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753 items found (1 item not approved), displaying 21 to 30.[First/Prev] 1, 2, 3, 4, 5, 6, 7, 8 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Complex N-glycan breakdown by gut Bacteroides involves an extensive enzymatic apparatus encoded by multiple co-regulated genetic loci Justina Briliūtė , Paulina A. Urbanowicz , Ana S. Luis , Arnaud Basle , Neil Paterson , Osmond Rebello , Jenifer Hendel , Didier A. Ndeh , Elisabeth C. Lowe , Eric C. Martens , Daniel I. R. Spencer , David N. Bolam , Lucy I. Crouch Nature Microbiology 39 DOI: 10.1038/s41564-019-0466-x Diamond Proposal Number(s): [13587, 18598] Show Abstract ▼ Abstract: Glycans are the major carbon sources available to the human colonic microbiota. Numerous N-glycosylated proteins are found in the human gut, from both dietary and host sources, including immunoglobulins such as IgA that are secreted into the intestine at high levels. Here, we show that many mutualistic gut Bacteroides spp. have the capacity to utilize complex N-glycans (CNGs) as nutrients, including those from immunoglobulins. Detailed mechanistic studies using transcriptomic, biochemical, structural and genetic techniques reveal the pathway employed by Bacteroides thetaiotaomicron (Bt) for CNG degradation. The breakdown process involves an extensive enzymatic apparatus encoded by multiple non-adjacent loci and comprises 19 different carbohydrate-active enzymes from different families, including a CNG-specific endo-glycosidase activity. Furthermore, CNG degradation involves the activity of carbohydrate-active enzymes that have previously been implicated in the degradation of other classes of glycan. This complex and diverse apparatus provides Bt with the capacity to access the myriad different structural variants of CNGs likely to be found in the intestinal niche.	Jun 2019
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Novel inhibitory function of the rhizomucor miehei lipase propeptide and three-dimensional structures of its complexes with the enzyme Olga V. Moroz , Elena Blagova , Verena Reiser , Rakhi Saikia , Sohel Dalal , Christian Isak Jørgensen , Vikram K. Bhatia , Lone Baunsgaard , Birgitte Andersen , Allan Svendsen , Keith S. Wilson Acs Omega 4, 9964 - 9975 DOI: 10.1021/acsomega.9b00612 Diamond Proposal Number(s): [7864, 9948] Open Access Show Abstract ▼ Abstract: Many proteins are synthesized as precursors, with propeptides playing a variety of roles such as assisting in folding or preventing them from being active within the cell. While the precise role of the propeptide in fungal lipases is not completely understood, it was previously reported that mutations in the propeptide region of the Rhizomucor miehei lipase have an influence on the activity of the mature enzyme, stressing the importance of the amino acid composition of this region. We here report two structures of this enzyme in complex with its propeptide, which suggests that the latter plays a role in the correct maturation of the enzyme. Most importantly, we demonstrate that the propeptide shows inhibition of lipase activity in standard lipase assays and propose that an important role of the propeptide is to ensure that the enzyme is not active during its expression pathway in the original host.	Jun 2019
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design William Farnaby , Manfred Koegl , Michael J. Roy , Claire Whitworth , Emelyne Diers , Nicole Trainor , David Zollman , Steffen Steurer , Jale Karolyi-oezguer , Carina Riedmueller , Teresa Gmaschitz , Johannes Wachter , Christian Dank , Michael Galant , Bernadette Sharps , Klaus Rumpel , Elisabeth Traxler , Thomas Gerstberger , Renate Schnitzer , Oliver Petermann , Peter Greb , Harald Weinstabl , Gerd Bader , Andreas Zoephel , Alexander Weiss-puxbaum , Katharina Ehrenhöfer-wölfer , Simon Wöhrle , Guido Boehmelt , Joerg Rinnenthal , Heribert Arnhof , Nicola Wiechens , Meng-ying Wu , Tom Owen-hughes , Peter Ettmayer , Mark Pearson , Darryl B. Mcconnell , Alessio Ciulli Nature Chemical Biology DOI: 10.1038/s41589-019-0294-6 Diamond Proposal Number(s): [14980] Show Abstract ▼ Abstract: Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploit cancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficient optimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1 induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, and pave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.	Jun 2019
I04-1-Macromolecular Crystallography (fixed wavelength)	HspB1 phosphorylation regulates its intramolecular dynamics and mechanosensitive molecular chaperone interaction with filamin C Miranda P. Collier , T. Reid Alderson , Carin P. De Villiers , Daisy Nicholls , Heidi Y. Gastall , Timothy Allison , Matteo T. Degiacomi , He Jiang , Georg Mlynek , Dieter O. Fürst , Peter F. M. Van Der Ven , Kristina Djinovic-carugo , Andrew J. Baldwin , Hugh Watkins , Katja Gehmlich , Justin L. P. Benesch Science Advances 5 DOI: 10.1126/sciadv.aav8421 Diamond Proposal Number(s): [12346] Open Access Show Abstract ▼ Abstract: Mechanical force–induced conformational changes in proteins underpin a variety of physiological functions, typified in muscle contractile machinery. Mutations in the actin-binding protein filamin C (FLNC) are linked to musculoskeletal pathologies characterized by altered biomechanical properties and sometimes aggregates. HspB1, an abundant molecular chaperone, is prevalent in striated muscle where it is phosphorylated in response to cues including mechanical stress. We report the interaction and up-regulation of both proteins in three mouse models of biomechanical stress, with HspB1 being phosphorylated and FLNC being localized to load-bearing sites. We show how phosphorylation leads to increased exposure of the residues surrounding the HspB1 phosphosite, facilitating their binding to a compact multidomain region of FLNC proposed to have mechanosensing functions. Steered unfolding of FLNC reveals that its extension trajectory is modulated by the phosphorylated region of HspB1. This may represent a posttranslationally regulated chaperone-client protection mechanism targeting over-extension during mechanical stress.	May 2019
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Rapid covalent-probe discovery by electrophile-fragment screening Efrat Resnick , Anthony Bradley , Jinrui Gan , Alice Douangamath , Tobias Krojer , Ritika Sethi , Paul P. