The exact biological role of mitochondrial supercomplexes remains debated, particularly their role in guiding redox proteins across membranes during energy conversion. We integrate multiscale modeling and single particle cryo-electron microscopy (cryo-EM) to examine electron transfer in mitochondrial supercomplexes composed of complexes III and IV (CIII and CIV). Using bioinformatic and entropy-based methods, we generated structural ensembles capturing conformations of CIII's disordered QCR6 hinge within the yeast CIII2CIV2 supercomplex. Molecular and Brownian Dynamics simulations reveal that these negatively charged hinge states electrostatically couple with redox proteins, promoting their binding and directional diffusion across the membrane on millisecond timescales. Rather than hindering transfer, disorder lowers the diffusion barrier. Anionic lipids reinforce this recognition by retaining a membrane pool of redox proteins when hinge length is critical. Cryo-EM models of ΔQCR6 show large rearrangements, yet maintain a robust electrostatic environment enabling surface-mediated transfer despite reduced charge. Overall, electron carriers confined on bioenergetic membranes follow a refolding-guided diffusion mechanism that enhances supercomplex energy conversion efficiency by nearly 30%.

The circadian clock is essential for maintaining cellular homeostasis and physiological fitness. At the molecular level, core clock proteins function via transcriptional-translational feedback loops in the cellular oscillator, and are highly regulated by post-translational modifications. Our unbiased screening of core clock proteins revealed that Cryptochrome 1 (CRY1), the central transcriptional repressor in the circadian clock, undergoes a novel post-translational modification known as S-acylation. We show that this reversible lipidation of CRY1 is required for its nuclear import and interaction with key clock components. Further, we mapped four cysteine residues as CRY1 S-acylation sites and identified DHHC3 as the primary protein acyltransferase for CRY1. Importantly, loss of CRY1 S-acylation, either via cysteine mutagenesis or genetic deletion of DHHC3, impaired CRY1 repressor function and consequently cellular circadian rhythms, suggesting that dynamic S-acylation couples cytoplasmic regulation of CRY1 and its transcriptional repressor function in the nucleus. Together, our findings identify S-acylation as a previously unknown post-translational modification of CRY1 critical for circadian clock function and establish DHHC3 as a pivotal circadian regulatory enzyme. Targeting CRY1 S-acylation or its regulatory enzymes may constitute an innovative therapeutic approach against clock-associated diseases.

To understand how gyrase interacts with DNA and selects a site of action, we created an ad hoc shape-based recognition methodology to ascertain the DNA sequence from cryoEM density maps as a string of purines and pyrimidines, which matched to the DNA minicircle sequence in our two previous cryoEM structures of negatively supercoiled DNA bound to E. coli gyrase. For one structure, the Gate- or G-segment sequence contains base-pair steps that are among the most flexible in the minicircle, facilitating the bend. The sequence flanking this G-segment is highly inflexible, preventing wrapping the β-pinwheel of gyrase. In the other structure, a flexible DNA minicircle sequence wraps a β-pinwheel of gyrase and the G-segment contains base-pair steps of average deformability. This work highlights how DNA sequence and deformability impact gyrase. It also demonstrates the utility of both identifying DNA sequences from cryoEM structures and assessing base-pair step deformability.

Calcium release through inositol 1,4,5-trisphosphate receptors (IP3Rs) is a fundamental signaling mechanism that regulates diverse cellular processes. Among the three mammalian IP3R isoforms, IP3R2 is widely expressed, yet its structural basis for activation and regulation remains unclear. Here, we report cryo-EM structures of mammalian IP3R2 in ligand-free (closed) and Ca2+/IP3/ATP-bound (activated) states at 3.3 Å and 3.6 Å resolution, respectively. These structures define the architecture of IP3R2 and reveal conformational transitions associated with channel activation. Although the IP3-binding pocket is conserved, subtype-specific differences in IP3 affinity likely arise from conformational dynamics of the regulatory ARM2 domain. Comparative analyses of IP3R isoforms identify subtype-specific allosteric networks and domain motions that underlie differential regulation. We further define the ATP-binding site and, through mutagenesis and electrophysiology, establish the structural basis for ATP modulation of channel activity. Together, these findings reveal mechanisms of IP3R2 activation and subtype-specific regulation, providing a framework for understanding isoform-dependent Ca2+ signaling.

Cerebellar ataxias are characterized by impaired motor coordination resulting from neuronal dysfunction within the cerebellum. The mechanisms underlying this pathology and its cerebellar-specific neurodegeneration remain unknown. We uncover how a gain-of-function canonical transient receptor potential member 3 (TRPC3) mutation, coupled with a cerebellum-specific isoform, stabilizes the channel's open state, resists the leading inhibitor Pyr3, and drives calcium-dependent cell death. Restoring calcium homeostasis by expressing a Purkinje cell calcium pump improves cell viability. Transgenic expression of the TRPC3 hypermorphic variant in Caenorhabditis elegans induces neurodegeneration, confirming its pathogenicity across species. Cryo-electron microscopy and molecular simulations reveal the structural basis for the stabilization of the cerebellar-specific TRPC3 variant in its open state and uncover a druggable allosteric inhibitory binding site. These findings provide an explanation for the vulnerability of cerebellar neurons in TRPC3-associated ataxias and highlight a site for therapeutic intervention.

