European Molecular Biology Laboratory

Bacterial proton pumps, proteorhodopsins (PRs), are a major group of light-driven membrane proteins found in marine bacteria. They are functionally and structurally distinct from archaeal and eukaryotic proton pumps. To elucidate the proton transfer mechanism by PRs and understand the differences to nonbacterial pumps on a molecular level, high-resolution structures of PRs’ functional states are needed. In this work, we have determined atomic-resolution structures of MAR, a PR from marine actinobacteria, in various functional states, notably the challenging late O intermediate state. These data and information from recent atomic-resolution structures on an archaeal outward proton pump bacteriorhodopsin and bacterial inward proton pump xenorhodopsin allow for deducing key universal elements for light-driven proton pumping. First, long hydrogen-bonded chains characterize proton pathways. Second, short hydrogen bonds allow proton storage and inhibit their backflow. Last, the retinal Schiff base is the active proton donor and acceptor to and from hydrogen-bonded chains.

Understanding how genetic variation impacts transcription factor (TF) binding remains a major challenge, limiting our ability to model disease-associated variants. Here, we used a highly controlled system of F 1 crosses with extensive genetic diversity to profile allele-specific binding of four TFs at several time points during Drosophila embryogenesis. Using a combined haplotype test, we identified 9%–18% of TF-bound regions impacted by genetic variation even for essential regulators. By expanding WASP (a tool for allele-specific read mapping) to examine indels, we increased detection of allelically imbalanced peaks by 30%–50%. This fine-grained “mutagenesis” can reconstruct functionalized binding motifs for all factors. To prioritize causal variants, we trained a convolutional neural network (Basenji) to accurately predict binding from DNA sequence. The model can also predict measured allelic imbalance for strong effect variants, providing a mechanistic interpretation for how the variant impacts binding. This reveals unexpected relationships between TFs, including potential cooperative pairs, and mechanisms of tissue-specific recruitment of the ubiquitous factor CTCF.

The European Photon and Neutron campus in Grenoble is a unique site, encompassing the European Synchrotron Radiation Facility Extremely Brilliant Source, the Institut Laue–Langevin, the European Molecular Biology Laboratory and the Institut de Biologie Structurale. Here, we present an overview of the structural biology beamlines, instruments and support facilities available on the EPN campus. These include advanced macromolecular crystallography using neutrons or X‐rays, small‐angle X‐ray or neutron scattering, cryogenic electron microscopy, and spectroscopy. These highly complementary experimental approaches support cutting‐edge research for integrated structural biology in our large user community. This article emphasizes our significant contributions to the field, outlines current advancements made and provides insights into our future prospects, offering readers a comprehensive understanding of the EPN campus's role in advancing integrated structural biology research.

In this contribution a novel polarization gratings aided common‐path Hilbert holotomography (CP‐HHT) is presented for label‐free 3D refractive index imaging. Addressing limitations in current holotomography methods, the extended space‐bandwidth product is leveraged through robust phase demodulation using the Hilbert spiral transform. The application of polarization diffraction gratings in this system enables fully tailored holographic settings such as fringe density and shear, allowing flexible hologram demodulation, while maintaining simplicity and robustness. The performance is tested using 3D‐printed (fabricated with two‐photon polymerization) brain phantom and fixed HeLa cells supplemented with cholesterol and oleic acids. Reconstruction analysis using the brain phantom indicates that the Hilbert method provides improved spatial resolution to the Fourier transform method in a significantly expanded measurement information content. This CP‐HHT approach demonstrates the unique (not possible by fluorescence) ability to measure the lipid droplets enriched with cholesterol and oleic acid and highlights that they exhibit measurable differences in their refractive index values. These findings suggest that this method is sensitive to variations in neutral lipid content, offering promising insights into lipid droplet heterogeneity and supporting its potential for label‐free sub‐cellular bioimaging applications, thus reinforcing the versatility and applicability of this CP‐HHT system in broader bioimaging applications.

