The metabotropic glutamate receptor 6 (mGlu6) is essential for synaptic communication of rod photoreceptors, and mutations in mGlu6 lead to a blinding disorder. However, its structural organization remains unknown. Here, we present the structure of agonist-bound mGlu6, revealing an asymmetric dimer arrangement in the absence of a G protein. This indicates that agonist binding alone can induce the homodimeric receptor asymmetry in metabotropic glutamate receptors and structurally prime mGlu6 for activation by pre-organizing the transmembrane domain dimer interface for G protein binding. The structure also identifies noncanonical interactions between the cysteine-rich domain and extracellular loop 2, forming a unique interface that likely stabilizes the activation state. Mutational analyses of this interface reveal its role in maintaining rapid Gαo activation and surface targeting. The structure also permits mechanistic investigation of congenital stationary night blindness and reveals diverse effects of pathogenic mutations on surface trafficking, Gαo coupling, and activation dynamics, including unexpected gain-of-function. These results provide critical insight into the intermediate asymmetric structure of mGlu6 and offer a molecular framework for understanding the pathogenesis of inherited retinal disorders.

TRPM8, a cold-activated ion channel, enables mammals to sense cooling agents such as menthol. While PIP2 is essential for menthol-induced activation of TRPM8, the precise cooperative mechanism and the specific binding mode of menthol have remained elusive. Here, we present cryo-EM structures of mouse TRPM8 in diverse conformations, including a PIP2-induced two-fold symmetric intermediate and an icilin-bound open state. Our results reveal that PIP2 binding initiates a symmetry-breaking event, priming the channel for activation through a noncanonical intermediate states. The subsequent binding of cooling agonists promotes a transition back to four-fold symmetry. Notably, we find that menthol stabilizes the PIP2-bound state, thereby overcoming channel desensitization, while icilin, in concert with calcium, stabilizes a fully open conformation. Together, these structures illuminate a stepwise activation pathway involving distinct symmetry transitions and define the cooperative allosteric mechanism by which PIP2 and cooling agonists gate the channel.

In recent years, cryo-electron microscopy structures of ion channels in complex with G proteins have been resolved, providing insights into the molecular mechanisms underlying the crosstalk between G protein-coupled receptors (GPCRs) and ion channels. Downstream signaling initiated by GPCR activation can indirectly modulate ion channel activity. Alternatively, the direct binding of Gα or Gβγ subunits to ion channels can directly regulate their ion conduction activity. Recent cryo-electron microscopy structures, such as TRPC5–Gαi3, GIRK–Gβγ and TRPM3–Gβγ, have elucidated these direct interactions and advanced our understanding of how Gα or Gβγ subunits activated by GPCRs modulate ion channel activity. In addition, the structure of the TRPV4–RhoA complex has revealed that small G proteins can also directly modulate ion channels. Understanding the physiological roles of these complexes will be critical for their potential use as pharmacological targets. Here we summarize the current knowledge of the interactions between ion channels and G proteins. Ion channels are crucial for cell communication, allowing ions to move across cell membranes. This study explores how G proteins, which are molecules that help to transmit signals inside cells, interact with ion channels. The authors focus on a specific type of ion channel called transient receptor potential (TRP) channels, which are involved in sensing changes in the environment. This research highlights how G proteins, made up of subunits such as Gα and Gβγ, can directly bind to these ion channels and influence their activity. Using advanced imaging techniques such as cryo-electron microscopy, the authors examined the structures of these complexes to understand how they work. They found that G proteins can either activate or inhibit ion channels by binding to them directly. This interaction is important for various cellular processes and could be linked to certain diseases when it goes wrong. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

