I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26794]
Abstract: ADP-ribosylation is an enzymatic process where an ADP-ribose moiety is transferred from NAD+ to an acceptor molecule. While ADP-ribosylation is well-established as a post-translational modification of proteins, rifamycin antibiotics are its only known small-molecule targets. ADP-ribosylation of rifampicin was first identified in Mycolicibacterium smegmatis, whose Arr enzyme transfers the ADP-ribose moiety to the 23-hydroxy group of rifampicin preventing its interaction with the bacterial RNA polymerase thereby inactivating the antibiotic. Arr homologues are widely spread among bacterial species and present in several pathogenic species often associated with mobile genetic elements. Inhibition of Arr enzymes offers a promising strategy to overcome ADP-ribosylation mediated rifamycin resistance. We developed a high-throughput activity assay which was applied to screen an in-house library of human ADP-ribosyltransferase-targeted compounds. We identified 15 inhibitors with IC50 values below 5 μM against four Arr enzymes from M. smegmatis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Mycobacteroides abscessus. The observed overall selectivity of the hit compounds over the other homologues indicated structural differences between the proteins. We crystallized M. smegmatis and P. aeruginosa Arr enzymes, the former in complex with its most potent hit compound with an IC50 value of 1.3 μM. We observed structural differences in the NAD+ binding pockets of the two Arr homologues explaining the selectivity. Although the Arr inhibitors did not sensitize M. smegmatis to rifampicin in a growth inhibition assay, the structural information and the collection of inhibitors provide a foundation for rational modifications and further development of the compounds.
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Jun 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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Valentina
Borlandelli
,
Wendy
Offen
,
Olga
Moroz
,
Alba
Nin-Hill
,
Nicholas
Mcgregor
,
Lars
Binkhorst
,
Akihiro
Ishiwata
,
Zachary
Armstrong
,
Marta
Artola
,
Carme
Rovira
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: GH127 and GH146 microorganismal retaining β-l-arabinofuranosidases, expressed by human gut microbiomes, feature an atypical catalytic domain and an unusual mechanism of action. We recently reported that both Bacteroides thetaiotaomicron BtGH146 and Bifidobacterium longum HypBA1 are inhibited by β-l-arabinofuranosyl cyclophellitol epoxide, supporting the action of a zinc-coordinated cysteine as a catalytic nucleophile, where in most retaining GH families, an aspartate or glutamate is employed. This work presents a panel of β-l-arabinofuranosyl cyclophellitol epoxides and aziridines as mechanism-based BtGH146/HypBA1 inhibitors and activity-based probes. The β-l-arabinofuranosyl cyclophellitol aziridines both inhibit and label β-l-arabinofuranosidase efficiently (however with different activities), whereas the epoxide-derived probes favor BtGH146 over HypBA1. These findings are accompanied by X-ray structural analysis of the unmodified β-l-arabinofuranosyl cyclophellitol aziridine in complex with both isozymes, which were shown to react by nucleophilic opening of the aziridine, at the pseudoanomeric carbon, by the active site cysteine nucleophile to form a stable thioether bond. Altogether, our activity-based probes may serve as chemical tools for the detection and identification of low-abundance β-l-arabinofuranosidases in complex biological samples.
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Dec 2023
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I04-Macromolecular Crystallography
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Adam M.
Thomas
,
Marta
Serafini
,
Emma K.
Grant
,
Edward A. J.
Coombs
,
Joseph P.
Bluck
,
Matthias
Schiedel
,
Michael A.
Mcdonough
,
Jessica K.
Reynolds
,
Bernadette
Lee
,
Michael
Platt
,
Vassilena
Sharlandjieva
,
Philip C.
Biggin
,
Fernanda
Duarte
,
Thomas A.
Milne
,
Jacob T.
Bush
,
Stuart J.
Conway
Diamond Proposal Number(s):
[18069]
Open Access
Abstract: Target validation remains a challenge in drug discovery, which leads to a high attrition rate in the drug discovery process, particularly in Phase II clinical trials. Consequently, new approaches to enhance target validation are valuable tools to improve the drug discovery process. Here, we report the combination of site-directed mutagenesis and electrophilic fragments to enable the rapid identification of small molecules that selectively inhibit the mutant protein. Using the bromodomain-containing protein BRD4 as an example, we employed a structure-based approach to identify the L94C mutation in the first bromodomain of BRD4 [BRD4(1)] as having a minimal effect on BRD4(1) function. We then screened a focused, KAc mimic-containing fragment set and a diverse fragment library against the mutant and wild-type proteins and identified a series of fragments that showed high selectivity for the mutant protein. These compounds were elaborated to include an alkyne click tag to enable the attachment of a fluorescent dye. These clickable compounds were then assessed in HEK293T cells, transiently expressing BRD4(1)WT or BRD4(1)L94C, to determine their selectivity for BRD4(1)L94C over other possible cellular targets. One compound was identified that shows very high selectivity for BRD4(1)L94C over all other proteins. This work provides a proof-of-concept that the combination of site-directed mutagenesis and electrophilic fragments, in a mutate and conjugate approach, can enable rapid identification of small molecule inhibitors for an appropriately mutated protein of interest. This technology can be used to assess the cellular phenotype of inhibiting the protein of interest, and the electrophilic ligand provides a starting point for noncovalent ligand development.
