Metrics details The rise of methicillin-resistant Staphylococcus aureus (MRSA) as a major public health threat underscores a critical need for new antibacterial strategies Quercetin is a naturally occurring flavonoid with a range of bioactivities including antibacterial activity against S aureus and binds to its potential molecular target is not well understood Understanding the interaction of quercetin with potential bacterial targets may provide crucial insights for developing modified derivatives with better drug-like properties To investigate potential targets of quercetin in S we employed a targeted subtractive proteomics approach which identified the glycosyltransferase MurG as a novel quercetin target Through rigorous molecular docking and extensive 250 ns molecular dynamics simulations quercetin was shown to bind stably to MurG suggesting a mechanism that interferes with the critical peptidoglycan biosynthesis pathway Molecular Mechanics Generalized Born Surface Area (MM-GBSA) analyses provided quantitative evidence of the complex’s stability indicating a strong and stable interaction with potential therapeutic implications Principal Component Analysis (PCA) further validated the reduction in MurG’s structural flexibility upon quercetin binding reinforcing the hypothesis that this interaction could effectively inhibit its biological function The identification of its interaction with MurG provides a foundation for the development of novel could also be adapted to target other resistant pathogens demonstrating broad applicability in the fight against antibiotic resistance the absence of crystal structures or structural data showing quercetin bound to MRSA-specific target limits the ability to visualize interactions at the molecular level as structural insights are essential for the rational design of quercetin-based therapies or their derivatives This approach not only advances our understanding of quercetin’s antibacterial mechanism but also refines hypotheses before experimental validation ensuring that computational predictions focus on strategically relevant targets a crucial enzyme in the peptidoglycan biosynthesis pathway was found to be a significant target for quercetin binding and interactions between quercetin and MurG were investigated using structural modelling and MD simulations to understand the stability and dynamics of the quercetin-MurG interaction These results suggest that quercetin can inhibit S leading to defects in peptidoglycan biosynthesis While quercetin itself may not be an ideal therapeutic this study provides critical insights that will guide the development of more effective This strain was selected due to its clinical relevance as a methicillin-resistant S aureus strain associated with nosocomial outbreaks as well as the availability of a complete and annotated proteome dataset for computational analyses This step clusters similar protein sequences and retains only unique representatives effectively reducing redundancy in the dataset this process ensures a streamlined and accurate dataset facilitating the identification of essential and druggable proteins during subsequent analyses including an e-value threshold of < 0.0001 and bit score > 100 to ensure the selection of high-confidence essential proteins we aimed to prioritize targets whose inhibition could significantly disrupt bacterial viability proteins confirmed to be co-crystallized with quercetin were categorized and organized according to protein family and the nature of their interaction with quercetin The database was then queried using "blastp" against a curated list of protein sequences known to bind quercetin which included structural PDB entries such as 6IJD The goal was to identify proteins within the MRSA252 proteome that not only exhibit significant sequence similarity to known quercetin-binding proteins but also possess potential binding sites for quercetin This step aimed to narrow down potential targets to those with a higher likelihood of interaction with quercetin based on their structural and functional similarities to previously characterized proteins was prepared with the LigPrep module in Schrödinger Maestro Active sites on MurG were identified by superimposing its structure, retrieved from AlphaFold-221 onto the structure of 8SFW—a glycosyltransferase known to interact with quercetin Subsequent double docking experiments involved separately docking quercetin and UDP into their predicted active sites on MurG using the Glide module of Schrödinger Maestro in the extra precision (XP) docking mode The Docked complex was exported in ‘.pdb’ format and 2D representations of protein–ligand interactions were generated using Discovery Studio Visualizer V The production phase of the MD simulations was executed over 250 ns yielding trajectories consisting of 1000 frames These comprehensive simulations provide valuable insights into the molecular interactions and structural dynamics critical for understanding the mechanistic basis of MurG inhibition by quercetin Post-simulation analyses further focused on evaluating several critical parameters to elucidate the structural and dynamic nuances of the MurG-quercetin complex These parameters included the radius of gyration (rGyr) for evaluating protein compactness and the molecular surface area (MolSA) alongside the polar surface area (PSA) to analyze the interaction surface the solvent-accessible surface area (SASA) was examined to gain insights into hydration shell dynamics and ligand accessibility The eigenvectors and eigenvalues derived from PCA elucidated the direction and magnitude of atom movements offering a granular view of structural variations over the course of the simulation thus enhancing our understanding of the dynamic behavior of the protein–ligand complex in comparison to apo form of the protein under study This approach allowed for a detailed visualization of the degree of conservation at the molecular level across different strains Outline of targeted subtractive proteomics methodology employed for filtering proteins from the proteome of S Superimposition of glycosyltransferases from plants co-crystallized with quercetin retrieved from protein data bank (PDB) (a) Role of MurG in Biosynthetic pathway of Peptidoglycan in S. aureus (b) Structural Alignment of MurG (AF-Q6GGZ0 from AlphaFold-2) with 8SFW (from PDB), suggestive of structural similarity amongst both proteins. This image was made with BioRender (https://biorender.com/) Interaction of quercetin and UDP with MurG (AF-Q6GGZ0 from AlphaFold-2) predicted from computationally Quantitative Assessment of Dynamic Interactions and Structural Stability in MurG Complexed with Ligands During MD Simulations c) detail the RMSD and RMSF for MurG in complex with quercetin d) display the RMSD and RMSF for MurG in complex with UDP These metrics elucidate the conformational stability and residue flexibility within MurG throughout the ligand binding process residues interacting with the ligands are marked with green vertical bars denoting crucial contact points that could influence ligand binding and efficacy This comprehensive analysis suggests a complex interplay between MurG and its ligands It highlights the potential of quercetin to stably bind within the active site of MurG potentially influencing its function and paving the way for further experimental validation of its inhibitory effects on the peptidoglycan biosynthesis pathway in S Time-resolved interaction profiles of MurG with (a) quercetin and (b) UDP showing the fluctuation in binding stability and key residues involved in ligand binding over the course of the simulation; interaction fraction profiles of MurG with (c) quercetin and (d) UDP Percentage of Protein–Ligand contacts and Ligand properties of quercetin and UDP while interacting with MurG MM-GBSA Energy Component Analysis of MurG-Ligand Interactions Violin plots illustrate the distribution of various energy components ΔGSolvation = Solvation energy and ΔGvdW = Van der Waals energy) The analysis revealed that the total binding energy which is a primary indicator of binding affinity becomes more negative throughout the simulation This suggests that the interaction between quercetin and MurG strengthens over time pointing to an increased binding affinity as the MD simulation progresses provides a granular view of how this interaction evolves but they begin to cluster more densely towards the lower end of this range in the second half indicating a growing stabilization of the ligand-enzyme complex ranging broadly from about − 20 to − 40 kcal/mol throughout the simulation This variability underscores that electrostatic interactions play a significant but fluctuating role in the binding process maintain relatively stable values between − 20 to − 40 kcal/mol consistently contributing to the interaction’s stability shows minor fluctuations but remains largely in the negative range suggesting that solvation dynamics generally favor the binding of quercetin to MurG Other energy components such as hydrogen bonds and packing energies exhibit less dramatic variations but align with the overall trend of increasing binding affinity across the simulation the data from the MM-GBSA analysis indicates that the interaction between quercetin and MurG not only strengthens over time as evidenced by the increasingly negative ΔGBind but also achieves greater stability due to contributions from various interaction forces including electrostatic The increasingly negative values of ΔGBind throughout the duration of the MD simulation significantly reinforce the inference that the binding affinity of quercetin to MurG improves and stabilizes over time Comprehensive Post-MD Simulation Assessment of MurG in Apo and Ligand-Bound States This figure evaluates the structural and dynamic properties of the MurG enzyme in its unbound (apo) form compared to its complexed state with ligands The panels provide insights into various aspects of protein behavior: (a) RMSD over time showcasing the conformational stability (c) Molecular Surface Area (MolSA) reflecting the overall molecular footprint (d) Solvent Accessible Surface Area (SASA) indicating exposure to the solvent (e) Polar Surface Area (PSA) measuring interactions with polar molecules and (f) Radius of Gyration (rGyr) depicting the compactness of the protein structure The PCA of the MurG enzyme (AF-Q6GGZ0) from a post-MD simulation highlights differences between its behavior in the unbound (apo) form and when complexed with ligands (Fig. 10). PCA is used here to emphasize variation and extract prominent patterns from the dataset. Post MD Simulation PCA of MurG Conformational Variability PCA results show the variance in protein conformations of MurG in both Apo- and ligand-bound states over the course of a MD simulation This analysis underscores the impact of ligand binding on the conformational behavior of MurG The upper section of Fig. 10 examines MurG in its apo form The PCA scatter plots display the first three principal components (PC1 with data points transitioning from blue to red likely indicating progression through the MD simulation or different conformational states within the simulation The scree plot shows the proportion of variance explained by each principal component PC1 and PC2 show a broad spread in conformations highlighting substantial conformational flexibility or diversity in MurG’s apo form PC3 also displays noticeable variation but to a lesser extent The scree plot emphasizes that the first few components account for a significant portion of the total variance suggesting these components capture major movements within the protein structure The lower section of Fig. 10 focuses on MurG in complex with the ligands The layout and color progression are similar to the upper section but likely denote interaction dynamics over time or various binding states of the ligands This section reveals a more clustered distribution of data points compared to the apo form indicating reduced conformational flexibility due to ligand binding The narrowed range of motion in PC1 and PC2 suggests a decrease in the diversity of protein conformations compared to the apo form The corresponding scree plot reveals a shift in variance distribution with less variance explained by the first three components (38.4% by PC3) highlighting that ligand binding influences the dynamics and potentially stabilizes specific conformations over others In summary, Fig. 10 demonstrates that the apo form of MurG exhibits greater conformational diversity and flexibility as evidenced by the broader distribution of PCA scores the complexed form with ligands displays a more restricted conformational space suggesting that ligand binding stabilizes specific protein structures potentially offering a more predictable and consistent interaction interface for therapeutic targeting This difference could be critical in understanding how ligand binding impacts the structural and functional dynamics of MurG (a) MSA of MurG protein sequences from various S The alignment reveals a significant degree of conservation within the binding site residues Only minor variations are observed in other parts of the protein (b) Superimposition of 3D structures of MurG proteins from various S The structural overlay demonstrates a remarkable overlap of the binding site residues across different strains confirming the structural conservation of the binding pocket This high level of conservation suggests that the MurG binding site is preserved across the global S To further substantiate these findings, the 3D structures of MurG from various strains were superimposed using ChimeraX. The resulting structural overlay (Fig. 11) demonstrated a remarkable