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30 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I24-Microfocus Macromolecular Crystallography	Botulinum neurotoxin A mutants with enhanced ganglioside binding show improved potency and altered ganglioside selectivity Geoffrey Masuyer , Andreas Rummel , Pal Stenmark Communications Chemistry 8 DOI: 10.1038/s42004-025-01569-0 Diamond Proposal Number(s): [11265] Open Access Show Abstract ▼ Abstract: Botulinum neurotoxins are the causative agents of botulism, a lethal paralytic disease, but are also one of the most commonly used therapeutics for the treatment of numerous neuromuscular conditions. These toxins recognise motor nerve terminals with high specificity and affinity by using a dual binding mechanism involving gangliosides and protein receptors. The initial recognition of gangliosides is crucial for the toxins’ potency. In this study, we employed a synaptosome-binding screening strategy to identify BoNT/A mutants with enhanced ganglioside-binding which translated into improved potency. X-ray crystallography and receptor-binding assays were used to elucidate the molecular mechanisms underlying the increased affinity or altered ganglioside selectivity of these mutants. Our findings provide a basis for the development of BoNT/A variants with enhanced therapeutic potential.	Jun 2025
I03-Macromolecular Crystallography	Structural and functional insights into Listeria monocytogenes phage endolysin PlyP100 - a promising food safety tool Karson R. Bateman , Emma Scaletti Hutchinson , Göran Widmalm , Michael J. Miller , Pal Stenmark Journal Of Biological Chemistry 12 DOI: 10.1016/j.jbc.2025.110295 Diamond Proposal Number(s): [21625] Open Access Show Abstract ▼ Abstract: Listeria monocytogenes is a ubiquitous, psychrotrophic human pathogen that can cause listeriosis, a serious illness for vulnerable populations. Some foods, such as Hispanic-style fresh cheeses like queso fresco, pose a specific risk because there are no widely accepted or available methods for L. monocytogenes mitigation that are both effective and able to maintain the properties of the products. Listeria-specific bacteriophages encode endolysins that are able to cleave the peptidoglycan layer of L. monocytogenes cells externally, showing promise as a potential solution to this problem. PlyP100, from the GRAS Listeria phage P100, is one such endolysin that can prevent the growth of L. monocytogenes in both lab culture conditions and a miniaturized queso fresco model. In this work, we aimed to understand the structural and functional properties of PlyP100. An AlphaFold prediction suggested the presence of three separate domains (D1, D2, and D3). By solving a crystal structure of D1 and assessing various domain truncations, we present evidence that D1 is responsible for catalytic activity, D3 is sufficient for cell wall binding, and D2 is necessary for full function of the enzyme against live cells. Additionally, we performed point mutations in D1 and compared PlyP100 to proteins with similar structures, including S. pneumoniae LytA and Listeria endolysin Ply511, in order to understand its specific enzymatic mechanism and target strain specificity. These insights into the structure and function of PlyP100 will aid future work aiming to engineer better endolysins as safe food antimicrobials.	May 2025
I04-Macromolecular Crystallography	Bacterial transcriptional repressor NrdR - a flexible multifactorial nucleotide sensor Inna Rozman Grinberg , Ornella Bimaï , Saher Shahid , Lena Philip , Markel Martinez-Carranza , Ipsita Banerjee , Daniel Lundin , Pal Stenmark , Britt-Marie Sjöberg , Derek T. Logan The Febs Journal DOI: 10.1111/febs.70037 Diamond Proposal Number(s): [21625] Open Access Show Abstract ▼ Abstract: NrdR is a bacterial transcriptional repressor consisting of a zinc (Zn)-ribbon domain followed by an ATP-cone domain. Understanding its mechanism of action could aid the design of novel antibacterials. NrdR binds specifically to two “NrdR boxes” upstream of ribonucleotide reductase operons, of which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, in the last of which we identified a new box. We show that E. coli NrdR (EcoNrdR) has similar binding strength to all three sites when loaded with ATP plus deoxyadenosine triphosphate (dATP) or equivalent diphosphate combinations. No other combination of adenine nucleotides promotes binding to DNA. We