Biology subjects

Smits, S.

Publications and source records attributed to Smits, S..

15 recordsLinked to original sources

bioRxiv2026

The multidrug transporter Pdr5 from Saccharomyces cerevisiae is an ergosterol-modulated efflux pump

The pleiotropic drug resistance ABC transporter Pdr5 and its homologs play a critical role in multidrug resistance of numerous fungi, including pathogens. Previous studies demonstrated that alterations in the composition of the plasma membrane in which Pdr5 is embedded, specifically the ergosterol content, affected transport activity. Here we report the cryo-EM map of Saccharomyces cerevisiae Pdr5 in complex with ATP/ADP at 2.2 [A] resolution, revealing densities on the surface of the transmembrane domains as well as inside the substrate-binding site, that were assigned as ergosterol, identified and quantified by GC-MS, and the sphingolipid M(IP)2C, identified via native MS. The ergosterol molecules are bound to the protein in well-defined grooves and unexpected locations. We found that the activity of Pdr5 follows a Gaussian-like distribution, increasing with the ergosterol-concentration to reach a maximum before declining at higher concentrations, but this modulation was not observed in the presence of cholesterol, the sterol analogue found in mammalian cells. Mutational analysis corroborates the role of a single ergosterol-binding site in the modulation of Pdr5 activity. Our structural analysis identifies a protein-lipid network that could serve as a center for lipid engagement linking the surrounding lipid environment to the substrate-binding region of Pdr5. These findings advance our understanding of Pdr5 function by demonstrating that the lipid environment is closely linked to Pdr5 structure and activity.

biochemistry↗

bioRxiv2026

Structure determination and dual targeting of a plant TACO1 identifies its ancient role as an organelle translation regulator

Ribosome stalling caused by polyproline (PPs) motifs is common. Their translation is enhanced by accessory proteins such as YebC in bacteria, whose homolog, TRANSLATIONAL ACTIVATOR OF CYTOCHROME C OXIDASE 1 (TACO1), aids the translation of mitochondria-encoded proteins. The prevalence of PP motifs across plastid-encoded genes and their impact on the translation of photosynthesis-relevant proteins remains unexplored. Equally, a translation-enhancer of PP motifs equivalent to TACO1 for plastid ribosomes has not been reported. Here, we show that plastid genomes encode 24 proteins with a minimum of one PP motif on average, half of which are conserved in their cyanobacterial homologs, and that the vast majority of eukaryotes, including plants, encode a single TACO1 that we demonstrate to be dually targeted to mitochondria and plastids of Marchantia polymorpha. We resolved the MpTACO1 structure at 2.34 [A] by X-ray crystallography and the flexibility by small-angle X-ray scattering. Through modelling, we demonstrate that MpTACO1 can fit into the peptidyl transfer centre of plant chlororibosomes in a similar manner as human TACO1 in the mitoribosome. The identification and structure determination of the first plastid-targeted YebC/TACO1 allows us to sketch a unified model for the function and evolution of this ancient family of ribosomal accessory proteins, underscoring their indispensable role in the translation of bioenergetic membrane proteins reaching back almost 4 billion years. HighlightsO_LIDozens of GC-rich polyproline (PP) encoding regions are retained by AT-rich genomes C_LIO_LIPP motif conservation hints at regulatory mechanisms and required translation pauses C_LIO_LIChloroplast targeting of a (mitochondrial) translation enhancer of PP motifs C_LIO_LIMpTACO1 structure at 2.34 [A] resolution demonstrates its high level of conservation C_LI

plant biology↗

bioRxiv2026

Context-dependent siderophore exploitability shapes microbial community structure

1)Siderophores are classically viewed as shared iron-scavenging public goods, yet their ecological roles in multispecies communities remain poorly defined. Here, we establish a synthetic microbial community to dissect how different siderophores, their uptake compatibility and spatial structure shape iron competition. Using Corynebacterium glutamicum as a model, we show that this siderophore non-producer accesses diverse xenosiderophores, including enterobactin secreted by Escherichia coli. However, exploitation was constrained and co-cultures converged to stable compositions. Dose-response experiments combined with mathematical modelling indicated that the producer retains more effective access to enterobactin than the exploiter. Presence of Pseudomonas putida altered this interaction, as it exploited enterobactin while producing pyoverdine, a siderophore inaccessible to the other community members that restricted their iron access. Across different cultivation scales, community dynamics was strongly influenced by spatial organization and initial composition. These findings identify siderophores as context-dependent iron-allocation agents that can promote microbial coexistence or exclusion.

