Signalling via the epidermal growth factor receptor (EGFR) is indispensable for morphogenesis and tissue homeostasis. It is activated by extracellular ligands, typically released from transmembrane precursors by proteolysis. Ligand shedding activity is provided by the conserved rhomboid intramembrane serine proteases in Drosophila, but by the unrelated ADAM family metalloproteases in mammals, leaving the functions of mammalian non-mitochondrial rhomboids underexplored. Using quantitative proteomics, we show that EGFR is the main endogenous substrate of the human rhomboid protease RHBDL2 in keratinocytes. By shedding the EGFR ectodomain, thus producing a decoy receptor, RHBDL2 suppresses EGFR signalling, limiting cell migration and invasion. Conspicuously, RHBDL2 activity is upregulated by elevated intracellular calcium concentration, a condition typical of keratinocyte differentiation. These effects are recapitulated in primary human keratinocytes, and human skin equivalents deficient in RHBDL2 display incomplete differentiation and are morphologically disordered compared to wild-type cells. We propose that context-specific fine-tuning of EGFR signalling and sensitivity to cross-talk from other signalling pathways could be important and hitherto overlooked roles of rhomboid proteases in mammals.

Most antibiotics are natural compounds or their derivatives, and bacteria have evolved defensive mechanisms to resist them. Many of these mechanisms are still poorly understood or unknown. This study reveals that in Bacillus subtilis, the transcription factor HelD increases resistance to rifampicin by protecting its target, RNA polymerase (RNAP). This protection is mediated by the HelD N-terminal domain that penetrates into RNAP to the close vicinity of the rifampicin binding pocket. Importantly, the bacterium detects low rifampicin levels using a unique regulatory system involving two convergent promoters with finely tuned kinetic properties. In the absence of rifampicin, the stronger antisense promoter inhibits transcription from the sense promoter. In the presence of subinhibitory rifampicin concentration, the antisense promoter is more likely to encounter rifampicin-bound RNAP. This relieves the repression from the sense promoter, increasing its transcription by almost two orders of magnitude, boosting helD expression. A similar two-promoter arrangement also controls the pps gene, which encodes a rifampicin-modifying enzyme. These findings define a widespread bacterial response system sensitive to rifampicin, as this dual-promoter architecture is conserved across many bacterial species and found upstream of genes potentially involved in rifampicin resistance, such as those for hydrolases, transporters, and transferases.