overlap of the binding site residues across the different S reinforcing the potential of MurG as a universal therapeutic target These findings underscore the relevance of MurG as a broadly applicable target for antibacterial strategies supporting the validity of targeting this enzyme across diverse S including those beyond the MRSA252 strain studied initially This broad applicability highlights the potential of MurG inhibitors in the development of new treatments for a wide range of S a novel paradigm for antibiotic discovery that leverages evolutionary conservation to repurpose plant-natural product interactions against bacterial pathogens By focusing on MRSA (rather than methicillin-susceptible S this study addresses the urgent need for therapies that circumvent conventional resistance mechanisms which focused on generic essential pathways without leveraging evolutionary mimicry thereby refining our understanding of its mode of action Our findings demonstrate that quercetin acts as a promising inhibitor of MurG in S supported by both subtractive proteomics and comprehensive molecular dynamics simulations By exploiting structural similarities between plant glycosyltransferases and bacterial MurG quercetin effectively competes with UDP-GlcNAc disrupting a critical step in peptidoglycan biosynthesis This mechanism diverges from previously proposed targets for quercetin and underscores the potential advantages of repurposing naturally occurring molecules against bacterial pathogens The observed stability of the quercetin–MurG complex suggests that bacterial cell wall assembly could be impeded by quercetin in a manner akin to synthetic MurG inhibitors but with the added advantage of utilizing a natural scaffold This novel insight into quercetin’s antibacterial mode of action thus paves the way for developing enhanced therapeutic strategies against MRSA and potentially other resistant pathogens Future work should prioritize experimental validation of quercetin’s inhibitory effects on MurG through rigorous in vitro and in vivo assays such as X-ray crystallography or site-directed mutagenesis of key residues would confirm the critical interactions identified in silico Such data would also guide rational design of quercetin-based analogs with improved pharmacokinetic properties possibly by leveraging structural parallels between plant and bacterial glycosyltransferases Assessing the synergistic potential of quercetin in combination with existing antibiotics may further illuminate its role in mitigating resistance this research highlights the utility of integrating computational screening with targeted enzymatic assays offering a blueprint for discovering new antibacterial agents that harness evolutionarily conserved architectures across distinct biological kingdoms The datasets analysed during the current study are publicly available and were accessed from the following databases: UniProt (for the Staphylococcus aureus strain MRSA252 proteome and the AlphaFold-2 Protein Structure Database (for the predicted structure of S Biofilm producing methicillin-resistant Staphylococcus aureus (MRSA) infections in humans: Clinical implications and management Staphylococcus aureus infections: Epidemiology Molecular and anti-microbial resistance (AMR) profiling of methicillin-resistant staphylococcus aureus (MRSA) from hospital and long-term care facilities (ltcf) environment Silva, V., Correia, S., Pereira, J. E., Igrejas, G. & Poeta, P. Surveillance and Environmental Risk Assessment of Antibiotics and AMR/ARGs Related with MRSA: One Health Perspective, 271–295 (2020). https://doi.org/10.1007/978-3-030-40422-2_13 Evaluation of broad-spectrum antibacterial efficacy of quercetin by molecular docking molecular dynamics simulation and in vitro studies Discovery of quercetin and its analogs as potent OXA-48 beta-lactamase inhibitors Quercetin 3-O-rutinoside mediated inhibition of PBP2a: Computational and experimental evidence to its anti-MRSA activity Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination aureus by targeting ClpP to protect mice from MRSA-induced lethal pneumonia Exploring optimal drug targets through subtractive proteomics analysis and pangenomic insights for tailored drug design in tuberculosis In silico subtractive proteomics approach for identification of potential drug targets in Staphylococcus saprophyticus an update of the database of essential genes that includes built-in analysis tools DrugBank 5.0: A major update to the DrugBank database for 2018 Complete genomes of two clinical Staphylococcus aureus strains: Evidence for the evolution of virulence and drug resistance CD-HIT Suite: A web server for clustering and comparing biological sequences UCSF ChimeraX: Tools for structure building and analysis OPLS3: A force field providing broad coverage of drug-like small molecules and proteins Highly accurate protein structure prediction with AlphaFold Karplus, M. & McCammon, J. A. Molecular dynamics simulations of biomolecules. Nat. Struct. Biol. 9, 646–652. https://doi.org/10.1038/nsb0902-646 (2002) Canonical dynamics: Equilibrium phase-space distributions How does solvation layer mobility affect protein structural dynamics? free energy calculation and molecular dynamics simulation studies of CDK2 inhibitors The Bio3D packages for structural bioinformatics The MurG glycosyltransferase provides an oligomeric scaffold for the cytoplasmic steps of peptidoglycan biosynthesis in the human pathogen Bordetella pertussis Inhibitors targeting on cell wall biosynthesis pathway of MRSA and immunoinformatics investigation to unveil a potential multi-epitope vaccine against schistosomiasis Subtractive proteomics to identify novel drug targets and reverse vaccinology for the development of chimeric vaccine against Acinetobacter baumannii Identification of potential drug targets by subtractive genome analysis of Escherichia coli O157:H7: An in silico approach Francois, P., Scherl, A., Hochstrasser, D. & Schrenzel, J. Proteomic Approach to Investigate MRSA, 179–199 (2007). https://doi.org/10.1007/978-1-59745-468-1_14 Leveraging big data bioinformatics approaches to extract knowledge from Staphylococcus aureus public omics data Whole-genome analysis uncovers loss of blaZ associated with carriage isolates belonging to methicillin-resistant Staphylococcus aureus (MRSA) clone ST5-VI in Cape Verde Antibiotic resistance and the MRSA problem Identification of active-site inhibitors of MurG using a generalizable high-throughput glycosyltransferase screen Murgocil is a highly bioactive staphylococcal-specific inhibitor of the peptidoglycan glycosyltransferase enzyme MurG Molecular basis of non-β-lactam antibiotics resistance in Staphylococcus aureus Download references gratefully acknowledges the Indian Council of Medical Research (ICMR) and the Department of Health Research (DHR) for awarding the Long-Term International Fellowship [12011/12/23-HR and INDO/FRC/452/Y-65/2023-24-IHD] which provided essential support for this work This research did not receive any specific grant from funding agencies in the public The facility of computational work represented in this paper was supported by—Network Program on Antimicrobial Resistance and One Health: Human Health Care Node (Gujarat University) [GSBTM/JD(R&D)/616/21-22/1236] by the Gujarat State Biotechnology Mission (GSBTM) Department of Microbiology & Biotechnology Peter Doherty Institute for Infection and Immunity Department of Biochemistry and Forensic Science Jignesh Prajapati; Formal analysis: Dweipayan Goswami Pidot; Writing—original draft: Dweipayan Goswami Pidot; Writing—review & editing: Dweipayan Goswami The authors declare no competing interests The research did not involve human or animal participants Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-025-90395-4 Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly. Join the conversation You can save this article by registering for free here. Or sign-in if you have an account Create an account or sign in to continue with your reading experience she travelled to her hometown of New Town in Burnpur friends and teenage crushes to tell the tale of two kitchens in her third cookbook Now a broadcaster and food and travel writer based in southwest England Gill has travelled to many parts of India and the world for various publications including The New York Times and The Telegraph Join Laura Brehaut on a weekly food adventure with mouth-watering reads delivered Fridays By signing up you consent to receive the above newsletter from Postmedia Network Inc The next issue of Cook This will soon be in your inbox Interested in more newsletters? Browse here. documenting her journey from Kashmir to Ladakh “is very close to my heart because it showcases me as a writer.” Romy Gill’s India enabled her to revisit her past like never before and go deeper into the experiences that shaped her as a chef I wanted to write really personal stories.” Gill’s parents were born in Punjab in northern India moved to the industrial New Town to work at the steel plant she shares more than 70 recipes inspired by her Bengali home Punjabi heritage and many other diverse influences from childhood friends to long train journeys Gill discovered that while much had changed They went to the shop where Sandhu used to buy chanachur (a popular Bengali snack mix) for parties her high school and the house where she was born but also it was very emotionally draining at the same time,” says Gill “Going back to both Punjab and Bengal opened so many things.” “I just couldn’t sit here and write,” adds Gill from her kitchen in Thornbury where she had just pulled a polenta cake with cherries out of the oven “I needed to be there to be able to write this book.” Drawing on her memories and phone calls with her late mother to cook the dishes she grew up with — such as momos stuffed parathas and samosas — helped ease her homesickness She started hosting supper clubs and opened the Michelin-recognized restaurant Romy’s Kitchen in 2013 Gill was appointed an MBE (Member of the Order of the British Empire) for her work in the food industry Gill credits her knack for cooking to her parents and grandparents she would visit their farmhouses in Punjab where they would plant vegetables and cook over an open fire She would marvel at how her nani (maternal grandmother) and dadi (paternal grandmother) made different dishes in different ways “Even if you’re Punjabi but from a different region they would make completely different things Gill took inspiration from her mother’s plant-based cooking Romy Gill’s India takes a broader view of the people and places that shaped her career as a chef “I want to write books that are close to my heart you change a cumin there or coriander here and create a new recipe I had to tell a story about these two kitchens.” Gill highlights that these are the dishes she cooks now — her recipes from home This is the food I grew up eating and that my parents and grandparents would eat,” says Gill “I wanted recipes that are meaningful to me I didn’t want recipes just for the sake of it.” “For me to write this book as a chef was very very important to showcase where I came from For the marinade: 1 tsp ground cumin 1 tsp ground coriander 1 tsp chili powder 1/2 tsp salt 1 tsp cornstarch plus extra for frying 200 g (7 oz) white onions peeled and finely chopped 3 large garlic cloves peeled and grated 15 g (1/2 oz) ginger root finely chopped 1/2 tsp tomato paste 1 tsp salt 1 tsp chili powder 2 tsp dried fenugreek leaves (kasoori methi) or use around 20 g (3/4 oz) fresh cilantro 1 tsp ground cumin 1 tsp ground coriander 1 tsp ground turmeric 50 g (2 oz/1/4 cup) plain yogurt 50 mL (1 3/4 fl oz/3 tbsp) table cream (18 per cent) place all the ingredients in a bowl and mix well Add the paneer and gently toss in the marinade to ensure it is well coated Leave to marinate at room temperature for 20-30 minutes heat the oil in a saucepan over a high heat Add the tomatoes and tomato purée and cook for a further 4-5 minutes over a medium heat chili powder and dried fenugreek leaves along with the remaining spices cream and 100 mL (3 1/2 fl oz/scant 1/2 cup) water heat a little oil in a frying pan and fry the marinated paneer until light brown on all sides add it to the sauce and cook for a further 5 minutes Remove from the heat and leave to rest for 5 minutes before serving with Indian flatbreads or the side dish of your choice You will need 6 wooden skewers soaked in cold water for 30 minutes cut into bite-size pieces 200 mL (7 fl oz/scant 1 cup) whipping cream (36 per cent or higher) 50 g (2 oz/1/4 cup) plain yogurt 100 g (3 1/2 oz) paneer then just crumble it) 1 tsp ground cumin 1 tsp ground coriander 1 tsp salt 1 tsp ground black pepper Small handful of fresh cilantro chopped Juice of 1/2 lemon 4 tsp oil 20 g (3/4 oz) melted butter To serve: Sirke wale pyaaz (pickled shallots recipe follows) Pudina aur dhaniya chutney (mint and cilantro chutney recipe follows) Kachumber (cucumber and tomato salad Place the chicken pieces in a large bowl with all of the remaining ingredients except the butter Mix well to combine and prick the meat gently with a fork or toothpick to allow the marinade to penetrate Cover the bowl and marinate in the fridge for at least 2-3 hours Thread the marinated chicken pieces onto the skewers place on a baking tray and cook under the hot grill for 5 minutes turn the skewers and brush again with the melted butter to ensure the skewers are evenly cooked on each side Return to the grill for a further 5 minutes spicy cilantro chutney and kachumber salad 15 small shallots 50 mL (1 3/4 fl oz/3 tbsp) distilled malt vinegar 1 tsp salt Peel the shallots and soak in a bowl of water for 15 minutes