microbiology↗

bioRxiv2026

ATPLyzer An advanced ratiometric multi-colour biosensor for long-term monitoring of ATP dynamics

Adenosine triphosphate (ATP) is a central molecule in cellular metabolism, serving as the primary energy currency that links catabolic and anabolic pathways. Monitoring intracellular ATP in vivo is essential for understanding the dynamics of metabolic states, as well as intracellular functions and intercellular interactions in health and disease. We report the design and application of ATPLyzer, a series of genetically encoded, ratiometric biosensors for the monitoring of ATP levels in living cells. The matryoshka design consists of an ATP-binding cassette linked to a circularly permutated GFP coupled with an internal large stokes shift reference fluorophore, allowing for single-wavelength excitation and ratiometric output. This design overcomes limitations of conventional biosensors, reliance on dual excitation wavelengths, and susceptibility to photobleaching. Multi-colour ATPLyzer variants with different dissociation constants were characterized in vitro, exhibiting high specificity for ATP over ADP. Monitoring ATP in Escherichia coli confirmed in vivo utility and revealed growth-phase and carbon-supply-dependent ATP dynamics. The ATPLyzer biosensor offers a robust and tuneable tool for minimally invasive, time-resolved monitoring of intracellular ATP dynamics.

bioengineering↗

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

Visualizing extracellular vesicle-mediated RNA transfer using a novel metabolic labeling approach

Extracellular vesicles (EVs) are key players in intercellular communication, facilitated by the transfer of RNA and other molecular cargo between cells. However, how EV-RNA is processed in recipient cells remains poorly understood, particularly the uptake and intracellular trafficking pathways leading to functional transfer remains elusive. Visualizing EV-RNA in recipient cells may help to address these knowledge gaps. Although EV labeling methods are well-established, tracking endogenous RNA cargo within EVs and in recipient cells is challenging due to its low abundance, requiring highly sensitive labeling methods. Existing RNA labeling methods often lack sensitivity, specificity for donor cell-derived RNA, or are limited to labeling long RNA species. In this study, we propose a novel metabolic labeling approach to track RNA in donor cells, EVs and recipient cells using 5-ethynyl uridine (5-EU). 5-EU is a nucleoside analog of uridine that is incorporated into nascent RNA and can be detected through click chemistry with a fluorescent azide. To enhance RNA labeling efficiency, we overexpressed Uridine-Cytidine Kinase 2 (UCK2) in MDA-MB-231 donor cells, which increased 5-EU incorporation into nascent RNA chains. This approach allowed visualization and quantification of RNA in isolated EVs. Furthermore, co-culturing MDA-MB-231 UCK2+ donor cells expressing a CD63-HaloTag fusion protein with HMEC1-eGFP+ recipient cells enabled visualization and quantification of intercellular transfer of EVs and their endogenous RNA cargo. This novel approach offers a sensitive and specific tool for studying physiological EV-RNA transfer, thereby advancing our understanding of the biological roles of EVs in intercellular communication.

cell biology↗

bioRxiv2025

A novel biosensor for ferrous iron developed via CoBiSe:A computational method for rapid biosensor design