The Molecular-Scale Biophysics Research Infrastructure (MOSBRI) project addressed the need for enhanced European-level coverage of molecular-scale biophysics, a field situated between atomic-resolution structural biology and cellular imaging. This initiative fostered community building through networking, training, and joint research activities, providing transnational access to 25 distinct technologies, including real-time biosensing, advanced spectroscopies, hydrodynamic, thermodynamic, and single-molecule approaches. Over its four-year span, MOSBRI attracted more than 250 applications for access and engaged nearly 1000 participants across 4 international conferences, 14 training schools, and 4 benchmarking actions. Key outcomes include the development of the eSPC software suite and the Molecular Biophysics Database (MBDB). The eSPC suite is a web-based, open-source platform offering user-friendly data analysis tools for techniques such as microscale thermophoresis (MST), differential scanning fluorimetry (DSF), dynamic light scattering (DLS), circular dichroism (CD), biolayer interferometry (BLI), and mass photometry (MP). Ongoing development focuses on integrating artificial intelligence via MCP servers for enhanced, high-throughput analysis, exemplified by the CheMelt tool for global analysis of chemical and thermal denaturation in DSF, which revealed that a reduced heat capacity of unfolding is a mechanism for achieving higher thermal stability in designed proteins. The MBDB serves as a public, FAIR (Findable, Accessible, Interoperable, Reusable) repository for raw biophysical data, standardizing formats and enabling cross-method searches for ITC, BLI, MST, and SPR data, with MP integration underway. It employs precise chemistry definitions and unique persistent identifiers. The formation of the Association of Resources for Biophysical Research in Europe (ARBRE) as a legal entity ensures the continuity of MOSBRI’s community-building efforts. Outcomes of the Molecular-Scale Biophysics Research Infrastructure (MOSBRI) project : focus on the Molecular Biophysics Database (MBDB) and the eSPC software suite This webinar will introduce the EU-funded Molecular-Scale Biophysics Research Infrastructure (MOSBRI) project, and two of its more outstanding outcomes: 1) The Molecular Biophysics Database (MBDB), which allows researchers using MST, ITC, BLI and SPR to deposit their raw measurement data with appropriate details about the actual experimental conditions. 2) The eSPC web-based data analysis software suite, which allows researchers to analyse data from several different technologies: MST, DSF, DLS, CD, BLI During the webinar, speakers from the European Biophysics Journal, Springer Nature and MOSBRI will introduce the tools, explain the rationale for using them, how to use them, and will be open to question answer. For more details about the special collection, please see here: https://link.springer.com/collections/idcdjihhdb Introduction to MOSBRI and its outcomes - focus of the special EBJ special issue dedicated to MOSBRI The Molecular-Scale Biophysics Research Infrastructure (MOSBRI; www.mosbri.eu), has been funded until October 2025 by the European Commission through its Horizon 2020 INFRAIA scheme, bringing together an integrated consortium of 13 academic centres of excellence and two industrial partners from 11 European countries. It carried out three types of activities: 1) Joint Research Activities enabling the emergence of novel methodologies and technologies (in partnership with instrument and software developers), the design, production and dissemination of standard reference proteins, the coining of proficiency accreditation schemes for biophysical techniques, and the establishment of data and metadata archival standards and a universal database for molecular-scale biophysics data; 2) Networking Activities such as the organization of international conferences and courses, and the creation with synergies with like-minded networks and associations, and 3) Trans-national access (TNA) provision to cutting-edge instrumentation and expertise. This talk will show how, over the last 4 years, MOSBRI has successfully contributed to consolidate the European molecular biophysics community. It will also focus on one of its publication outcomes, a special issue of the European Biophysics Journal: https://link.springer.com/collections/idcdjihhdb Most of MOSBRI activities are currently continued in the frame of the Association of Resources for Biophysical Research in Europe (ARBRE; https://www.arbre-biophysics.eu/), a new member of the European Biophysical Societies' Association (EBSA). The eSPC web-based data analysis software suite, which allows researchers to analyse data from several different technologies: MST, DSF, DLS, CD, BLI, MP The characterisation of biomolecules and their interactions is key to understanding how biological processes operate at the molecular level and to developing new therapies or biotechnological products. For this purpose, a wide range of biophysical techniques, such as circular dichroism (CD), differential scanning fluorimetry (DSF), microscale thermophoresis (MST), biolayer interferometry (BLI), mass photometry (MP), and dynamic light scattering (DLS), can be employed. The interpretation of the generated data usually requires specialized software provided with the instruments or the need to write code. To democratise access to advanced analysis tools and increase the reproducibility of the data analysis step, we have developed the open-source eSPC platform, available at https://spc.embl-hamburg.de. The eSPC platform contains user-friendly and interactive modules, freely available for academia, that allow assessment of biomolecular interactions, complex formation, sample size, homogeneity, and stability. The Molecular Biophysics Database (MBDB) The Molecular Biophysics Database (MBDB) Jan Dohnálek1, Terezie Prchalová1, Jitka Plucarová1, Tim Kadlec1, Jan Stránský1, Andrea S. Veiga2, Juan Sabín2, Miroslav Šimek3, Emil Dandanell Agerschou1 1 Institute of Biotechnology of the Czech Academy of Sciences, v.v.i., Průmyslová 595, 252 50 Vestec, Czech Republic 2 AFFINImeter, Software 4 Science Developments, Edificio Emprendia, Campus Vida, 15782 Santiago de Compostela, Spain 3 Czech Education and Scientific NETwork (CESNET), Generála Píky 430/26, 160 00 Prague 6, Czech Republic dohnalek@ibt.cas.cz With the number of methods of molecular biophysics growing every year, there are still very limited resources for storage of raw data with annotation of experimental conditions for their reuse. The Molecular Biophysics Database (MBDB) stores raw data files together with metadata descriptions. The design of the database makes measurement results Findable, Accessible, Interoperable and Reusable. The metadata for individual data sets consist of a general part and a method-specific part. The general part defines, in a unique way, descriptors for key parameters common for different experimental techniques, e.g. source organism, identity of individual molecules, including chemicals, with reference to external databases and unique identifiers for the most relevant types. The method-specific part is devoted to the metadata special for a particular technique (such as MST, BLI, etc.). This approach enables searching across different techniques and allows direct comparisons of results, e.g. interaction parameters, for the same molecular system measured by different techniques. The Molecular Biophysics Database (MBDB) is built using the Invenio repository platform technology (https://inveniosoftware.org/) and JSON as the key representation format of metadata, in collaboration with the CESNET data storage team and their hardware resources. MBDB was launched in January 2025 (https://mbdb-data.org/) and is ready for deposition of raw measurement files with rich metadata describing experimental details for the techniques MST, BLI, SPR and ITC [1]. We invite researchers to deposit their raw data and make them publicly available, with each record receiving a DOI identifier. We acknowledge support by the Horizon 2020 programme of EU (MOSBRI, no. 101004806) and by MEYS (CIISB support, LM2023042 and the OP JAK programme – project NRP, no. CZ.02.01.01/00/23_014/0008787). 1. Agerschou ED, Prchalová T, Šimek M, Malý M, Stránský J, Strnad M, Santisteban-Veiga A, Williams MA, Sabín J, Dohnálek J. Molecular Biophysics Database (MBDB) makes raw measurements findable and reusable. Eur Biophys J. 2025 Aug 10.

Introduction:The bacterial phosphotransferase system (PTS) transports and phosphorylates sugars. Some PTS proteins share structural motifs with rifampicin phosphotransferases (RPHs), which inactivate rifampicin by phosphorylation. This homology suggests that the PTS may represent an evolutionary ancestor of the multi domain RPHs, though direct biochemical evidence has been lacking. Methods:Bacillus subtilis strains lacking genes encoding PTS proteins were evaluated in growth assays in the absence/presence of rifampicin; liquid chromatography-mass spectrometry was used to monitor the ability of B. subtilis PTS proteins to phosphorylate rifampicin; thermophoresis was employed to characterize protein-rifampicin interactions. Results:Deletion of B. subtilis ptsH, ptsI genes (encoding PTS proteins: HPr and EI) or rphT (encoding RphT-B. subtilis RPH) impaired growth in the presence of rifampicin. In vitro, the PTS complex (HPr, EI, MtlF, and PckA) phosphorylated rifampicin, with EI alone sufficient for this activity. However, no rifampicin phosphorylation by EI was detected in vivo. Heterologous expression of rphT then strongly increased rifampicin resistance, while ptsH/ptsI expression did not. Conclusion:This study shows that part of the PTS, protein EI, can phosphorylate rifampicin, supporting its evolutionary link to RPHs. We also establish that RphT, a putative rifampicin phosphotransferase misannotated as phosphoenolpyruvate synthase (Pps), is a bona fide rifampicin-modifying enzyme in B. subtilis. Finally, we demonstrate that derepressing RphT or its horizontal transfer confers high-level resistance to rifampicin.

Tissue regeneration requires tight control of immune cell behavior, yet the mechanisms that restrain immune-driven regenerative responses remain poorly defined. Here, we identify the rhomboid intramembrane serine protease Rhbdl2 as a critical regulator of regeneration in zebrafish. We generated rhbdl2 mutants by CRISPR-Cas9 and found that it does not affect normal development, but triggers enhanced regenerative growth following injury, accompanied by increased macrophage accumulation at the wound site, which is accompanied by increased early apoptosis and proliferation. Proteomic analyses reveal increased Rac2 protein levels in rhbdl2 mutants, indicating dysregulated immune signaling. Functionally, Rac2 morpholino oligonucleotides-mediated knockdown in rhbdl2 mutant larvae suppresses the elevated macrophage recruitment and enhanced tissue regenerative phenotype. Together, these findings uncover Rhbdl2 as an immune checkpoint that constrains macrophage-driven enhanced regeneration, with vast implications for inflammatory disease, fibrosis, and tumor-immune interactions.

Open science is now established as an important paradigm for publicly funded research. The main principle being that to ensure best use of research data and integrity of the scientific process the information from experiments should be made widely and freely available. However, dedicated technical infrastructure to enable useful access to comprehensive experimental information in molecular biophysics is lacking, in particular in regard to repositories for raw measurement data. The Molecular Biophysics Database (MBDB) was created to fill this gap. The MBDB provides a common and extensible framework to store and access raw measurement data from a growing number of biophysical methods, currently including bio-layer interferometry, isothermal titration calorimetry, surface plasmon resonance, and microscale thermophoresis, with additional methods planned for the future. Alongside the raw measurement data from these methods, a rich set of metadata to enable data reuse is captured in accordance with the FAIR data management principles. An overview of the data models and technologies that were used to create the MBDB is presented here.

The activity of the light-oxygen-voltage/helix-turn-helix (LOV-HTH) photoreceptor EL222 is regulated through protein-protein and protein-DNA interactions, both triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV-visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using noncanonical amino acids. Kinetic experiments provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently bound N5 protonated, and noncovalently bound hydroquinone forms of FMN, respectively. Only subtle structural differences were observed between the monomeric forms of all three EL222 species (dark, lit1, and lit2). While the dark state is largely monomeric, both lit states undergo monomer-dimer exchange. Furthermore, molecular modeling revealed differential dynamics and interdomain separation times arising from the three FMN states (oxidized, adduct, and reduced). Unexpectedly, all three EL222 species can associate with DNA, but only upon blue-light irradiation, a high population of stable complexes is obtained. Overall, we propose a model of EL222 activation where photoinduced changes in the FMN moiety shift the population equilibrium toward an open conformation that favors self-association and DNA-binding.

The activity of the transcription factor EL222 is regulated through protein-chromophore adduct formation, interdomain dynamics, oligomerization and protein-DNA interactions, all triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV/visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using non-canonical amino acids. The observation of only subtle atomic displacements between crystal structures of EL222 with and without blue-light back-illumination, was confirmed by NMR data indicating no major changes in secondary structure and fold compactness. Kinetic experiments in solution provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently-bound N5 protonated, and non-covalently-bound hydroquinone forms of FMN, respectively. Molecular modeling revealed differential dynamics and domain separation times arising from the three FMN states (oxidized, adduct, and reduced). Furthermore, while the dark state is largely monomeric, both lit states undergo slow monomer-dimer exchange. The photoinduced loss of α-helicity, seen by infrared difference spectroscopy, was ascribed to dimeric EL222 species. Unexpectedly, NMR revealed that all three EL222 species (dark, lit1, lit2) can associate with DNA to some extent, but only under illumination a high population of stable complexes is obtained. Overall, we propose a refined model of EL222 photo-activation where photoinduced changes in the oxidation state of FMN and thioadduct formation shift the population equilibrium towards an open conformation that favors self-association and DNA-binding. ### Competing Interest Statement The authors have declared no competing interest.

Rhomboid proteases play a variety of physiological roles, but rhomboid protease inhibitors have been mostly developed for the E. coli model rhomboid GlpG. In this work, we screened different electrophilic scaffolds against the human mitochondrial rhomboid PARL and found 4-oxo-β-lactams as submicromolar inhibitors. Multifaceted computations suggest explanations for the activity at the molecular scale and provide models of covalently bound complexes. Together with the straightforward synthesis of the 4-oxo-β-lactam scaffold, this may pave the way toward selective, nonpeptidic PARL inhibitors.

In mycobacteria, σA is the primary sigma factor. This essential protein binds to RNA polymerase (RNAP) and mediates transcription initiation of housekeeping genes. Our knowledge about this factor in mycobacteria is limited. Here, we performed an unbiased search for interacting partners of Mycobacterium smegmatis σA. The search revealed a number of proteins; prominent among them was MoaB2. The σA-MoaB2 interaction was validated and characterized by several approaches, revealing that it likely does not require RNAP and is specific, as alternative σ factors (e.g., closely related σB) do not interact with MoaB2. The structure of MoaB2 was solved by X-ray crystallography. By immunoprecipitation and nuclear magnetic resonance, the unique, unstructured N-terminal domain of σA was identified to play a role in the σA-MoaB2 interaction. Functional experiments then showed that MoaB2 inhibits σA-dependent (but not σB-dependent) transcription and may increase the stability of σA in the cell. We propose that MoaB2, by sequestering σA, has a potential to modulate gene expression. In summary, this study has uncovered a new binding partner of mycobacterial σA, paving the way for future investigation of this phenomenon.IMPORTANCEMycobacteria cause serious human diseases such as tuberculosis and leprosy. The mycobacterial transcription machinery is unique, containing transcription factors such as RbpA, CarD, and the RNA polymerase (RNAP) core-interacting small RNA Ms1. Here, we extend our knowledge of the mycobacterial transcription apparatus by identifying MoaB2 as an interacting partner of σA, the primary sigma factor, and characterize its effects on transcription and σA stability. This information expands our knowledge of interacting partners of subunits of mycobacterial RNAP, providing opportunities for future development of antimycobacterial compounds.

