eLife: latest articles by subject

Experimental verification of the error minimization theory using non-standard genetic codes constructed in vitro

ichihashi@bio.c.u-tokyo.ac.jp (Norikazu Ichihashi) — Mon, 13 Jul 2026 00:00:00 +0000

All living systems use an almost identical standard genetic code (SGC), in which 20 amino acids are assigned non-randomly. According to the error minimization theory, amino acids are arranged to minimize the mutational effect on protein function, while experimental verification remains limited. Here, we constructed 10 non-standard genetic codes (non-SGCs) in vitro by reassigning three amino acids (Ala, Ser, and Leu) in vacant codons of the minimal genetic code consisting of 21 tRNAs. Most of these non-SGCs have a higher cost of amino acid replacement than the SGC, calculated based on three amino acid properties: polar requirement (PR), molecular volume (MV), and hydropathy index (HI). The protein function of three reporter genes expressed using these non-SGCs decreased similarly when random mutations were introduced into the genes, implying that the effect of mutations was similar across all the non-SGCs tested here. This result provides direct experimental evidence that mutational robustness does not significantly change in individual reporter protein activity within the range of mutational cost tested in this study (Cost_PR: 5.29–5.77, Cost_MV: 1848–2348, and Cost_HI: 3.27–5.10), which covers approximately 18.4% (PR), 37.6% (MV), and 50.8% (HI) of the possible cost range achievable among one million randomly-generated genetic codes.

Dynamic assembly of malate dehydrogenase–citrate synthase multienzyme complex in the mitochondria

tobata2@unl.edu (Connor Pedersen) — Wed, 08 Jul 2026 00:00:00 +0000

The tricarboxylic acid (TCA) cycle enzymes malate dehydrogenase (MDH1) and citrate synthase (CIT1) form a multienzyme complex, referred to as a metabolon, that channels intermediate oxaloacetate between their reaction centers. Given that the MDH1–CIT1 metabolon enhances pathway reactions in vitro, its dynamic assembly is hypothesized to contribute to TCA cycle regulation in response to cellular metabolic demands. Here, we demonstrated that yeast mitochondrial MDH1 and CIT1 dissociated when aerobic respiration was suppressed by the Crabtree effect and associated when the respiratory activity was enhanced by acetate. Pharmacological TCA cycle inhibition dissociated the complex, whereas electron transport chain inhibition enhanced the interaction. The multienzyme complex assembly was related to the mitochondrial matrix acidification and oxidation, as well as cellular levels of malate, fumarate, and citrate. These factors significantly affected the MDH1–CIT1 complex affinity in vitro. Especially, variations in buffer pH within the physiological pH range between 6.0 and 7.0 in the mitochondrial matrix significantly impacted the MDH1–CIT1 affinity. These results demonstrate the dynamic association and dissociation of the MDH1–CIT1 metabolon and its relationship with respiratory activity, supporting metabolon dynamics as an integral factor in metabolic regulation governed by multiple factors such as mitochondrial pH and metabolite levels.

Profiling of terminating ribosomes reveals translational control at stop codons

sq38@cornell.edu (Leiming Dong) — Tue, 07 Jul 2026 00:00:00 +0000

Accurate termination of protein synthesis is paramount for the integrity of the cellular proteome, yet the dynamics and fidelity of ribosome termination remain poorly understood. Here, we establish a profiling strategy to capture terminating ribosomes in mammalian cells and reveal a substantial heterogeneity in ribosome pausing at individual stop codons. We identify a sequence motif upstream of the stop codon that promotes termination pausing, a finding supported by massively parallel reporter assays. Unexpectedly, reduced termination pausing increases the likelihood of stop codon slippage, giving rise to proteins with heterogeneous C-terminal extensions. Mechanistically, we show that sequence-dependent termination pausing is consistent with post-decoding mRNA scanning by the 3′ end of 18 S rRNA. We further uncover tissue-specific patterns of termination pausing that correlate with the stoichiometry of Rps26, which potentially modulates mRNA:rRNA interactions. Together, these results suggest termination pausing as a distinct translational signature shaped by mRNA sequence contexts, ribosome heterogeneity, and cell type-specific translational control.

Non-canonical amino acid incorporation enables minimally disruptive labeling of stress granule and TDP-43 proteinopathy

jiouw@jhu.edu (Hao Chen) — Fri, 03 Jul 2026 00:00:00 +0000

We report a minimally disruptive labeling strategy for stress granule protein, G3BP Stress Granule Assembly Factor 1 (G3BP1), and ALS-linked protein, TAR DNA-binding protein 43 (TDP-43), using the fluorescent non-canonical amino acid Anap. By integrating the genetic code expansion (GCE) with rational site selection, we achieved precise incorporation of Anap that preserves protein structure and function. In live cells and neurons, Anap labeling faithfully recapitulated localization, stress-induced dynamics, and recovery behavior, outperforming conventional fluorescent tags, and enabling physiologically relevant visualization of protein pathobiology.