Genetically encoded biosensors enable monitoring of metabolite dynamics in living organisms. We present CoBiSe, a computational approach using Constraint Network Analysis to identify optimal insertion sites for reporter modules in molecular recognition elements (MREs). Applied to the iron-binding protein DtxR from Corynebacterium glutamicum, CoBiSe identified a flexible connective loop (residues 138-150) for inserting the reporter module, resulting in IronSenseR, a novel ratiometric biosensor for ferrous iron (Fe{superscript 2}). IronSenseR demonstrates high specificity for Fe{superscript 2} with dissociation constants of 1.55 {+/-} 0.08 {micro}M (FeSO4) and 2.44 {+/-} 0.28 {micro}M (FeCl2), while showing no binding to Fe3 and other divalent cations. In vivo assessment in Escherichia coli, Pseudomonas putida and Corynebacterium glutamicum confirmed IronSenseRs capability to detect changes in the intracellular iron pool. The creation of IronSenseR underlines that, by reducing search space and eliminating labor-intensive screening, CoBiSe streamlines biosensor development and enables precise creation of next-generation biosensors for diverse metabolites.

bioengineering↗

bioRxiv2025

A critical role of sux cistron-mediated sucrose uptake for virulence of the rice blight pathogen Xanthomonas oryzae pv. oryzae

The virulence of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight (BB) of rice, critically depends on the activation of SWEET sucrose uniporters of the host. To date, the role of SWEET-released sucrose for virulence remains unclear. We here identified the sux locus of Xoo, consisting of a LacI-type repressor (SuxR), an outer membrane TonB-like porin (SuxA), an inner membrane MFS H+-symporter (SuxC), and a cytosolic sucrose hydrolase (SuxB). Structural and functional analyses demonstrate that SuxB has exclusive sucrose hydrolase activity. Mutant analyses show that the transporter SuxC and the sucrose hydrolase are necessary for growth of bacteria on sucrose, while SuxA is not essential, likely due to the ability of other porins to transport sucrose across the outer membrane. Consistent with a role of SuxR as a sucrose repressor, transcriptome studies show sucrose-dependent regulation of the suxA/suxB genes. Besides a role of sucrose for reproduction, we found that sucrose promotes motility, EPS production, biofilm formation, and virulence. Notably, the SuxC sucrose H+-symporter and the sucrose hydrolase SuxB were required for full virulence of Xoo on indica and japonica rice varieties. Our findings indicate that pathogen-induced sucrose efflux via SWEETs provides sucrose to Xoo, that Xoo uses the sux gene cluster to acquire and utilize sucrose, and that sucrose promotes bacterial fitness and xylem colonization. Significance StatementUnderstanding disease mechanisms is critical for developing strategies to protect plants against infections. Bacterial blight, a major threat to global rice production, is caused by Xanthomonas oryzae pv. oryzae strains that hijack rice SWEET sucrose uniporters. Xanthomonas oryzae pv. oryzae deploys a set of molecular "keys", TAL effectors, to trigger host sucrose release. Sugars could serve as nutrients for the bacteria or prime host defense. Here, we provide evidence that the pathogens ability to attack and grow depends on sucrose uptake. We explore how crucial virulence functions, such as motility, EPS production, and biofilm formation, depend on the activity of the sucrose utilization system, supporting a key role of sucrose for the nutrition of the bacteria in the xylem.

microbiology↗

bioRxiv2025

Molecular Insights into CLD Domain Dynamics and Toxin Recruitment of the HlyA E. coli T1SS

Escherichia coli is a Gram-negative opportunistic pathogen causing nosocomial infections through the production of various virulence factors. Type 1 secretion systems (T1SS) contribute to virulence by mediating one-step secretion of unfolded substrates directly into the extracellular space, bypassing the periplasm. A well-studied example is the hemolysin A (HlyA) system, which secretes the HlyA toxin in an unfolded state across the inner and outer membranes. T1SS typically comprise a homodimeric ABC transporter (HlyB), a membrane fusion protein (HlyD), and the outer membrane protein TolC. Some ABC transporters in T1SS also contain N-terminal C39 peptidase or peptidase-like (CLD) domains implicated in substrate interaction. Recent cryo-EM studies have resolved the inner-membrane complex as a trimer of HlyB homodimers with associated HlyD protomers. However, a full structural model including TolC remains unavailable. We present the first complete structural model of the HlyA T1SS, constructed using template- and MSA-based information and validated by SAXS. Molecular dynamics simulations provide insights into the function of the CLD domains, which are partially absent from existing cryo-EM structures. These domains may modulate transport by stabilizing specific conformations of the complex. Simulations with a C-terminal fragment of HlyA indicate that toxin binding occurs in the occluded conformation of HlyB, potentially initiating substrate transport through a single HlyB protomer before transitioning to an inward-facing state. HlyA binding also induces allosteric effects on HlyD, altering key residues involved in TolC recruitment. These results indicate how substrate recognition and transport are coupled and may support the development of antimicrobial strategies targeting the T1SS.

biophysics↗

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↗

bioRxiv2022

Integrative modeling of guanylate binding protein dimers

Guanylate binding proteins (GBPs) are interferon-{gamma}-activated large GTPases, effective against intracellular pathogens like Toxoplasma gondii. Their host-protective functions require oligomerization, however, the oligomer structures have not been completely resolved yet. Here, we provide dimer models for hGBP1 and the murine GBPs 2 and 7 (mGBP2 and mGBP7) based on integrative modeling that involves the crystal structure of the G domain dimer of hGBP1, cross-linking mass spectrometry (XL-MS), small angle X-ray scattering (SAXS), protein-protein docking, and molecular dynamics (MD) simulations of hGBP1, mGBP2 and mGBP7. We first compare the sequences and protein dynamics of the monomeric hGBP1, mGBP2, and mGBP7, finding that the M/E domain of all three proteins is highly mobile featuring a hinge movement, yet this motion is less pronounced in mGBP7 while its GTPase (G) domain is more flexible. These differences can be explained by the variations in the sequences between mGBP7 and hGBP1/mGBP2 and extend to their dimers. While hGBP1 and its close orthologue mGBP2 dimerize via their G domains, mGBP7 shows a variety of possible dimer structures, among them parallel and crossed-stalk conformations. The G domain is only partly involved in mGBP7 dimerization, which provides a rational why mGBP7, unlike hGBP1 and mGBP2, can dimerize in the absence of GTP. The different GBP dimer structures, which still exhibit hinge movements to certain degrees, are expected to encode diverging functions, such as a destabilization of pathogenic membranes or fusion of the parasitophorous vacuole membrane with the autophagic machinery.

biochemistry↗

bioRxiv2021

A new twist in ABC transporter mediated multidrug resistance - Pdr5 is a drug/proton co-transporter

The two major efflux pump systems are involved in multidrug resistance (MDR): (i) ATP binding cassette (ABC) transporters and (ii) secondary transporters. While the former use binding and hydrolysis of ATP to facilitate export of cytotoxic compounds, the latter utilize electrochemical gradients to expel their substrates. Pdr5 from Saccharomyces cerevisiae is a prominent member of eukaryotic ABC transporters that are involved in MDR and used as a frequently studied model system. Although investigated for decades, the underlying molecular mechanisms of transport and specificity remain elusive. Here, we provide electrophysiological data on reconstituted Pdr5 demonstrating that this MDR efflux pump does not only actively translocate its substrates across the lipid bilayer, but generates a proton motif force in the presence of Mg2+-ATP and substrates by acting as a proton/drug co-transporter. Importantly, a strictly substrate dependent co-transport of protons was also observed in in vitro transport studies using Pdr5-enriched plasma membranes. Similar observations have not yet been reported for any other MDR efflux pump. We conclude from these results that the mechanism of MDR conferred by Pdr5 and likely other transporters is more complex than the sole extrusion of cytotoxic compounds and involves secondary coupled processes suitable to increase the effectiveness.

biochemistry↗

bioRxiv2020

Mucin-derived O-glycans supplemented to diet mitigate diverse microbiota perturbations

