Biology subjects

Bange, G.

Publications and source records attributed to Bange, G..

At least 19 recordsLinked to original sources

bioRxiv2026

The diadenosine tetraphosphate hydrolase YqeK controls fitness, biofilm formation, staphyloxanthin production and virulence in Staphylococcus aureus

Diadenosine tetraphosphate (Ap4A) is a nucleotide metabolite, which is degraded by the YqeK hydrolase in Staphylococcus aureus in vitro. In this study, we analyzed the phenotypes of the yqeK mutant under stress, antibiotics, biofilm and macrophage infection conditions to investigate the functions of Ap4A in S. aureus COL. Using nucleotide metabolomics, we confirmed that Ap4A levels are 105-fold higher in the yqeK mutant, accompanied by lower adenylate and guanylate nucleotide pools. The yqeK mutant showed a delayed growth in LB, TSB and RPMI medium, and decreased survival under lethal oxidative and quinone stress. Transcriptome analysis revealed the upregulation of the sspABC operon and the CodY, T-box Met and G-box regulons indicating increased amino acids and GTP biosynthesis, whereas the AgrA, Fur, PurR, and T-box Cys regulons were downregulated in the yqeK mutant. These gene expression changes could be restored to WT level in the yqeK complemented strain, resulting also in a DNA damage response as revealed by the induction of LexA regulon members and mobile genetic elements (pathogenicity island SAPI3 and prophage L54a). Moreover, the yqeK mutant showed enhanced biofilm formation, higher intracellular iron levels and lower staphyloxanthin levels. Using infection assays, we demonstrated a decreased survival of the yqeK mutant inside J774A.1 murine macrophages, supporting a link between Ap4A and pathogenicity regulation via Agr-controlled virulence factors in S. aureus. Future research should be directed to understand how Ap4A regulates nucleotide, iron and amino acid metabolism as well as biofilm and virulence phenotypes in S. aureus. IMPORTANCE: S. aureus is an important human pathogen, which can cause life-threatening infections especially in immunocompromised patients. Due to the prevalence of multidrug resistant strains, the search for new drug targets is an urgent goal. Diadenosine tetraphosphate (Ap4A) has been shown to contribute to stress responses, antibiotic resistance, biofilm development and virulence in bacteria. In this work, we showed that Ap4A is upregulated upon deletion of yqeK encoding the Ap4A hydrolase in S. aureus COL. Moreover, the yqeK mutant was impaired in growth and survival during oxidative stress and after infection of murine macrophages, indicating that Ap4A contributes to the host-pathogen interactions in S. aureus. Transcriptome analyses revealed alterations of the nucleotide, amino acid and iron metabolism as well as the downregulation of Agr-controlled cytotoxins, contributing to the lower virulence of the yqeK mutant. Altogether, our results provide leads for the design of inhibitors against YqeK to combat S. aureus infections.

microbiology↗

bioRxiv2026

NUDT2 loss defines quantitative limits for dinucleoside polyphosphate action on the cGAS-STING-TBK1 axis

NUDT2 is an emerging candidate for therapeutic intervention in cancer, and its inhibition or loss is known to elevate adenosine-containing dinucleoside polyphosphates (ApnNs), including diadenosine tetraphosphate (Ap4A). Ap4A is a stress- and immune-associated nucleotide metabolite proposed to act as a second messenger, raising the possibility that NUDT2 targeting may unintentionally affect important nucleotide-sensitive signaling pathways. One such pathway is cGAS-STING signaling, a central innate immune axis that detects cytosolic DNA, produces the nucleotide second messenger 2'3'-cGAMP, and drives type I interferon responses. Because cGAS-STING also contributes to antitumor immunity and is being pharmacologically targeted in cancer, we asked whether sustained Ap4A accumulation perturbs this pathway. We systematically evaluated Ap4A and related dinucleoside polyphosphates across the cGAS-STING-TBK1 axis using biophysical, enzymatic, structural, and cellular approaches. Contrary to a previous model, STING did not bind Ap4A, Ap3A, or Ap4G, despite robust binding of canonical cyclic dinucleotides. Although cGAS bound these nucleotides with micromolar affinities, DNA-activated cGAMP synthesis was inhibited only at high, supra-substrate ratios. Similarly, TBK1 inhibition required extreme Ap4A ratios beyond physiologically relevant levels. In a THP-1 cell model, NUDT2 knockout caused strong Ap4A accumulation, but the resulting intracellular dinucleoside polyphosphate levels remained below the ratios required to inhibit cGAS or TBK1 in vitro. This study thus distinguishes biochemical possibility from physiological relevance and argues that NUDT2-linked Ap4A accumulation is unlikely to directly compromise cGAS-STING pathway activity.

biochemistry↗

bioRxiv2026

Discovery of a Cell-Permeable Gi-Signaling Inhibitor

Heterotrimeric G proteins regulate diverse physiological processes, yet selective small molecule modulators for the Gi subfamily remain scarce. Here, we employed native metabolomics to screen a fungal culture collection for G protein binders, which led to the discovery of N-hydroxyapiosporamide (NHAP), a fungal specialized metabolite that functionally inhibits Gi-signaling. NHAP selectively binds Gi1 and diminishes Gi1-mediated GTP turnover in biochemical assays. In primary ventricular cardiomyocytes, NHAP largely reversed acetylcholine-induced negative inotropy, demonstrating functional Gi-signaling blockade in a physiological context without acute cytotoxicity. This work establishes NHAP as a first-in-class Gi-selective inhibitor, providing a cell-permeable chemical scaffold for the optimization of next-generation agents to control Gi-signaling in cell-based systems and, ultimately, to target Gi1-driven pathologies.

biochemistry↗

bioRxiv2026

Amino acid Mutations at the Interface of Sudan Virus VP40 alter transport and budding

The matrix protein VP40 of orthoebolaviruses coordinates virion release and downregulates viral RNA synthesis through distinct oligomeric states, including dimers, octamers, and filamentous assemblies. To dissect the contributions of two oligomeric interface residues, L117 and W95, in the Sudan virus (SUDV) VP40 (sVP40), we created variants carrying alanine substitutions and assessed their structural and functional properties. sVP40 L117A failed to form dimers and was predominantly monomeric showing increased structural flexibility, reduced thermal stability together with loss of plasma membrane transport, budding activity, and the ability to regulate viral RNA synthesis. VP40 W95A preserved dimerization but also exhibited increased structural flexibility and reduced thermal stability. Functionally, sVP40 W95A more strongly inhibited viral RNA synthesis and markedly enhanced budding. However, in a transcription- and replication-competent virus-like particle (trVLP) assay, trVLPs produced with sVP40 W95A induced substantially reduced reporter activity in target cells, indicating impaired particle infectivity or functionality and suggesting possible defects in minigenome packaging, entry, or early post-entry steps. These results demonstrate that mutations at key oligomerization interfaces exert distinct structural and functional effects and highlight the requirement for precise oligomerization in coordinating sVP40s dual roles in genome regulation and virion release. By defining the contributions of L117 and W95, this study advances mechanistic understanding of sVP40 function and identifies processes that may serve as targets for antiviral intervention. ImportanceSudan virus (SUDV) causes regular outbreaks in Sub-Sahara Africa with unusually high lethality rates. However, in contrast to the more often occurring Zaire ebolavirus (EBOV), no monoclonal antibodies or vaccines are available and SUDV is generally understudied. The matrix protein VP40 is responsible for the downregulation of viral genome replication and transcription as well as budding. Here, we present structural and functional characterization of the SUDV VP40 interface residues L117 and W95 and show that while both amino acids are crucial for VP40s structural integrity, their functional effects are dramatically different ranging from complete abolishment to improving regulatory and budding activities.

microbiology↗

bioRxiv2025

Structural basis of quinone-sensing by the MarR-type repressor MhqR in Staphylococcus aureus

The MarR-family regulator MhqR of Staphylococcus aureus (SaMhqR) was previously characterized as quinone-sensing repressor of the mhqRED operon. Here, we resolved the crystal structures of apo-SaMhqR and the 2-methylbenzoquinone (MBQ)-bound SaMhqR complex. AlphaFold3 modelling was used to predict the structure of the SaMhqR in complex with its operator DNA. In the DNA-bound SaMhqR state, S65 and S66 of an allosteric 3-4 loop adapted a helically wound conformation to elongate helix 4 for optimal DNA binding. Key residues for MBQ interaction were identified as F11, F39, E43, and H111, forming the MBQ-binding pocket. MBQ binding prevented the formation of the extended helix 4 in the allosteric loop, leading to steric clashes with the DNA. Molecular dynamics (MD) simulations revealed an increased intrinsic dynamics within the allosteric loop and the {beta}1/{beta}2-wing regions after MBQ binding, to prevent DNA binding. Using mutational analyses, we validated that F11, F39, and H111 are required for quinone sensing in vivo, whereas S65 and S66 of the allosteric loop and D88, K89, V91 and Y92 of the {beta}1/{beta}2-wing are essential for DNA binding in vitro and in vivo. In conclusion, our structure-guided modelling and mutational analyses identified a quinone-binding pocket of SaMhqR and the mechanism of SaMhqR inactivation, which involves local structural rearrangements of an allosteric loop and a high intrinsic dynamics to prevent DNA interactions. Our results provide novel insights into the redox-mechanism of the conserved SaMhqR repressor, that functions as an important determinant of quinone and antimicrobial resistance in S. aureus. IMPORTANCES. aureus is a major human pathogen, which can cause life-threatening infections in humans. However, treatment options are limited due to the prevalence of antimicrobial resistant isolates in the hospital and the community. The MarR-type repressor SaMhqR was described to control resistance towards quinones and quinone-like antimicrobials. However, the redox-regulatory mechanism of SaMhqR by quinones was unknown. In this work, we explored the DNA-binding and quinone-sensing mechanism of SaMhqR and identified a quinone-binding pocket and an allosteric loop, which facilitates DNA binding activity via a helical wound conformation and adapts an unstructured coiled conformation upon quinone binding to inhibit DNA binding. A similar mechanism has been recently discovered for regulation of uric acid resistance by UrtR family repressors (1). Our results contribute to a better understanding of antimicrobial resistance regulation, which can be exploited for future drug-design to eradicate multidrug-resistant S. aureus.

microbiology↗

bioRxiv2025

Controlled Delivery and Light-Induced Release of Magic Spot Nucleotides in Escherichia coli

The "magic spot nucleotides" (MSNs) ppGpp and pppGpp (also: (p)ppGpp) are bacterial alarmones central to the conserved stringent response, a stress adaptation mechanism that helps bacteria adapt to stress conditions and hostile environments. Current strategies to manipulate MSN levels rely mainly on genetic or environmental approaches, which are slow and lack temporal control. Chemical tools such as photocaged MSN analogues could provide such temporal control over MSN levels. However, the high negative charge of MSNs prevents spontaneous passage through the complex bacterial cell envelope. Here, we report the synthesis of photocaged, clickable, and isotope-labeled MSN analogues and their delivery into Escherichia coli comparing different approaches. A cyclodextrin-based synthetic nucleotide transporter provides particular advantages. Upon 400 nm irradiation, these probes were photo-released inside living cells, where we tracked their conversion from pppGpp to ppGpp by capillary electrophoresis mass spectrometry and studied their ability to alter growth in a (p)ppGpp0 mutant. This work provides the first demonstration that highly charged, photocaged MSNs can traverse the bacterial envelope, be photo-released intracellularly, and be metabolically tracked in real time. These probes lay the foundation for spatially and temporally controlled studies of MSN function and of other highly negatively charged metabolites in bacteria.

biochemistry↗

bioRxiv2025

Exploration of the proxiOME of large subunit ribosomal proteins reveals Acl1 and Bcl1 as cooperating dedicated chaperones of Rpl1

