I04-Macromolecular Crystallography
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Pyung-Gang
Lee
,
Linxiang
Yin
,
Xin
Wei
,
Jingyuan
Shi
,
Geoffrey
Masuyer
,
Travis G.
Wentz
,
Pengsheng
Chen
,
Ying
Xu
,
Junjie
Liang
,
Haonan
Zhang
,
Sara
Persson Kosenina
,
Briallen
Lobb
,
Michael
Mansfield
,
Sarjeet S.
Gill
,
Sabine
Pellett
,
Pal
Stenmark
,
Andrew C.
Doxey
,
Min
Dong
Open Access
Abstract: Insecticidal bacterial proteins play key roles in insect-bacteria interactions and have been used as biopesticides. Here, we identify two insecticidal proteins in Paeniclostridium ghonii, designated PG-toxin 1 (PG1) and PG-toxin 2 (PG2), which are homologs of botulinum neurotoxins (BoNTs). Unlike BoNTs, PG1 and PG2 contain two separate proteins: One is the protease light chain (LC), and the other is the heavy chain containing the translocation domain and the receptor binding domain. Crystal and cryo–electron microscopy structures show a conserved BoNT-like architecture but without an interchain disulfide bond. Functional characterizations establish that the LCs of PG1 and PG2 cleave insect synaptosomal–associated protein 25 (SNAP25), but not human or rat SNAP25, and microinjection of PG1 and PG2 caused paralysis and death in Drosophila and Aedes mosquitoes. These findings identified unique two-component BoNT-like insecticidal proteins, revealing insights into the evolution of the BoNT family of toxins, and broadening our understanding of bacteria that can be used for biopest controls.
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Nov 2025
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I03-Macromolecular Crystallography
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Emily C.
Hank
,
Nicholas D.
D'Arcy-Evans
,
Emma R.
Scaletti
,
Carlos
Benítez-Buelga
,
Olov
Wallner
,
Florian
Ortis
,
Kaixin
Zhou
,
Liuzhen
Meng
,
Alicia
Del Prado
,
Patricia
Calvo
,
Ingrid
Almlöf
,
Elisée
Wiita
,
Karen
Nierlin
,
Sara
Kosenina
,
Andreas
Krämer
,
Alice
Eddershaw
,
Mario
Kehler
,
Maeve
Long
,
Ann-Sofie
Jemth
,
Holly
Dawson
,
Josephine
Stewart
,
Adam
Dickey
,
Mikhael E.
Astorga
,
Marek
Varga
,
Evert J.
Homan
,
Martin
Scobie
,
Stefan
Knapp
,
Leandro
Sastre
,
Pal
Stenmark
,
Miguel
De Vega
,
Thomas
Helleday
,
Maurice
Michel
Diamond Proposal Number(s):
[15806, 21625]
Open Access
Abstract: Bifunctional DNA glycosylases employ an active site lysine or the N-terminus to form a Schiff base with an abasic (AP) site base excision repair intermediate. For 8-oxoguanine DNA glycosylase 1 (OGG1), cleaving this reversible structure is the rate-determining step in the initiation of 8-oxoguanine (8-oxoG) repair in DNA. Evolution has led OGG1 to use a product-assisted catalysis approach, where the excised 8-oxoG acts as a Brønsted base for cleavage of a Schiff base intermediate. However, the physicochemical properties of 8-oxoG significantly limit the inherent enzymatic turnover leading to a weak, cellularly absent, AP lyase activity. We hypothesized that chemical synthesis of purine analogues enables access to complex structures that are suitable as product-like catalysts. Herein, the nucleobase landscape is profiled for its potential to increase OGG1 Schiff base cleavage. 8-Substituted 6-thioguanines emerge as potent and selective scaffolds enabling OGG1 to cleave AP sites opposite any canonical nucleobase by β-elimination. This effectively broadens the enzymatic substrate scope of OGG1, shaping a complete, artificial AP-lyase function. In addition, a second class of compounds, 6-substituted pyrazolo-[3,4-d]-pyrimidines, stimulate OGG1 function at high pH, while thioguanines govern enzymatic control at acidic pH. This enables up to 20-fold increased enzyme turnover and a de novo OGG1 β-elimination in conditions commonly not tolerated. The tool compounds employed here are non-toxic in cells and stimulate the repair of AP sites through a natural, APE1 dependent pathway, as opposed to previously reported β,δ-lyase stimulator TH10785.
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Oct 2025
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[11265]
Open Access
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.
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Jun 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[21625]
Open Access
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.
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May 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[21625]
Open Access
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.
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Mar 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[21625]
Open Access
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.
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Feb 2025
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I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[15806]
Open Access
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.
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Aug 2024
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[21625]
Open Access
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.
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Jun 2024
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[21625]
Open Access
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.
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Sep 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[15806, 21625]
Open Access
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.
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Sep 2022
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