then place in a bowl with the vinegar and salt Transfer the pickled shallot to a sterilized glass jar with an airtight lid These pickled shallots will keep for up to one week when stored in the fridge 50 g (2 oz) fresh mint leaves 30 g (1 oz) fresh cilantro roughly chopped with stems 1 medium white onion peeled and roughly chopped 2 green chilies 1 small raw mango peeled and diced into 1-cm (1/2-in) cubes Juice of 1 lemon 2 tsp sugar 1 tsp salt 2 tbsp olive oil Place all the ingredients along with 75 mL (2 1/2 fl oz/5 tbsp) water in a blender or food processor and blitz to a fine paste Transfer the chutney to a sterilized glass jar with an airtight lid This chutney will keep for up to 5 days when stored in the fridge seeds removed and flesh finely chopped 1 medium red onion peeled and finely chopped 20 g (3/4 oz) fresh cilantro finely chopped 1/2 tsp salt 1/2 tsp coarsely ground black pepper Juice of 1 lime Mix together all the vegetables with the fresh cilantro 30 mL (1 fl oz/2 tbsp) canola oil 1 tsp panch phoron (see note) 3 dried red chilies 1 dried bay leaf 15 g (1/2 oz) ginger root peeled and grated 500 g (1 lb 2 oz) pumpkin skin on and diced into 2-cm (3/4-in) cubes 1 tsp salt 1 tsp granulated or superfine sugar 1 tsp chili powder 1 tsp ground turmeric 1 tsp ground coriander 1 tsp ground cumin 1 tsp Bengali garam masala (recipe follows) add the ginger and cook over a high heat for 1 minute then cover the pan with a lid and cook for 10 minutes sprinkle over the garam masala and serve with boiled rice or fried poori 20 green cardamom seeds 10-12 cloves 2 x 5-cm (2-in) cinnamon sticks Place the spices in a blender or food processor and blitz to a powder you can use ground spices — but you should still blitz them in a blender or food processor so they’re well combined Transfer the garam masala to a sterilized glass jar with an airtight lid This garam masala will keep for up to 6 months when stored in a cool transmission or republication strictly prohibited This website uses cookies to personalize your content (including ads), and allows us to analyze our traffic. Read more about cookies here. By continuing to use our site, you agree to our Terms of Use and Privacy Policy You can manage saved articles in your account Sie haben erfolgreich Ihre Einwilligung in die Nutzung von Transfermarkt mit Tracking und Cookies widerrufen. Sie können sich jetzt zwischen dem Contentpass-Abo und der Nutzung mit personalisierter Werbung, Cookies und Tracking entscheiden. The purpose of the scholarship is to motivate and support high school students who have a deeper interest in IT The scholarships were awarded to Loore Johanna Jaup consisting of representatives of TalTech and MURG selected three MURG students with excellent study results in science who will receive a scholarship in the amount of 200 euros per month from September to June 2025 The scholarships were handed over by Rector Tiit Land and Vice-Rector Hendrik Voll "We decided to give scholarships to three good MURG 11th grade students All three are extremely good in science and take part in extracurriculum activities Karl Robin Volmer is building a solar car in the Solaride team Gregor Siimon is an active leader in the field of robotics Loore Johanna Jaup is a big fan of architecture and wants to continue her studies in the TalTech architecture programme I wish success and happiness to all scholarship recipients," said Hendrik Voll A total of 10 qualifying applications were received from young people with outstanding results students in the current 10th grade who will receive the scholarship from January to June can apply for the scholarship Metrics details Peptidoglycan is a major component of the bacterial cell wall and thus a major determinant of cell shape Its biosynthesis is initiated by several sequential reactions catalyzed by cytoplasmic Mur enzymes metabolize molecules not present in eukaryotes and are structurally and biochemically tractable although many Mur inhibitors have been developed few have shown promising antibacterial activity prompting the hypothesis that within the cytoplasm Mur enzymes could exist as a complex whose architecture limits access of small molecules to their active sites This suggestion is supported by the observation that in many bacteria and pairs of these genes often are fused to generate a single polypeptide we explored this genetic arrangement in the human pathogen Bordetella pertussis and show that MurE and MurF are expressed as a single and analytical centrifugation (AUC) revealed that the MurE–MurF fusion displays an elongated MurE–MurF interacted with the peripheral glycosyltransferase MurG which formed discrete oligomers resembling 4- or 5-armed stars in EM images The oligomeric structure of MurG may allow it to play a bona fide scaffolding role for a potential Mur complex facilitating the efficient conveyance of peptidoglycan-building blocks toward the inner membrane leaflet Our findings shed light on the structural determinants of a peptidoglycan formation complex involving Mur enzymes in bacterial cell wall formation Schematic diagram of the cytoplasmic and membrane-related steps of peptidoglycan biosynthesis The scheme includes structures of macromolecules from different bacterial species: MurA (1NAW); MurB (1MBT); MurC (1J6U); MurD (4BUC); MurE (4BUB); MurF (3ZL8); MurG (1F0K); MraY (4J72) which brings up questions regarding a catalytic advantage for the cell in their association while still underlining the potential relevance of a multi-protein complex the structural nature of such a scaffold has not been studied to date we characterized the scaffolding role of MurG from the human pathogen Bordetella pertussis by examining its oligomeric state and stoichiometry both in vitro and on bacterial membranes MurG is dimeric in the presence of detergents but oligomerizes into higher order species (tetramers and above) in their absence Negative staining electron microscopy (EM) images of MurG oligomers revealed isolated particles that resemble 4- or 5- pointed stars and the different forms of MurG recognize MurE-MurF with similar affinities These results shed light onto the structural determinants of a peptidoglycan formation complex involving Mur enzymes that plays a role in cell wall formation in a pathogenic species but that can be applicable to all walled bacteria but to date the structural determinants and functional significance of this arrangement have not been reported Given the fact that MurG has been reported to serve as a potential scaffold for other peptidoglycan biosynthesis enzymes and that this function could be impacted by its oligomeric form we set out to characterize MurG both in vitro and in cellulo MurG adopts different oligomeric states in bacterial membranes and in vitro (A) Western Blot of MurG present in purified E coli membranes treated with increasing concentrations of DMP (lanes 1–4) Molecular weight markers are shown to the right of the blot and the approximate location of monomers (1X) to hexamers (6X) is shown on the left (B) SDS-PAGE of MurG overexpressed and purified as a soluble protein in presence of detergent was treated with increasing concentrations of EGS (ethylene glycol bis(succinimidyl succinate)) Biophysical characterization of purified MurG (A) SEC analysis of MurG performed on a Superose 6 10/300 column (B) Analytical ultracentrifugation sedimentation profiles of MurG in buffer containing DDM: experimental and fitted values (top panel) and residuals (lower panel) The profiles correspond to absorbance measurements at 250 nm made in buffer containing DDM during 8 h of sedimentation Data obtained in interference (J) and absorbance at 280 nm are not shown for simplicity (C) Corresponding c(s) distributions of MurG in buffer without detergents or in the presence of DM or DDM (1.2 x CMC) we observed a large asymmetrical main peak with a max s-value of 5.1 +/− 0.05 S (s20,w of 7.24 S +/− 0.1 S) The broad distribution of s values suggests a mixture of different oligomers which could include a tetramer as well as forms of lower and higher molecular mass the gradient tubes were fractionated and different fractions were observed by negative stain EM Negative-stain electron microscopy of MurG and MurE-MurF samples (A) Representative micrographs of MurG and (B) MurE-MurF Below: gallery of ab initio class averages of MurG (left) and MurE-MurF (right) The capability to form tetramers or higher order species as well as the presence of flexible regions could play a role in the ability of MurG to serve as a framework for other Mur ligases in order to expand our study of a Mur ligase complex scaffolded by MurG we set out to characterize a potential interaction partner as well as the interaction between the different Mur enzymes (A) Scheme of MurE-MurF employed in this study (B) SEC-MALS chromatograms of MurE-MurF monomers and dimers showing readings from the light scattering detector The discontinuous lines indicate the calculated molecular masses of the two species Sedimentation velocity AUC analyses of MurE-MurF from B Superposition of experimental and fitted sedimentation velocity AUC profiles obtained during 8 h of sedimentation at 42,000 rpm and 20 °C for monomeric (A) and dimeric (B) MurE-MurF at 40 µM One of five acquired profiles is shown for clarity (C) Superposition of the c(s) distribution profiles for the monomer and dimeric species of MurE-MurF both having been tested at 3 different concentrations and include the extrapolated s20,w at infinite dilution The f/fmin estimates were slightly larger than those derived from Rh but nevertheless confirmed that the dimer is more elongated as compared to the monomer The dimeric MurE-MurF sample showed additional minor contributions (≈20%) which could be indicative of dissociation events as well as larger species at or above 7.4 S (s20,w = 9.3 S which could correspond to larger aggregates SAXS analysis of monomeric and dimer MurE-MurF (A) The radially averaged scattered X-ray intensity (I) was plotted as function of the scattering angle (s) Scattering patterns for MurE-MurF monomer and dimer (blue and red respectively) were recorded at different concentrations but the two curves are related to the highest concentrations only (B) Plot of pair-distance distribution function for the MurE-MurF monomer (light blue) and dimer (red) (C) SAXS envelope of the MurE-MurF monomer The individual crystal structures of MurE and MurF from T maritima (PDB codes: 4BUB and 3ZL8) (approx This observation suggests that MurE-MurF could contain flexible regions that cannot be well fitted using the rigid crystallographic models these observations indicate how this elongated flexible arrangement could favor interactions with other Mur enzymes within a higher order complex Many class averages are well defined and elongated which could represent individual subdomains L- or S-like forms indicate flexibility and a potential mixture of conformational states (A) The meso-A2pm-adding activity (MurE) of the MurE-MurF fusion protein was tested individually using ATP meso-A2pm and UM-[14C]dipeptide as substrates The kinetics of consumption of the latter radiolabeled substrate (■) and formation of the UM-[14C]tripeptide product (▲) were followed as described in Materials and Methods (B) The same assay mixture was supplemented with D-Ala-D-Ala so that the intermediate UM-tripeptide MurE product could subsequently be used by MurF to generate radiolabeled UM-pentapeptide (MurE-MurF coupled assay) The respective amounts of the UM-dipeptide substrate and of the intermediate (UM-tripeptide) and final (UM-pentapeptide) reaction products (■ respectively) were determined at different times and it is conceivable that oligomerization of Mur ligases could play a role in regulation of enzyme activity and channeling of intermediates towards later steps in the pathway MurE-MurF interacts with both forms of MurG (A) Dot Blot assays performed on a nitrocellulose membrane where MurG was blotted in increasing concentrations and subsequently incubated with a solution of MurE-MurF Negative control: an unrelated bacterial protein (B) MST binding curves were fitted to derive the KD between MurG and MurE-MurF (in blue) in the presence (left) or absence (right) of DM The curves related to the experimental control (LAMTOR4/5 unrelated to peptidoglycan biosynthesis) are in red Solid lines represent linear extrapolations Bottom: MST traces corresponding to the titration of MurG (in blue) and the control protein (in red) against 50 nM of MurE-MurF-FITC in the presence and absence of DM right) the dose-dependent curves did not allow the attainment of a clear plateau possibly due to the fact that the MurG sample is not composed of a single oligomeric species we were able to measure an apparent KD value of 0.97 ± 0.25 μM that is in the range of the KD obtained for dimeric MurG This indicates that both forms of MurG can interact with MurE-MurF providing further evidence of the existence of a multi-Mur complex in the bacterial cytoplasm involving the recognition of multiple partners could be facilitated through oligomerization These observations suggest that the last step of Lipid II biosynthesis occurs in specific sites of the cell where the concentration of MurG would be high enough for self-association It is conceivable thus that Mur ligase flexibility may be a necessary element for association with different intracellular partners during either cell division or cell wall elongation facilitating binding/release events in accordance with the peptidoglycan biosynthesis requirement of the cell cycle A potential structural stabilization of the MurE-MurF bifunctional protein in the presence of ligands is still under investigation This work thus provides an initial framework towards the understanding of cytoplasmic complexes that regulate and facilitate peptidoglycan biosynthesis during different phases of the cell cycle which could lead the way to the eventual employment of Mur enzymes as de facto targets for antibiotic development pertussis (strain ATCC 9797/DSM No 5571) was amplified using conventional PCR methods and cloned into a pET15b vector (Novagen) in frame with a thrombin-cleavable N-terminal 6-histidine tag The murG gene from the same strain was cloned into a pET30b vector modified in order to express the protein with a TEV-cleavable N-terminal Strep-tag sequence coli RIL cells carrying the expression vector for the MurE-MurF fusion protein were grown at 37 °C in Luria-Bertani medium (LB) supplemented with 100 mg/l ampicillin and 34 mg/l chloramphenicol Expression was induced by the addition of IPTG to a final concentration of 0.2 mM at OD600nm = 0.7 A.U. and the cells were harvested by centrifugation (6,000 rpm coli BL21(DE3) cells carrying the MurG expression vector were grown at 37 °C in LB medium supplemented with 50 mg/l kanamycin Over-expression was induced by the addition of IPTG to a final concentration of 1 mM at OD600nm = 0.8 A.U. and the cells were collected by centrifugation (6,000 rpm Cells were resuspended in buffer A (25 mM HEPES pH 7.5 5 mM β-mercaptoethanol) supplemented with protease inhibitors cocktail (Sigma-Aldrich) and 200 μg/ml lysozyme (Fluka) Cell lysis was carried out by passing the sample three times through a pre-cooled cell disruptor (Constant Systems) at 15,000 psi The soluble fraction was obtained by centrifugation at 18,000 rpm for 1 h at 4 °C and was loaded onto a pre-equilibrated 5 ml Ni-NTA column (Qiagen) After a washing step to eliminate the unbound proteins the hexahistidine-tagged protein was eluted from the column with buffer B (25 mM HEPES pH 7.5 The fractions containing MurE-MurF were pooled and further purified by size-exclusion chromatography (SEC The protein-containing fractions were collected The protein was concentrated in a 50 kDa cut off concentrator (Vivaspin) Cells were resuspended in buffer D (25 mM HEPES pH 7.5 5 mM DTT) supplemented with 0.5% (w/v) DDM a protease inhibitor cocktail (Sigma-Aldrich) and 200 μg/ml lysozyme (Fluka) Cell lysis was carried out by passing the sample four times through a precooled cell disruptor at 15,000 psi Cell debris were removed by centrifugation at 18,000 rpm for 1 h at 4 °C and the cleared lysate was loaded on pre-equilibrated 5 ml Strep-trap column (Qiagen) After a washing step in buffer D supplemented with 0.01% (w/v) DDM the Strep-fused protein was eluted in buffer D supplemented with 0.01% (w/v) DDM and 2.5 mM d-desthiobiotin Fractions containing purified MurG were analyzed by SDS-PAGE MurG was over-expressed in cells as described before and 4.4 g of cell pellet were re- suspended in ice-cold 25 mM HEPES pH 7.5 The sample was passed three times through a refrigerated cell disruptor at 15,000 psi Unbroken cells and debris were removed by low-speed centrifugation (10,000 rpm The supernatant was collected and the membrane portion pelleted by ultracentrifugation at 40,000 rpm for 1 h at 4 °C and re-suspended in 25 mM HEPES pH 7.5 100 μl of the re-suspended membranes were incubated with increasing concentrations of DMP (from 0.1 to 5 mM) and incubated for 30 min at room temperature Cross-linked samples were quenched by addition of 50 mM Tris-HCl pH 8.0 and were loaded on a SDS-PAGE gradient gel (4–20%) In order to detect MurG by Western blotting samples were transferred to a nitrocellulose membrane for 1.5 h at a constant voltage of 300 V the membrane was blocked for one hour with shaking at room temperature in 3% BSA PBS-T (PBS-Tween 0.05%) The membranes were incubated overnight at 4 °C with shaking with anti-MurG primary antibodies (mouse they were incubated with secondary antibodies (anti-mouse) for 1 h at room temperature The signal was developed using SIGMA FASTDAB tablets with Metal Enhancer according to manufacturer’s instructions Cross-linking experiments were performed with EGS (ethylene glycol bis(succinimidyl succinate); Thermo Scientific) solubilized in DMSO MurG was prepared in buffer containing HEPES pH 7.5 200 mM NaCl and 0.11% DM and mixed with increasing concentrations of EGS (0.1 The reaction was incubated at room temperature for 30 min and afterwards quenched by adding Tris-HCl buffer pH 8.0 at a final concentration of 50 mM The reaction was incubated for additional 15 min and 15 μl of the reaction were loaded on a SDS-PAGE gradient gel (4–20%) MurG was extracted from the bacterial membrane and purified by affinity chromatography as described above; subsequently buffer exchange was carried out on a Superose 6 10/300 column (GE) with buffer C either in the absence or in the presence of detergents (DDM and DM) at a concentration corresponding to 1.2 x CMC 500 μl of sample at a concentration of 2 mg/ml were centrifuged at 13,000 rpm for 20 min at 4 °C and injected onto the column The sample was eluted at a flow rate of 0.5 ml/min at 4 °C Solution density (ρ = 1.036 g/ml) and viscosity (η = 1.259 cp) values were measured experimentally at 20 °C on an Anton Paar DMA 5000 density meter and an Anton Paar AMVn rolling ball viscometer Putative oligomeric states were derived from s considering the Svedberg equation: The experiment was performed at the biophysical analysis platform of the ISBG A Shimadzu Prominence HPLC system equipped with a Superose 6 SEC column was connected in line with a mini DAWN-TREOS and a DynaPro NANOSTAR static and dynamic light scattering instrument (Wyatt Technologies an Optilab rEX refractometer (Wyatt Technologies) and a UV-Vis detector SPD-M20A The light scattering and refractive index detectors were calibrated following the manufacturer’s instructions The SEC column was equilibrated with buffer C and 45–100 μl of each sample were injected with a flow rate of 0.5 ml/min Data from the three detectors were imported by the Astra V software version 5.4.3.20 (Wyatt Technologies) and processed following the manufacturer’s guidelines The experiment was performed at room temperature For the analysis of MurE-MurF the ΔLS and the ΔRI signals were collected and the experimental mass was derived from derived from the calculated from the sequence dn/dc value of 0.185 ml/g for MurE-MurF The standard MurE activity assay measured the formation of radiolabeled UM-L-Ala-γ-D-Glu-meso-A2pm in a reaction mixture (40 μl) containing 100 mM Tris-HCl pH 8.6 and pure enzyme (20 μl of an appropriate dilution in buffer E (20 mM potassium phosphate The standard MurF activity assay measured the formation of radiolabeled UM-L-Ala-γ-D-Glu-meso-A2pm-D-Ala-D-Ala in a reaction mixture (40 μl) containing 100 mM Tris-HCl pH 8.6 0.1 mM UM-L-[14 C]Ala-γ-D-Glu-meso-A2pm (400 Bq) and pure enzyme (20 μl of an appropriate dilution in buffer E) In this assay that measured the formation of the two radiolabeled products UM-tripeptide and UM-pentapeptide the reaction mixture (40 μl) contained 100 mM Tris-HCl pH 8.6 After incubation for 40 min (5 to 40 min in some assays) at 37 °C these reactions were terminated by the addition of glacial acetic acid (8 μl) the radioactive substrates and products were separated on a Nucleosil 100 C18 5U column (150 × 4.6 mm; Alltech France) using 50 mM ammonium formate pH 4.0 Radioactivity was detected with a flow detector (model LB506- C1; Berthold) using the Quicksafe Flow 2 scintillator (Zinsser Analytic) at 0.6 ml/min and quantification was performed with the Radiostar software (Berthold) MurG purified either in the presence of 1.2 x CMC DDM or in its absence was spotted onto a nitrocellulose membrane at different concentrations (from 50 ng to 1 μg) in a final volume of 2 μl The positive and the negative control samples were spotted at 0.8 μg the membrane was blocked in PBS-Tween 0.05% (PBS-T) supplemented with 1% Bovine Serum Albumin (BSA) for 1 h at room temperature with agitation the membrane was incubated overnight at 4 °C in a PBS-T solution containing 0.1 mg/mL of MurE-MurF the membrane was stained with anti-MurE-MurF primary antibodies at a dilution of 1:500 overnight at 4 °C then incubated with anti-rabbit secondary antibodies for 1 h at room temperature Detection was performed with SIGMA FASTTM DAB tablets with Metal Enhancer MurE-MurF was incubated overnight with a 3-fold molar excess of FITC in labelling buffer (25 mM HEPES pH 7.5 Excess FITC was removed by using a PD-10 desalting column and fractions containing the FITC labeled MurE-MurF were pooled and used for the assay Protein concentration was quantified by measuring the absorbance at 280 nm using a Nanodrop a fixed concentration of labeled MurE-MurF (50 µM) was mixed with serial/increasing concentrations of purified MurG ranging from 0.3 nM to 26 µM in a final buffer containing 25 mM HEPES pH 7.5 5% glycerol and 0.17% w/v n-decyl-β-D-maltopyranoside (DM) The samples were inserted into MST standard capillaries and measurements were performed using a Nano Temper Monolith NT.115 instrument equipped with a blue-red detector Detection of labeled protein was performed using the blue LED-filter (excitation at 495 nM) at 22 °C On and Off laser times were set at 5 and 25 s Data sets were processed and analyzed using the MO affinity software version 2.1 (NanoTemper Technologies was plotted against analyte concentration to generate the binding curve labeled MurE-MurF was incubated in increasing concentrations of an unrelated eukaryotic protein The experiment with the oligomeric form of MurG was performed using a fixed concentration of labeled MurE-MurF (50 µM) which was mixed with serial/increasing concentrations of purified MurG ranging from 1 nM to 37 µM in a final buffer containing 25 mM HEPES pH 7.5 The samples were inserted into MST Premium capillaries and measurements were performed using a Nano Temper Monolith NT.115 instrument equipped with a blue-red detector In order to stabilize oligomeric MurG for negative stain electron microscopy, GraFix was carried out on purified MurG24 50 μl of sample at a concentration of 1.6 mg/ml were applied to the top of a 1 ml centrifuge tube (Beckman) containing 10–30% (v/v) glycerol and 0–0.15% (v/v) glutaraldehyde gradient prepared in 25 mM HEPES pH 7.5 and 300 mM NaCl Gradients were ultracentrifuged for 14 h at 50,000 rpm and 4 °C the gradient was fractionated from the top of the ultracentrifugation tube and the single 25 μl fractions were transferred to a 96-well plate Fractions were tested using a Bradford assay and subsequently analyzed by negative stain EM in order to detect the most homogeneous sample 3 μl of sample were applied to the clean side of carbon on a carbon–mica interface and stained with 2% sodium silicotungstate Micrographs were recorded on a FEI Tecnai T12 microscope operated at 120 kV with a Gatan Orius 1000 camera Images were recorded at a nominal magnification of 29,000 x resulting in a pixel size of 2.22 Å architecture and biosynthesis in Escherichia coli Bridging cell wall biosynthesis and bacterial morphogenesis Purification and characterization of the bacterial MraY translocase catalyzing the first membrane step of peptidoglycan biosynthesis MurJ is the flippase of lipid-linked precursors for peptidoglycan biogenesis Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane SEDS proteins are a widespread family of bacterial cell wall polymerases Bacterial cell wall biogenesis is mediated by SEDS and PBP polymerase families functioning semi-autonomously A central role for PBP2 in the activation of peptidoglycan polymerization by the bacterial cell elongation machinery Purification and biochemical characterization of Mur ligases rom Staphylococcus aureus Cytoplasmic steps of peptidoglycan biosynthesis Structural insights into protein-protein interactions involved in bacterial cell wall biogenesis Identification and partial characterization of a novel UDP-N-acetylenolpyruvoylglucosamine reductase/UDP-N-acetylmuramate:L-alanine ligase fusion enzyme from Verrucomicrobium spinosum DSM 4136 Functional and biochemical analysis of Chlamydia trachomatis MurC an enzyme displaying UDP-N-acetylmuramate:amino acid ligase activity Positioning cell wall synthetic complexes by the bacterial morphogenetic proteins MreB and MreD The essential peptidoglycan glycosyltransferase MurG forms a complex with proteins involved in lateral envelope growth as well as with proteins involved in cell division in Escherichia coli MreB and MurG as scaffolds for the cytoplasmic steps of peptidoglycan biosynthesis Membrane interaction of the glycosyltransferase MurG: a special role for cardiolipin Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains The 1.9 Å crystal structure of Escherichia coli MurG a membrane-associated glycosyltransferase involved in peptidoglycan biosynthesis The kinetic characterization of Escherichia coli MurG using synthetic substrate analogues Sedimentation velocity analytical ultracentrifugation for intriniscally disordered proteins GraFix: stabilization of fragile macromolecular complexes for single particle cryo-EM Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases Light scattering and the absolute characterization of macromolecules The concentration dependence of sedimentation Analytical Ultracentrifugation in biochemistry and polymer science eds Harding PRIMUS: a Windows PC-based system for small-angle scattering data analysis Determination of the regularization parameter in indirect transform methods using perceptual criteria Structural characterization of proteins and complexes using small-angle X-ray solution scattering a program for rapid ab-initio shape determination in small angle scattering Uniqueness of ab initio