Transient receptor potential (TRP) ion channels form heteromers through combinatorial associations of distinct subunits, contributing to the diversity of TRP channel functions. Among them, TRPC5, which forms a heteromer with TRPC1, represents an attractive pharmaceutical target for treating anxiety and depression. Here, we present the cryo-electron microscopy structure of the human TRPC1/C5 heteromer, composed of one TRPC1 subunit and three TRPC5 subunits. The incorporation of TRPC1 into the heteromer disrupts the C4 symmetry of the TRPC5 homotetramer, resulting in a distinct ion conduction pathway characterized by an asymmetrically constricted selectivity filter and an asymmetric lower gate. The TRPC1/C5 heteromer displays recognizable structural features compared to the TRPC1/C4 heteromer, including a noncanonically tilted coiled-coil domain and a distinct intersubunit interactions. Furthermore, we elucidate the structures of human TRPC5 bound to the TRPC1/4/5-specific agonist, (-)-Englerin A. Our findings establish a foundation for exploring the diversity of heteromeric TRP channels and pave the way for targeting TRPC1/C5 as a therapeutic strategy.

β-arrestins (βarrs) play a crucial role in regulating G protein-coupled receptor (GPCR) signaling and trafficking. Canonically, interactions of βarr with the phosphorylated intracellular GPCR-tail induce a multi-step conformational transition that results in the activation of βarr. Depending on the specific interaction pattern with the receptor, βarrs adopt multiple conformational states, each tightly linked to a specific functional outcome of βarr recruitment. Despite its physiological relevance, the structural determinants of βarr activation remain poorly understood. Using a combination of molecular dynamics simulations, biochemical and cell-based experiments, we reveal how specific interactions with a chemokine receptor 7 (CXCR7) promote the unbinding of the βarr2 C-tail-a crucial step in arrestin activation. Importantly, we observe that the expulsion of the C-tail is promoted by the displacement of a conserved arginine residue (Arg394) within the βarr polar core, which we dub "the arginine switch." Our study uncovers a role for the arginine switch that, upon engagement, destabilizes the polar core as a crucial step in the CXCR7-induced βarr activation.

Quantitative measurement of protein-protein interactions (PPIs) within living cells is vital for understanding their cellular functions at the molecular level and for applications in synthetic biology, protein engineering, and drug discovery. Although several techniques have been developed to measure PPI strength in vitro, direct measurement of PPI strength within living bacterial cells remains challenging. Here, a method for quantitatively measuring PPIs by determining the dissociation constant (Kd) in living E. coli using fluorescence resonance energy transfer (FRET), a technique termed KD-FRET, is reported. It is found that the direct excitation of the acceptor fluorophore among spectral crosstalks primarily results in non-interacting pairs exhibiting an apparent Kd, leading to false-positive signals. KD-FRET proves highly effective in quantifying various PPI Kd values, including both heterologous and homologous pairs. Moreover, KD-FRET enables the quantification of Kd for interaction pairs that are unmeasurable in vitro owing to their instability under standard buffer conditions. KD-FRET is successfully applied in the development of a novel synthetic biology tool to enhance naringenin production in E. coli and lycopene production in S. cerevisiae by precisely engineering metabolic pathway. These results demonstrate the potential of KD-FRET as a powerful tool for studying PPIs in their native cellular environments.

The inherent O2 sensitivity of Ni─Fe carbon monoxide dehydrogenases (CODHs), crucial for rapid CO to CO2 interconversion, presents substantial challenges for industrial application. Transforming CO/CO2, a prevalent anthropogenic air pollutant, into valuable carbon chemicals either directly or through intermediate steps via biocatalytic methods offers a promising pathway to achieve net-zero emissions across industries and the environment. However, completely eliminating oxygen from industrial biotransformations, especially under ambient conditions, is exceedingly onerous. Here, we engineered variants of the CODH2 from Carboxydothermus hydrogenoformans (ChCODH2) with dual blocking at both the O2 entrance and near the active site, effectively sealing the tunnel against atmospheric O2 levels (20%). The O2-tunnel engineered A559W/V610H variant demonstrated a marked improvement in air stability, with a half-life of 24.6 h compared to the wild type's 2.4 h. Crystallographic snapshots of this air-viable variant after 24 h of exposure revealed the robust integrity of the fortified FeS and NiFeS clusters. Additionally, electro-enzymatic reactions corroborated its CO/CO2 conversion capability even in ubiquitous air. These findings, which address the O2 sensitivity of anaerobic enzymes caused by O2-induced metal cluster collapse, enhance their potential for biological CO/CO2 transformations in O2-rich environments, thereby broadening their industrial viability and applicability.