Structural insights into the recruitment of viral type 2 IRES to ribosomal preinitiation complex for protein synthesis

hussain@iisc.ac.in (Deepakash Das) — Thu, 25 Jun 2026 00:00:00 +0000

Picornaviruses employ internal ribosome entry sites (IRESs) in their genomic RNA to hijack the host’s translational machinery. The picornavirus, encephalomyocarditis virus, employs a type 2 IRES present in its 5’ untranslated region (5’UTR) and requires 43S ribosomal preinitiation complex (PIC), the central domain of eukaryotic initiation factor (eIF) 4G, eIF4A, and an essential ITAF (IRES trans-acting factor)-polypyrimidine tract binding protein 1 (PTB1) to form 48S PIC. In this study, we have used cryo-electron microscopy (cryo-EM) to determine the structure of encephalomyocarditis virus (EMCV) IRES-bound mammalian 48S PIC in a scanning-arrested closed state at the start codon. The EMCV IRES domains contact initiator tRNA (tRNA_i) and 40S head at the inter-subunit interface, which reveals an altogether unique mechanism used by viruses to capture host translational machinery for its protein synthesis. The tRNA_i is held away from the 40S body in contrast to canonical cap-dependent translation while the domain I apical region of EMCV IRES mimics 28S rRNA of 60S to interact with 40S ribosomal head proteins uS13 and uS19. The structural analysis accounts for numerous previously reported biochemical studies on type 2 IRES and shows how type 2 IRES interacts with 43S PIC to form 48S PIC. This study provides mechanistic insights for understanding EMCV IRES-mediated translation initiation, which could be extrapolated to other IRESs sharing similar motifs and factor requirements, including type 1 viral IRESs.

Systematic characterisation of site-specific proline hydroxylation using hydrophilic interaction chromatography and mass spectrometry

Sonia.Rocha@liverpool.ac.uk (Angus I Lamond) — Thu, 25 Jun 2026 00:00:00 +0000

We have developed a robust workflow to identify proline hydroxylation sites in proteins, combining hydrophilic interaction chromatography (HILIC) enrichment and high-resolution nano-liquid chromatography-mass spectrometry (LC-MS) with refining and filtering parameters during data analysis. Using this approach, we have combined data from cell lines treated with either the prolyl hydroxylase (PHD) inhibitor, Roxadustat (FG-4592), or with the proteasome inhibitor MG-132, or with a DMSO control, to identify a total of 4993 and 3247 proline hydroxylation sites, respectively, in HEK293 and RCC4 cells. Of these, 1954 (HEK293) and 1253 (RCC4) high-confidence non-collagen sites were inhibited by FG-4592. Hydroxylated peptides showed consistent characteristics across both datasets, including enrichment in more hydrophilic HILIC fractions and distinct charge and mass distributions compared to unmodified or oxidised peptides. The intensity of the diagnostic hydroxyproline immonium ion varied with MS collision energy, peptide concentration, and adjacent amino acid sequence. Using synthetic peptides, we demonstrate that combining LC retention time with optimised MS parameters enables reliable site identification, even with multiple proline residues present. Proteins with FG-4592-inhibited hydroxylation sites were enriched for roles in RNA metabolism, mRNA splicing, and cell cycle regulation, including the phosphatase 1 regulatory subunit Repo-Man (CDCA2).

Correction: SEC24A deficiency lowers plasma cholesterol through reduced PCSK9 secretion

Wed, 24 Jun 2026 00:00:00 +0000

Exploration of precision coregulator TR-FRET identifies diverse signatures for LXR ligands relevant to discovery of nonlipogenic ABCA1 inducers

grjthatcher@arizona.edu (Anandhan Annadurai) — Mon, 22 Jun 2026 00:00:00 +0000

APOE4, the major genetic risk factor for Alzheimer’s disease (AD), and ATP-binding cassette-A1 (ABCA1), required for lipidation of APOE are gene products of the liver X receptor (LXR) receptor. LXR agonists have been validated in animal models as therapeutics for AD, atherosclerosis, and many other diseases. Clinical progress has been thwarted by unwanted hepatic lipogenesis. Structurally diverse LXR ligands were profiled in coregulator TR-FRET (CRT) assays analyzing ligand-induced coactivator recruitment, coactivator selectivity, corepressor dissociation, and LXR isoform selectivity. A multiplex CRT assay was developed to measure synchronous ligand-induced displacement of corepressor by coactivator. Potency for coactivator recruitment to LXRβ correlated with induction of ABCA1 in human astrocytoma cells. Correlation with lipogenic activation of sterol response element (SRE) in hepatocarcinoma cells, was more complex. CRT response was diverse revealing ligands with theoretical full agonist, partial agonist, antagonist, inverse agonist, and other signatures within the same chemical series, suggesting the scope for precision CRT to guide nonlipogenic LXR agonist design.