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Oct 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Jin
Gan
,
Jelle
De Vries
,
Jimmy J. L. L.
Akkermans
,
Yassene
Mohammed
,
Rayman T. N.
Tjokrodirijo
,
Arnoud H.
De Ru
,
Robbert Q.
Kim
,
David A.
Vargas
,
Vito
Pol
,
Rudi
Fasan
,
Peter A.
Van Veelen
,
Jacques
Neefjes
,
Hans
Van Dam
,
Huib
Ovaa
,
Aysegul
Sapmaz
,
Paul P.
Geurink
Diamond Proposal Number(s):
[19800]
Open Access
Abstract: Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is often overexpressed during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, LN5P45, comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue. LN5P45 shows outstanding target engagement and proteome-wide selectivity in living cells. Importantly, LN5P45 as well as other OTUB2 inhibitors strongly induce monoubiquitination of OTUB2 on lysine 31. We present a route to future OTUB2-related therapeutics and have shown that the OTUB2 inhibitor developed in this study can help to uncover new aspects of the related biology and open new questions regarding the understanding of OTUB2 regulation at the post-translational modification level.
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Aug 2023
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I03-Macromolecular Crystallography
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Angelina R.
Sekirnik
,
Jessica K.
Reynolds
,
Larissa
See
,
Joseph P.
Bluck
,
Amy R.
Scorah
,
Cynthia
Tallant
,
Bernadette
Lee
,
Katarzyna B.
Leszczynska
,
Rachel L.
Grimley
,
R. Ian
Storer
,
Marta
Malattia
,
Sara
Crespillo
,
Sofia
Caria
,
Stephanie
Duclos
,
Ester M.
Hammond
,
Stefan
Knapp
,
Garrett M.
Morris
,
Fernanda
Duarte
,
Philip C.
Biggin
,
Stuart J.
Conway
Open Access
Abstract: TRIM33 is a member of the tripartite motif (TRIM) family of proteins, some of which possess E3 ligase activity and are involved in the ubiquitin-dependent degradation of proteins. Four of the TRIM family proteins, TRIM24 (TIF1α), TRIM28 (TIF1β), TRIM33 (TIF1γ) and TRIM66, contain C-terminal plant homeodomain (PHD) and bromodomain (BRD) modules, which bind to methylated lysine (KMen) and acetylated lysine (KAc), respectively. Here we investigate the differences between the two isoforms of TRIM33, TRIM33α and TRIM33β, using structural and biophysical approaches. We show that the N1039 residue, which is equivalent to N140 in BRD4(1) and which is conserved in most BRDs, has a different orientation in each isoform. In TRIM33β, this residue coordinates KAc, but this is not the case in TRIM33α. Despite these differences, both isoforms show similar affinities for H31–27K18Ac, and bind preferentially to H31–27K9Me3K18Ac. We used this information to develop an AlphaScreen assay, with which we have identified four new ligands for the TRIM33 PHD-BRD cassette. These findings provide fundamental new information regarding which histone marks are recognized by both isoforms of TRIM33 and suggest starting points for the development of chemical probes to investigate the cellular function of TRIM33.
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Sep 2022
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I04-Macromolecular Crystallography
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Cécile
Exertier
,
Federico
Sebastiani
,
Ida
Freda
,
Elena
Gugole
,
Gabriele
Cerutti
,
Giacomo
Parisi
,
Linda Celeste
Montemiglio
,
Maurizio
Becucci
,
Cristiano
Viappiani
,
Stefano
Bruno
,
Carmelinda
Savino
,
Carlotta
Zamparelli
,
Massimiliano
Anselmi
,
Stefania
Abbruzzetti
,
Giulietta
Smulevich
,
Beatrice
Vallone
Diamond Proposal Number(s):
[21741]
Open Access
Abstract: We produced a neuroglobin variant, namely, Ngb CDless, with the excised CDloop- and D-helix, directly joining the C- and E-helices. The CDless variant retained bis-His hexacoordination, and we investigated the role of the CDloop–D-helix unit in controlling the CO binding and structural dynamics by an integrative approach based on X-ray crystallography, rapid mixing, laser flash photolysis, resonance Raman spectroscopy, and molecular dynamics simulations. Rapid mixing and laser flash photolysis showed that ligand affinity was unchanged with respect to the wild-type protein, albeit with increased on and off constants for rate-limiting heme iron hexacoordination by the distal His64. Accordingly, resonance Raman spectroscopy highlighted a more open distal pocket in the CO complex that, in agreement with MD simulations, likely involves His64 swinging inward and outward of the distal heme pocket. Ngb CDless displays a more rigid overall structure with respect to the wild type, abolishing the structural dynamics of the CDloop–D-helix hypothesized to mediate its signaling role, and it retains ligand binding control by distal His64. In conclusion, this mutant may represent a tool to investigate the involvement of CDloop–D-helix in neuroprotective signaling in a cellular or animal model.
