I03-Macromolecular Crystallography
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Dóra
Laczi
,
Sofia Schönbauer
Huamán
,
Taylah
Andrews-Clark
,
Stephen M.
Laidlaw
,
Eidarus
Salah
,
Leo
Dumjahn
,
Petra
Lukacik
,
Hani
Choudhry
,
Martin A.
Walsh
,
Miles W.
Carroll
,
Christopher J.
Schofield
,
Lennart
Brewitz
Diamond Proposal Number(s):
[27088]
Open Access
Abstract: Nirmatrelvir is a substrate-related inhibitor of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) main protease (Mpro) that is clinically used in combination with ritonavir to treat COVID-19. Derivatives of nirmatrelvir, modified at the substrate P2-equivalent position, have been developed to fine-tune inhibitor properties and are now in clinical use. We report the synthesis of nirmatrelvir derivatives with a (R)-4,4-dimethyl-4-silaproline (silaproline) group at the P2-equivalent position. Mass spectrometry (MS)-based assays demonstrate that silaproline-bearing nirmatrelvir derivatives efficiently inhibit isolated recombinant Mpro, albeit with reduced potency compared to nirmatrelvir. Investigations with SARS-CoV-2 infected VeroE6 cells reveal that the silaproline-bearing inhibitors with a CF3 group at the P4-equivalent position inhibit viral progression, implying that incorporating silicon atoms into Mpro inhibitors can yield in vivo active inhibitors with appropriate optimization. MS and crystallographic studies show that the nucleophilic active site cysteine residue of Mpro (Cys145) reacts with the nitrile group of the silaproline-bearing inhibitors. Substituting the electrophilic nitrile group for a non-activated terminal alkyne shifts the inhibition mode from reversible covalent inhibition to irreversible covalent inhibition. One of the two prochiral silaproline methyl groups occupies space in the S2 pocket that is unoccupied in Mpro:nirmatrelvir complex structures, highlighting the value of sila-derivatives in structure-activity-relationship (SAR) studies. The combined results highlight the potential of silicon-containing molecules for inhibition of Mpro and, by implication, other nucleophilic cysteine enzymes.
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Apr 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[21773]
Open Access
Abstract: There is an urgent need for new antibiotics. FabF (3-oxoacyl-[acyl-carrier-protein] synthase 2), which catalyses the rate limiting condensation reaction in the fatty acid synthesis II pathway, is an attractive target. Very few inhibitors of FabF are known and most are derived from natural products. In an effort to further explore the chemical space of FabF ligands, we have carried out fragment screening by X-ray crystallography against an intermediated state-mimicking variant of P. aeruginosa FabF (PaFabF C164Q). This screen has resulted in 48 hits out of which 16 bind in or close to the malonyl-CoA or fatty acid binding site or an adjacent dimer interface. None of the closer investigated fragments were active in a binding assay, but the same was the case for fragments derived from a potent FabF inhibitor. For hit optimization, we focused on the two fragments binding close to the catalytic residues of FabF. Different strategies were followed in the optimization process: exploration of commercially available analogues, fragment merging, virtual screening of a combinatorial make-on-demand space, and design and in-house synthesis of analogues. In total, more than 90 analogues of the hit compounds were explored, and for 10 of those co-crystal structures could be determined. The most potent ligand was discovered using manual structure-based design and has a binding affinity of 65 μM. This data package forms a strong foundation for the development of more potent and diverse FabF inhibitors.
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Apr 2025
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Krios I-Titan Krios I at Diamond
Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Nattapong
Sanguankiattichai
,
Balakumaran
Chandrasekar
,
Yuewen
Sheng
,
Nathan
Hardenbrook
,
Werner W. A.
Tabak
,
Margit
Drapal
,
Farnusch
Kaschani
,
Clemens
Grünwald-Gruber
,
Daniel
Krahn
,
Pierre
Buscaill
,
Suzuka
Yamamoto
,
Atsushi
Kato
,
Robert
Nash
,
George
Fleet
,
Richard
Strasser
,
Paul D.
Fraser
,
Markus
Kaiser
,
Peijun
Zhang
,
Gail M.
Preston
,
Renier A. L.