Nucleases of the S1/P1 family have important applications in biotechnology and molecular biology. We have performed structural analyses of SmNuc1 nuclease from Stenotrophomonas maltophilia, including RNA cleavage product binding and mutagenesis in a newly discovered flexible Arg74-motif, involved in substrate binding and product release and likely contributing to the high catalytic rate. The Arg74Gln mutation shifts substrate preference towards RNA. Purine nucleotide binding differs compared to pyrimidines, confirming the plasticity of the active site. The enzyme-product interactions indicate a gradual, stepwise product release. The activity of SmNuc1 towards c-di-GMP in crystal resulted in a distinguished complex with the emerging product 5 '-GMP. This enzyme from an opportunistic pathogen relies on specific architecture enabling high performance under broad conditions, attractive for biotechnologies.

Rhomboid intramembrane serine proteases have been implicated in several pathologies, and emerge as attractive pharmacological target candidates. The most potent and selective rhomboid inhibitors available to date are peptidyl α-ketoamides, but their selectivity for diverse rhomboid proteases and strategies to modulate it in relevant contexts are poorly understood. This gap, together with the lack of suitable in vitro models, hinders ketoamide development for relevant eukaryotic rhomboid enzymes. Here we explore the structure-activity relationship principles of rhomboid inhibiting ketoamides by medicinal chemistry and enzymatic in vitro and in-cell assays with recombinant rhomboid proteases GlpG, human mitochondrial rhomboid PARL and human RHBDL2. We use X-ray crystallography in lipidic cubic phase to understand the binding mode of one of the best ketoamide inhibitors synthesized here containing a branched terminal substituent bound to GlpG. In addition, to extend the interpretation of the co-crystal structure, we use quantum mechanical calculations and quantify the relative importance of interactions along the inhibitor molecule. These combined experimental analyses implicates that more extensive exploration of chemical space at the prime side is unexpectedly powerful for the selectivity of rhomboid inhibiting ketoamides. Together with variations in the peptide sequence at the non-prime side, or its non-peptidic alternatives, this strategy enables targeted tailoring of potent and selective ketoamides towards diverse rhomboid proteases including disease-relevant ones such as PARL and RHBDL2.

AbstractMycobacterial HelD is a transcription factor that recycles stalled RNAP by dissociating it from nucleic acids and, if present, from the antibiotic rifampicin. The rescued RNAP, however, must disengage from HelD to participate in subsequent rounds of transcription. The mechanism of release is unknown. We show that HelD from Mycobacterium smegmatis forms a complex with RNAP associated with the primary sigma factor σA and transcription factor RbpA but not CarD. We solve several structures of RNAP-σA-RbpA-HelD without and with promoter DNA. These snapshots capture HelD during transcription initiation, describing mechanistic aspects of HelD release from RNAP and its protective effect against rifampicin. Biochemical evidence supports these findings, defines the role of ATP binding and hydrolysis by HelD in the process, and confirms the rifampicin-protective effect of HelD. Collectively, these results show that when HelD is present during transcription initiation, the process is protected from rifampicin until the last possible moment.

Abstract Rifampicin is a clinically important antibiotic that binds to, and blocks the DNA/RNA channel of bacterial RNA polymerase (RNAP). Stalled, nonfunctional RNAPs can be removed from DNA by HelD proteins; this is important for maintenance of genome integrity. Recently, it was reported that HelD proteins from high G+C Actinobacteria, called HelR, are able to dissociate rifampicin-stalled RNAPs from DNA and provide rifampicin resistance. This is achieved by the ability of HelR proteins to dissociate rifampicin from RNAP. The HelR-mediated mechanism of rifampicin resistance is discussed here, and the roles of HelD/HelR in the transcriptional cycle are outlined. Moreover, the possibility that the structurally similar HelD proteins from low G+C Firmicutes may be also involved in rifampicin resistance is explored. Finally, the discovery of the involvement of HelR in rifampicin resistance provides a blueprint for analogous studies to reveal novel mechanisms of bacterial antibiotic resistance.