I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459]
Open Access
Abstract: There is an urgent need for improved malaria vaccine immunogens. Invasion of erythrocytes by Plasmodium falciparum is essential for its life cycle, preceding symptoms of disease and parasite transmission. Antibodies which target PfRH5 are highly effective at preventing erythrocyte invasion and the most potent growth-inhibitory antibodies bind a single epitope. Here we use structure-guided approaches to design a small synthetic immunogen, RH5-34EM which recapitulates this epitope. Structural biology and biophysics demonstrate that RH5-34EM is correctly folded and binds neutralising monoclonal antibodies with nanomolar affinity. In immunised rats, RH5-34EM induces PfRH5-targeting antibodies that inhibit parasite growth. While PfRH5-specific antibodies were induced at a lower concentration by RH5-34EM than by PfRH5, RH5-34EM induced antibodies that were a thousand-fold more growth-inhibitory as a factor of PfRH5-specific antibody concentration. Finally, we show that priming with RH5-34EM and boosting with PfRH5 achieves the best balance between antibody quality and quantity and induces the most effective growth-inhibitory response. This rationally designed vaccine immunogen is now available for use as part of future malaria vaccines, alone or in combination with other immunogens.
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Sep 2024
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[29074]
Open Access
Abstract: Aurora-A is an essential cell-cycle kinase with critical roles in mitotic entry and spindle dynamics. These functions require binding partners such as CEP192 and TPX2, which modulate both kinase activity and localisation of Aurora-A. Here we investigate the structure and role of the centrosomal Aurora-A:CEP192 complex in the wider molecular network. We find that CEP192 wraps around Aurora-A, occupies the binding sites for mitotic spindle-associated partners, and thus competes with them. Comparison of two different Aurora-A conformations reveals how CEP192 modifies kinase activity through the site used for TPX2-mediated activation. Deleting the Aurora-A-binding interface in CEP192 prevents centrosomal accumulation of Aurora-A, curtails its activation-loop phosphorylation, and reduces spindle-bound TPX2:Aurora-A complexes, resulting in error-prone mitosis. Thus, by supplying the pool of phosphorylated Aurora-A necessary for TPX2 binding, CEP192:Aurora-A complexes regulate spindle function. We propose an evolutionarily conserved spatial hierarchy, which protects genome integrity through fine-tuning and correctly localising Aurora-A activity.
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Sep 2024
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Garry
Dolton
,
Anna
Bulek
,
Aaron
Wall
,
Hannah
Thomas
,
Jade R.
Hopkins
,
Cristina
Rius
,
Sarah A. E.
Galloway
,
Thomas
Whalley
,
Li Rong
Tan
,
Théo
Morin
,
Nader
Omidvar
,
Anna
Fuller
,
Katie
Topley
,
Md Samiul
Hasan
,
Shikha
Jain
,
Nirupa
D’souza
,
Thomas
Hodges-Hoyland
,
Owen B.
Spiller
,
Deborah
Kronenberg-Versteeg
,
Barbara
Szomolay
,
Hugo A.
Van Den Berg
,
Lucy C.
Jones
,
Mark
Peakman
,
David K.
Cole
,
Pierre J.
Rizkallah
,
Andrew K.
Sewell
Diamond Proposal Number(s):
[10462, 18812]
Open Access
Abstract: CD8+ T cells destroy insulin-producing pancreatic β cells in type 1 diabetes through HLA class I–restricted presentation of self-antigens. Combinatorial peptide library screening was used to produce a preferred peptide recognition landscape for a patient-derived T cell receptor (TCR) that recognized the preproinsulin-derived (PPI-derived) peptide sequence LWMRLLPLL in the context of disease risk allele HLA A*24:02. Data were used to generate a strong superagonist peptide, enabling production of an autoimmune HLA A*24:02–peptide–TCR structure by crystal seeding. TCR binding to the PPI epitope was strongly focused on peptide residues Arg4 and Leu5, with more flexibility at other positions, allowing the TCR to strongly engage many peptides derived from pathogenic bacteria. We confirmed an epitope from Klebsiella that was recognized by PPI-reactive T cells from 3 of 3 HLA A*24:02+ patients. Remarkably, the same epitope selected T cells from 7 of 8 HLA A*24+ healthy donors that cross-reacted with PPI, leading to recognition and killing of HLA A*24:02+ cells expressing PPI. These data provide a mechanism by which molecular mimicry between pathogen and self-antigens could have resulted in the breaking of self-tolerance to initiate disease.