present crystal structures of EcoNrdR–ATP–dATP and EcoNrdR–ADP–dATP, which are the first high-resolution crystal structures of an NrdR. We have also determined cryo-electron microscopy structures of DNA-bound EcoNrdR–ATP–dATP and novel filaments of EcoNrdR–ATP. Tetrameric forms of EcoNrdR involve alternating interactions between pairs of Zn-ribbon domains and ATP-cones. The structures reveal considerable flexibility in relative orientation of ATP-cones vs Zn-ribbon domains. The structure of DNA-bound EcoNrdR–ATP–dATP shows that significant conformational rearrangements between ATP-cones and Zn-ribbons accompany DNA binding while the ATP-cones retain the same relative orientation. In contrast, ATP-loaded EcoNrdR filaments show rearrangements of the ATP-cone pairs and sequester the DNA-binding residues of NrdR such that they are unable to bind to DNA. Our results, in combination with a previous structural and biochemical study, point to highly flexible EcoNrdR structures that, when loaded with the correct nucleotides, adapt to an optimal promoter-binding conformation.	Mar 2025
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Virtual fragment screening for DNA repair inhibitors in vast chemical space Andreas Luttens , Duc Duy Vo , Emma R. Scaletti , Elisée Wiita , Ingrid Almlöf , Olov Wallner , Jonathan Davies , Sara Kosenina , Liuzhen Meng , Maeve Long , Oliver Mortusewicz , Geoffrey Masuyer , Flavio Ballante , Maurice Michel , Evert Homan , Martin Scobie , Christina Kalderén , Ulrika Warpman Berglund , Andrii V. Tarnovskiy , Dmytro S. Radchenko , Yurii S. Moroz , Jan Kihlberg , Pål Stenmark , Thomas Helleday , Jens Carlsson Nature Communications 16 DOI: 10.1038/s41467-025-56893-9 Diamond Proposal Number(s): [21625] Open Access Show Abstract ▼ Abstract: Fragment-based screening can catalyze drug discovery by identifying novel scaffolds, but this approach is limited by the small chemical libraries studied by biophysical experiments and the challenging optimization process. To expand the explored chemical space, we employ structure-based docking to evaluate orders-of-magnitude larger libraries than those used in traditional fragment screening. We computationally dock a set of 14 million fragments to 8-oxoguanine DNA glycosylase (OGG1), a difficult drug target involved in cancer and inflammation, and evaluate 29 highly ranked compounds experimentally. Four of these bind to OGG1 and X-ray crystallography confirms the binding modes predicted by docking. Furthermore, we show how fragment elaboration using searches among billions of readily synthesizable compounds identifies submicromolar inhibitors with anti-inflammatory and anti-cancer effects in cells. Comparisons of virtual screening strategies to explore a chemical space of 1022 compounds illustrate that fragment-based design enables enumeration of all molecules relevant for inhibitor discovery. Virtual fragment screening is hence a highly efficient strategy for navigating the rapidly growing combinatorial libraries and can serve as a powerful tool to accelerate drug discovery efforts for challenging therapeutic targets.	Feb 2025
I04-Macromolecular Crystallography	Activity of botulinum neurotoxin X and its structure when shielded by a non-toxic non-hemagglutinin protein Markel Martinez-Carranza , Jana Skerlova , Pyung-Gang Lee , Jie Zhang , Ajda Krc , Abhishek Sirohiwal , Dave Burgin , Mark Elliott , Jules Philippe , Sarah Donald , Fraser Hornby , Linda Henriksson , Geoffrey Masuyer , Ville R. I. Kaila , Matthew Beard , Min Dong , Pal Stenmark Communications Chemistry 7 DOI: 10.1038/s42004-024-01262-8 Diamond Proposal Number(s): [15806] Open Access Show Abstract ▼ Abstract: Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein (NTNH) with which they form a 300 kDa complex, to resist acidic and proteolytic attack from the digestive tract. We have previously identified a new botulinum neurotoxin serotype, BoNT/X, that has unique and therapeutically attractive properties. We present the cryo-EM structure of the BoNT/X-NTNH/X complex and the crystal structure of the isolated NTNH protein. Unexpectedly, the BoNT/X complex is stable and protease-resistant at both neutral and acidic pH and disassembles only in alkaline conditions. Using the stabilizing effect of NTNH, we isolated BoNT/X and showed that it has very low potency both in vitro and in vivo. Given the high catalytic activity and translocation efficacy of BoNT/X, low activity of the full toxin is likely due to the receptor-binding domain, which presents very weak ganglioside binding and exposed hydrophobic surfaces.	Aug 2024
