectivity filter. This work provides a structural foundation to understanding the regulation of epithelial Ca 2+ uptake and its role in pathophysiology.

Only two polyethylene glycol terephthalate (PET)-degrading enzymes have been reported, and their mechanism for the biochemical degradation of PET remains unclear. To identify a novel PET-degrading enzyme, a putative cutinase gene (cut190) was cloned from the thermophile Saccharomonospora viridis AHK190 and expressed in Escherichia coli Rosetta-gami B (DE3). Mutational analysis indicated that substitution of Ser226 with Pro and Arg228 with Ser yielded the highest activity and thermostability. The Ca²⁺ion enhanced the enzyme activity and thermostability of the wild-type and mutant Cut190. Circular dichroism suggested that the Ca²⁺changes the tertiary structure of the Cut190 (S226P/R228S), which has optimal activity at 65–75 °C and pH 6.5–8.0 in the presence of 20 % glycerol. The enzyme was stable over a pH range of 5–9 and at temperatures up to 65 °C for 24 h with 40 % activity remaining after incubation for 1 h at 70 °C. The Cut190 (S226P/R228S) efficiently hydrolyzed various aliphatic and aliphatic-co-aromatic polyester films. Furthermore, the enzyme degraded the PET film above 60 °C. Therefore, Cut190 is the novel-reported PET-degrading enzyme with the potential for industrial applications in polyester degradation, monomer recycling, and PET surface modification. Thus, the Cut190 will be a useful tool to elucidate the molecular mechanisms of the PET degradation, Ca²⁺activation, and stabilization.

Achieving a high nutritional value of food often involves fortifying microorganisms (such as bacteria and yeast) used in baking and dairy industry with essential elements. The aim of this study was to investigate the effect of a pulsed electric field (PEF) on the penetration and accumulation of Ca2+ and Mg2+ ions into model membranes of the food-grade yeast Saccharomyces cerevisiae. Simplified model membranes (monolayers and liposomes) were constructed using the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). The Langmuir monolayer technique, dynamic light scattering (DLS) and microelectrophoresis were employed to characterize the physicochemical properties of the model membranes investigated. The results showed significant molecular-level differences in the interactions of the selected cations with lipid monolayers and bilayers in liposome structures. Both cations deeply penetrated the membrane’s hydrophilic region, yet two competing effects were evident: expansion induced by hydrated Mg2+ and condensation driven by Ca2+ bridging. Furthermore, the application of PEF increased the concentration of ions absorbed by the liposomes. Specifically, optimized PEF parameters resulted in cation accumulation within the model membranes, ranging from 6 to 13%. This finding correlates well with the increased Ca2+ and Mg2+ uptake observed in real yeast cells, providing a deeper understanding of the cell membrane-environment interface and the underlying processes.

Voltage-gated calcium (Ca V ) channels catalyse rapid, highly selective influx of Ca 2+ into cells despite a 70-fold higher extracellular concentration of Na + . How Ca V channels solve this fundamental biophysical problem remains unclear. Here we report physiological and crystallographic analyses of a calcium selectivity filter constructed in the homotetrameric bacterial Na V channel Na V Ab. Our results reveal interactions of hydrated Ca 2+ with two high-affinity Ca 2+ -binding sites followed by a third lower-affinity site that would coordinate Ca 2+ as it moves inward. At the selectivity filter entry, Site 1 is formed by four carboxyl side chains, which have a critical role in determining Ca 2+ selectivity. Four carboxyls plus four backbone carbonyls form Site 2, which is targeted by the blocking cations Cd 2+ and Mn 2+ , with single occupancy. The lower-affinity Site 3 is formed by four backbone carbonyls alone, which mediate exit into the central cavity. This pore architecture suggests a conduction pathway involving transitions between two main states with one or two hydrated Ca 2+ ions bound in the selectivity filter and supports a ‘knock-off’ mechanism of ion permeation through a stepwise-binding process. The multi-ion selectivity filter of our Ca V Ab model establishes a structural framework for understanding the mechanisms of ion selectivity and conductance by vertebrate Ca V channels. X-ray crystal structures of a voltage-gated Na + channel mutated to be highly Ca 2+ selective provide a framework for understanding the mechanisms of ion selectivity and conductance in vertebrate voltage-gated Ca 2+ channels. Super-selectivity of the calcium channel Voltage-gated calcium (Ca V ) channels show remarkable selectivity, facilitating the import of Ca 2+ from an extracellular medium where the Na + concentration is 70-fold higher than that of Ca 2+ . How Ca V channels solve this fundamental biophysical problem remains unclear. This study presents several X-ray crystal structures of the full-length tetrameric bacterial Na V channel engineered through mutations to act as a highly selective voltage-gated calcium channel. The structures reveal that there are three Ca 2+ -binding sites in the pore: two high-affinity Ca 2+ -binding sites, and a lower-affinity site that Ca 2+ occupies as it moves through the pore. The Ca 2+ ions remain nearly fully hydrated as they pass through the channel, which is completely contrary to the textbook picture of fully dehydrated metal ions binding to amino acid side chains or backbone carbonyl groups lining the selectivity filter.

The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of murine TMEM16F (mTMEM16F) that underlie its function as a lipid scramblase and an ion channel. The cryo-EM data of mTMEM16F in absence and presence of Ca2+ define the ligand-free closed conformation of the protein and the structure of a Ca2+-bound intermediate. Both conformations resemble their counterparts of the scrambling-incompetent anion channel mTMEM16A, yet with distinct differences in the region of ion and lipid permeation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, both functions appear to be mediated by alternate protein conformations that are at equilibrium in the ligand-bound state.

TRPM2 is critically involved in diverse physiological processes including core temperature sensing, apoptosis, and immune response. TRPM2’s activation by Ca2+ and ADP ribose (ADPR), an NAD+-metabolite produced under oxidative stress and neurodegenerative conditions, suggests a role in neurological disorders. We provide a central concept between triple-site ligand binding and the channel gating of human TRPM2. We show consecutive structural rearrangements and channel activation of TRPM2 induced by binding of ADPR in two indispensable locations, and the binding of Ca2+ in the transmembrane domain. The 8-Br-cADPR—an antagonist of cADPR—binds only to the MHR1/2 domain and inhibits TRPM2 by stabilizing the channel in an apo-like conformation. We conclude that MHR1/2 acts as a orthostatic ligand-binding site for TRPM2. The NUDT9-H domain binds to a second ADPR to assist channel activation in vertebrates, but not necessary in invertebrates. Our work provides insights into the gating mechanism of human TRPM2 and its pharmacology.