Single-step in vitro reconstitution of the Escherichia coli ribosome mediated by two GTPase factors, EngA and ObgE

yshimizu@riken.jp (Aya Sato) — Thu, 18 Jun 2026 00:00:00 +0000

When Escherichia coli ribosomes are assembled in vitro, manipulation of incubation temperature and magnesium ion concentration has been an essential procedure, which is a crucial step for the assembly of active large subunits. The present study tackles this issue to develop a single-step procedure, which can be performed in near-physiological conditions, where cell-free protein synthesis is active. We found that GTPase factors EngA and ObgE can complement the changes in temperature and magnesium ion concentrations. In the presence of these factors, both the ribosome assembly and translation processes were successfully integrated in the reconstituted cell-free protein synthesis system. Furthermore, we found that these GTPase factors can reassemble the ribosomes to an active state, whose structure was disrupted by EDTA chelation of magnesium ions, indicating that these two factors can reversibly induce the ribosome structure to an intact state. The findings are essential for the bottom-up construction of synthetic cells.

Nuclear CK1δ as a critical determinant of PER:CRY complex dynamics and circadian period

michael.brunner@bzh.uni-heidelberg.de (Axel CR Diernfellner) — Mon, 15 Jun 2026 00:00:00 +0000

The mammalian circadian clock is governed by a feedback loop in which the transcription activator CLOCK:BMAL1 induces expression of its inhibitors, PERs and CRYs, which form a complex with CK1δ, the main circadian kinase. However, the spatiotemporal dynamics of this feedback loop and the precise role of CK1δ remain incompletely understood. Using an inducible overexpression system, we show that nuclear availability of CK1δ is limited by both rapid nuclear degradation and active export of unassembled kinase, while cytoplasmic kinase is readily available for association with PERs. We demonstrate that CK1δ-mediated phosphorylation may disrupt PER2–CRY1 interaction, thereby resulting in cytoplasmic PER2 dimers containing substoichiometric amounts of CRY1. Analysis of endogenous PER2 localization in the context of an intact circadian clock reveals that PER2 accumulates in the cytoplasm late in the circadian cycle. Based on these findings, we propose that cytoplasmic accumulation of PER:CRY:CK1δ complexes contributes to the clearance of nuclear PER2, while the CK1δ-dependent release of CRY1 into the nucleus may sustain CLOCK:BMAL1 repression on DNA, supporting the transition from the early to the late repressive phase.

Cell size modulates ferroptosis susceptibility

ez225@cam.ac.uk (Evgeny Zatulovskiy) — Wed, 10 Jun 2026 00:00:00 +0000

Size is a fundamental property of cells that influences many aspects of their physiology. This is because cell size sets the scale for all subcellular components and drives changes in the composition of the proteome. Given that large and small cells differ in their biochemical composition, we hypothesized that they should also differ in how they respond to signals and make decisions. Here, we investigated how cell size affects the susceptibility of human cells to cell death. We found that large cells are more resistant to ferroptosis caused by system x_c^- inhibition. Ferroptosis is a type of cell death characterized by the iron-dependent accumulation of toxic lipid peroxides. This process is opposed by cysteine-dependent lipid peroxide detoxification mechanisms. We found that larger cells exhibit higher concentrations of the cysteine-containing metabolite glutathione and lower concentrations of membrane lipid peroxides. Mechanistically, this can be explained by the fact that larger cells had lower concentrations of an enzyme that enriches cellular membranes with peroxidation-prone polyunsaturated fatty acids, ACSL4, and increased concentrations of the glutathione-producing enzymes glutamate-cysteine ligase and glutathione synthetase, the iron-chelating protein ferritin, and the lysosomal protease cathepsin B, which can catabolize cysteine-rich extracellular proteins to produce additional cystine for fueling the synthesis of glutathione. Taken together, our results highlight the significant impact of cell size on cellular function and survival, revealing a size-dependent vulnerability to ferroptosis that could influence therapeutic strategies based on this cell death pathway.