In eukaryotes, most newly synthesized ribosomal proteins (r-proteins) need to rapidly and safely get into the nucleus to reach their assembly site on pre-ribosomal particles. However, only for few r-proteins tailored support mechanisms involving so-called dedicated chaperones could so far be revealed. Here, with the primary aim of identifying novel dedicated chaperones, we performed TurboID-based proximity labelling with all 46 large subunit r-proteins of Saccharomyces cerevisiae, which unveiled the fungi-specific Acl1 and the conserved Bcl1 as candidate dedicated chaperones of Rpl1. We show that the functionally cooperating Acl1 and Bcl1 both directly interact with Rpl1, form a trimeric Acl1-Rpl1-Bcl1 complex, and enable the nuclear import of Rpl1. Moreover, our crystal structure of the minimal Acl1-Rpl1 complex reveals how Acl1s ankyrin repeat domain shields a positively charged rRNA-binding surface of Rpl1. Our proximity labelling approach also permitted to establish novel interactions between four r-proteins and distinct importins and to illuminate r-protein neighbourhoods on successive pre-60S particles. Additionally, reciprocal proximity labelling with the known dedicated chaperones indicates that almost all appear to be transiently associated with pre-ribosomal particles. Our study provides for the first time comprehensive insight into the physical proximities of large subunit r-proteins along their entire life cycle. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/677003v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1167c6corg.highwire.dtl.DTLVardef@1429c23org.highwire.dtl.DTLVardef@18f7ecorg.highwire.dtl.DTLVardef@13ccdfb_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

biochemistry↗

bioRxiv2025

Molecular basis of ParA ATPase activation by the CTPase ParB during bacterial chromosome segregation

DNA segregation by bacterial ParABS systems is mediated by transient tethering interactions between nucleoid-bound dimers of the ATPase ParA and centromere (parS)-associated complexes of the clamp-forming CTPase ParB. The lifetime of these interactions is limited by the ParB-dependent activation of ParA ATPase activity. Here, we elucidate the functional interplay between ParA and ParB in the model bacterium Myxococcus xanthus. We demonstrate that the N-terminal ParA-binding motif of ParB associates with a conserved bipartite binding pocket at the ParA dimer interface, in a manner dependent on ParB clamp closure. Moreover, we show that ParB and non-specific DNA interact cooperatively with ParA and synergistically induce structural changes at its Walker A and Walker B motifs that correlate with the activation of ParA ATPase activity. These results advance our understanding of the mechanism underlying DNA transport by the ParABS system and may help to unravel the mode of action of related cargo-positioning systems.

microbiology↗

bioRxiv2025

Specificities of Chemosensory Receptors in the Human Gut Microbiota

The human gut is rich in metabolites and harbors a complex microbial community, yet the sensory repertoire of its commensal bacteria remains largely uncharacterized. Here we systematically mapped ligand specificities of extracytoplasmic sensory domains from twenty members of the human gut microbiota, with a primary focus on the abundant and physiologically important class of Clostridia. We identified diverse metabolites as specific stimuli for three major functional classes of transmembrane receptors. We further characterized novel subsets of sensors belonging to the Cache superfamily, specific for lactate, dicarboxylic acids, and for uracil and short-chain fatty acids (SCFAs), respectively, and investigated the evolution of their ligand specificity. Structural and biochemical analysis of the newly described dCache_1UR domain revealed an independent binding of uracil and SCFA at distinct modules. Altogether, we could identify or predict specificities for over a half of the Cache-type chemotactic sensors in the selected gut commensals, with the carboxylic acids representing the largest class of ligands. Among those, the most commonly found specificities were for lactate and formate, indicating particular importance of these metabolites in the human gut microbiome and consistent with their observed beneficial impact on the growth of selected bacterial species.

microbiology↗

bioRxiv2024

Allosteric Regulation of Pyruvate Kinase Enables Efficient and Robust Gluconeogenesis by Preventing Metabolic Conflicts and Carbon Overflow