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Sep 2024
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[25108, 32728]
Open Access
Abstract: The HECT E3 ubiquitin ligases 1 (WWP1) and 2 (WWP2) are responsible for the ubiquitin-mediated degradation of key tumour suppressor proteins and are dysregulated in various cancers and diseases. Here we expand their limited inhibitor space by identification of NSC-217913 displaying a WWP1 IC50 of 158.3 µM (95% CI = 128.7, 195.1 µM). A structure-activity relationship by synthesis approach aided by molecular docking led to compound 11 which displayed increased potency with an IC50 of 32.7 µM (95% CI = 24.6, 44.3 µM) for WWP1 and 269.2 µM (95% CI = 209.4, 347.9 µM) for WWP2. Molecular docking yielded active site-bound poses suggesting that the heterocyclic imidazo[4,5-b]pyrazine scaffold undertakes a π-stacking interaction with the phenolic group of tyrosine, and the ethyl ester enables strong ion-dipole interactions. Given the therapeutic potential of WWP1 and WWP2, we propose that compound 11 may provide a basis for future lead compound development.
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Sep 2024
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Nucleotidyltransferases (NTases) control diverse physiological processes, including RNA modification, DNA replication and repair, and antibiotic resistance. The Mycobacterium tuberculosis NTase toxin family, MenT, modifies tRNAs to block translation. MenT toxin activity can be stringently regulated by diverse MenA antitoxins. There has been no unifying mechanism linking antitoxicity across MenT homologues. Here we demonstrate through structural, biochemical, biophysical and computational studies that despite lacking kinase motifs, antitoxin MenA1 induces auto-phosphorylation of MenT1 by repositioning the MenT1 phosphoacceptor T39 active site residue towards bound nucleotide. Finally, we expand this predictive model to explain how unrelated antitoxin MenA3 is similarly able to induce auto-phosphorylation of cognate toxin MenT3. Our study reveals a conserved mechanism for the control of tuberculosis toxins, and demonstrates how active site auto-phosphorylation can regulate the activity of widespread NTases.
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Sep 2024
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I04-Macromolecular Crystallography
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Qin
Su
,
Max
Louwerse
,
Rob F.
Lammers
,
Elmer
Maurits
,
Max
Janssen
,
Rolf G.
Boot
,
Valentina
Borlandelli
,
Wendy A.
Offen
,
Daniël
Linzel
,
Sybrin P.
Schröder
,
Gideon J.
Davies
,
Herman S.
Overkleeft
,
Marta
Artola
,
Johannes M. F. G.
Aerts
Open Access
Abstract: GBA2, the non-lysosomal β-glucosylceramidase, is an enzyme involved in glucosylceramide metabolism. Pharmacological inhibition of GBA2 by N-alkyl iminosugars is well tolerated and benefits patients suffering from Sandhoff and Niemann–Pick type C diseases, and GBA2 inhibitors have been proposed as candidate-clinical drugs for the treatment of parkinsonism. With the ultimate goal to unravel the role of GBA2 in (patho)physiology, we sought to develop a GBA2-specific activity-based probe (ABP). A library of probes was tested for activity against GBA2 and the two other cellular retaining β-glucosidases, lysosomal GBA1 and cytosolic GBA3. We show that β-D-arabinofuranosyl cyclitol aziridine (β-D-Araf aziridine) reacts with the GBA2 active site nucleophile to form a covalent and irreversible bond. Fluorescent β-D-Araf aziridine probes potently and selectively label GBA2 both in vitro and in cellulo, allowing for visualization of the localization of overexpressed GBA2 using fluorescence microscopy. Co-staining with an antibody selective for the lysosomal β-glucosylceramidase GBA1, shows distinct subcellular localization of the two enzymes. We proffer our ABP technology for further delineating the role and functioning of GBA2 in disease and propose the β-D-Araf aziridine scaffold as a good starting point for the development of GBA2-specific inhibitors for clinical development.
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Sep 2024
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I04-Macromolecular Crystallography
I23-Long wavelength MX
I24-Microfocus Macromolecular Crystallography
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David
Hollingworth
,
Frances
Thomas
,
Dana A.
Page
,
Mohamed A.
Fouda
,
Raquel Lopez-Rios
De Castro
,
Altin
Sula
,
Vitaliy B.
Mykhaylyk
,
Geoff
Kelly
,
Martin B.
Ulmschneider
,
Peter C.
Ruben
,
Bonnie A.
Wallace
Diamond Proposal Number(s):
[23853]
Open Access
Abstract: Neuronal hyperexcitability is a key element of many neurodegenerative disorders including the motor neuron disease Amyotrophic Lateral Sclerosis (ALS), where it occurs associated with elevated late sodium current (INaL). INaL results from incomplete inactivation of voltage-gated sodium channels (VGSCs) after their opening and shapes physiological membrane excitability. However, dysfunctional increases can cause hyperexcitability-associated diseases. Here we reveal the atypical binding mechanism which explains how the neuroprotective ALS-treatment drug riluzole stabilises VGSCs in their inactivated state to cause the suppression of INaL that leads to reversed cellular overexcitability. Riluzole accumulates in the membrane and enters VGSCs through openings to their membrane-accessible fenestrations. Riluzole binds within these fenestrations to stabilise the inactivated channel state, allowing for the selective allosteric inhibition of INaL without the physical block of Na+ conduction associated with traditional channel pore binding VGSC drugs. We further demonstrate that riluzole can reproduce these effects on a disease variant of the non-neuronal VGSC isoform Nav1.4, where pathologically increased INaL is caused directly by mutation. Overall, we identify a model for VGSC inhibition that produces effects consistent with the inhibitory action of riluzole observed in models of ALS. Our findings will aid future drug design and supports research directed towards riluzole repurposing.