Correction for ‘(Thio)chromenone derivatives exhibit anti-metastatic effects through selective inhibition of uPAR in cancer cell lines: discovery of an uPAR-targeting fluorescent probe’ by So-Young Chun et al., Chem. Commun., 2025, https://doi.org/10.1039/D4CC05907G.

G protein-coupled receptors (GPCRs), the key regulators of cellular signaling, transduce signals through G proteins or arrestins. G protein- or arrestin-mediated signal transduction induces distinct functional consequences, and therefore the molecular mechanisms of the interaction between GPCR-G protein or GPCR-arrestin have been of great interest. While the mechanism of arrestin binding to GPCRs with a phosphorylated C-terminal tail (C-tail) is well understood, little is known about arrestin interactions with GPCRs that possess a short C-tail and a long phosphorylated intracellular loop 3 (ICL3). Here, we explore the interaction between β-arrestin 2 (βarr2) and the dopamine receptor D2 (D2R), a critical receptor for brain function, which features an exceptionally long ICL3 but no C-tail. Using cell-based assays, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and biochemical methods, we identify key phosphorylated residues in D2R ICL3 for βarr2 recruitment and observe unique conformational changes in βarr2 upon binding the phosphorylated D2R ICL3 peptide (D2Rpp). Our results provide new insights into structural dynamics of GPCR-arrestin complex, especially in understanding the D2R-βarr2 interaction.

A class of (thio)chromenone derivatives has been identified as suitable ligands for uPAR, a glycoprotein with a prognostic value in a large number of human cancers. The (thio)chromenone agents actively inhibited the binding of uPAR to uPA with a binding affinity of 18.6 nM, reducing cell migration in the wound healing assay by up to 40% without apparent cell motility. The discovery of an uPAR-targeting fluorescent probe was also made in this study that can selectively bind to the membrane uPAR, providing valuable molecular insights into the role of uPAR in cancer metastasis. This study should serve as a basis for the development of new uPAR-targeting agents that can control the metastatic potential of cancer cells with minimal cytotoxicity.

GPR179, an orphan class C GPCR, is expressed at the dendritic tips of ON-bipolar cells in the retina. It plays a pivotal role in the initial synaptic transmission of visual signals from photoreceptors, and its deficiency is known to be the cause of complete congenital stationary night blindness. Here, we present the cryo-electron microscopy structure of human GPR179. Notably, the transmembrane domain (TMD) of GPR179 forms a homodimer through the TM1/7 interface with a single inter-protomer disulfide bond, adopting a noncanonical dimerization mode. Furthermore, the TMD dimer exhibits architecture well-suited for the highly curved membrane of the dendritic tip and distinct from the flat membrane arrangement observed in other class C GPCR dimers. Our structure reveals unique structural features of GPR179 TMD, setting it apart from other class C GPCRs. These findings provide a foundation for understanding signal transduction through GPR179 in visual processing and offers insights into the underlying causes of ocular diseases. GPR179, an orphan class C GPCR crucial for visual signaling, is expressed at dendritic tips in the retina. Here, authors present cryo-EM structure of GPR179, revealing its dimerization mode and curvature.

Formate dehydrogenase (FDH) is critical for the conversion between formate and carbon dioxide. Despite its importance, the structural complexity of FDH and difficulties in the production of the enzyme have made elucidating its unique physicochemical properties challenging. Here, we purified recombinant Methylobacterium extorquens AM1 FDH (MeFDH1) and used cryo-electron microscopy to determine its structure. We resolved a heterodimeric MeFDH1 structure at a resolution of 2.8 Å, showing a noncanonical active site and a well-embedded Fe-S redox chain relay. In particular, the tungsten bis-molybdopterin guanine dinucleotide active site showed an open configuration with a flexible C-terminal cap domain, suggesting structural and dynamic heterogeneity in the enzyme.