Van Der Hoorn
Diamond Proposal Number(s):
[21004, 29812, 28713]
Abstract: The extracellular space (apoplast) in plants is a key battleground during microbial infections. To avoid recognition, the bacterial model phytopathogen Pseudomonas syringae pv. tomato DC3000 produces glycosyrin. Glycosyrin inhibits the plant-secreted β-galactosidase BGAL1, which would otherwise initiate the release of immunogenic peptides from bacterial flagellin. Here, we report the structure, biosynthesis, and multifunctional roles of glycosyrin. High-resolution cryo–electron microscopy and chemical synthesis revealed that glycosyrin is an iminosugar with a five-membered pyrrolidine ring and a hydrated aldehyde that mimics monosaccharides. Glycosyrin biosynthesis was controlled by virulence regulators, and its production is common in bacteria and prevents flagellin recognition and alters the extracellular glycoproteome and metabolome of infected plants. These findings highlight a potentially wider role for glycobiology manipulation by plant pathogens across the plant kingdom.
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Apr 2025
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I03-Macromolecular Crystallography
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Kalvis
Brangulis
,
Jill
Malfetano
,
Ashley L.
Marcinkiewicz
,
Alan
Wang
,
Yi-Lin
Chen
,
Jungsoon
Lee
,
Zhuyun
Liu
,
Xiuli
Yang
,
Ulrich
Strych
,
Dagnija
Tupina
,
Inara
Akopjana
,
Maria-Elena
Bottazzi
,
Utpal
Pal
,
Ching-Lin
Hsieh
,
Wen-Hsiang
Chen
,
Yi-Pin
Lin
Diamond Proposal Number(s):
[35587]
Open Access
Abstract: Borrelia burgdorferi (Bb) causes Lyme disease (LD), one of the most common vector-borne diseases in the Northern Hemisphere. Here, we solve the crystal structure of a mutated Bb vaccine antigen, CspZ-YA that lacks the ability to bind to host complement factor H (FH). We generate point mutants of CspZ-YA and identify CspZ-YAI183Y and CspZ-YAC187S to trigger more robust bactericidal responses. Compared to CspZ-YA, these CspZ-YA mutants require a lower immunization frequency to protect mice from LD-associated inflammation and bacterial colonization. Antigenicity of wild-type and mutant CspZ-YA proteins are similar, as measured using sera from infected people or immunized female mice. Structural comparison of CspZ-YA with CspZ-YAI183Y and CspZ-YAC187S shows enhanced interactions of two helices adjacent to the FH-binding sites in the mutants, consistent with their elevated thermostability. In line with these findings, protective CspZ-YA monoclonal antibodies show increased binding to CspZ-YA at a physiological temperature (37 °C). In summary, this proof-of-concept study applies structural vaccinology to enhance intramolecular interactions for the long-term stability of a Bb antigen while maintaining its protective epitopes, thus promoting LD vaccine development.
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Apr 2025
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[31336]
Open Access
Abstract: Pathogens have evolved diverse strategies to counteract host immunity. Ubiquitylation of lipopolysaccharide (LPS) on cytosol-invading bacteria by the E3 ligase RNF213 creates ‘eat me’ signals for antibacterial autophagy, but whether and how cytosol-adapted bacteria avoid LPS ubiquitylation remains poorly understood. Here, we show that the enterobacterium Shigella flexneri actively antagonizes LPS ubiquitylation through IpaH1.4, a secreted effector protein with ubiquitin E3 ligase activity. IpaH1.4 binds to RNF213, ubiquitylates it and targets it for proteasomal degradation, thus counteracting host-protective LPS ubiquitylation. To understand how IpaH1.4 recognizes RNF213, we determined the cryogenic electron microscopy structure of the IpaH1.4–RNF213 complex. The specificity of the interaction is achieved through the leucine-rich repeat of IpaH1.4, which binds the RING domain of RNF213 by hijacking the conserved RING interface required for binding to ubiquitin-charged E2 enzymes. IpaH1.4 also targets other E3 ligases involved in inflammation and immunity through binding to the E2-interacting face of their RING domains, including the E3 ligase LUBAC that is required for the synthesis of M1-linked ubiquitin chains on cytosol-invading bacteria downstream of RNF213. We conclude that IpaH1.4 has evolved to antagonize multiple antibacterial and proinflammatory host E3 ligases.