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Sep 2024
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[28516]
Open Access
Abstract: Recognition of ubiquitin (Ub) is often mediated by small Ub binding domains such as the Ubiquitin Interacting Motif (UIM). Most Ub binding events are low affinity interactions, and designing stronger binders for Ub can be challenging. We here report the design of a short crosslinked coiled coil (CC) which is conformationally and chemically stable, and which can act as a scaffold to present the key binding residues from known UIM sequences. Doing so gives rise to a hybrid CC peptide that reconciles the important features of both UIM and CC sequences. We show by fluorescence polarization assays that this crosslinked ‘CC–UIM’ peptide exhibits enhanced binding to Ub compared to the original UIM sequence. Furthermore, we report a crystal structure of this peptide in complex with Ub. These studies show that preorganization of a small number of important binding residues onto a stable helical scaffold can be a successful strategy for binder design.
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Sep 2024
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I04-Macromolecular Crystallography
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Kaitlyn M.
Logan
,
Will
Kaplan
,
Vladimir
Simov
,
Hua
Zhou
,
Derun
Li
,
Luis
Torres
,
Gregori J.
Morriello
,
John J.
Acton
,
Barbara
Pio
,
Yi-Heng
Chen
,
Mitchell H.
Keylor
,
Rebecca
Johnson
,
Solomon D.
Kattar
,
Ryan
Chau
,
Xin
Yan
,
Michael
Ardolino
,
Cayetana
Zarate
,
Karin M.
Otte
,
Rachel L.
Palte
,
Tina
Xiong
,
Spencer E.
Mcminn
,
Shishi
Lin
,
Santhosh F.
Neelamkavil
,
Ping
Liu
,
Jing
Su
,
Laxminarayan G.
Hegde
,
Janice D.
Woodhouse
,
Lily Y.
Moy
,
Paul J.
Ciaccio
,
Jennifer
Piesvaux
,
Matthias
Zebisch
,
Clare
Henry
,
John
Barker
,
Harold B.
Wood
,
Matthew E.
Kennedy
,
Erin F.
Dimauro
,
Matthew J.
Fell
,
Peter H.
Fuller
Diamond Proposal Number(s):
[20021]
Abstract: Inhibition of leucine-rich repeat kinase 2 is a genetically supported mechanism for the treatment of Parkinson’s disease. We previously disclosed the discovery of an indazole series lead that demonstrated both safety and translational risks. The safety risks were hypothesized to be of unknown origin, so structural diversity in subsequent chemical matter was prioritized. The translational risks were identified due to a low brain Kpu,u in nonhuman primate studies, which raised concern over the use of an established peripheral biomarker as a surrogate for central target engagement. Given these challenges, the team sought to leverage structure- and property-based drug design and expanded efflux transporter profiling to identify structurally distinct leads with enhanced CNS drug-likeness. Herein, we describe the discovery of a “reinvented” indazole series with improved physicochemical properties and efflux transporter profiles while maintaining excellent potency and off-target kinase selectivity, which resulted in advanced lead, compound 23.
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Sep 2024
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Pragya
Parashara
,
Bethan
Medina-Pritchard
,
Maria Alba
Abad
,
Paula
Sotelo-Parrilla
,
Reshma
Thamkachy
,
David
Grundei
,
Juan
Zou
,
Christos
Spanos
,
Chandni Natalia
Kumar
,
Claire
Basquin
,
Vimal
Das
,
Zhaoyue
Yan
,
Asma Abdullah
Al-Murtadha
,
David A.
Kelly
,
Toni
Mchugh
,
Axel
Imhof
,
Juri
Rappsilber
,
A. Arockia
Jeyaprakash
Diamond Proposal Number(s):
[25233]
Abstract: Accurate chromosome segregation requires the attachment of microtubules to centromeres, epigenetically defined by the enrichment of CENP-A nucleosomes. During DNA replication, CENP-A nucleosomes undergo dilution. To preserve centromere identity, correct amounts of CENP-A must be restored in a cell cycle–controlled manner orchestrated by the Mis18 complex (Mis18α-Mis18β-Mis18BP1). We demonstrate here that PLK1 interacts with the Mis18 complex by recognizing self-primed phosphorylations of Mis18α (Ser54) and Mis18BP1 (Thr78 and Ser93) through its Polo-box domain. Disrupting these phosphorylations perturbed both centromere recruitment of the CENP-A chaperone HJURP and new CENP-A loading. Biochemical and functional analyses showed that phosphorylation of Mis18α and PLK1 binding were required to activate Mis18α-Mis18β and promote Mis18 complex-HJURP interaction. Thus, our study reveals key molecular events underpinning the licensing role of PLK1 in ensuring accurate centromere inheritance.
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Sep 2024
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