The c-SN6j/TRC105 antibody does not recognize murine or rhesus endoglin.

List of antibodies and reagents used in the study.

There are limited therapeutic options for patients with advanced sarcomas, which leads to dismal outcomes for children and adults. Although chimeric antigen receptor (CAR) T cells hold promise for treating advanced sarcomas, this approach is constrained by a paucity of effective targets. Our previous clinical study identified endoglin (ENG/CD105), a TGFβ coreceptor, as a target of the endogenous immune response in a patient with sarcoma who exhibited an exceptional response to HER2-targeted CAR T-cell therapy. ENG is expressed on various sarcomas, cancer-associated fibroblasts, and neoangiogenic vessels and therefore offers comprehensive tumor targeting. Furthermore, ENG knockout in sarcoma cells reduces their invasiveness, highlighting its potential as a therapeutic target. Accordingly, we designed a second-generation human ENG-targeting CAR molecule signaling through the CD28 endodomain and retrovirally transduced primary human T cells with this CAR. ENG CAR T cells exhibited strong antigen-specific cytokine release, robust proliferation, memory formation, and cytotoxic function against various sarcoma cell lines. Their cytotoxicity remained unaffected by the presence of soluble ENG or its natural ligand, bone morphogenetic protein-9. Furthermore, ENG CAR T cells disrupted multicellular tumor spheroids in vitro, overcoming tumor compactness and the stromal barrier created by cancer-associated fibroblasts, which are critical challenges in sarcoma CAR T-cell therapy. In orthotopic xenograft models of sarcomas, ENG CAR T-cell treatment resulted in control of tumor growth and metastasis, leading to survival extension. In summary, our study describes the involvement of ENG in sarcoma metastasis and validates our human ENG CAR T cells as a potential therapeutic for advanced sarcomas.

ENG CAR design and molecular modeling.

The bacterial flagellar filament acts as a propeller to drive most bacterial swimming. The filament is made of flagellin, known as FliC in Escherichia coli. FliC consists of four domains, the highly conserved core D0 and D1 domains and the hypervariable outer D2 and D3 domains. The size and structure of the outer domains vary, being completely absent in some bacterial species. Here, we sought to identify outer domains from various species that are compatible with the ability of E. coli K-12 FliC to form filaments capable of supporting motility. We calculated a phylogeny of 210 representative flagellin amino acid sequences and generated a series of FliC variants, including outer domain-deleted forms and 11 chimeric FliC mutants using domains from E. coli K-12, Salmonella Typhimurium, Pseudomonas aeruginosa, Collimonas fungivorans, Helicobacter mustelae, and Mesorhizobium sp. ORS3359 in various combinations. Notably, two of the chimeric fliC mutants rescued motility in a fliC-disrupted E. coli K-12 strain, both of which contained the S. Typhimurium D2 domain. Overall, we demonstrate that, while most FliC chimeras did not support motility, interchangeability of the outer domains can produce filaments that provide motility, providing insights to guide the design of synthetic flagellins.IMPORTANCEFlagellin is a key protein forming the filament of the bacterial flagellar motor which powers most bacterial swimming. Flagellin can have hypervariable domains which can alter motility in different environments and provide immune evasion. Here we engineered two flagellin chimeras that could drive motility. This indicates that the flagellin outer domains can be exchanged, to some degree, allowing us to refine rational design approaches for engineering of bacterial swimming. Our work shows the challenges to overcome when combining flagellins from different species and provides evidence that domain-switched flagellins can form filaments.

Bacterial flagella drive motility in many species, likely including the last bacterial common ancestor1,2. Knowledge of flagellar assembly and function has mainly come from studies of Escherichia coli and Salmonella enterica, which have simple flagellar motors3-7. However, most flagellated bacteria possess complex motors with unique, species-specific adaptations whose mechanisms and evolution remain largely unexplored8-10. Here, we deploy a multidisciplinary approach to build a near-complete model of the flagellar motor in Campylobacter jejuni, revealing its remarkable complexity in architecture and composition. We identify an E-ring around the MS-ring, a periplasmic cage with two distinctive conformations, and an intricate interaction network between the E-ring and cage. These scaffolds play critical roles in stabilizing and regulating 17 torque-generating stator complexes for optimal motility. In-depth evolutionary analyses uncover the ancient origin and prevalence of the E-ring in flagellated species of the domain Bacteria as well as a unique exaptation of type IV pili components PilMNOPQF in the ancestral motor of the phylum Campylobacterota. Collectively, our studies reveal novel mechanisms of assembly and function in complex flagellar motors and shed light on the evolution of flagella and modern bacterial species.

Rhabdomyosarcoma cell lines with transcript expression of ENG, CD44, CD70, and CD90 from the CCLE public release 24Q4 with fusion status and histology.