shape determination in small-angle scattering UCSF Chimera–a visualization system for exploratory research and analysis CRYSOL - A program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins Protein-protein interaction regulates the direction of catalysis and electron transfer in a redox enzyme complex The biosynthesis of peptidoglycan lipid-linked intermediates Functional analysis of the cytoskeleton protein MreB from Chlamydophila pneumoniae The murG gene of Escherichia coli codes for the UDP-N-acetylglucosamine: N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase involved in the membrane steps of peptidoglycan synthesis Crystal structure of the Pseudomonas aeruginosa MurG:UDP-GlcNAc substrate complex Structural and enzymatic analysis of TarM glycosyltransferase from Staphylococcus aureus reveals an oligomeric protein specific for the glycosylation of wall teichoic acid Hetero-oligomeric interactions between early glycosyltransferases of the dolichol cycle Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources The tubulin homologue FtsZ contributes to cell elongation by guiding cell wall precursos synthesis in Caulobacter crescentus Natural strategies for the spatial optimization of metabolism in synthetic biology Monitoring peripheral protein oligomerization on biological membranes Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling Calculations and publication-quality illustrations for analytical ultracentrifugation data Analytical ultracentrifuge for the characterization of detergents in solution Calculation of partial specific volumes and other volumetric of detergents and lipids AUC and small-angle scattering for membrane proteins Guinier peak analysis for visual and automated inspection of small-angle X-ray scattering data Phenyl thiazolyl urea and carbamate derivatives as new inhibitors of bacterial cell-wall biosynthesis EMAN: semi-automated software for high-resolution single-particle reconstructions RELION: Implementation of a Bayesian approach to cryo-EM structure determination Download references We acknowledge the platforms of the Grenoble Instruct-ERIC center (ISBG :UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB) We wish to thank Daniel Maragno Trindade (LNBio Campinas) for help with MST experiments and Didier Blanot (I2BC) for discussions on enzyme kinetic assays The electron microscope facility is supported by the Rhône-Alpes Region the Fondation pour la Recherche Médicale (FRM) the Centre National de la Recherche Scientifique (CNRS) the Commissariat à l’Énergie Atomique et aux Énergies Alternatives (CEA) and the GIS-Infrastructures en Biologie Santé et Agronomie (IBISA) We acknowledge support from the Laboratoire International Associé BACWALL (CNRS) 2017/12436-9 from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and ANR-13-BSV8-0015-01 (Agence Nationale de la Recherche) to A.D was supported by doctoral grants from the ANR and the Fondation pour la Recherche Médicale (FRM) FDT20160435484 was supported by doctoral grant 2013/02451-0 from FAPESP Brazilian Biosciences National Laboratory (LNBio) Universidade Estadual de Campinas (UNICAMP) Institute for Integrative Biology of the Cell (I2BC) Univ Paris-Sud and Université Paris-Saclay Delphine Patin & Dominique Mengin-Lecreulx participated in protein purification as well as biochemical and biophysical analyses participated in AUC experiments and data analysis Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-019-40966-z a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science Metrics details Peptidoglycan is a carbohydrate with a cross-linked structure that protects the cytoplasmic membrane of bacterial cells from damage The mechanism of peptidoglycan biosynthesis involves the main synthesizing enzyme glycosyltransferase MurG which is known as a potential target for antibiotic therapy Many MurG inhibitors have been recognized as MurG targets but high toxicity and drug-resistant Escherichia coli strains remain the most important problems for further development the discovery of selective MurG inhibitors has been limited to the synthesis of peptidoglycan-mimicking compounds such as virtual screening using molecular docking drug likeness ADMET proprieties predictions to identify potential natural products (NPs) for Escherichia coli We conducted a screening of 30,926 NPs from the NPASS database 20 of these compounds successfully passed the potency and their validation was further confirmed through molecular docking The best three hits and the standard were chosen for further MD simulations up to 400 ns and energy calculations to investigate the stability of the NPs-MurG complexes The analyses of MD simulations and total binding energies suggested the higher stability of NPC272174 The potential compounds can be further explored in vivo and in vitro for promising novel antibacterial drug discovery Peptidoglycan component synthesis (PCS) cycle catalyzed by MraY Numbers from (1–8) represent common antibiotics targeting the synthesis of bacterial cell walls Overlays of the crystal structures of the MurG enzyme from E PDB code: 1nlm) and Pseudomonas aeruginosa (orange pdb: 3s2u) complexed with UDP-GlcNAc (grey) in the gorge region cell wall drugs are significant antimicrobial agents and it is important to find new agents for this class to prevent the modification of resistance to these inhibitors only a few methods can be used to treat infections caused by highly resistant gram-negative strains we aimed to search novel MurG enzyme inhibitors employing a structure based virtual screening method from databases pursued by ADMET estimation and MD simulations via in silico methods All biological compounds were downloaded in SDF file and used for optimization for a new virtual testing process In silico screening method utilized in the study The cocrystal ligand was used to define the active site within a 10 Å radius during the docking calculations the FRED default parameters were employed to calculate the binding energies of the compounds against the MurG enzyme Ten docked conformations were generated for each ligand using the above-mentioned docking procedure the best five hit compounds with the lowest binding energy were selected for molecular simulation study Superimposition of the UDP_GlcNAc native ligand and the top five hits interacted in the MurG active site Showing the binding poses in a groove between the two domains the last 20 ns of the simulations trajectories of the standard UDP_GlcNAc and three NPs from the top five structures (NPC727174 and NPC259098) complexes were chosen for energy analysis MM-PBSA was applied to predict the average binding free energies using a Python script the output file summary_energy.dat was obtained and contains the total binding energy of all energetic components (ΔGTotal) including the polar solvation energy to calculate the average contribution of the residues to the binding energy the Python script MmPbSaDecomp.py was used including the binding energy for each residue were plotted to show the energy contribution of each significant amino acid residue with its energy The "Supplementary Python scripts S1 and S2" provide details on the average binding energy calculation and contribution of residues to the binding energy play a crucial role in determining the likelihood of a compound entering clinical trials and ultimately becoming a successful drug These properties encompass Molecular Weight (MW) which assesses a compound's size and its ability to cross biological membranes; Number of Rotatable Bonds (nRot) which evaluates a compound's flexibility and its susceptibility to unwanted transformations in the body; Number of Hydrogen Bond Acceptors (nHBacc) which gauges a compound's capacity to interact with and bind to proteins; Number of Hydrogen Bond Donors (nHBDon) which predicts a compound's potential to be metabolized by enzymes; and Topological Polar Surface Area (TPSA) which measures a compound's overall polarity and its ability to interface with water and other polar molecules compounds with values within the following acceptable criteria are considered to have a higher likelihood of proving successful drug candidates: MW < 500 g/mol These criteria serve as valuable guidelines for early-stage drug discovery efforts helping researchers identify compounds with favorable physicochemical properties that are more likely to progress through the drug development pipeline and ultimately reach patients Docking structures of the best five NPs interacted with MurG binding site of E while MurG residues are depicted in thin-stick with olive colour Comparing the interacting residues involved in binding interaction for the top five compounds we observe some similarities and differences Both NPC272174 and NPC170742 interact with ARG164 but NPC170742 also has additional interactions with ILE245 and a pi-sulfur bond with MET248 NPC272174 has a pi-donor H-bond with ASN128 and π–π T-shaped interaction with HIS19 Both NPC117260 and NPC277205 interact with SER192 as well as PHE244 via π–π stacked interactions but NPC117260 also interacts with GLN288 and GLN289 via hydrogen bonds and MET248 via a π-sulfur bond NPC277205 has an additional π–π stacked interaction with MET248 and π-alkyl interactions with LEU265 as well as π-alkyl contacts with ILE245 and MET248 Although the binding scores for the top five compounds are relatively close the differences in their specific interactions with amino acid residues may account for differences in their overall binding and biological activities The various types of interactions play significant roles in ligand–protein binding contributing to the specificity and strength of the interaction help stabilize the complex and contribute to the specificity of the interaction Π–π stacking contributes to the strength of the interaction and helps to orient the ligand in the binding site π-alkyl interactions help increase the affinity of the ligand for the protein by reducing the overall energy of the complex π-donor hydrogen bonds contribute to the specificity of the interaction and stabilize the complex while π-sulfur interactions aid to orient the NPs in the binding site and contribute to the strength of the interaction RMSD curve of the complexes of (a) NPC272174 and standard UDP_GlcNAc and standard UDP_GlcNAc backbone atoms complexed with MurG enzyme of E Rg profile of the compounds (a) NPC272174 and standard UDP_GlcNAc and standard UDP_GlcNAc complexed with MurG enzyme of E Fluctuation profile (a) RMSF plot of the NPC272174 and standard UDP_GlcNAc compounds and standard UDP_GlcNAc compounds complexed with MurG enzyme during the simulation time (c) Amino acid residues involve the highest RMSF value in the ligands-MurG complex structures From the contribution of the calculated energy components of the binding free energies in the NPC727174, NPC259098, and the standard UDP_GlcNAc as shown in Table 7 the main driving force for the binding interactions is electrostatic and van der Waals interactions The polar solvation contributed unfavourably to the binding of the ligand to MurG enzyme and SASA interactions contribute favourably towards the binding of all ligands to MurG enzyme and are compensated by the large polar solvation energy Energetic components per-residue decomposition of (a) the standard UDP_GlcNAc (b) NPC727174 complexes with MurG enzyme of E Overlays of the conformational dynamics snapshot for NPC272174-MurG complex taken at different simulation times The conformation colors corresponding to the appropriate time are highlighted Many studies identified inhibitors for the antibiotic target MurG enzyme the nucleus structure for large MurG enzyme inhibitors is restricted to peptidoglycan-mimicking scaffolds thus simulating the resistance of drugs in the E the development of new MurG potential inhibitors has increased to a favorable approach We analyzed the best five hits from the NPASS database of natural products as potential MurG inhibitors followed by the drug-likeness assessment using a computational prediction method The results indicated that the promising NPs bind with an increased affinity mimicking the binding of the UDP_GlcNAc substrate The best five hits form H-bond interaction with important amino acid residues of ARG164 A molecular dynamics simulation study of the best three candidate natural compounds complexed with MurG demonstrated strong stability of NPC272174 to the enzyme structure upon comparing the MM-PBSA binding free energy values for the three NPs the results suggest that the complexation of NPC272174 to the MurG is more favorable the identified NPs suggest that the potential NPC272174 compound is a promising novel scaffold inhibitor for the MurG protein in E All data generated or analysed during this study are included in this published article [and its supplementary information files] Bélanger, L. et al. 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Res. 22, 2692–2705. https://doi.org/10.1007/s00044-012-0262-0 (2013) Download references Amneh Shtaiwi gratefully acknowledges the financial support received from the Deanship of Graduate Studies Scientific Research Shafi Ullah Khan is the Recipient of the WINNINGNormandy Program supported by the Normandy Region and this project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 101034329 Abdelouahid Samadi thanks the United Arab Emirates University and Zayed Center for Health Sciences for the financial grants Strategic Research Program (Grant G00003680) for support We also thank OpenEye Scientific Software for providing a free academic license to perform in silico studies Interdisciplinary Research Unit for Cancer Prevention and Treatment Université de Caen Normandie Inserm Anticipe UMR 1086 Laboratory of Analytical and Molecular Chemistry writing—original draft preparation; S.U.K.: conceptualization writing-original draft; A.S.: formal analysis All authors gave final approval for publication and agreed to be held accountable for the work performed therein Download citation DOI: https://doi.org/10.1038/s41598-024-57702-x Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology Metrics details Maintenance of bacterial cell shape and resistance to osmotic stress by the peptidoglycan (PG) renders PG biosynthetic enzymes and precursors attractive targets for combating bacterial infections we elucidate the effects of lipid substrates on the PG membrane enzymes MraY We show that dimerization of MraY is coupled with binding of the carrier lipid substrate undecaprenyl phosphate (C55-P) we demonstrate the use of native MS for biosynthetic reaction monitoring and find that the passage of substrates and products is controlled by the relative binding affinities of the different membrane enzymes we provide a molecular view of how PG membrane enzymes convey lipid precursors through favourable binding events and highlight possible opportunities for intervention Understanding the working mechanisms of existing antibiotics and the development of novel targets is critical to overcoming the problem of antibiotic resistance This entails the deployment of novel strategies and methodologies to track core biosynthetic pathways with molecular-level precision as a prelude to testing antibiotic candidates Owing to the limitations of existing methodologies the identity and structural roles of interfacial ligands/lipids is a subject of speculation we apply native mass spectrometry (MS) approaches combined with coarse-grained (CG) and atomistic molecular dynamic (MD) simulations to study the effects of lipid substrates on the PG biosynthetic membrane enzymes MraY Our data reveal a monomer-dimer equilibrium for MraY and that binding of C55-P at the interfacial sites mediates dimerisation we recapitulate the enzymatic activities of MraY and MurG to capture the effect of an antibiotic By probing the relative affinities of MraY MurG and MurJ for the peptidoglycan precursor lipids we find that although MraY can bind its own reaction product lipid I MurG and MurJ exhibit a strong preference for their respective natural substrates we provide an in-depth molecular view of the core reactions in the biosynthesis of peptidoglycan precursors and present an approach for antibiotic screening while each circle represents an individual measurement value Source data are provided as a Source data file our data suggest that the hydrophobic tails of tunicamycin can also stabilise the MraY dimer Together these results suggest that MraY forms a more stable noncovalent complex with the lipid substrates as a dimer and that the lipid-like ligands are providing structural stability to the MraY oligomer Simulating C55-P with an MraY protomer (see Methods) further indicates that C55-P can bind to MraY via Arg-340 with appreciably lower affinity (koff = 2.1 ± 0.3 µs−1) than when at the interfacial sites When bound to the interfacial sites of the MraY dimer a C55-P molecule will have its phosphate group on the cytoplasmic face of the membrane and will bridge the two MraY protomers the C55-P tail will make extensive contact with both protomers these data indicate that MraY can interact with both C55-P and lipid I at multiple sites and suggests a putative role of C55-P and lipid I in conferring stability to the MraY dimer Peaks exclusive to (MraY)2(C55-P) and (MraY)2(lipid I)(C55-P) are highlighted these data confirm predictions of C55-P binding residues from MD simulations and highlight in particular the significant role of R340 in coordinating the C55-P molecules bound to MraY at the interfacial sites the dimer (15+) and lipid-bound charge states are highlighted (red) c The same lipid I synthesis reaction as above but performed in the presence of an increasing concentration of tunicamycin The number and intensity of synthetic lipid I adducts decrease with increasing tunicamycin concentration Excess substrate and inhibitor molecules were removed via buffer exchange prior to measurements At the lowest concentration of tunicamycin tested the number and intensity of lipid I adducts decreased compared with spectra recorded in the absence of the antibiotic Increasing tunicamycin concentrations to 125 and 250 µM led to further depletion of the lipid I product reflecting effective inhibition of MraY-mediated lipid I synthesis by tunicamycin we have captured enzymatic activities of MraY and this approach can be used to monitor antibiotic inhibition of enzymes that exhibit a measurable binding affinity for their synthetic product red and blue sticks and the membrane phosphates as gold spheres with the GlcNAc-coordinating residues as coloured sticks: Phe-21 in orange This pose might represent an enzymatic intermediate state whereby GlcNAc has just been added to lipid I to form lipid II which will now be released from the active site we observed on average 2.15 ± 0.5 lipid I molecules bound with high affinity to MurG sites Analysis of koff from these simulations reveals that lipid II is less strongly bound to MurG than lipid I by a factor of about 2-3 (koff = 0.70 ± 1.03 µs-1 for lipid I and koff = 1.91 ± 0.30 µs−1 for lipid II) at this site The MD results are consistent with the apparent Kd determined by native MS experiments above This further confirms our initial assignment of the endogenous substrate bound to MraY as lipid I These observations imply that MurJ has a higher affinity for lipid II Together these results validate our hypothesis that differential binding affinities of carrier lipid for these enzymes present a driving force for their onward passage to the flippase MurJ for export across the bacterial cytoplasmic membrane to enable the maturation of a nascent PG MraY exists in a monomer-dimer equilibrium with dimerisation favoured in the presence of endogenous C55-P and lipid I MraY binds lipid substrates to stabilise its dimeric state Synthesis of lipid I via MraY can be effected in vitro from C55-P and UDP-MurNAc-pentapeptide and a change in mass confirms that endogenous lipid I was displaced by its biosynthetic counterpart Tunicamycin binds to MraY and inhibits lipid I formation MurG binds to lipid I with a higher affinity than MraY to drive the passage of lipid I along the pathway and forms lipid II in the presence of UDP-GlcNAc The flippase MurJ then outcompetes MurG and MraY to bind lipid II Lipid II is flipped into the periplasm where its polar disaccharide peptide head is incorporated into the peptidoglycan polymer stabilisation of the MraY dimer by C55-P and lipid I is intriguing in that it is an oligomer mediated by its own lipid substrate and catalysed product C55-P was shown to interact with MraY close to the groove surrounding TM9b How C55-P is recruited from the membrane to the proposed site remains unclear in the absence of a MraY structure with C55-P or a similar precursor The hydrophobic chain of C55-P extends beyond the bilayer thickness of the membrane and suffers from limited rotational freedom C55-P bound to MraY with its tail at the shallow groove proposed to accommodate the tunicamycin tail likely bends to allow the phosphate headgroup to access the coordinating aspartate residue This is consistent with our MD simulation data that reveal relatively weaker C55-P binding near the proposed active site than at the interfacial sites While this indicates that MraY can interact with C55-P at multiple sites with different affinities it raises a question regarding the role of C55-P molecules whose phosphate groups bind adjacent to the dimer interface Given the observed connection between dimerisation and the interfacial binding of endogenous C55-P and lipid I binding of these molecules at the interfacial sites likely provides structural stability to MraY dimer We therefore ascribe structural roles to the interfacial lipids bound to MraY dimers This is particularly clear when we run simulations of lipid II binding to MurG wherein we observed interactions with the same residues of MurG that coordinate with GlcNAc in the x-ray crystal structure We propose that this site could represent the binding region of lipid I for optimal synthesis of lipid II we showed by native MS that MurG can bind simultaneously to lipid I and lipid II implying a possible coordinating role in the synthesis and release of the lipid II product We also found that MurG binds to lipid I in preference to the monomeric form of MraY and that MurJ exhibited a higher affinity for lipid II than MurG This is an indication that the relative affinity of lipid substrates for their respective downstream enzymes presents a driving force for the lipid II precursor towards the flippase MurJ for onward translocation across the inner membrane the development of lipophilic molecules that tilt binding affinity gradients of PG precursor lipids away from their respective biosynthetic enzyme presents new opportunities for cell wall directed interventions to combat antimicrobial resistance coli MurJ were generated on a modified pET15b vector and contain a C-terminal GFP-His6 and His6 fusions the plasmid was transformed into chemically competent E Plasmids for MurG and MurJ were transformed into C43(DE3) E Cells were grown at 37 °C to OD600nm 0.6–0.8 and protein expression was induced by the addition of 1 mM IPTG cells were harvested by centrifugation at 5000 × g and stored at −80 °C until required Cell paste was thawed on ice and resuspended in a buffer containing 20 mM Tris-HCl and EDTA-free protease inhibitor cocktail tablets (Roche) The cells were then disrupted by 4–5 passes over a microfluidizer (Microfluidics) at 20,000 psi Non-lysed cells and debris were pelleted by centrifugation at 20,000 × g for 20 min and the clarified lysate was retained MBP-MraY was extracted from the clarified lysates by incubation with 1% DDM for 60 min Nonsolubilised aggregates were removed by centrifugation at 20,000×g for 1 h and the supernatant was loaded on a 5-mL HisTrap HP column preequilibrated in buffer A (20 mM Tris-HCl (pH 8.0) After washing with buffer B (20 mM Tris-HCl (pH 8.0) the protein was eluted from the column with a linear gradient of buffer C (20 mM Tris-HCl passed over a PD-10 desalting column (GE Healthcare) and digested overnight with HRV3C protease (Merck) MraY was isolated from the digestion mixture by size-exclusion chromatography (SEC) using a Superdex S200 increase column The SEC buffer was 20 mM Tris-HCl (pH 8.0) All MraY variants (wild type and mutants) were expressed and purified at least three times under the same conditions starting with single colonies from a freshly transformed C41(DE3) host strain the clarified cell lysates were ultra-centrifuged at 140,000×g to pellet the membranes The proteins were solubilized from the membrane fractions with 20 mM Tris-HCl 20% glycerol supplemented with 2% DDM for 2 h for UppP and with 2% DDM and 2% OGNG for 16 h for MurJ at 4 °C The insoluble matrix was removed by centrifugation and the supernatant was purified over 5-mL HisTrap HP column Proteins were either used immediately or flash-frozen in liquid nitrogen and stored at −80 °C Protein concentration was measured using a Biomate UV detector at 280 nm using extinction coefficients calculated from their predicted amino acid sequences C55-P was dissolved in methanol at a final concentration of 1 mM methanol was removed under a gentle stream of nitrogen and the lipid film resuspended in SEC buffer Stock solutions of 1 mM tunicamycin and 1 mM UM5 the protein was incubated with the desired concentration of ligand the mixture was buffer-exchanged into MS buffer and diluted then to a final protein concentration of 10 µM before measurements Lipid I reaction mixture was made by incubating 30 µM MraY and 5 mM MgCl2 in a buffer containing 20 mM Tris-HCl (pH 8.0) Lipid II reaction mixture was made as for lipid I but the mixture contained 30 µM MurG and 100 µM UDP-GlcNAc Samples were incubated overnight at 4 °C and then exchanged into 0.05% LDAO 200 mM ammonium acetate (“MS buffer”) using a centrifugal buffer exchange device (Micro Bio-Spin 6 the proteins were buffer-exchanged into LDAO as it provides sufficient stability for all the three enzymes MraY Of note detergents used to study MraY (C8E4 and OG) caused precipitation of MurG and MurJ Relative binding affinities were obtained from deconvoluted spectra by dividing the intensity of ligand-bound protein peaks by the sum of the intensities of ligand-bound and ligand-free proteins peaks All measurements were performed at least three times and yielded similar results where Bmax is the maximum specific binding we could not deduce the low-affinity Kd because a much higher concentrations of lipid I or lipid II caused a drastic reduction in spectral quality and potential interference from non-specific binding events through the electrospray process The proteins were buffer-exchanged into 200 mM ammonium acetate supplemented with 0.05% (w/v) LDAO Concentrations were determined and 10 μM aliquots were prepared by dilution with the same buffer and 10 μM lipid II were also prepared in the same buffer Samples were prepared by mixing volumes of desired protein and ligand such that the final protein concentration in each case is 5 μM The same optimised instrument settings (see below) with 100 V in the HCD cell were used to measure all the samples All experiments were repeated three times from newly prepared stock solutions Standard deviations were calculated from at least five observed charge states in three independent experiments additional systems were built with 62% POPE Quantification of