Purified zymogens reveal mechanisms of snake venom metalloproteinase auto-activation

imre.berger@bristol.ac.uk (Alastair Poole) — Wed, 10 Jun 2026 00:00:00 +0000

Snake venoms contain diverse mixtures of toxins that evolved to incapacitate prey, but in humans, they cause extensive pathology following snakebite envenomation. In viper venom, some of the most potent toxins are the haemorrhagic and coagulopathic snake venom metalloproteinases (SVMPs). Because venoms contain an SVMP cocktail and due to their cytotoxicity, SVMP characterisations have been hampered by the lack of purified enzymes. By incorporating their prodomain, which blocks the active SVMP site, we overcame their cytotoxicity and enabled recombinant production of zymogens from all three structurally variable SVMP classes (PI, PII, and PIII) using our baculovirus/insect cell expression system. Zymogens were auto-activated by incubation with Zn²⁺ ions, resulting in prodomain cleavage, PII disintegrin cleavage and PIII prodomain proteolysis. Auto-activated SVMPs were characterised using protein substrate degradation, platelet aggregation and blood coagulation assays, benchmarked to native venom-purified SVMP. Our recombinant zymogen production protocol is generically applicable for the expression of SVMPs, unlocking biomedical use in haematology and discovery of novel snakebite therapeutics.

Effects of residue substitutions on the cellular abundance of proteins

lindorff@bio.ku.dk (Kresten Lindorff-Larsen) — Thu, 28 May 2026 00:00:00 +0000

Multiplexed assays of variant effects (MAVEs) make it possible to measure the functional impact of all possible single amino acid residue substitutions in a protein in a single experiment. Combination of variant effect data from several such experiments provides the opportunity to conduct large-scale analyses of variant effect scores measured across proteins, but can be complicated by variations in the phenotypes that are probed across experiments. Thus, using variant effect datasets obtained with similar MAVE techniques can help reveal general rules governing the effects of amino acid variation for a single molecular phenotype. In this work, we accordingly combined data from six individual variant abundance by massively parallel sequencing (VAMP-seq) experiments and analysed a total of 31,614 variant effect scores reporting solely on the impact of single amino acid residue substitutions on the cellular abundance of proteins. Using our combined variant effect dataset, we derived and analysed a collection of amino acid substitution matrices describing the average impact on cellular abundance of all residue substitution types in different structural environments. We found that the substitution matrices predict the cellular abundance of protein variants with surprisingly high accuracy when given structural information only in the form of whether a residue is buried or exposed. We thus propose our substitution matrix-based predictions as strong baselines for future abundance model development.

Isolation of small extracellular vesicles from small volumes of blood plasma using size exclusion chromatography and density gradient ultracentrifugation

kongfang@gmail.com (Andrew SW Wong) — Fri, 22 May 2026 00:00:00 +0000

Small extracellular vesicles (sEVs) are heterogeneous biological vesicles released by cells under both physiological and pathological conditions. Due to their potential as valuable diagnostic and prognostic biomarkers in human blood, there is a pressing need to develop effective methods for isolating high-purity sEVs from the complex milieu of blood plasma, which contains abundant plasma proteins and lipoproteins. Size exclusion chromatography (SEC) and density gradient ultracentrifugation (DGUC) are two commonly employed isolation techniques that have shown promise in addressing this challenge. In this study, we aimed to determine the optimal combination and sequence of SEC and DGUC for isolating sEVs from small plasma volumes, in order to enhance both the efficiency and purity of the resulting isolates. To achieve this, we compared sEV isolation using two combinations: SEC-DGUC and DGUC-SEC, from unit volumes of 500 μL plasma. Both protocols successfully isolated high-purity sEVs; however, the SEC-DGUC combination yielded higher sEV protein and RNA content. We further characterized the isolated sEVs obtained from the SEC-DGUC protocol using flow cytometry and mass spectrometry to assess their quality and purity. In conclusion, the optimized SEC-DGUC protocol is efficient, highly reproducible, and well suited for isolating high-purity sEVs from small blood volumes.

MORC2 mediates transcriptional regulation through liquid-liquid phase separation

huangcd@ustc.edu.cn (Chao Wang) — Wed, 20 May 2026 00:00:00 +0000

MORC2 is a chromatin-associated ATPase essential for transcriptional silencing and genome stability, yet the biophysical principles governing its regulatory activity remain elusive. Here, we demonstrate that full-length MORC2 undergoes biomolecular condensation to form dynamic nuclear assemblies, a process fundamentally required for its repressor function. Endogenous MORC2 forms discrete, dynamic condensates in neurons from Morc2a^EGFP chimeric mice, supporting the physiological relevance of these assemblies in vivo. Mechanistically, a 3.1 Å crystal structure of coiled-coil 3 (CC3) identifies a dimeric scaffold that serves as a structural hub, while multivalent ‘sticker’ interactions between an intrinsically disordered region (IDR) and a newly defined IDR-binding domain (IBD) drive condensation. We show that DNA acts as a molecular scaffold that triggers MORC2 condensation, which in turn allosterically stimulates its ATPase activity. Critically, by employing a ‘killswitch’ strategy to decouple assembly from internal fluidity, we reveal that only dynamic MORC2 condensates, not static aggregates or condensation-deficient mutants, can restore transcriptional regulation in MORC2-knockout cells. Furthermore, pathogenic variants linked to CMT2Z and SMA differentially perturb these material properties and enzymatic turnover, providing a mechanistic link between condensate dysregulation and human neuropathies. Together, our findings establish a DNA-templated condensation mechanism for MORC2 and provide a molecular framework for understanding how the material state of chromatin-associated machinery dictates gene regulation and disease pathogenesis.