The perception of innocuous temperatures is crucial for thermoregulation. The TRP ion channels TRPV1 and TRPM2 have been implicated in warmth detection, yet their precise roles remain unclear. A key challenge is the low prevalence of warmth-sensitive sensory neurons, comprising fewer than 10% of rodent dorsal root ganglion (DRG) neurons. Using calcium imaging of >20,000 cultured mouse DRG neurons, we uncovered distinct contributions of TRPV1 and TRPM2 to warmth sensitivity. TRPV1’s absence – and to a lesser extent absence of TRPM2 – reduces the number of neurons responding to warmth. Additionally, TRPV1 mediates the rapid, dynamic response to a warmth challenge. Behavioural tracking in a whole-body thermal preference assay revealed that these cellular differences shape nuanced thermal behaviours. Drift diffusion modelling of decision-making in mice exposed to varying temperatures showed that TRPV1 deletion impairs evidence accumulation, reducing the precision of thermal choice, while TRPM2 deletion increases overall preference for warmer environments that wildtype mice avoid. It remains unclear whether TRPM2 in DRG sensory neurons or elsewhere mediates thermal preference. Our findings suggest that different aspects of thermal information, such as stimulation speed and temperature magnitude, are encoded by distinct TRP channel mechanisms.

Channelrhodopsins (ChRs) have emerged as major optogenetics tools, particularly in neuroscience. Despite their importance, the molecular mechanism of ChR opening remains elusive. Moreover, all reported structures of ChRs correspond to either a closed or an early intermediate state and lack the necessary level of detail owing to the limited resolution. Here we present the structures of the closed and open states of a cation-conducting ChR, OLPVR1, from Organic Lake phycodnavirus, belonging to the family of viral ChRs solved at 1.1- and 1.3-Å resolution at physiologically relevant pH conditions (pH 8.0). OLPVR1 was expressed in Escherichia coli and crystallized using an in meso approach, and the structures were solved by X-ray crystallography. We also present the structure of the OLPVR1 protonated state at acidic pH (pH 2.5) at 1.4-Å resolution. Together, these three structures elucidate the molecular mechanisms of the channel’s opening and permeability in detail. Extensive functional studies support the proposed mechanisms. Channel opening is controlled by isomerization of the retinal cofactor, triggering protonation of proton acceptors and deprotonation of proton donors located in the three gates of the channel. The E51 residue in the core of the central gate (similar to E90 of ChR2 from Chlamydomonasreinhardtii) plays a key role in the opening of the channel. E51 flips out of the gate and towards the proton acceptor D200 (D253 in ChR2 in C.reinhardtii), establishing a hydrogen bond between them. Despite differences in subfamilies of ChRs, they share a common gate–cavity architecture, suggesting that they could have similar general gating mechanisms. These results enabled us to design viral rhodopsin with improved properties for optogenetic applications. The structural data and mechanisms might also be helpful for better understanding other ChRs and their engineering.

Long dismissed as mere genomic parasites, transposable elements (TEs) are now recognized as major drivers of genome evolution. TEs serve as a source of cell-type specific cis-regulatory elements, influencing gene expression and observable phenotypes. However, the precise TE regulatory roles in different contexts remain largely unexplored and the impact of TEs on transcriptional regulatory networks and contribution to disease risk is likely deeply underestimated. Using a multimapper-aware strategy, we systematically characterised the epigenetic profile of TEs in the brain. This analysis revealed that MER57E3, a primate-specific TE subfamily, exhibits strong enrichment for active, and absence of repressive, histone modifications across six brain cell types. MER57E3 copies are predominantly located near zinc finger genes and enriched for homeodomain motifs recognized by brain-specific transcription factors, including GBX1 and BSX. Upon CRISPR interference (CRISPRi) targeting specific MER57E3 copies, RNA-seq analysis demonstrated downregulation of the key neurogenesis-related genes PAX6 and NEUROG2. Our data indicate that members of the MER57E3 TE subfamily regulate the expression of critical neurogenesis genes during neural progenitor cell (NPC) development. Moreover, this study emphasises the importance of characterising TEs, offering new insights into how their epigenetic dysregulation may contribute to pathogenesis of neurodevelopmental disorders.