the number of C55-PP bound to lipid I and II was done using the Gromacs tool gmx select Production simulations were run using 2 fs time steps for 3 × 300 ns (MraY-C55P and MraY alone (C55-P removed)) 3 × 100 ns (lipid II in membrane) or relaxation for 1 ns (MraY-lipid I) Further information on research design is available in the Nature Research Reporting Summary linked to this article Clinical and economic impact of antibiotic resistance in developing countries: a systematic review and meta-analysis Antimicrobial resistance: a global emerging threat to public health systems Core steps of membrane-bound peptidoglycan biosynthesis: recent advances Regulation of peptidoglycan synthesis and remodelling An oldie but a goodie - cell wall biosynthesis as antibiotic target pathway Development of new drugs for an old target: the penicillin binding proteins Penicillin-binding proteins and beta-lactam resistance Resistance to antibiotics targeted to the bacterial cell wall The membrane steps of bacterial cell wall synthesis as antibiotic targets Cell-wall recycling of the gram-negative bacteria and the nexus to antibiotic resistance mraY is an essential gene for cell growth in Escherichia coli Phospho-N-acetyl-muramyl-pentapeptide translocase from Escherichia coli: catalytic role of conserved aspartic acid residues The 1.9 A crystal structure of Escherichia coli MurG The bacterial lipid II flippase MurJ functions by an alternating-access mechanism Direct observation of the influence of cardiolipin and antibiotics on lipid II binding to MurJ an essential membrane enzyme for bacterial cell wall synthesis MraY–antibiotic complex reveals details of tunicamycin mode of action Crystal structure of the Pseudomonas aeruginosa MurG: UDP-GlcNAc substrate complex Chemical logic of MraY inhibition by antibacterial nucleoside natural products Structural insights into inhibition of lipid I production in bacterial cell wall synthesis Membrane proteins bind lipids selectively to modulate their structure and function The role of the detergent micelle in preserving the structure of membrane proteins in the gas phase Analyzing native membrane protein assembly in nanodiscs by combined non-covalent mass spectrometry and synthetic biology Studies on novel bacterial translocase I inhibitors physico-chemical properties and structure elucidation of A-500359 A and mureidomycin A: inhibition of phospho-N-acetylmuramyl-pentapeptide translocase from Escherichia coli Structures of bacterial MraY and human GPT provide insights into rational antibiotic design PyLipID: a python package for analysis of protein-lipid interactions from molecular dynamics simulations GlcNAc-1-P-transferase-tunicamycin complex structure reveals basis for inhibition of N-glycosylation The role of interfacial lipids in stabilizing membrane protein oligomers Structural lipids enable the formation of functional oligomers of the eukaryotic purine symporter UapA Quantitative high-performance liquid chromatography analysis of the pool levels of undecaprenyl phosphate and its derivatives in bacterial membranes Minimal exposure of lipid II cycle intermediates triggers cell wall antibiotic resistance a member of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily catalyzing the first membrane step of peptidoglycan biosynthesis Enzymatic synthesis of lipid II and analogues Deciphering the metabolism of undecaprenyl-phosphate: the bacterial cell-wall unit carrier at the membrane frontier Lipid II is an intrinsic component of the pore induced by nisin in bacterial membranes Interaction between two murein (peptidoglycan) synthases High-resolution mass spectrometry of small molecules bound to membrane proteins Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles Computational lipidomics with insane: a versatile tool for generating custom membranes for molecular simulations Resistance of Gram-positive bacteria to nisin is not determined by Lipid II levels A mass-spectrometry-based approach to distinguish annular and specific lipid binding to membrane proteins Developing a coarse-grained model for bacterial cell walls: evaluating mechanical properties and free energy barriers Molecular dynamics with coupling to an external bath Polymorphic transitions in single crystals: a new molecular dynamics method GROMACS: a message-passing parallel molecular dynamics implementation 2 interactions with an ensemble of cholesterol molecules CG2AT2: an enhanced fragment-based approach for serial multi-scale molecular dynamics simulations Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone ϕ ψ and side-chain χ1 and χ2 dihedral angles CHARMM-GUI: a web-based graphical user interface for CHARMM and CHARMM/OpenMM simulations using the CHARMM36 additive force field Oluwole, A. et al. Peptidoglycan biosynthesis is driven by lipid transfer along enzyme-substrate affinity gradients. figshare https://doi.org/10.6084/m9.figshare.19403852.v2 (2022) Download references We thank Eefjan Breukink (University of Utrecht) for the generous gift of lipid II laboratory is supported by a Medical Research Council (MRC) programme grant (MR/V028839/1) on which J.R.B is a Researcher Co-Investigator J.R.B holds a Royal Society University Research Fellowship and is a Research Fellow at Wolfson College Research in the WV laboratory is funded by the BBSRC grant BB/R017409/1 (to W.V.) laboratory is funded by Wellcome (208361/Z/17/Z) the MRC (MR/S009213/1) and BBSRC (BB/P01948X/1 and the University of Warwick Scientific Computing Research Technology Platform for computational access Physical and Theoretical Chemistry Laboratory The Kavli Institute for Nanoscience Discovery School of Life Sciences and Department of Chemistry wrote the paper with input from all authors is a cofounder of and consultant at OMass Therapeutics The remaining authors declare no competing interests Nature Communications thanks Iain Campuzano, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-022-29836-x Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research a survey show of Rosler’s work from 1965 to the present “All her work still feels very immediate and urgent especially when it comes to the omnipresent power of the media to shape public opinion and influence private reality,” Alexander continues Rosler took a break from completing several new pieces for the Jewish Museum show to invite PIN–UP into her Greenpoint brownstone where we discussed her trailblazing work on housing and the built environment and how she continues to bring the big issues home from the series House Beautiful: Bringing the War Home Courtesy of the artist and Mitchell-Innes & Nash (1989); Times Square Spectacolor animation Installation view of If you can’t afford to live here Taken from PIN–UP 25 Fall Winter 2018/19 Slide "+(i+1)+" of "+$(set).length+": "+$(this).find("figcaption")[0].innerHTML+" Tim Theriault sat on Market Street across from the Portsmouth Brewery writing the set list for a show that night with the band Heavens to Murgatroid he watched as the line out the door grew longer and longer as showtime approached for the band commonly referred to as "Murgatroid," or sometimes just "Murg." That was one of many packed shows downtown in the 1990s for Murgatroid a Portsmouth power pop trio that once topped the WFNX chart with its song "Electromagnetic Mind." the group is gearing up for two sold-out shows at 3S Artspace Oct They will reunite on a double bill with Fly Spinach Fly to commemorate 20 years since the Elvis Room closed on Congress Street which happen every three to five years or so bring back memories of a grittier Portsmouth and a hopeful music scene Thanks to Gravity and other bands in the 1990s played relentlessly in places like the Elvis Room the Portsmouth Gas Light Company and other venues 'Why couldn't Portsmouth be a Seattle?’" said Theriault referring to the booming grunge scene of the 1990s who founded the band with bassist Tim McCoy in 1990 said the scene had an eclectic sound and enough talent to qualify as more than a "little brother" to bigger nearby scenes like in Boston "The only missing piece would be somebody to take what we were doing to the next level The four reuniting for the two October shows is considered to be the strongest lineup for Murgatroid by the band Murg had already put out its first album "!" and signed to Prospective Records based in Minnesota when Theriault joined the band bringing a love of classic rock guitar to Murg's power pop sound The songwriting was mainly handled by Twombly who along with McCoy loved pop and punk-oriented rock and bands like the Jam and the Who McCoy booked and promoted shows while stocking local music stores with the band's demo tape and later CDs Fans could tell where the band was playing by spotting its 1979 GMC school bus from the Exeter School District painted purple and considered a "fifth member," said Twombly One of the pinnacle moments for the band was holding the No 1 spot on the popular Boston radio station WFNX for several weeks The band toured frequently around the Northeast and played a total of 1,500 shows according to McCoy They opened for Modern English at the Portsmouth Music Hall as well as Dee Dee Ramone and the Box Tops That core group had put out Murg's second album "Cyclop Pop," and he said Theriault was a big part of the Murg sound McCoy said the band took its natural course as other members wanted to explore other musical projects as well The members of Murgatroid are still involved in music today Theriault plays full time and has worked with artists like Sully Erna of Godsmack McCoy has continued to play with original acts like his band the Papercuts Twombly also plays locally in a cover band called Acoustic Radio McCoy believes some venues like the Press Room have kept Portsmouth original music scene alive but he believes those kinds of rooms are becoming more scarce up-and-coming music scene reminiscent of Portsmouth "Now Portsmouth feels like it's got bigger things happening as far as the bigger shows," said McCoy "But the regular kind of sweaty rock and roll nights Murg members remember the Elvis Room shows as always packed inside the small venue which served no alcohol and allowed underage patrons Twombly said it was good for Portsmouth at the time to have a venue for teens to get in so young people could experience rock shows typically held in 21+ clubs He recalled giving teens shirts and CDs and seeing them "blown away." "They don't have experiences like seeing bands up close," said Twombly Murg members say the band has not missed a beat since last playing together McCoy said he saw some songs in a new light with years removed from last playing them like "Hannah the Palindrome" and "Jokes on You." Jordan and Twombly said seeing both shows sell out in October demonstrates the group left its mark on Portsmouth "It validates what we've done all these years." "Elvis Room 20, A Retrospective Art Exhibit" in the 3S Lobby Gallery is on exhibit: Oct. 4-27 with an opening reception on Friday, Oct. 4 from 5-8 p.m. It is free and open to the public. 3S is located at 319 Vaughan St, Portsmouth. These concerts are all ages. Doors open at 7 p.m./shows 8 p.m. Ticket prices vary. For more information, go to www.3sarts.org or check out https://elvisroom.com Marinated with the goodness of ginger-garlic On the final day of the extended transfer window Thomas Murg left the club at his own request but with Marcel Ritzmaier we brought a former Eredivisie winner and midfield all-rounder to Hütteldorf Thomas Murg joined SK Rapid in the spring of 2016 and since then the attacking-midfielder amassed 163 competitive appearances for the club over the course of six seasons The 25-year-old expressed his desire to move abroad and the club have granted permission for that to happen Murg has thus moved to PAOK Thessaloniki despite having two years left on his current contract The two parties have agreed not to disclose the transfer fee involved Zoran Barisic: “Thomas Murg informed us of a very good offer from abroad and declared his express intention to accept that offer because he has been an important offensive player for us over the years but at the same time we respect his decision and have complied with his personal request to allow him to take this step in his career We wish him all the best in his new challenge and thank him for the effort that he put in whilst at this club.” Thomas Murg: “It was a wonderful time at SK Rapid with many great years that gave me unforgettable moments as a part of this team I have decided to take a step in a new direction which I am convinced will help me grow and gain experience as a footballer I would like to thank all the Rapid players as well as the club staff for all the great support I have received at Rapid over the years Managing Director of Sport Zoran Barisic with new signing Marcel Ritzmaier The midfield position vacated by Murg's departure doesn’t remain empty though because we can already announce a new squad member at SK Rapid: Marcel Ritzmaier The 27-year-old joins on a year-long loan deal from English Championship side Barnsley FC “We are very happy that we have managed to gain the services of Marcel Ritzmaier He’s a player who has had a great footballing education and we hope that he can help the team from the very first moments!” Marcel Ritzmaier moved from SKA Carinthia to PSV Eindhoven in the Netherlands the Styrian-born player rose through the ranks of the U19s to the senior squad making his professional debut in the Eredivisie in September 2012 After going out on loan to several Eredivisie clubs he finally ended up at WAC in the summer of 2018 In Carinthia he developed into a top performer in a very successful side In January 2020 he linked up with his former WAC coach Gerhard Struber by moving to Barnsley Marcel Ritzmaier on his new club: “I’m very happy that I’ve finally arrived in Vienna It’s a great honour for me to wear the Green and White jersey for the upcoming season My anticipation and excitement for the challenges ahead for the fans and for the whole club is huge!” Die grün-weiße Videoplattform – jetzt Rapid schauen Seit November 2019 für iOS und Android gratis erhältlich He immediately found a role in the team and promised muh but was plagued by injuries that set him back he made his own personal «restart» worked hard and he is now an important cog in Razvan Lucescu’s team The Austrian has made 110 appearances so far in the black and white jersey scoring 18 goals and contributing 9 assists The player and PAOK reached an agreement on his future and proceeded to renew his contract Murg will be with us at least until June 30 We deliver! 