Identification and classification of ion channels across the tree of life provide functional insights into understudied CALHM channels

wei.lu@northwestern.edu (Kennady Boyd) — Mon, 18 May 2026 00:00:00 +0000

The ion channel (IC) genes encoded in the human genome play fundamental roles in cellular functions and disease, and are one of the largest classes of druggable proteins. However, limited knowledge of the diverse molecular and cellular functions carried out by ICs presents a major bottleneck in developing selective chemical probes for modulating their functions in disease states. The wealth of sequence data available on ICs from diverse organisms provides a valuable source of untapped information for illuminating the unique modes of channel regulation and functional specialization. However, the extensive diversification of IC sequences and the lack of a unified resource present a challenge in effectively using existing data for IC research. Here, we perform integrative mining of available sequence, structure, and functional data on 419 human ICs across disparate sources, including extensive literature mining by leveraging advances in LLMs to annotate and curate the full complement of the ‘channelome’. We employ a well-established orthology inference approach to identify and extend the IC orthologs across diverse organisms to above 48,000. We show that the depth of conservation and taxonomic representation of IC sequences can further be translated to functional similarities by clustering them into functionally relevant groups, which can be used for downstream functional prediction on understudied members. We demonstrate this by delineating co-conserved patterns characteristic of the understudied family of the calcium homeostasis modulator (CALHM) family of ICs. Through mutational analysis of co-conserved residues altered in human diseases and electrophysiological studies, we show that these evolutionarily constrained residues play an important role in channel gating functions. Thus, by providing new tools and resources for performing large comparative analyses on ICs, this study addresses the unique needs of the IC community and provides the groundwork for accelerating the functional characterization of dark channels for therapeutic intervention.

The role of ATP synthase subunit e (ATP5I) in mediating the metabolic and antiproliferative effects of metformin in cancer cells

sp.gravel@umontreal.ca (Ana Maria Duman) — Fri, 15 May 2026 00:00:00 +0000

Here, we identify the subunit e of F₁F₀-ATP synthase (ATP5I) as a target of metformin, a first-in-class antidiabetic biguanide. ATP5I maintains the stability of F₁F₀-ATP synthase dimers, which is crucial for shaping cristae morphology. We demonstrate that ATP5I interacts with a biguanide analogue in vitro, and disabling its expression by CRISPR–Cas9 in pancreatic cancer cells leads to the same phenotype as biguanide-treated cells, including mitochondrial morphology alterations, reduction of the NAD⁺/NADH ratio, inhibition of oxidative phosphorylation (OXPHOS), rescue of respiration by uncouplers, and a compensatory increase in glycolysis. Notably, metformin disrupts F₁F₀-ATP synthase oligomerization, leading to the accumulation of vestigial assembly intermediates in pancreatic and osteosarcoma cancer cells, a phenotype also observed upon ATP5I inactivation in pancreatic cancer cells. Moreover, ATP5I knockout (KO) cells exhibit resistance to the antiproliferative effects of biguanides, but reintroduction of ATP5I rescues the metabolic and antiproliferative effects of metformin and phenformin. Finally, a genome-wide CRISPR screening in NALM-6 lymphoma cells revealed that metformin-treated cells exhibit genetic interaction profiles similar to those observed with the F₁F₀-ATP synthase inhibitor oligomycin, but not with the complex I inhibitor rotenone. This provides unbiased support for the relevance of the newly proposed target.

How membranes shape up for lipid transfer

tendo@cc.kyoto-su.ac.jp (Takashi Hirashima) — Thu, 07 May 2026 00:00:00 +0000

The extraction of a phospholipid called phosphatidic acid from the mitochondrial outer membrane is regulated by the curvature of this membrane.

