Biology subjects

Steinchen, W.

Publications and source records attributed to Steinchen, W..

At least 19 recordsLinked to original sources

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↗

bioRxiv2026

Conformational dynamics of the membrane-anchored foldase LipH from Pseudomonas aeruginosa governs recognition and release of its client lipase

The lipase LipA from Pseudomonas aeruginosa is an extracellular enzyme that plays an important role in bacterial infections. Prior its export via the type II secretion system, LipA requires the cognate membrane-anchored foldase LipH for maturation in the periplasm. Though structural studies elucidated the architecture of the LipH:lipase complex, how the full-length, membrane-tethered foldase recognizes, folds, and releases its client has remained barely understood. Here, we combine in silico and in vitro analysis to resolve the conformational dynamics and function of the full-length LipH in a membrane context. Simulations reveal that the membrane-anchored LipH is highly dynamic, sampling a broad ensemble of conformations. The chaperoning cavity is transiently closed or occluded by both the proximal membrane and the linker polypeptide, which is further confirmed by structural analysis. Despite the steric hindrance, the full-length LipH reconstituted into lipid-based nanodiscs and amphipols efficiently activates LipA, though it displays substantially reduced affinity for the client. We propose that the negatively charged membrane promotes release of the folded client, enabling multiple chaperoning cycles. Hydrogen/deuterium exchange analysis reveals that the MD2 domain of LipH in engaged in stable interactions with the lipase, whereas MD1 contacts are transient. Consistently, LipH readily captures N-terminal fragments of LipA, indicating that initial recognition relies on local interactions via the MD2 domain. Together, our results show how membrane coupling and intrinsic conformational plasticity modulate the function of the steric chaperone, and suggest that the membrane-anchored LipH balances capture, folding, and release of the client LipA to enable its efficient secretion.

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↗

bioRxiv2024

Mechanism of cooperative strigolactone perception by the MAX2 ubiquitin ligase-receptor-substrate complex

Strigolactones (SLs) are a group of plant hormones that regulate various aspects of plant growth and development. Additionally, SLs exuded into the soil promote symbiotic relationships with arbuscular mycorrhizal fungi and stimulate the germination of parasitic plants such as Striga hermonthica. The binding and hydrolysis of SLs by their receptors (D14 in Arabidopsis and HTL in Striga) promote the ubiquitination of transcriptional repressors by Skp1-cullin-F-box (SCF)-type E3 ubiquitin ligases. The mechanistic link between SL perception by D14/HTL and substrate recognition by the E3 remains unclear. We identified an E3-HTL-substrate complex that is sufficiently stable for cryogenic electron microscopy. This complex, composed of SKP1 (ASK1) and substrate (SMAX1) from Arabidopsis, and Striga F-box (MAX2) and SL receptor (HTL7), reveals that the substrate engages in a bidentate association through its N and D2 domains. This interaction, which is both conformationally and compositionally dynamic, directly and allosterically stabilises the MAX2-(SL)HTL7 complex and affects the positioning of ASK1 relative to MAX2. This dynamic positioning influences the proximity between the substrate D2 domain and the ubiquitin-conjugated E2 enzyme. This work advances our understanding of how E3 ligases in plants translate hormone perception into genetic adaptations.

biophysics↗

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

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 fungal protein Jps1 facilitates unconventional protein secretion through a direct phosphoinositide interaction

Protein secretion is indispensable for essential cellular processes in eukaryotic cells, contributing significantly to nutrient acquisition, defense or communication. Alternative pathways bypassing the endomembrane system collectively referred to as unconventional secretion are gaining increasing attention. A number of important molecules such as cytokines, fibroblast growth factor or viral proteins are being exported through these mechanistically diverse pathways. In the fungal model Ustilago maydis, cytokinesis-dependent unconventional secretion mediates export of the chitinase Cts1 via the fragmentation zone. This membrane-rich compartment is formed during cytokinesis between mother and daughter cells. Recently, we identified Jps1, a previously uncharacterized protein, as a crucial factor for Cts1 localization and export. Combining biochemical experiments and in vivo studies, we here uncover two pivotal features of Jps1: dimerization and phosphoinositide (PIP) binding. Our findings reveal that a conserved structural core domain mediates homodimerization, while surrounding flexible variable regions suggest potential diversification in different basidiomycete species. Jps1 does not harbor a canonical PIP-binding domain but instead specificity of the interaction with the preferred PIP PI(4,5)P2 is determined by basic residues. Importantly, loss of PI(4,5)P2 binding specificity results in mis-localisation, morphological defects and reduced extracellular Cts1 activity, particularly at low cell densities. This discovery sheds light on previously unknown key features of Jps1, elucidating its role in supporting Cts1 secretion, and representing a crucial step towards understanding the broader implications of unconventional secretion in eukaryotic cells.

biochemistry↗

bioRxiv2024

Layered entrenchment maintains essentiality in protein-protein interactions.