I03-Macromolecular Crystallography	Kinetic and structural characterization of NUDT15 and NUDT18 as catalysts of isoprene pyrophosphate hydrolysis Emma Scaletti , Judith E. Unterlass , Ingrid Almlöf , Tobias Koolmeister , Karl S. Vallin , Despina Kapsitidou , Viktoriia Tsuber , Thomas Helleday , Pal Stenmark , Ann-Sofie Jemth The Febs Journal 39 DOI: 10.1111/febs.17202 Diamond Proposal Number(s): [21625] Open Access Show Abstract ▼ Abstract: Isoprene pyrophosphates play a crucial role in the synthesis of a diverse array of essential nonsterol and sterol biomolecules and serve as substrates for posttranslational isoprenylation of proteins, enabling specific anchoring to cellular membranes. Hydrolysis of isoprene pyrophosphates would be a means to modulate their levels, downstream products, and protein isoprenylation. While NUDIX hydrolases from plants have been described to catalyze the hydrolysis of isoprene pyrophosphates, homologous enzymes with this function in animals have not yet been reported. In this study, we screened an extensive panel of human NUDIX hydrolases for activity in hydrolyzing isoprene pyrophosphates. We found that human nucleotide triphosphate diphosphatase NUDT15 and 8-oxo-dGDP phosphatase NUDT18 efficiently catalyze the hydrolysis of several physiologically relevant isoprene pyrophosphates. Notably, we demonstrate that geranyl pyrophosphate is an excellent substrate for NUDT18, with a catalytic efficiency of 2.1 × 105 m−1·s−1, thus making it the best substrate identified for NUDT18 to date. Similarly, geranyl pyrophosphate proved to be the best isoprene pyrophosphate substrate for NUDT15, with a catalytic efficiency of 4.0 × 104 M−1·s−1. LC–MS analysis of NUDT15 and NUDT18 catalyzed isoprene pyrophosphate hydrolysis revealed the generation of the corresponding monophosphates and inorganic phosphate. Furthermore, we solved the crystal structure of NUDT15 in complex with the hydrolysis product geranyl phosphate at a resolution of 1.70 Å. This structure revealed that the active site nicely accommodates the hydrophobic isoprenoid moiety and helped identify key binding residues. Our findings imply that isoprene pyrophosphates are endogenous substrates of NUDT15 and NUDT18, suggesting they are involved in animal isoprene pyrophosphate metabolism.	Jun 2024
I03-Macromolecular Crystallography	Structural and functional insights into the Pseudomonas aeruginosa glycosyltransferase WaaG and the implications for lipopolysaccharide biosynthesis Emma R. Scaletti , Pontus Pettersson , Joan Patrick , Patrick J. Shilling , Robert Gustafsson Westergren , Daniel O. Daley , Lena Mäler , Göran Widmalm , Pal Stenmark Journal Of Biological Chemistry 280 DOI: 10.1016/j.jbc.2023.105256 Diamond Proposal Number(s): [21625] Open Access Show Abstract ▼ Abstract: The glycosyltransferase WaaG in Pseudomonas aeruginosa (PaWaaG) is involved in synthesis of the core region of lipopolysaccharides. It is a promising target for developing adjuvants that could help in the uptake of antibiotics. Herein we have determined structures of PaWaaG in complex with the nucleotide-sugars UDP-glucose, UDP-galactose and UDP-GalNAc. Structural comparison with the homologue from Escherichia coli (EcWaaG) revealed five key differences in the sugar binding pocket. Solution-state NMR analysis showed that wildtype PaWaaG specifically hydrolyzes UDP-GalNAc and unlike EcWaaG, does not hydrolyze UDP-glucose. Furthermore, we found that a PaWaaG mutant (Y97F/T208R/N282A/T283A/T285I) designed to resemble the EcWaaG sugar binding site, only hydrolyzed UDP-glucose, underscoring the importance of the identified amino acids in substrate specificity. However, neither wildtype PaWaaG nor the PaWaaG mutant capable of hydrolyzing UDP-glucose was able to complement an E. coli ΔwaaG strain, indicating that more remains to be uncovered about the function of PaWaaG in vivo. This structural and biochemical information will guide future structure-based drug design efforts targeting PaWaaG.	Sep 2023