Natural xanthones as α-Mangostin induce vasorelaxation involving key gating residues in the S6 domain of BK channels

m.musinszki@physiologie.uni-kiel.de (Marianne A Musinszki) — Wed, 06 May 2026 00:00:00 +0000

Polyphenolic compounds are widely explored for health benefits, including hypertension, but their active ingredients, molecular targets, and mechanisms remain poorly defined. We identify the xanthone Mangostin from Garcinia mangostana as a potent modulator of several potassium channels, with large-conductance K⁺ (BK) channels as its primary target for vasorelaxation. Mangostin-activated BK channels as α subunits alone, in complexes with vascular β1 subunits, and in reconstituted BKα/β1–Ca_v nanodomains. It shifted BK voltage activation to more negative potentials by antagonizing channel closure and promoting channel opening without markedly altering Ca²⁺ sensitivity. Docking, competition, single-channel analysis, and mutagenesis localized the binding site in the pore cavity below the SF, involving gating-critical S6 residues I308, L312, and A316, and suggest that Mangostin stays bound in closed and open states. These findings establish BK channel activation as the core molecular mechanism driving Mangostin’s vascular effects and define its structural mode of action, informing nutraceutical safety assessment and BK-targeted drug design.

Towards a unified molecular mechanism for ligand-dependent activation of NR4A-RXR heterodimers

douglas.kojetin@vanderbilt.edu (Douglas J Kojetin) — Thu, 30 Apr 2026 00:00:00 +0000

A subset of nuclear receptors (NRs) function as permissive heterodimers with retinoid X receptor (RXR), defined by transcriptional activation in response to RXR agonist ligands. Permissive NR-RXR activation is generally understood to operate through a classical pharmacological mechanism in which RXR agonist binding enhances coactivator recruitment to the heterodimer. However, we previously demonstrated that transcriptional activation of permissive Nurr1-RXRα (NR4A2-NR2B1) heterodimers by an RXR ligand set, which included pharmacological RXR agonists and selective Nurr1-RXRα agonists that function as antagonists of RXRα homodimers, is explained by a non-classical activation mechanism involving ligand-binding domain (LBD) heterodimer dissociation (Yu et al., 2023). Here, we extend mechanistic ligand profiling of the same RXR ligand set to the evolutionarily related Nur77-RXRγ (NR4A1-NR2B3) heterodimer. Biochemical and NMR protein-protein interaction profiling, together with cellular transcription studies, indicate that activation of Nur77-RXRγ transcription by the RXR ligand set, which lacks selective Nur77-RXRγ agonists, is consistent with contributions from both classical pharmacological activation and LBD heterodimer dissociation. However, reanalysis of our previously published data for Nurr1-RXRα revealed that inclusion of selective Nurr1-RXRα agonists was essential for elucidating the LBD heterodimer dissociation mechanism. Together, our findings highlight the importance of using a more functionally diverse RXR ligand set to define the mechanism of Nur77-RXRγ activation and to further evaluate whether LBD heterodimer dissociation represents a shared activation mechanism among NR4A-RXR heterodimers relevant to neurodegenerative and inflammatory diseases.

Interrogating the structure and function of the human voltage-gated proton channel (hH_v1) with a fluorescent noncanonical amino acid

zagotta@uw.edu (Emerson M Carmona) — Tue, 28 Apr 2026 00:00:00 +0000

The human voltage-gated proton channel (hH_v1) is a dimer of voltage-sensor domains (VSDs) containing highly selective proton permeation pathways in each monomer. In addition to voltage, hH_v1 is regulated by other stimuli, including pH gradients, mechanical forces, and ligands, such as Zn²⁺. Aside from the VSDs, this membrane protein contains an N-terminal domain and a C-terminal coiled-coil domain (CC) formed between the monomers. To address the need for direct measurements of conformational rearrangements in hH_v1, we developed a Förster resonance energy transfer (FRET) approach to measuring the conformational rearrangements in full-length hH_v1 purified from E. coli. We used genetic code expansion (GCE) to generate a library of 14 full-length hH_v1 constructs, each incorporating the fluorescent noncanonical amino acid acridon-2-ylalanine (Acd) at a different site throughout the various structural domains. Following the expression and purification of these hH_v1-Acd proteins, we found that 12 sites yielded stable and functional proton-permeable channels. The fluorescence properties of Acd at each site showed small site-specific differences. Furthermore, we measured site-specific FRET efficiencies from tryptophan (Trp) and tyrosine (Tyr) to Acd in the hH_v1-Acd proteins and found results consistent with correct folding in detergent micelles. Finally, the addition of Zn²⁺ produced reversible changes in FRET, with affected residues clustered on the intracellular side of the channel.