Glycolysis and gluconeogenesis are reciprocal metabolic pathways that utilize different carbon sources. Pyruvate kinase catalyzes the irreversible final step of glycolysis, yet the physiological function of its regulation is poorly understood. Through metabolomics and enzyme kinetics studies, we discovered that pyruvate kinase activity is inhibited during gluconeogenesis in the soil bacterium Bacillus subtilis. This regulation involves an extra C-terminal domain (ECTD) of pyruvate kinase, which is essential for autoinhibition and regulation by metabolic effectors. Introducing a pyruvate kinase mutant lacking the ECTD into B. subtilis resulted in defects specifically under gluconeogenic conditions, including inefficient carbon utilization, slower growth, and decreased resistance to the herbicide glyphosate. These defects are not caused by the phosphoenolpyruvate-pyruvate-oxaloacetate futile cycle. Instead, we identified two significant metabolic consequences of pyruvate kinase dysregulation during gluconeogenesis: increased carbon overflow into the medium and failure to expand glycolytic intermediates such as phosphoenolpyruvate (PEP). In silico analysis revealed that in wild-type cells, an expanded PEP pool enabled by pyruvate kinase regulation is critical for the thermodynamic feasibility of gluconeogenesis. Our findings underscore the importance of allosteric regulation during gluconeogenesis in coordinating metabolic flux, efficient energy utilization, and antimicrobial resistance.

microbiology↗

bioRxiv2024

Frequent transitions in self-assembly across the evolution of a central metabolic enzyme

Many enzymes assemble into homomeric protein complexes comprising multiple copies of one protein. Because structural form is usually assumed to follow function in biochemistry, these assemblies are thought to evolve because they provide some functional advantage. In many cases, however, no specific advantage is known and, in some cases, quaternary structure varies among orthologs. This has led to the proposition that self-assembly may instead vary neutrally within protein families. The extent of such variation has been difficult to ascertain because quaternary structure has until recently been difficult to measure on large scales. Here, we employ mass photometry, phylogenetics, and structural biology to interrogate the evolution of homo-oligomeric assembly across the entire phylogeny of prokaryotic citrate synthases - an enzyme with a highly conserved function. We discover a menagerie of different assembly types that come and go over the course of evolution, including cases of parallel evolution and reversions from complex to simple assemblies. Functional experiments in vitro and in vivo indicate that evolutionary transitions between different assemblies do not strongly influence enzyme catalysis. Our work suggests that enzymes can wander relatively freely through a large space of possible assemblies and demonstrates the power of characterizing structure-function relationships across entire phylogenies.

evolutionary biology↗

bioRxiv2024

Membrane binding properties of the cytoskeletal protein bactofilin

Bactofilins are a widespread family of cytoskeletal proteins with important roles in bacterial morpho-genesis, chromosome organization and motility. They polymerize in a nucleotide-independent manner, forming non-polar filaments that are typically associated with the cytoplasmic membrane. Membrane binding was suggested to be mediated by a short N-terminal peptide, but the underlying mechanism and the conservation of this interaction determinant among bacteria remain unclear. Here, we use the bacto-filin homolog BacA of the stalked bacterium Caulobacter crescentus as a model to analyze the membrane-binding behavior of bactofilins. Based on site-directed mutagenesis of the N-terminal region, we identify the full membrane-targeting sequence of BacA (MFSKQAKS) and pinpoint amino acid residues that are critical for its function in vivo and in vitro. Molecular dynamics simulations then provide detailed insight into the molecular mechanism underlying the membrane affinity of this peptide. Collectively, these analyses reveal a delicate interplay between the water exclusion of hydrophobic N-terminal residues, the arrangement of the peptide within the membrane and the electrostatic attraction between positively charged groups in the peptide and negative charges in the phospholipid molecules. A comprehensive bio-informatic analysis shows that the composition and properties of the membrane-targeting sequence of BacA are conserved in numerous bactofilin homologs from diverse bacterial phyla. Importantly, our findings reveal cooperative effects between the membrane-binding and polymerization activities of BacA. Moreover, they demonstrate that both of these activities critically contribute to the recruitment of the BacA client protein PbpC, a membrane-bound cell wall synthase that uses a conserved peptide in its N-terminal cytoplasmic tail to interact with BacA assemblies. Finally, we show that PbpC can functionally replace the endogenous membrane-targeting sequence of BacA when provided at elevated levels in trans, indicating that client proteins can make a significant contribution to the membrane association of bacto-filin polymers. Together, these results unravel the mechanistic underpinnings of membrane binding by bactofilin homologs, thereby illuminating a previously obscure but important aspect in the biology of this cytoskeletal protein family.