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Subscribe to Adweek newsletters Subscribe now for unlimited access to exclusive insider reporting I want to receive emails from Adweek about products services and events that they feel may be of interest to me I want to receive emails from Adweek on behalf of carefully-selected third party partners about products By submitting your information you agree to Adweek's Terms of Use and Privacy Policy and you will begin receiving our newsletters I understand by creating an account, I agree to Adweek’s Terms of Use and Privacy Policy and that I may review and update my marketing preferences at any time A code has been sent to your email address By subscribing you agree to Adweek’s Terms of Use and Privacy Policy Automatic Renewal and Cancellation By subscribing, you agree to the terms of sale, including the  Cancellation and Refund Policy You authorize Adweek.com to charge your credit/debit card at the annual subscription price at the amount noted above now and in time to  automatically renew your Adweek.com subscription every year before the start of each new 12-month term unless you tell us to stop.  Renewal rates are subject to change at any time with or without notice your service will continue without interruption The cancellation goes into effect at the start of the following billing cycle contact customer service at 844.674.8161 (U.S.) or 845.267.3007 (Outside U.S.) Adweek is the leading source of news and insight serving the brand marketing ecosystem «In the first half we played very well In the second half we conceded goals quickly The final result of 3-0 is definitely a heavy one but it does not reflect the true image of the match.» On the fact that now there are a lot of clubs chasing the top places and if this creates extra anxiety: now all the teams are close to each other in the standings The aim is to make a fresh start with the match against Panetolikos We have games we can take and our goal is only victories.» to fight hard and to become even more effective We have to score goals in the phases we create because in every game we create many opportunities.» On Shinji Kagawa and his arrival at the club; He is an excellent player who will help us a lot He did his first training session with us and I think he will help us a lot He plays in the ‘Number 10’ position and as an winger And so this means I’m also competing with him This makes me happy because there is healthy competition and this is something that improves you as a footballer and as a team.» Shortly before the January transfer window slammed shut SK Rapid's efforts to continuously improve the squad bore fruit as the club made an exciting announcement the club has captured one of Austria's top young midfield talents with the 21-year old midfielder making the switch from SV Ried to Hütteldorf The versatile Murg has signed a contract tying him to the club until 2019 and contains no release clauses After thrashing out a deal with his hitherto employers the deal was confirmed after Murg successfully completed a medical and underwent routine examinations Trainer Zoran Barisic is naturally delighted with his newest recruit: "Thomas Murg was a player both the club and me personally were after and is just what we need as he was already showing just how talented he was during his time with GAK With this signing we've added another unpredictable element to our side and he is a player that fits perfectly into our squad."Sporting Director Andreas Müller sees the signing in similar terms: "Firstly I'd like to thank SV Ried who conducted the negotiations in a hard but fair and professional manner Thomas Murg fits our philosophy perfectly: he is a young Austrian player who has already been proving his quality over a long period but who also has enormous potential to develop and improve further."Thomas Murg began his career in his native Styria where he made his competitive debut for GAK at the tender age of 15 he had already made over 50 first team appearances and had netted on no less than 13 occasions After winning many international caps at various youth levels Murg made the switch to the violet half of Vienna where he continued to excel under the highly rated Peter Stöger now the successful coach of German Bundesliga outfit 1 Under Stöger the young Murg continued to pick up appearances in the top flight of Austrian football whilst he additionally went on to make 4 appearances in the UEFA Champions League In summer 2014 Murg made a surprise move to SV Ried for whom he has scored 8 times in 44 appearances Murg will join a growing band of Styrians on the Rapid books (along with Sonnleitner Alar and Kainz) and can't wait to pull on the famous Green & White jersey: "I've been impressed for a long time now with Rapid’s continued development and their policy of looking to bring in young Austrian players I’m convinced that I'm a very good fit for this squad and having played against Rapid I know that the atmosphere the fans create is something special Perhaps certain members of the crowd might be a bit sceptical due to me having played for their great rivals but I can promise that from day one I’m going give everything to ensure that I offer a contribution to the success of the club Although I would never say a negative word about another club I’m looking forward to the first derby as I do have a couple of scores to settle with Austria Vienna that is history and now all my focus and energy is on Rapid a club with which I want to start winning trophies as soon as possible."(gw) It is with great sadness that we relay the news that architect Peter Shelton has died of cancer at the age of 67 A star distiller of big ideas into simple gestures he formed a dynamic duo with Lee Mindel (the two had been classmates at Penn) in 1978 shortly after graduating from Pratt quickly built a reputation for its thoughtful modernism and knack with rich materials You may also recall Shelton’s cameo on Project Runway when he and wife Laura Bennett (a season-three finalist) welcomed Tim Gunn into the Manhattan loft they shared with six children and and several pets While the best way to sample these is to raid a friend or relative's place in the city, many local shops and halwais (cooks) in bazaars have also been serving up sumptuous winter comfort foods for many years We asked city gourmands about their go-to for a winter delicacy and their recommendations are worth bookmarking Jaipur) is in the heart of Johari bazaar amidst all the hustle and bustle The shop has a variety of gajaks and if you’re a true gajak fan then the gud ki gajak is the one to pick.” Insia Lacewalla Samode Haveli“I love the Khad Murg at Samode Haveli (Jorawar Singh Gate It’s a great experience sitting at the palace’s courtyard restaurant and digging into this hot feast.” Insia Lacewalla Gajak at Ishwarji Gajak WaleRaghav Modi“Since I live in the walled city I am spoilt for choice when it comes to food we empty a lot of gajak boxes from Ishwar Ji (279 We also gift them around and everyone seems to love them too My number one would be gud ki gajak and the second best is revri.” Namokar Jain Jaipur) is one of the popular sweet shops in Jaipur and rightly so The best way to enjoy the Gond laddoos is to grab a plate at the shop and dig into them.” Insia Lacewalla Chokhi Dhani “Chokhi Dani (12 Miles Tonk Road via Vatika Jaipur) is one of those touristy places you’ll always end up going to when in Jaipur You must try their baajre ki khichdi with some boora I also ask for some gur on the side to eat with my khichdi Use of this website constitutes acceptance of our Terms of Service (updated April 1 2021) and Policy and Cookie Statement (updated April 1 The material on this site may not be reproduced except with the prior written permission of Condé Nast PAOK did not get off to the best of starts in the Europa League group stage since the opening match against Omonia on home soil ended in a draw despite the fact that PAOK were on top for long spells The main protagonist for the Double-headed Eagle was Thomas Murg In only his second game for the club – and his first start – the summer signing found the back of the net And in the voting for the Fans’ Man of the Match award the Austrian received 37,59% of the vote to win the individual title Thomas Murg was born on November 14 1994 in Voitsberg Austria and at just age six he joined the youth academy at Bärnbach where he made his professional debut in 2010 making 49 appearances and scoring 13 goals in the Austrian lower divisions before then making a big step up and joining Austria Vienna There he made 19 appearances and scored two goals playing for both the reserve and first teams while he also won a league title in the 2012-13 season establishing himself as one of the league’s best attacking midfielders In January 2016 Rapid Vienna came calling and signed him Murg immediately took on a leading role in the team and has played a major part in the team’s rise to prominence in recent years Murg made 163 appearances in a Rapid shirt scoring 35 goals and contributing another 40 assists The officials of the Federal Capital Territory Administration (FCTA) accompanied by heavily armed security personnel in the early hours of Tuesday seals the Headquarter building of the Federal Ministry of Works and Housing located at Mabushi in Abuja Other places shut by the FCTA officials, who were armed with court orders also include the popular murg plaza located just opposite the UTC market at Area 10 and the Federal Character Commission and still counting The Director of the Abuja Environment Protection Board (AEPB) Engineer Osilama Braimah who is also the Chairman of the Special Debt Taskforce in company of the Senior Special Assistant to the FCT Minister on Monitoring Inspection and Enforcement Comrade Ikharo Attah led the seal up operation which was carried out before dawn on Tuesday Speaking to newsmen on the breakdown of debt owed the agency Departments and Agencies are owing to the tune of 10 billion Naira and this he said is not acceptable hinted that some ministries are making frantic efforts to pay while others show signs of on willingness “The board got court orders to seal the premises” he said the exercise which has just begun will cover all public and private offices owing the board A senior magistrate court sitting in Wuse II Abuja had served the defendants with summons to appear before the court on March 30th “The debtors includes: Federal Ministry of  Works — N9,998,625.00; Federal Ministry  of  Defence  –N17,220,775.00; Federal  Character  Commission—N10,128,906.25; Civil service  Commission–2,451,649.50; and Revenue  Mobilization and Fiscal Commission—21,683,750.00 Federal  Ministry of  Health —N14,204,843.75; Federal Ministry of  Trade & Investment—N19,222,287.50; Federal Ministry of Education  Hqtrs –N25,838,275 ; and Nigeria Security and Civil Defence Corps Wuse–N16,583,031.25” warned the public against attempting to bribe AEPB officials as he urged them to pay into government coffers as stated in the bill they received.  the Senior Special Assistant to the FCT Minister on Monitoring,  Inspection and Enforcement said the FCT Minister is committed to the full recovery of all funds owed the Administration “We are just starting with the Abuja Environmental Protection Board,  seeing that most offices and persons generate waste and are not willing to pay to the collection and evacuation of such waste Government establishments are expected to lead by examples Now that we have started with ourselves,  everyone will be certain that there would be no sacred cow ” The FCT Administration is starting with the debtors of Abuja Environmental Protection Board (AEPB).Very soon we will move to the FCT Waterboard, the Abuja Geographic information system (AGIS) and other FCTA revenue generatiing agencies ” The FCT as you know is growing beyond expectation and plan Government must do all to march infrastructure with population growth and expansions The FCT Administration needs all the money it can get to bridge the infrastructural gap Our drive for revenue is at its peak at the moment”  called on all those who are owing to the FCT in whatever way to immediately hurry and pay up while adding that more public and privately owned properties would be sealed up in the coming days and weeks Toyin Adebayo is dedicated professional with over a decade experience of covering news and current events My background lies in gathering and analyzing facts about newsworthy events through stories My reports have featured in many National Dailies and currently with INDEPENDENT Newspaper a member of the Nigeria Union of Journalist Barde Will Deliver Gombe To PDP In 2023 — Group Amnesty For Dariye, Iyame: Presidential Aspirant, Ayom Hails Buhari TV Independent on YouTube and IndependentNgr (Facebook