Distinct mechanisms of inhibition of Kv2 potassium channels by tetraethylammonium and RY785

jfg4wrk@gmail.com (Esam A Orabi) — Mon, 27 Apr 2026 00:00:00 +0000

Voltage-gated K⁺ channels play central roles in human physiology, in health, and disease. A repertoire of inhibitors that are both potent and specific would, therefore, be of great value. RY785 has been described as promising in this regard, as it selectively inhibits channels in the Kv2 subfamily with high potency. Its mechanism of action has not yet been determined at the molecular level, but functional studies indicate it differs from those of less specific inhibitors, such as quaternary-ammonium compounds or aminopyridines. To examine this mechanism at the single-molecule level, we have carried out a series of all-atom molecular dynamics simulations based on the structure of the Kv2.1 channel in the ion-conducting state. The simulations demonstrate both RY785 and tetraethylammonium spontaneously enter the channel interior through the cytoplasmic gate, but with distinct effects. Tetraethylammonium binds to a site adjacent to the selectivity filter, on the pore axis, thus blocking the flow of K⁺ ions. RY785, by contrast, binds to the channel walls, off-axis, and allows K⁺ flow while the gate remains open. This observation indicates RY785 inhibits Kv2.1 by fostering the occlusion of the gate, through a network of hydrophobic interactions therein, explaining why it also modulates the voltage-sensing mechanism of the channel, 3 nanometers away.

A lipoprotein partner for the Escherichia coli outer membrane protein TolC

bfl20@cam.ac.uk (Andrzej Harris) — Wed, 15 Apr 2026 00:00:00 +0000

The outer membrane protein TolC from Escherichia coli belongs to an extensive superfamily whose members are found throughout the didermal, Gram-negative bacterial lineages. The protein serves as an activated exit duct in multi-drug efflux pumps and protein secretion machinery. Many TolC homologues bear a lipid modification on the N-terminus that embeds into the inner leaflet of the outer membrane and appears to have been a conserved feature; however, the moiety is absent entirely in the E. coli TolC. We have discovered that the E. coli lipoprotein YbjP interacts extensively with the periplasmic surface of TolC and its N-terminal lipid moiety is embedded in the membrane, mimicking the intramolecular and modification-membrane interactions seen in TolC homologues. Here, we present cryo-EM structures of the MacA-MacB-TolC and AcrA-AcrB-TolC tripartite pumps complexed to YbjP. Although the association occurs spontaneously both in vitro and in vivo, the YbjP-TolC interaction is not required for efflux activity under standard laboratory conditions. YbjP may contribute to stabilising the orientation and distribution of TolC in the outer membrane, as well as the expression of transporters for tryptophan and cyclic peptide toxins.

Investigating the native functions of [NiFe]-CODH through genomic context analysis

henrik.land@kemi.uu.se (Henrik Land) — Tue, 07 Apr 2026 00:00:00 +0000

Carbon monoxide dehydrogenases containing nickel-iron active sites ([NiFe]-CODHs) catalyze the reversible oxidation of CO to CO₂, representing key targets for biocatalytic CO₂ reduction. Despite dramatic differences in catalytic rates and O₂ tolerance between CODH variants, the molecular basis for this functional diversity remains poorly understood. We applied comparative genomics and synteny analysis to investigate the biochemical roles of CODH clades A-F using 1376 CODH and 1545 hybrid cluster protein sequences. Around 30% of genomes encode multiple CODH isoforms. Analysis revealed distinct gene clustering patterns correlating with biochemical function. Clades A, E, and F exhibit a degree of distributional exclusivity. Clades C and D frequently co-occur with active CODHs, suggesting auxiliary roles. Operon architecture analysis revealed functional specialization: clade A links to acetyl-CoA synthase; clades A, E, and F contain essential maturation machinery (CooC, CooJ, CooT) correlating with catalytic activity; clade B associates with transporters; clade C with electron transfer partners; clade D with transcriptional regulators. High CODH-HCP co-occurrence (except clade A) suggests functional or environmental interdependency. These findings establish clades A, E, and F as primary biocatalyst targets while defining regulatory functions for clades C and D, providing a genomics framework for predicting CODH phenotypes.

Transforming a fragile protein helix into an ultrastable scaffold via a hierarchical AI and chemistry framework

pengz@nju.edu.cn (Bin Zheng) — Thu, 02 Apr 2026 00:00:00 +0000

The rational design of proteins that maintain structural integrity under concurrent thermal, mechanical, and chemical stress remains a challenge in molecular engineering. We present a hierarchical framework that transforms an α-helical domain into an ultrastable scaffold by integrating AI-guided design with foundational chemical principles. This approach progresses from global architectural reinforcement, using multiple AI tools to create a stabilized four-helix bundle, to local chemical tuning, where AlphaFold3 guides the installation of salt bridges and metal-coordination motifs. A computational pipeline using physics-based screening such as molecular dynamics simulations efficiently distilled millions of designs into a minimal candidate set. The resulting α-helical proteins exhibit unprecedented multi-axis stability, with mechanical unfolding forces exceeding 200 pN, thermal resilience>100°C, and high resistance to chemical denaturants. By systematically dissecting the contributions of hydrophobic packing, electrostatics, and metal coordination, we establish a general blueprint for imparting extreme robustness. This work bridges AI-driven structural generation with chemical precision, advancing the creation of durable proteins for mechanistic studies and synthetic biology.