Microbiota-accessible carbohydrates (MACs) are powerful modulators of microbiota composition and function. These substrates are often derived from diet, such as complex polysaccharides from plants or human milk oligosaccharides (HMOs) during breastfeeding. Host-derived mucus glycans on gut-secreted mucin proteins may serve as a continuous endogenous source of MACs for resident microbes; here we investigate the potential role of purified, orally-administered mucus glycans in maintaining a healthy microbial community. In this study, we liberated and purified O-linked glycans from porcine gastric mucin and assessed their efficacy in shaping the recovery of a perturbed microbiota in a mouse model. We found that porcine mucin glycans (PMGs) and HMOs enrich for taxonomically similar resident microbes. We demonstrate that PMGs aid recovery of the microbiota after antibiotic treatment, suppress C. difficile abundance, delay the onset of diet-induced obesity, and increase relative abundance of resident Akkermansia muciniphila. In silico analysis revealed that genes associated with mucus utilization are abundant and diverse in prevalent gut commensals and rare in enteric pathogens, consistent with these glycan-degrading capabilities being selected for during host development and throughout evolution of the host-microbe relationship. Importantly, we identify mucus glycans as a novel class of prebiotic compounds that can be used to mitigate perturbations to the microbiota and provide benefits to host physiology.

microbiology↗

bioRxiv2020

Mechanism of BceAB-type transporter: Resistance by lipid II flipping

Treatment of bacterial infections are the great challenge of our era due to the evolved resistance mechanisms against antibiotics. The Achilles heel of bacteria is the cell wall especially during the needs of its synthesis and cell division. Here lipid II is an essential cell wall precursor component synthesized in the cytosol and flipped into the outer leaflet of the membrane prior to its incorporation into the cell wall. Compounds targeting the cell wall or its biosynthesis precursors have been around for decades and have been used as antibiotics against bacterial infections like meningitis, pneumonia and endocarditis. Antimicrobial peptides (AMPs) have proven to be a promising weapon against multiresistant bacteria. However, the Bacitracin efflux (BceAB)-type ATP binding cassette transporters expressed in the membrane of human pathogenic bacteria have been shown to confer resistance to these alternative antibiotics, thereby hampering their medical development. In Streptococcus agalactiae COH1 the BceAB-type transporter NsrFP (SaNsrFP) confers high-level resistance against the antimicrobial peptide nisin, a member of the lantibiotic subfamily. We showed that SaNsrFP provides a novel resistance mechanism by flipping lipid II back into the cytosol, thereby preventing the binding of nisin as well as other lipid II targeting compounds. This is intriguing since a relatively simple reaction mediates resistance to human pathogenic bacteria to lipid II targeting antibiotics, regardless of their structure. Significance StatementThe ABC-transporter NsrFP from Streptococcus agalactiae (SaNsrFP) belongs to the BceAB-type transporters. Several BceAB-type transporters are known to confer resistance against multiple antimicrobial peptides. In this study a new resistance mechanism was identified, which is based on the reduction of the number of cell wall precursor lipid II molecules on the cell surface mediated by SaNsrFP. SaNsrFP flips lipid II, which are considered to be the target for many antibiotics, back into the cytoplasm. With this newly gained knowledge about the resistance mechanism of BceAB-type transporters, novel strategies can be established to overcome or bypass this resistance in human pathogenic bacteria.

microbiology↗

bioRxiv2019

The Object Space Task reveals a dissociation between semantic-like and episodic-like memory in a mouse model of Kleefstra Syndrome

Kleefstra syndrome is a disorder caused by a mutation in the EHMT1 gene characterized in humans by general developmental delay, mild to severe intellectual disability and autism. Here, we characterized semantic- and episodic-like memory in the Ehmt1+/- mouse model using the Object Space Task. We combined conventional behavioral analysis with automated analysis by deep-learning networks, a session-based computational learning model and a trial-based classifier. Ehmt1 +/- mice showed more anxiety-like features and generally explored objects less, but the difference decreased over time. Interestingly, when analyzing memory-specific exploration, Ehmt1 +/- show increased expression of semantic-like memory, but a deficit in episodic-like memory. A similar dissociation of semantic and episodic memory performance has been previously reported in humans with autism. Using our automatic classifier to differentiate between genotypes, we found that semantic-like memory features are better suited for classification than general exploration differences. Thus, detailed behavioral classification with the Object Space Task produced a more detailed behavioral phenotype of the Ehmt1 +/- mouse model.\n\nOne Sentence SummaryEhmt1 +/- mice show decreased exploration and episodic-like memory but increased semantic-like memory In the Object Space Task. (143 of 150)

neuroscience↗