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43 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Discovery of mono-ADP ribosylating toxins with high structural homology to Pseudomonas exotoxin A Geoffrey Masuyer , Alistair Taverner , Julia Mackay , Ana Rita Lima Marques , Yuye Wang , Tom Hunter , Keyi Liu , Randall J. Mrsny Communications Biology 8 DOI: 10.1038/s42003-025-07845-y Diamond Proposal Number(s): [23269, 21265] Open Access Show Abstract ▼ Abstract: Mono-ADP-ribosyl transferase (mART) proteins are secreted virulence factors produced by several human pathogens, the founding member being diphtheria toxin (DT). Pseudomonas aeruginosa can also secrete a mART toxin, known as exotoxin A (PE), but with an organization of its three functional domains (receptor, translocation, and enzymatic elements) that is opposite to DT. Two additional PE-like toxins (PLTs) have been identified from Vibrio cholerae and Aeromonas hydrophila, suggesting more PLT family members may exist. Database mining discovered six additional putative homologues, considerably extending this group of PLTs across a wide range of bacterial species. Here, we examine sequence and structural information for these new family members with respect to previously identified PLTs. The X-ray crystal structures of four new homologues show the conservation of critical features responsible for structure and function. This study shows the potential of these newly described toxins for the development of novel drug delivery platforms. Additionally, genomic analysis suggests horizontal gene transfer to account for the wide distribution of PLTs across a range of eubacteria species, highlighting the need to monitor emerging pathogens and their virulence factors.	Mar 2025
Krios III-Titan Krios III at Diamond	Stepwise recruitment of chaperone Hsc70 by DNAJB1 produces ordered arrays primed for bursts of amyloid fibril disassembly Jim Monistrol , Joseph G. Beton , Erin C. Johnston , Thi Lieu Dang , Bernd Bukau , Helen R. Saibil Communications Biology 8 DOI: 10.1038/s42003-025-07906-2 Diamond Proposal Number(s): [20287] Open Access Show Abstract ▼ Abstract: The Hsp70 chaperone system is capable of disassembling pathological aggregates such as amyloid fibres associated with serious degenerative diseases. Here we examine the role of the J-domain protein co-factor in amyloid disaggregation by the Hsc70 system. We used cryo-EM and tomography to compare the assemblies with wild-type DNAJB1 or inactive mutants. We show that DNAJB1 binds regularly along α-synuclein amyloid fibrils and acts in a 2-step recruitment of Hsc70, releasing DNAJB1 auto-inhibition before activating Hsc70 ATPase. The wild-type DNAJB1:Hsc70:Apg2 complex forms dense arrays of chaperones on the fibrils, with Hsc70 on the outer surface. When the auto-inhibition is removed by mutating DNAJB1 (ΔH5 DNAJB1), Hsc70 is recruited to the fibrils at a similar level, but the ΔH5 DNAJB1:Ηsc70:Apg2 complex is inactive, binds less regularly to the fibrils and lacks the ordered clusters. Therefore, we propose that 2-step activation of DNAJB1 regulates the ordered assembly of Hsc70 on the fibril. The localised, dense packing of chaperones could trigger a cascade of recruitment and activation to give coordinated, sequential binding and disaggregation from an exposed fibril end, as previously observed in AFM videos. This mechanism is likely to be important in maintaining a healthy cellular proteome into old age.	Mar 2025
I04-Macromolecular Crystallography	Characterization of a second class Ie ribonucleotide reductase Juliane John , Daniel Lundin , Rui M. Branca , Rohit Kumar , Vivek Srinivas , Hugo Lebrette , Martin Högbom Communications Biology 8 DOI: 10.1038/s42003-025-07565-3 Diamond Proposal Number(s): [29948] Open Access Show Abstract ▼ Abstract: Class I ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides under oxic conditions. The R2 subunit provides a radical required for catalysis conducted by the R1 subunit. In most R2s the radical is generated on a tyrosine via oxidation by an adjacent metal site. The discovery of a metal-free R2 defined the new