Stall force measurement of the kinesin-3 motor KIF1A using a programmable DNA origami nanospring

iwakim@nict.go.jp (Hiroko Furumoto) — Wed, 25 Mar 2026 00:00:00 +0000

DNA origami technology is a method for designing and constructing nanoscale structures using DNA, and it is being applied across various fields. This technology was advanced by developing the nanospring (NS), a fluorescently visible molecular spring that quantifies forces through its extension and has been used to measure myosin-generated forces. This study aims to measure the force exerted by the kinesin-3 motor protein KIF1A, mutations of which cause KIF1A-associated neurological disorder (KAND) and are associated with reduced force and motility. Unlike kinesin-1, KIF1A detaches easily under perpendicular loads, which can occur in optical tweezers experiments. By applying force parallel to the microtubule using the NS, we were able to precisely measure the stall force even for KAND mutants, for which such measurements are typically challenging. This result highlights the potential of the NS as a new tool for force spectroscopy in biophysics.

Superoxide dismutases maintain niche homeostasis in stem cell populations

devanjan@bhu.ac.in (Aishwarya Chhatre) — Mon, 23 Mar 2026 00:00:00 +0000

Reactive oxygen species (ROS), predominantly derived from mitochondrial respiratory complexes, have emerged as key molecules influencing cell fate decisions like maintenance and differentiation. These redox-dependent events are mainly considered to be cell intrinsic in nature; on the contrary, our observations indicate involvement of these oxygen-derived entities as intercellular communicating agents. In Drosophila male germline, Germline Stem Cells (GSCs) and neighbouring Cyst Stem Cells (CySCs) maintain differential redox thresholds where CySCs have higher redox state compared to the adjacent GSCs. Disruption of the redox equilibrium between the two adjoining stem cell populations by depleting Superoxide Dismutases (SODs), especially Sod1, results in deregulated niche architecture and loss of GSCs, which was mainly attributed to loss of contact-based receptions and uncontrolled CySC proliferation due to ROS-mediated activation of self-renewing signals. Our observations hint towards the crucial role of differential redox states where CySCs containing higher ROS function not only as a source of their own maintenance cues but also serve as non-autonomous redox moderators of GSCs. Our findings underscore the complexity of niche homeostasis and predicate the importance of intercellular redox communication in understanding stem cell microenvironments.

Dietary sulfur amino acid restriction elicits a cold-like transcriptional response in inguinal but not epididymal white adipose tissue of male mice

pmr96@cornell.edu (Aylin S Gueller) — Tue, 17 Mar 2026 00:00:00 +0000

About 1 billion people are living with obesity worldwide. GLP-1-based drugs have massively transformed care, but long-term consequences are unclear in part due to reductions in energy expenditure with ongoing use. Diet-induced thermogenesis (DIT) and cold exposure (CE) raise EE via brown adipose tissue (BAT) activation and beiging of white adipose tissue (WAT). Methionine restriction (MetR) is a candidate DIT stimulus, but its EE effect has not been benchmarked against CE, nor have their tissue-level interactions been defined. In a 2×2 design (Control vs. MetR; room temperature, RT: 22°C vs. CE: 4°C for 24 hr), we used male C57BL/6 N mice to benchmark MetR-induced thermogenesis against CE and mapped how diet and temperature interact across tissues. Bulk RNA-seq profiled liver, iBAT, iWAT, and eWAT. Differential expression was modeled with main effects and a diet × temperature interaction. KEGG GSEA was used to assess pathway-level enrichment. MetR increased EE at RT and shifted fuel use towards lipid oxidation, supporting MetR as a bona fide DIT stimulus. CE elevated EE across diets and blunted diet differences. Transcriptomic responses were tissue-specific: in liver, CE dominated gene induction while MetR and CE cooperatively repressed genes. The combination enriched glucagon/AMPK-linked and core metabolic pathways. In iBAT, CE dominated thermogenic and lipid-oxidation programs with minimal MetR contribution. In iWAT, MetR and CE acted largely additively with high concordance, enhancing fatty-acid degradation, PPAR signaling, thermogenesis, and TCA cycle pathways. In eWAT, robust co-dependent and synergistic differential expression emerged only with MetR+CE. MetR is a genuine DIT stimulus that remodels metabolism in a tissue-specific manner. Our study provides a tissue-resolved transcriptomic resource that benchmarks diet-induced (MetR) against cold-induced thermogenesis and maps their interactions across liver, iBAT, iWAT, and eWAT.