Protein complexes composed of strictly essential subunits are abundant in nature and arise through the gradual complexification of ancestral precursor proteins followed by their co-evolution with the newly recruited components. Essentiality arises during co-evolution by the accumulation of changes that are tolerated in the complex state but would be deleterious for the standalone complex components. While this theoretical framework to explain how essentiality arises has been proposed long ago, it is unclear which factors cause essentiality to persist over evolutionary timescales. In this work we show that the central enzyme of photosynthesis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), rapidly started to depend on a newly recruited interaction partner through multiple, genetically distinct mechanisms that affect stability, solubility, and catalysis. We further demonstrate that layering multiple mechanisms of essentiality can lead to the persistence of essentiality, even if any given mechanism reverts through chance or selection. More broadly, our work highlights that new interaction partners can drastically re-shape which substitutions are tolerated in the proteins they are recruited into. This can lead to the rapid evolution of multi-layered essentiality through the exploration of areas of sequence space that are only accessible in the complex state.

biochemistry↗

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

Archaeal self-activating GPN-loop GTPases involve a lock-switch-rock mechanism for GTP hydrolysis

Three GPN-loop GTPases, GPN1-GPN3, are central to the maturation and trafficking of eukaryotic RNA polymerase II. This GTPase family is widely represented in archaea but typically occurs as single paralogs. Structural analysis of the GTP- and GDP-bound states of the Sulfolobus acidocaldarius GPN enzyme (SaGPN) showed that this central GPN-loop GTPase adopts two distinct quaternary structures. In the GTP-bound form the {gamma}-phosphate induces a tensed dimeric arrangement by interacting with the GPN region that is relaxed upon hydrolysis to GDP. Consequently, a rocking-like motion of the two protomers causes a major allosteric structural change towards the roof-like helices. Using a lock-switch-rock (LSR) mechanism, homo- and heterodimeric GPN-like GTPases are locked in the GTP-bound state and undergo large conformational changes upon GTP hydrolysis. A{Delta} saGPN strain of S. acidocaldarius was characterized by impaired motility and major changes in the proteome underscoring its functional relevance for S. acidocaldarius in vivo. Significance StatementGPN-loop GTPases have been found to be crucial for eukaryotic RNA polymerase II assembly and nuclear trafficking. Despite their ubiquitous occurrence in eukaryotes and archaea the mechanism by which these self-activating GTPases mediate their function is unknown. Our study on an archaeal representative from Sulfolobus acidocaldarius showed that these dimeric GTPases undergo large-scale conformational changes upon GTP hydrolysis, which can be summarized as a lock-switch-rock mechanism. The observed requirement of SaGPN for motility appears to be due to its large footprint on the archaeal proteome.

biochemistry↗

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

Crosstalk between regulatory elements in the disordered TRPV4 N-terminus modulateslipid-dependent channel activity

Intrinsically disordered regions (IDRs) are essential for membrane receptor regulation but often remain unresolved in structural studies. TRPV4, a member of the TRP vanilloid channel family involved in thermo- and osmosensation, has a large N-terminal IDR of approximately 150 amino acids. With an integrated structural biology approach, we analyze the structural ensemble of the TRPV4 IDR and identify a network of regulatory elements that modulate channel activity in a hierarchical lipid-dependent manner through transient long-range interactions. A highly conserved autoinhibitory patch acts as a master regulator by competing with PIP2 binding to attenuate channel activity. Molecular dynamics simulations show that loss of the interaction between the PIP2-binding site and the membrane reduces the force exerted by the IDR on the structured core of TRPV4. This work demonstrates that IDR structural dynamics are coupled to TRPV4 activity and highlights the importance of IDRs for TRP channel function and regulation.

biophysics↗

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↗

bioRxiv2022

Structural and functional analysis of YopR and identification of an additional key component of the SPβ phage lysis-lysogeny management system