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Optimization of N-piperidinyl-benzimidazolone derivatives as potent and selective inhibitors of 8-oxo guanine DNA glycosylase 1 Olov Wallner , Armando Cázares-Körner , Emma R. Scaletti , Geoffrey Masuyer , Tove Bekkhus , Torkild Visnes , Kirill Mamonov , Florian Ortis , Thomas Lundbäck , Maria Volkova , Tobias Koolmeister , Elisée Wiita , Olga Loseva , Monica Pandey , Evert Homan , Carlos Benítez-Buelga , Jonathan Davies , Martin Scobie , Ulrika Warpman Berglund , Christina Kalderén , Pal Stenmark , Thomas Helleday , Maurice Michel Chemmedchem DOI: 10.1002/cmdc.202200310 Diamond Proposal Number(s): [15806, 21625] Open Access Show Abstract ▼ Abstract: 8-oxo Guanine DNA Glycosylase 1 is the initiating enzyme within base excision repair and removes oxidized guanines from damaged DNA. Since unrepaired 8-oxoG could lead to G:C→T:A transversion, base removal is of utmost importance for cells to ensure genomic integrity. For cells with elevated levels of reactive oxygen species this dependency is further increased. In the past we and others have validated OGG1 as a target for inhibitors to treat cancer and inflammation. Here, we present the optimization campaign that led to the broadly used tool compound TH5487. Based on results from a small molecule screening campaign, we performed hit to lead expansion and arrived at potent and selective substituted N -piperidinyl-benzimidazolones. Using X-ray crystallography data, we describe the surprising binding mode of different members of the class. Potent members adopt a chair within the N -Piperidinyl-linker, while a boat conformation was found for weaker analogues.	Sep 2022
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Small-molecule activation of OGG1 increases oxidative DNA damage repair by gaining a new function Maurice Michel , Carlos Benítez-Buelga , Patricia A. Calvo , Bishoy M. F. Hanna , Oliver Mortusewicz , Geoffrey Masuyer , Jonathan Davies , Olov Wallner , Sanjiv Kumar , Julian J. Albers , Sergio Castañeda-Zegarra , Ann-Sofie Jemth , Torkild Visnes , Ana Sastre-Perona , Akhilesh N. Danda , Evert J. Homan , Karthick Marimuthu , Zhao Zhenjun , Celestine N. Chi , Antonio Sarno , Elisée Wiita , Catharina Von Nicolai , Anna J. Komor , Varshni Rajagopal , Sarah Müller , Emily C. Hank , Marek Varga , Emma R. Scaletti , Monica Pandey , Stella Karsten , Hanne Haslene-Hox , Simon Loevenich , Petra Marttila , Azita Rasti , Kirill Mamonov , Florian Ortis , Fritz Schömberg , Olga Loseva , Josephine Stewart , Nicholas D’arcy-Evans , Tobias Koolmeister , Martin Henriksson , Dana Michel , Ana De Ory , Lucia Acero , Oriol Calvete , Martin Scobie , Christian Hertweck , Ivan Vilotijevic , Christina Kalderén , Ana Osorio , Rosario Perona , Alexandra Stolz , Pal Stenmark , Ulrika Warpman Berglund , Miguel De Vega , Thomas Helleday Science 376, 1471 - 1476 DOI: 10.1126/science.abf8980 Diamond Proposal Number(s): [15806, 21625] Show Abstract ▼ Abstract: Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed β,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.	Jun 2022
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Pharmacological targeting of MTHFD2 suppresses acute myeloid leukemia by inducing thymidine depletion and replication stress Nadilly Bonagas , Nina M. S. Gustafsson , Martin Henriksson , Petra Marttila , Robert Gustafsson , Elisée Wiita , Sanjay Borhade , Alanna C. Green , Karl S. A. Vallin , Antonio Sarno , Richard Svensson , Camilla Göktürk , Therese Pham , Ann-Sofie Jemth , Olga Loseva , Victoria Cookson , Nicole Kiweler , Lars Sandberg , Azita Rasti , Judith E. Unterlass , Martin Haraldsson , Yasmin Andersson , Emma R. Scaletti , Christoffer Bengtsson , Cynthia B. J. Paulin , Kumar Sanjiv , Eldar Abdurakhmanov , Linda Pudelko , Ben Kunz , Matthieu Desroses , Petar Iliev , Katarina Färnegårdh , Andreas Krämer , Neeraj Garg , Maurice Michel , Sara Häggblad , Malin Jarvius , Christina Kalderén , Amanda Bögedahl Jensen , Ingrid Almlöf , Stella Karsten , Si Min Zhang , Maria Häggblad , Anders Eriksson , Jianping Liu , Björn Glinghammar , Natalia Nekhotiaeva , Fredrik Klingegård , Tobias Koolmeister , Ulf Martens , Sabin Llona-Minguez , Ruth Moulson , Helena Nordström , Vendela Parrow , Leif Dahllund , Birger Sjöberg , Irene L. Vargas , Duy Duc Vo , Johan Wannberg , Stefan Knapp , Hans E. Krokan , Per I. Arvidsson , Martin Scobie , Johannes Meiser , Pal Stenmark , Ulrika Warpman Berglund , Evert J. Homan , Thomas Helleday Nature Cancer 3, 156 - 172 DOI: 10.1038/s43018-022-00331-y Open Access Show Abstract ▼ Abstract: The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors.	Feb 2022

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