Biological nitrogen fixation is an important source of new nitrogen, influencing ocean fertility and carbon uptake. While recently documented in Arctic waters, its role in the Southern Ocean remains uncertain. We measured nitrogen fixation along the Western Antarctic Peninsula and at Palmer Station over two austral summer months. Rates from ¹⁵N2 assay were below conservative detection limits but detectable under less stringent detection thresholds. Continuous acetylene reduction assay provided further support. nifH gene sequencing identified Gammaproteobacteria as the dominating identified diazotrophs, while Epsilonproteobacteria contributed disproportionally to nifH expression when putative nitrogen fixation was highest. Combined with environmental observations, we hypothesize that vertical water mixing resuspended sediments into the water column and contributed to the limited nitrogen fixation. Given the sporadic and low rates, further research is needed to determine whether nitrogen fixation plays a minor role or represents an overlooked process with biogeochemical significance in the Southern Ocean.

Iron restriction is a critical pathomechanism underlying the Anemia of Inflammation and an innate immune response limiting the replication of extracellular pathogens. During infections, innate immune cells detect pathogen‐associated molecular patterns (PAMPs) and produce proinflammatory cytokines. Among these, interleukin (IL)‐6 is detected by hepatocytes, where it activates the production of the iron‐regulated hormone hepcidin that inhibits iron export from macrophages. Consequently, macrophages accumulate iron and hypoferremia (low plasma iron) develops. Whether Toll‐like receptors (TLRs) expressed on hepatocytes directly recognize PAMPs and contribute to hepcidin upregulation is still an open question. Stimulation of primary murine hepatocytes with a panel of PAMPs targeting TLRs 1–9 revealed that the TLR5 ligand flagellin and the TLR2:TLR6 ligand FSL1 upregulated hepcidin. Hepcidin was also induced upon treatment with heat‐killed Staphylococcus aureus (HKSA) and Brucella abortus (HKBA). The hepcidin response to flagellin, FSL1, HKSA, and HKBA started at an early time point, was independent of autocrine regulation by IL‐6, and occurred through the TLR‐mitogen‐activated protein kinase (MAPK) axis. By analyzing a macrophage:hepatocyte co‐culture, we additionally show that the hepcidin response was dependent on TLR2:TLR6 expression in hepatocytes and independent of macrophage cytokine secretion. Ex vivo liver perfusion of mice with FSL1 and HKSA further revealed that PAMPs and pathogens can pass the sinusoidal barrier and reach hepatocytes to cause hepcidin upregulation in a TLR2:TLR6‐dependent manner. We conclude that hepatocytes can directly recognize PAMPs and pathogens and promote hepcidin upregulation in a macrophage and cytokine‐independent manner. This positions hepatocytes in the spotlight as potential direct drivers of iron restriction.

Single alpha-helices (SAHs) are protein regions with unique mechanical properties, forming long stable monomeric helical structures in solution. To date, only a few naturally occurring SAH regions have been extensively characterized, primarily from myosins, leaving the structural and dynamic variability of SAH regions largely unexplored. Drebrin (developmentally regulated brain protein) contains a predicted SAH segment with unique sequence characteristics, including aromatic residues within the SAH region and a preference for arginine over lysine in its C-terminal half. Using and NMR spectroscopy, combined with SAXS measurements, we demonstrate that the Drebrin-SAH is helical and monomeric in solution. NMR resonance assignment required specific 4D techniques to resolve severe signal overlap resulting from the low complexity and largely helical conformation of the sequence. To further characterize its structure, we generated a structural ensemble consistent with Cα, Cβ chemical shifts and SAXS data, revealing a primarily extended structure with non-uniform helicity. Our results suggest that dynamic rearrangement of salt bridges and potential transient cation-π interactions contribute to the formation and stabilization of both helical and non-helical local conformational states.