Prophages need to tightly control their lifestyle to either be maintained within the host genome or enter the lytic cycle. The SP{beta} prophage present in the genome of Bacillus subtilis 168 was recently shown to possess an arbitrium system defining its replication stage. Using an historic B. subtilis strain harboring the heat-sensitive SP{beta} c2 mutant, we analyzed a key component of the lysis-lysogeny decision system called YopR, which is critical for maintenance of lysogeny. Here, we demonstrate that the heat-sensitive SP{beta} c2 phenotype is due to a single nucleotide exchange in the yopR gene, rendering the encoded YopRG136E protein temperature sensitive. Structural characterization of YopR revealed that the protein is a DNA-binding protein with an overall fold like tyrosine recombinases. Biochemical and functional analyses indicate that YopR has lost the recombinase function and the G136E exchange impairs its higher order structure and DNA binding activity. We further show that the heat-inducible SP{beta} excision of the c2 mutant still depends on the serine recombinase SprA. Finally, an evolution experiment identified the YosL protein of unknown function as a novel component of the lysis-lysogeny management system, as the presence of yosL is crucial for the induction of the lytic cycle of SP{beta}.

microbiology↗

bioRxiv2022

Structural and functional analysis of the cerato-platanin-like effector protein Cpl1 suggests diverging functions in smut fungi

Plant pathogenic fungi are causative agents of the majority of plant diseases and can lead to severe crop loss in infected populations. Fungal colonization is achieved by combining different strategies, such as avoiding and counteracting the plant immune system and manipulating the host metabolome. Of major importance are effector proteins secreted by the fungi that fulfill diverse functions to support the infection process. Most of these proteins are highly specialized and structural and biochemical information is often absent. Here, we present the atomic structures of the cerato-platanin-like protein Cpl1 from Ustilago maydis and its homolog Uvi2 from Ustilago hordei. Both proteins adopt a double-{Psi}-{beta}-barrel architecture reminiscent of cerato-platanin proteins, a class so far not described in smut fungi. Our structure-function analysis shows that Cpl1 binds to soluble chitin fragments via two extended grooves at the dimer interface of the two monomer molecules. This carbohydrate-binding mode has not been observed previously and expands the repertoire of chitin-binding proteins. Cpl1 localizes to the cell wall of U. maydis and specifically enriches cell-wall degrading and -decorating proteins during maize infection. The architecture of Cpl1 harboring four surface exposed loop regions supports the idea that it might play a role in spatial coordination of these proteins. While deletion of cpl1 has only mild effects on the virulence of U. maydis, a recent study showed that deletion of uvi2 strongly impairs U. hordei virulence. Our structural comparison between Cpl1 and Uvi2 reveals sequence variations in the loop regions which might explain a diverging function.

biochemistry↗

bioRxiv2021

A GGDEF domain serves as a spatial on-switch for a phosphodiesterase by direct interaction with a polar landmark protein

In bacteria, the monopolar localization of enzymes and protein complexes can result in a bi-modal distribution of enzyme activity between the dividing cells and heterogeneity of cellular behaviors. In Shewanella putrefaciens, the multidomain hybrid diguanylate cyclase/phosphodiesterase PdeB, which degrades the secondary messenger c-di-GMP, is located at the flagellated cell pole. Here we show how PdeB polar recruitment is mediated by direct interaction between the inactive diguanylate cyclase (GGDEF) domain of PdeB and the C-terminal FimV domain of the polar landmark protein HubP. We demonstrate that this interaction is crucial for full function of PdeB as a phosphodiesterase. Thus, the GGDEF domain serves as a spatially controlled on-switch that effectively restricts PdeBs activity to the flagellated cell pole. We further show that PdeB regulates abundance and activity of at least two crucial surface-interaction factors, the BpfA surface adhesion protein and the MSHA type IV pilus. The heterogeneity in c-di-GMP concentrations that is generated by differences in abundance and temporal polar appearance of PdeB as well as by bi-modal distribution after cell fission orchestrates the population behavior with respect to cell-surface interaction and environmental spreading. SignificancePhenotypic heterogeneity benefits the proliferation of microbial populations in changing environments. Such heterogeneity can be created by recruitment of enzymatic activity to specific cellular compartments, e.g., the cell pole. Here we show how a GGDEF domain of a multidomain phosphodiesterase has adopted the function as a spatial on-switch that is specifically activated upon direct interaction with a polar landmark protein.

microbiology↗