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Jul 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Ali
Nejatie
,
Elizabeth
Steves
,
Nick
Gauthier
,
Jamie
Baker
,
Jason
Nesbitt
,
Stephen A.
Mcmahon
,
Verena
Oehler
,
Nicholas J.
Thornton
,
Benjamin
Noyovitz
,
Kobra
Khazaei
,
Brock W.
Byers
,
Wesley F.
Zandberg
,
Tracey M.
Gloster
,
Margo M.
Moore
,
Andrew J.
Bennet
Abstract: Sialidases catalyze the release of sialic acid from the terminus of glycan chains. We previously characterized the sialidase from the opportunistic fungal pathogen, Aspergillus fumigatus, and showed that it is a Kdnase. That is, this enzyme prefers 3-deoxy-d-glycero-d-galacto-non-2-ulosonates (Kdn glycosides) as the substrate compared to N-acetylneuraminides (Neu5Ac). Here, we report characterization and crystal structures of putative sialidases from two other ascomycete fungal pathogens, Aspergillus terreus (AtS) and Trichophyton rubrum (TrS). Unlike A. fumigatus Kdnase (AfS), hydrolysis with the Neu5Ac substrates was negligible for TrS and AtS; thus, TrS and AtS are selective Kdnases. The second-order rate constant for hydrolysis of aryl Kdn glycosides by AtS is similar to that by AfS but 30-fold higher by TrS. The structures of these glycoside hydrolase family 33 (GH33) enzymes in complex with a range of ligands for both AtS and TrS show subtle changes in ring conformation that mimic the Michaelis complex, transition state, and covalent intermediate formed during catalysis. In addition, they can aid identification of important residues for distinguishing between Kdn and Neu5Ac substrates. When A. fumigatus, A. terreus, and T. rubrum were grown in chemically defined media, Kdn was detected in mycelial extracts, but Neu5Ac was only observed in A. terreus or T. rubrum extracts. The C8 monosaccharide 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) was also identified in A. fumigatus and T. rubrum samples. A fluorescent Kdn probe was synthesized and revealed the localization of AfS in vesicles at the cell surface.
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Nov 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12579]
Open Access
Abstract: Natural products and their analogues are often challenging to synthesize due to their complex scaffolds and embedded functional groups. Solely relying on engineering the biosynthesis of natural products may lead to limited compound diversity. Integrating synthetic biology with synthetic chemistry allows rapid access to much more diverse portfolios of xenobiotic compounds, which may accelerate the discovery of new therapeutics. As a proof-of-concept, by supplementing an Escherichia coli strain expressing the violacein biosynthesis pathway with 5-bromo-tryptophan in vitro or tryptophan 7-halogenase RebH in vivo, six halogenated analogues of violacein or deoxyviolacein were generated, demonstrating the promiscuity of the violacein biosynthesis pathway. Furthermore, 20 new derivatives were generated from 5-brominated violacein analogues via the Suzuki–Miyaura cross-coupling reaction directly using the crude extract without prior purification. Herein we demonstrate a flexible and rapid approach to access a diverse chemical space that can be applied to a wide range of natural product scaffolds.
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Oct 2021
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I24-Microfocus Macromolecular Crystallography
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Viktor
Mojr
,
Mohammad
Roghanian
,
Hedvig
Tamman
,
Duy Dinh
Do Pham
,
Magdalena
Petrová
,
Radek
Pohl
,
Hiraku
Takada
,
Katleen
Van Nerom
,
Hanna
Ainelo
,
Julien
Caballero-Montes
,
Steffi
Jimmy
,
Abel
Garcia-Pino
,
Vasili
Hauryliuk
,
Dominik
Rejman
Diamond Proposal Number(s):
[23248]
Abstract: While alarmone nucleotides guanosine-3′,5′-bisdiphosphate (ppGpp) and guanosine-5′-triphosphate-3′-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3′,5′-bisdiphosphate) and pppApp (adenosine-5′-triphosphate-3′-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3′-azido-3′-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.
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Sep 2021
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