In the canonical TGF-β signaling pathway, SMADs are transducers of receptor-activated signals. These proteins contain two well-structured domains (MH1 and MH2 domains) connected by a long, functional, unstructured linker. Beyond the physiological role of TGF-β signaling, its dysregulation is implicated in several pathologies, with many disease-causing mutations clustering within the SMAD4 MH2 domain. Here, we have discovered and structurally characterized (via X-ray crystallography) two nanobodies (Nbs) against SMAD4, with applications to study the effects of SMAD4 in TGF-β signaling. We found that NbA1 binds a region of SMAD4 MH2 domain that overlaps the R-SMAD interaction surface, preventing the formation of SMAD4/R-SMAD complexes, whereas NbA7 binding allows the formation of functional complexes. Applications of these two Nbs comprise detection of SMAD4 complexes, enabling study of the impact of disease-associated variants on complex formation, facilitating high-resolution cryo-EM structure determination, and opening avenues for therapeutic and diagnostic development.

Background Acute myeloid leukemia (AML) is an aggressive malignancy associated with poor prognosis, high relapse rates, and resistance to standard therapies. While chimeric antigen receptor (CAR) T-cell therapies targeting cell surface proteins offer potential, their efficacy in AML is limited due to the overlap of target protein expression between leukemic and healthy hematopoietic cells. To address these challenges, we utilized proteomics and surface glycoproteomics screening approaches to identify novel cell surface proteins specifically upregulated in AML patients. Leveraging these discoveries, we generated antigen specific antibodies (scFvs) and engineered third-generation CAR-T cells to target the identified AML-specific proteins, thereby enhancing and redirecting CAR-T cell efficacy against AML. Materials and Methods To identify AML-specific surface proteins, we performed LC/LC-MS on total and isolated surface glycoproteomes from AML patient samples. Antigen-specific antibodies (scFvs) for the discovered targets were then generated using a phage display library. The scFvs were incorporated into a GMP-compliant third-generation CAR backbone and expressed in primary human T cells by gammaretroviral transduction. The functionality and specificity of the engineered CAR-T cells were assessed through co-culture assays at 1:1 and 1:4 effector-to-target ratios. Anti-tumor efficacy was evaluated against antigen positive tumor cell lines in vitro. Results Detailed analysis of proteomics and glycoproteomics datasets identified 9 upregulated surface proteins in leukemia stem cells and AML, including 5 novel targets reported here for the first time. We produced 19 unique scFvs targeting extracellular domain epitopes that in CAR format, showed robust 20–80% CAR expression in engineered primary human T cells. CARs evaluated against CD206, ITGA4 and LILRB1 antigens targeted and eliminated antigen-positive tumor cells after 24 h at 1:1 and 1:4 effector-to-target ratios. Notably, anti-CD206 CAR-T cells eradicated nearly all K562 antigen-positive cells within 72 hours of co-culture. We observed a 10–15% increase in production of effector cytokines TNF-α and IFN-γ from both CD4⁺ and CD8⁺ CAR-T cells compared to non-transduced T cells. Functional differences between CARs incorporating different scFvs were found allowing their further evaluation and selection. Conclusions We present an approach to enrich surface glycoproteins in AML patients and identify AML-specific antigens through detailed proteomics analysis. The utility of these identified targets was evaluated by engineering CAR-T cells with precise targeting capabilities. These CAR-T cells, equipped with unique scFvs, demonstrated anti-tumor efficacy against antigen-positive AML cell lines in vitro. This approach paves the way for the discovery of new AML targets while effectively leveraging current immunotherapeutic tools.J.J. Brysting: None. S.M. Abdin: None. F. Schuler: None. Y. Liu: None. E. Cetin: None. B. Ince: None. J. Frenz: None. A.K. Jayavelu: None. J.J. Schwarz: None. T. Sauer: None. J.B. Zaugg: None. C. Müller-Tidow: None.