RNR subclass Ie. In R2e, three of the otherwise strictly conserved metal-binding glutamates in the active site are substituted. Two variants have been found, VPK and QSK. To date, the VPK version has been the focus of biochemical characterization. Here we characterize a QSK variant of R2e. We analyse the organismal distribution of the two R2e versions and find dozens of organisms relying solely on the QSK RNR for deoxyribonucleotide production. We demonstrate that the R2eQSK of the human pathogen Gardnerella vaginalis (Bifidobacterium vaginale) modifies the active site-adjacent tyrosine to DOPA. The amount of modified protein is shown to be dependent on coexpression with the other proteins encoded in the RNR operon. The DOPA containing R2eQSK can support ribonucleotide reduction in vitro while the unmodified protein cannot. Finally, we determined the first structures of R2eQSK in the unmodified and DOPA states.	Feb 2025
I03-Macromolecular Crystallography	Carbonic anhydrase 2-derived drug-responsive domain regulates membrane-bound cytokine expression and function in engineered T cells Mara C. Inniss , Sean G. Smith , Dan Jun Li , Benjamin Primack , Dexue Sun , Grace Y. Olinger , Kerri-Lynn Sheahan , Theresa Ross , Meghan Langley , Violet Young , Andres Alvarado , Shabnam Davoodi , Jiefei Geng , Michael Schebesta , Michelle L. Ols , Jeremy Tchaicha , Jan Ter Meulen , Dhruv K. Sethi Communications Biology 8 DOI: 10.1038/s42003-024-07410-z Open Access Show Abstract ▼ Abstract: Adoptive cell therapies (ACT) have shown reduced efficacy against solid tumor malignancies compared to hematologic malignancies, partly due to the immunosuppressive nature of the tumor microenvironment (TME). ACT efficacy may be enhanced with pleiotropic cytokines that remodel the TME; however, their expression needs to be tightly controlled to avoid systemic toxicities. Here we show T cells can be armored with membrane-bound cytokines with surface expression regulated using drug-responsive domains (DRDs) developed from the 260-amino acid protein human carbonic anhydrase 2 (CA2). The CA2-DRD can be stabilized in vitro and in vivo with the FDA-approved small-molecule CA2 inhibitor acetazolamide (ACZ). We develop conditional degrons using library-based screening of mutants and show characterization of one DRD using crystallography and molecular dynamics (MD) simulations. Using protein-engineering solutions to increase the valency of DRDs fused to the cargo we have developed “modulation hubs” and show tight regulation of membrane-bound cytokines IL2, IL12, IL15, IL21, IL23, and IFNα in genetically engineered T cells. Finally, CA2-DRD regulated IL12 mediates regulated efficacy in a solid tumor model. Regulation of pleotropic cytokines potentially paves the way to safely use these powerful cytokines in ACT for cancer treatment.	Jan 2025
B21-High Throughput SAXS	GPR180 is a new member of the Golgi-dynamics domain seven-transmembrane helix protein family Sarah-Ana Mitrovic , Chamalee Demalgiriya-Gamage , Lisa-Maria Winter , Tobias Kiechle , Rebecca Ebenhoch , Heike Neubauer , Birgit Stierstorfer , Lee Frego , Christian Wolfrum , Sophia Reindl , Herbert Nar Communications Biology 7 DOI: 10.1038/s42003-024-07260-9 Diamond Proposal Number(s): [26256] Open Access Show Abstract ▼ Abstract: GOLD domain seven-transmembrane helix (GOST) proteins form a new protein family involved in trafficking of membrane-associated cargo. They share a characteristic extracellular/luminal Golgi-dynamics (GOLD) domain, possibly responsible for ligand recognition. Based on structural homology, GPR180 is a new member of this protein family, but little is known about the cellular role of GPR180. Here we show the X-ray structure of the N-terminal domain of GPR180 (1.9 Å) and can confirm the homology to GOLD domains. Using cellular imaging we show the localization of GPR180 in intracellular vesicular structures implying its exposure to acidic pH environments. With Hydrogen/Deuterium Exchange-Mass Spectrometry (HDX-MS) we identify pH-dependent conformational changes, which can be mapped to a putative ligand binding site in the transmembrane region. The results reveal GPR180’s role in intracellular vesicles and offer insights into the pH-dependent function of this conserved GOST protein.	Dec 2024
B22-Multimode InfraRed imaging And Microspectroscopy	Identification of tauopathy-associated lipid signatures in Alzheimer’s disease mouse brain using label-free chemical imaging Hao Meng , Alicia Elliott , Jessica Mansfield , Michelle Bailey , Mark Frogley , Gianfelice Cinque , Julian Moger , Nick Stone , Francesco Tamagnini , Francesca Palombo Communications Biology 7 DOI: 10.1038/s42003-024-07034-3 Diamond Proposal Number(s): [27207] Open Access Show Abstract ▼ Abstract: There is cumulative evidence that lipid metabolism plays a key role in the pathogenesis of various neurodegenerative disorders including Alzheimer’s disease (AD). Visualising lipid content in a non-destructive label-free manner can aid in elucidating the AD phenotypes towards a better understanding of the disease. In this study, we combined multiple optical molecular-specific methods, Fourier transform infrared (FTIR) spectroscopic imaging, synchrotron radiation-infrared (SR-IR) microscopy, Raman and stimulated Raman scattering (SRS) microscopy, and optical-photothermal infrared (O-PTIR) microscopy with multivariate data analysis, to investigate the biochemistry of brain hippocampus in situ using a mouse model of tauopathy (rTg4510). We observed a significant difference in the morphology and lipid content between transgenic (TG) and wild type (WT) samples. Immunohistochemical staining revealed some degree of microglia co-localisation with elevated lipids in the brain. These results provide new evidence of tauopathy-related dysfunction in a preclinical study at a subcellular level.	Oct 2024
I03-Macromolecular Crystallography	Biochemical and crystallographic studies of l,d-transpeptidase 2 from Mycobacterium tuberculosis with its natural monomer substrate Mariska De Munnik , Pauline A. Lang , Karina Calvopina , Patrick Rabe , Jurgen Brem , Christopher J. Schofield Communications Biology 7 DOI: 10.1038/s42003-024-06785-3 Open Access Show Abstract ▼ Abstract: The essential L,D-transpeptidase of Mycobacterium tuberculosis (LdtMt2) catalyses the formation of 33 cross-links in cell wall peptidoglycan and is a target for development of antituberculosis therapeutics. Efforts to inhibit LdtMt2 have been hampered by lack of knowledge of how it binds its substrate. To address this gap, we optimised the isolation of natural disaccharide tetrapeptide monomers from the Corynebacterium jeikeium bacterial cell wall through overproduction of the peptidoglycan sacculus. The tetrapeptides were used in binding / turnover assays and biophysical studies on LdtMt2. We determined a crystal structure of wild-type LdtMt2 reacted with its natural substrate, the tetrapeptide monomer of the peptidoglycan layer. This structure shows formation of a thioester linking the catalytic cysteine and the donor substrate, reflecting an intermediate in the transpeptidase reaction; it informs on the mode of entrance of the donor substrate into the LdtMt2 active site. The results will be useful in design of LdtMt2 inhibitors, including those based on substrate binding interactions, a strategy successfully employed for other nucleophilic cysteine enzymes.	Sep 2024
I08-Scanning X-ray Microscopy beamline (SXM)	Nanoscale synchrotron x-ray analysis of intranuclear iron in melanised neurons of Parkinson's substantia nigra Jake Brooks , James Everett , Emily Hill , Kharmen Billimoria , Christopher M. Morris , Peter J. Sadler , Neil Telling , Joanna F. Collingwood Communications Biology 7 DOI: 10.1038/s42003-024-06636-1 Diamond Proposal Number(s): [29042, 24534] Open Access Show Abstract ▼ Abstract: Neuromelanin-pigmented neurons of the substantia nigra are selectively lost during the progression of Parkinson’s disease. These neurons accumulate iron in the disease state, and iron-mediated neuron damage is implicated in cell death. Animal models of Parkinson’s have evidenced iron loading inside the nucleoli of nigral neurons, however the nature of intranuclear iron deposition in the melanised neurons of the human substantia nigra is not understood. Here, scanning transmission x-ray microscopy (STXM) is used to probe iron foci in relation to the surrounding ultrastructure in melanised neurons of human substantia nigra from a confirmed Parkinson’s case. In addition to the expected neuromelanin-bound iron, iron deposits are also associated with the edge of the cell nucleolus. Speciation analysis confirms these deposits to be ferric (Fe3+) iron. The function of intranuclear iron in these cells remains unresolved, although both damaging and protective mechanisms are considered. This finding shows that STXM is a powerful label-free tool for the in situ, nanoscale chemical characterisation of both organic and inorganic intracellular components. Future applications are likely to shed new light on incompletely understood biochemical mechanisms, such as metal dysregulation and morphological changes to cell nucleoli, that are important in understanding the pathogenesis of Parkinson’s.	Aug 2024
I04-Macromolecular Crystallography	Peri active site catalysis of proline isomerisation is the molecular basis of allomorphy in β-phosphoglucomutase F. Aaron Cruz-Navarrete , Nicola J. Baxter , Adam J. Flinders , Anamaria Buzoianu , Matthew J. Cliff , Patrick J. Baker , Jonathan P. Waltho Communications Biology 7 DOI: 10.1038/s42003-024-06577-9 Diamond Proposal Number(s): [24447, 31850] Open Access Show Abstract ▼ Abstract: Metabolic regulation occurs through precise control of enzyme activity. Allomorphy is a post-translational fine control mechanism where the catalytic rate is governed by a conformational switch that shifts the enzyme population between forms with different activities. β-Phosphoglucomutase (βPGM) uses allomorphy in the catalysis of isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Herein, we describe structural and biophysical approaches to reveal its allomorphic regulatory mechanism. Binding of the full allomorphic activator β-glucose 1,6-bisphosphate stimulates enzyme closure, progressing through NAC I and NAC III conformers. Prior to phosphoryl transfer, loops positioned on the cap and core domains are brought into close proximity, modulating the environment of a key proline residue. Hence accelerated isomerisation, likely via a twisted anti/C4-endo transition state, leads to the rapid predominance of active cis-P βPGM. In contrast, binding of the partial allomorphic activator fructose 1,6-bisphosphate arrests βPGM at a NAC I conformation and phosphoryl transfer to both cis-P βPGM and trans-P βPGM occurs slowly. Thus, allomorphy allows a rapid response to changes in food supply while not otherwise impacting substantially on levels of important metabolites.	Jul 2024
I03-Macromolecular Crystallography	Structural and functional insights underlying recognition of histidine phosphotransfer protein in fungal phosphorelay systems Francisco Paredes-Martinez , Lluís Eixerés , Sara Zamora-Caballero , Patricia Casino Communications Biology 7 DOI: 10.1038/s42003-024-06459-0 Diamond Proposal Number(s): [14739, 20229] Open Access Show Abstract ▼ Abstract: In human pathogenic fungi, receiver domains from hybrid histidine kinases (hHK) have to recognize one HPt. To understand the recognition mechanism, we have assessed phosphorelay from receiver domains of five hHKs of group III, IV, V, VI, and XI to HPt from Chaetomium thermophilum and obtained the structures of Ct_HPt alone and in complex with the receiver domain of hHK group VI. Our data indicate that receiver domains phosphotransfer to Ct_HPt, show a low affinity for complex formation, and prevent a Leu-Thr switch to stabilize phosphoryl groups, also derived from the structures of the receiver domains of hHK group III and Candida albicans Sln1. Moreover, we have elucidated the envelope structure of C. albicans Ypd1 using small-angle X-ray scattering which reveals an extended flexible conformation of the long loop αD–αE which is not involved in phosphotransfer. Finally, we have analyzed the role of salt bridges in the structure of Ct_HPt alone.	Jul 2024

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