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Apr 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[35587]
Open Access
Abstract: Lyme disease, caused by the spirochete Borrelia burgdorferi, is transmitted to mammalian hosts during the feeding process of infected Ixodes ticks. Our previous studies demonstrated that the paralogous gene family 12 (PFam12) consisting of five members (BBK01, BBG01, BBH37, BBJ08, and BB0844) are non-specific DNA-binding proteins. PFam12 proteins share 31–69% sequence identity, are located either on the surface or within the periplasm and are upregulated as the tick starts its blood meal. The crystal structure of BBK01 revealed that the protein forms a homodimer, which is potentially critical for DNA binding. In this study, we determined the crystal structure of another PFam12 member, BBH37, to gain a better insight into this unique paralogous family. Although BBK01 dimerization is mediated by its C-terminal region and is thought to be critical for DNA binding, BBH37 forms dimers through an alternative mechanism where a unique disulfide bond is involved. We found that BBH37 is still able to interact with DNA with micromolar affinity. Molecular dynamics simulations and site-directed mutagenesis was conducted to characterize these unique DNA binding proteins. This study highlights the structural diversity within the PFam12, demonstrating that despite significant differences in dimerization mechanisms, these proteins retain their DNA-binding capability.
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Apr 2025
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Plasmodium falciparum plasmepsin X (PMX) has become a target of choice for the development of new antimalarial drugs due to its essential role across the parasite life cycle. Here we describe the 1.7Å crystallographic structure of PMX noncovalently bound to a potent macrocyclic peptidomimetic inhibitor (7k) possessing a hydroxyethylamine (HEA) scaffold. Upon 7k binding, the enzyme adopts a novel conformation, with significant involvement of the S2’S2 loop (M526-H536) and the S2 flap (F311-G314). This results in partial closure of the active site with widespread interactions in both the prime (S’) and the non-prime (S) sites of PMX. The catalytic aspartate residues D266 and D467 directly interact with the HEA pharmacophore. Docking of a 7k derivative, compound 7a, highlights a region in the S3 pocket near the S3 flexible loop (H242-F248) that may be key for ligand stabilisation. The dynamic nature of PMX and its propensity to undergo distinct types of induced fit upon inhibitor binding enables generation of potent inhibitors that target this essential malarial aspartic protease.
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Mar 2025
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Virginie
Will
,
Lucile
Moynie
,
Elise
Si Ahmed Charrier
,
Audrey
Le Bas
,
Lauriane
Kuhn
,
Florian
Volck
,
Johana
Chicher
,
Hava
Aksoy
,
Morgan
Madec
,
Cyril
Antheaume
,
Gaëtan L. A.
Mislin
,
Isabelle J.
Schalk
Diamond Proposal Number(s):
[33133]
Abstract: Iron is essential for bacterial growth, and Pseudomonas aeruginosa synthesizes the siderophores pyochelin (PCH) and pyoverdine to acquire it. PCH contains a thiazolidine ring that aids in iron chelation but is prone to hydrolysis, leading to the formation of 2-(2-hydroxylphenyl)-thiazole-4-carbaldehyde (IQS). Using mass spectrometry, we demonstrated that PCH undergoes hydrolysis and oxidation in solution, resulting in the formation of aeruginoic acid (AA). This study used proteomic analyses and fluorescent reporters to show that AA, dihydroaeruginoic acid (DHA), and PCH induce the expression of femA, a gene encoding the ferri-mycobactin outer membrane transporter in P. aeruginosa. Notably, the induction by AA and DHA was observed only in strains unable to produce pyoverdine, suggesting their weaker iron-chelating ability compared to that of pyoverdine. 55Fe uptake assays demonstrated that both AA-Fe and DHA-Fe complexes are transported via FemA; however, no uptake was observed for PCH-Fe through this transporter. Structural studies revealed that FemA is able to bind AA2-Fe or DHA2-Fe complexes. Key interactions are conserved between FemA and these two complexes, with specificity primarily driven by one of the two siderophore molecules. Interestingly, although no iron uptake was noted for PCH through FemA, the transporter also binds PCH-Fe in a similar manner. These findings show that under moderate iron deficiency, when only PCH is produced by P. aeruginosa, degradation products AA and DHA enhance iron uptake by inducing femA expression and facilitating iron transport through FemA. This provides new insights into the pathogen’s strategies for iron homeostasis.