microbiology↗

bioRxiv2024

Discovery of a synthetic small molecule targeting the central regulator of Salmonella pathogenicity

The enteric pathogen Salmonella enterica serovar Typhimurium relies on the activity of effector proteins to invade, replicate, and disseminate into host epithelial cells and other tissues, thereby causing disease. Secretion and injection of effector proteins into host cells is mediated by dedicated secretion systems, which hence represent major virulence determinants. Here, we report the identification of a synthetic small molecule with drug-like properties, C26, which suppresses the secretion of effector proteins, and consequently hinders bacterial invasion of eukaryotic cells. C26 binds to and inhibits HilD, the transcriptional regulator of the major secretion systems. While sharing the same binding pocket as the previously described long-chain fatty acid ligands, C26 inhibits HilD with a unique binding mode and a distinct mechanism. We provide evidence for target engagement within infected eukaryotic cells and present analogs with improved potency and suitability as scaffolds to develop anti-virulence agents against Salmonella infections in humans and animals.

microbiology↗

bioRxiv2024

The role of chloroplast SRP54 domains and its C-terminal tail region in post- and cotranslational protein transport in vivo

In the chloroplast, the 54 kDa subunit of the signal recognition particle (cpSRP54) is involved in the posttranslational transport of the light-harvesting chlorophyll a/b-binding proteins (LHCPs) and the cotranslational transport of plastid-encoded subunits of the photosynthetic complexes to the thylakoid membrane. It forms a high-affinity complex with plastid-specific cpSRP43 for posttranslational transport, while a ribosome-associated pool coordinates its cotranslational function. CpSRP54 constitutes a conserved multidomain protein, comprising a GTPase (NG) and a methionine-rich (M) domain linked by a flexible region. It is further characterized by a plastid-specific C-terminal tail region containing the cpSRP43-binding motif. To characterize the physiological role of the various regions of cpSRP54 in thylakoid membrane protein transport, we generated Arabidopsis thaliana cpSRP54 knockout (ffc1-2) lines producing truncated cpSRP54 variants or a GTPase point mutation variant. Phenotypic characterization of the complementation lines demonstrated that the C-terminal tail region of cpSRP54 plays an important role specifically in posttranslational LHCP transport. Furthermore, we show that the GTPase activity of cpSRP54 plays an essential role in the transport pathways for both nuclear-as well as plastid-encoded proteins. In addition, our data revealed that plants expressing cpSRP54 without the C-terminal region exhibit a strongly increased accumulation of a photosystem I assembly intermediate. HighlightThis study elucidates the important role of the chloroplast SRP subunit, cpSRP54, in the biogenesis of both the LHC antenna and the cores of photosystem I and II in vivo.

plant biology↗

bioRxiv2024

Polar confinement of a macromolecular machine by an SRP-type GTPase

The SRP-type GTPase FlhF, along with its regulator FlhG, orchestrates the localization and quantity of flagella in bacteria. Our study reveals that FlhF anchors developing flagellar structures to the polar landmark protein HubP/FimV, thereby restricting their formation to the cell pole. Specifically, the GTPase domain of FlhF interacts with HubP, while an as-yet-uncharacterized structured domain at the N-terminus of FlhF binds to FliG. This FlhF-bound FliG subsequently engages with the MS-ring protein FliF, but not with the C-ring proteins FliM/FliN. Consequently, FlhFs interaction with HubP/FliG recruits a functional FliF/FliG complex to the pole, while FlhGs modulation of FlhF controls FliGs interaction with FliM/FliN, thereby regulating the progression of flagellar assembly at the pole. Significance statementFlagella serve as bacterial locomotion organelles, with their number and location, known as the flagellation pattern, being species-specific and among the earliest taxonomic criteria in microbiology. Bacteria replicate their flagellation pattern with each cell division. Flagella localization and abundance depends on the SRP-type GTPase FlhF, together with its regulator FlhG. Our study clarifies the mechanism through which FlhF coordinates the polar positioning of the flagellum, working in tandem with the polar landmark protein HubP and aiding in the assembly of flagellar MS-ring/C-ring components at the cellular pole.