PIEZO channels are mechanosensitive ion channels conserved from plants to humans, yet structures exist for only a few mammalian orthologs. We define the structural and functional diversity of Caenorhabditis elegans PEZO-1, a single gene with extensive alternative splicing, by determining cryo-electron microscopy structures of three representative isoforms: G (full length), K (lacking the pore-distal N-terminal blade), and L (missing most of the blade). PEZO-1G displays mechanically evoked currents yet adopts a compact, semi-flattened conformation that significantly differs from the mammalian domes. The blades exhibit a three-step slope architecture stabilized by inter-blade latching among transmembrane helical units, yielding a circular, steering-wheel-like arrangement. A wider cap enables distinct blade-cap contacts that stabilize a “toggle-down” conformation. Isoform K also exhibits mechanically evoked currents, indicating that the pore-distal N-terminal blade is dispensable for mechanoactivation. Computational membrane-deformation modeling indicates that the isoforms impose distinct curvatures on the bilayer. Our findings indicate an evolutionarily distinct architecture for PEZO-1.

Activation induced cytokine release and proliferation of ENG CAR-T.

ENG expression in sarcoma tissues and cell lines.

Many bacteria swim by the rotation of the bacterial flagellar motor (BFM). The BFM is powered by proton translocation across the inner membrane through the heteroheptameric MotA5MotB2 protein complex. Two periplasmic domains of MotB are critical in activating BFM rotation: (1) the peptidoglycan (PG) binding domain that anchors MotB in the PG layer and (2) the plug domain that modulates the proton flow. Existing cytoplasmic fluorescent probes have been shown to negatively affect motor rotation and switching. Here, we inserted a fluorescent probe in the periplasm near the plug of MotB to circumvent issues with cytoplasmic probes and for possible use in observing the mechanism of plug-based regulation of proton flow. We inserted green fluorescent protein and improved light-oxygen-voltage (LOV), a fluorescent version of the LOV domain, in four periplasmic locations in MotB. Insertions near the plug retained motility but showed limited fluorescence for both fluorophores. Additional short, flexible glycine-serine linkers improved motility but did not improve brightness. Further optimization is necessary to improve the fluorescence of these periplasmic probes.

Efficacy of ENG CAR-T in orthotopic xenograft models of sarcoma.

Light is a key environmental component influencing many biological processes, particularly in prokaryotes such as archaea and bacteria. Light control techniques have revolutionized precise manipulation at molecular and cellular levels in recent years. Bacteria, with adaptability and genetic tractability, are promising candidates for light control studies. This review investigates the mechanisms underlying light activation in bacteria and discusses recent advancements focusing on light control methods and techniques for controlling bacteria. We delve into the mechanisms by which bacteria sense and transduce light signals, including engineered photoreceptors and light-sensitive actuators, and various strategies employed to modulate gene expression, protein function, and bacterial motility. Furthermore, we highlight recent developments in light-integrated methods of controlling microbial responses, such as upconversion nanoparticles and optical tweezers, which can enhance the spatial and temporal control of bacteria and open new horizons for biomedical applications.

ABSTRACT Powered by ion transport across the cell membrane, conserved ion-powered rotary motors (IRMs) drive bacterial motility by generating torque on the rotor of the bacterial flagellar motor. Homologous heteroheptameric IRMs have been structurally characterized in ion channels such as Tol/Ton/Exb/Gld, and most recently in phage defense systems such as Zor. Functional stator complexes synthesized from chimeras of PomB/MotB (PotB) have been used to study flagellar rotation at low ion-motive force achieved via reduced external sodium concentration. The function of such chimeras is highly sensitive to the location of the fusion site, and these hybrid proteins have thus far been arbitrarily designed. To date, no chimeras have been constructed using interchange of components from Tol/Ton/Exb/Gld and other ion-powered motors with more distant homology. Here, we synthesized chimeras of MotAB, PomAPotB, and ExbBD to assess their capacity for cross-compatibility. We generated motile strains powered by stator complexes with B-subunit chimeras. This motility was further optimized by directed evolution. Whole-genome sequencing of these strains revealed that motility-enhancing residue changes occurred in the A-subunit and at the peptidoglycan binding domain of the B-unit, which could improve motility. Overall, our work highlights the complexity of stator architecture and identifies the challenges associated with the rational design of chimeric IRMs. IMPORTANCE Ion-powered rotary motors (IRMs) underpin the rotation of one of nature’s oldest wheels, the flagellar motor. Recent structures show that this complex appears to be a fundamental molecular module with diverse biological utility where electrical energy is coupled to torque. Here, we attempted to rationally design chimeric IRMs to explore the cross-compatibility of these ancient motors. We succeeded in making one working chimera of a flagellar motor and a non-flagellar transport system protein. This had only a short hybrid stretch in the ion-conducting channel, and function was subsequently improved through additional substitutions at sites distant from this hybrid pore region. Our goal was to test the cross-compatibility of these homologous systems and highlight challenges arising when engineering new rotary motors.