Connexin 36 (Cx36) forms interneuronal gap junctions, establishing electrical synapses for rapid synaptic transmission. In disease conditions, inhibiting Cx36 gap junction channels (GJCs) is beneficial, as it prevents abnormal synchronous neuronal firing and apoptotic signal propagation, mitigating seizures and progressive cell death. Here, we present cryo-electron microscopy structures of human Cx36 GJC in complex with known channel inhibitors, such as mefloquine, arachidonic acid, and 1-hexanol. Notably, these inhibitors competitively bind to the binding pocket of the N-terminal helices (NTH), inducing a conformational shift from the pore-lining NTH (PLN) state to the flexible NTH (FN) state. This leads to the obstruction of the channel pore by flat double-layer densities of lipids. These studies elucidate the molecular mechanisms of how Cx36 GJC can be modulated by inhibitors, providing valuable insights into potential therapeutic applications. Connexin 36 (Cx36) forms neuronal gap junctions essential for electrical synapses. Here, the authors reveal cryo-EM structures of human Cx36 bound to inhibitors, showing how the inhibitors induce conformational changes that block the channel pore.

Fe‒S cluster-harboring enzymes, such as carbon monoxide dehydrogenases (CODH), employ sophisticated artificial electron mediators like viologens to serve as potent biocatalysts capable of cleaning-up industrial off-gases at stunning reaction rates. Unraveling the interplay between these enzymes and their associated mediators is essential for improving the efficiency of CODHs. Here we show the electron mediator-interaction site on ChCODHs (Ch, Carboxydothermus hydrogenoformans) using a systematic approach that leverages the viologen-reactive characteristics of superficial aromatic residues. By enhancing mediator-interaction (R57G/N59L) near the D-cluster, the strategically tailored variants exhibit a ten-fold increase in ethyl viologen affinity relative to the wild-type without sacrificing the turn-over rate (kcat). Viologen-complexed structures reveal the pivotal positions of surface phenylalanine residues, serving as external conduits for the D-cluster to/from viologen. One variant (R57G/N59L/A559W) can treat a broad spectrum of waste gases (from steel-process and plastic-gasification) containing O2. Decoding mediator interactions will facilitate the development of industrially high-efficient biocatalysts encompassing gas-utilizing enzymes.

This study introduces a novel approach for CO2 reduction to formate using the recombinant formate dehydrogenase 1 (MeFDH1) from Methylorubrum extorquens AM1 as biocatalyst, addressing challenges in activity, productivity, and long-term stability of enzyme. We demonstrate that immobilized MeFDH1 supported by electrochemical reaction system enhances formate production and stability, achieving over 1.7 M concentration with an initial rate of 20 mM/h and near-unity Faradaic efficiency for over 200 hours. In further, the reusability of immobilized MeFDH1 was obtained without significant declination of productivity and selectivity. The electrochemical study of MeFDH1 found the product inhibition in continuous CO2 conversion. To overcome this challenge and build efficient process, the integration of a flow reactor system and in-situ separation unit further improved the system's performance and scalability. This advancement in enzymatic CO2 conversion suggests the potential of biocatalysis towards addressing global warming through sustainable chemical synthesis.

Abstract HEPN–MNT, a type VII TA module, comprises the HEPN toxin and the MNT antitoxin, which acts as a nucleotidyltransferase that transfers the NMP moiety to the corresponding HEPN toxin, thereby interfering with its toxicity. Here, we report crystal structures of the Legionella pneumophila HEPN–MNT module, including HEPN, AMPylated HEPN, MNT, and the HEPN–MNT complex. Our structural analysis and biochemical assays, suggest that HEPN is a metal-dependent RNase and identify its active site residues. We also elucidate the oligomeric state of HEPN in solution. Interestingly, L. pneumophila MNT, which lacks a long C-terminal α4 helix, controls the toxicity of HEPN toxin via a distinct binding mode with HEPN. Finally, we propose a comprehensive regulatory mechanism of the L. pneumophila HEPN–MNT module based on structural and functional studies. These results provide insight into the type VII HEPN–MNT TA system.