microbiology↗

bioRxiv2023

The virulence regulator VirB from Shigella flexneri uses a CTP-dependent switch mechanism to activate gene expression

The transcriptional antisilencer VirB acts as a master regulator of virulence gene expression in the human pathogen Shigella flexneri. It binds defined sequences (virS) upstream of VirB-dependent promoters and counteracts their silencing by the nucleoid-organizing protein H-NS. However, its precise mode of action remains unclear. Notably, VirB is not a classical transcription factor but related to DNA partitioning proteins of the ParB family, which have recently been recognized as DNA-sliding clamps using CTP binding and hydrolysis to control their DNA entry gate. Here, we show that VirB binds CTP, embraces DNA in a clamp-like fashion upon its CTP-dependent loading at virS sites and slides laterally on DNA after clamp closure. Mutations that prevent CTP binding block the loading of VirB clamps in vitro and the formation of VirB nucleoprotein complexes in vivo. Thus, VirB represents a CTP-dependent molecular switch that uses a loading-and-sliding mechanism to control transcription during bacterial pathogenesis.

microbiology↗

bioRxiv2023

HilE represses the activity of HilD via a mechanism distinct from that of intestinal long-chain fatty acids.

The expression of virulence factors essential for the invasion of host cells by Salmonella enterica is tightly controlled by a network of transcription regulators. The AraC/XylS transcription factor HilD is the main integration point of environmental signals into this regulatory network, with many factors affecting HilD activity. Long chain fatty acids (LCFAs), which are highly abundant throughout the host intestine directly bind to, and repress HilD, acting as environmental cues to coordinate virulence gene expression. The regulatory protein HilE also negatively regulates HilD activity, through a protein-protein interaction. Both of these regulators inhibit HilD dimerisation, preventing HilD from binding to target DNA. We investigated the structural basis of these mechanisms of HilD repression. LCFAs bind to a conserved pocket in HilD, in a comparable manner to that reported for other AraC/XylS regulators, whereas HilE forms a stable heterodimer with HilD by binding to the HilD dimerisation interface. Our results highlight two distinct mechanisms by which HilD activity is repressed, which could be exploited for the development of new antivirulence leads.

biochemistry↗

bioRxiv2022

Peptide-mediated inhibition of the transcriptional regulator Elongin BC induces apoptosis in cancer cells

Inhibition of protein-protein interactions (PPIs) via designed peptides is an effective strategy to interfere with their biological functions. The Elongin BC heterodimer (ELOB/C) is involved in transcription elongation and protein turnover by PPIs that involve the so-called BC-box. ELOB and ELOC are commonly upregulated in cancer and essential for cancer cell growth, making them attractive drug targets. However, no strategy has been established to inhibit their functions in cells, so far. Here, we report a peptide that mimics a high-affinity BC-box and tightly binds to the ELOB/C dimer (kD = 0.45 {+/-} 0.03 nM). Our peptide blocks the association of ELOB/C with its interaction partners, both in vitro and in the cellular environment. Cancer cells treated with this peptide inhibitor show decreased cell viability, altered cell cycle and increased apoptosis. Therefore, our work proposes that blocking the BC-box binding pocket of ELOB/C is a feasible strategy to impair the function of the ELOB/C heterodimer and inhibit cancer cell growth. Our peptide inhibitor promises novel mechanistic insights into the biological function of the ELOB/C dimer and offers a starting point for therapeutics linked to ELOB/C dysfunction.

cancer biology↗