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Mar 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[28534]
Abstract: Objective: Along with rising resistance to antimalarials, the emergence of insecticide resistance in Anopheles mosquito species also remains a serious concern. Here, we reveal two potent compounds that show larvicidal and endectocidal activity against malaria vectors, Anopheles culicifacies and Anopheles stephensi, respectively. Methods: We investigated larvicidal activity of two inhibitors against III-instar larvae of Anopheles culicifacies. The survival and fertility of adult female Anopheles stephensi mosquitoes were assessed. Additionally, we purified recombinant prolyl-tRNA synthetase of Anopheles culicifacies and performed enzyme-based assays and structural analysis with the two inhibitors. Results: Our study reveals that the Anopheles culicifacies prolyl-tRNA synthetase (AcProRS) is potently inhibited by halofuginone (HFG) and an ATP mimetic (L95). The evaluation of larvicidal activity of HFG against Anopheles culicifacies III-instar larvae showed a dose-dependent increase in mortality. In adult female Anopheles stephensi mosquitoes, ingestion of HFG via artificial blood feeding resulted in impaired ovary development, reduced egg laying, and decreased overall survival. The potent enzymatic inhibition of AcProRS thus drives the killing of larvae. The co-crystal structure of AcProRS with inhibitors provides a structural basis for improving their potency as future larvicides. Conclusion: Our data suggest the potential for repositioning halofuginone (HFG) and pyrrolidine-based ATP-mimetics (L95) as larvicides. Targeting the vector-encoded aminoacyl-tRNA synthetases provides a new focus for developing effective agents that can control multiple mosquitoe-borne infectious diseases like malaria and dengue.
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Mar 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Shawn C.
Massoni
,
Nicola J.
Evans
,
Ingo
Hantke
,
Colleen
Fenton
,
James H.
Torpey
,
Katherine M.
Collins
,
Ewelina M.
Krysztofinska
,
Janina H.
Muench
,
Arjun
Thapaliya
,
Santiago
Martínez-Lumbreras
,
Sé
Hart Ferrell
,
Celia
Slater
,
Xinyue
Wang
,
Ruth
Fekade
,
Sandra
Obwar
,
Siyu
Yin
,
Alishba
Vazquez
,
Christopher B.
Prior
,
Kürşad
Turgay
,
Rivka L.
Isaacson
,
Amy H.
Camp
Diamond Proposal Number(s):
[13597]
Abstract: Bacterial protein degradation machinery consists of chaperone–protease complexes that play vital roles in bacterial growth and development and have sparked interest as novel antimicrobial targets. ClpC–ClpP (ClpCP) is one such chaperone–protease complex, recruited by adaptors to specific functions in the model bacterium Bacillus subtilis and other Gram-positive bacteria, including the pathogens Staphylococcus aureus and Mycobacterium tuberculosis. Here we have identified a new ClpCP adaptor protein, MdfA (metabolic differentiation factor A; formerly YjbA), in a genetic screen for factors that help drive B. subtilis toward metabolic dormancy during spore formation. A knockout of mdfA stimulates gene expression in the developing spore, while aberrant expression of mdfA during vegetative growth is toxic. MdfA binds directly to ClpC to induce its oligomerization and ATPase activity, and this interaction is required for the in vivo effects of mdfA. Finally, a cocrystal structure reveals that MdfA binds to the ClpC N-terminal domain at a location analogous to that on the M. tuberculosis ClpC1 protein where bactericidal cyclic peptides bind. Altogether, our data and that of an accompanying study by Riley and colleagues support a model in which MdfA induces ClpCP-mediated degradation of metabolic enzymes in the developing spore, helping drive it toward metabolic dormancy.
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Mar 2025
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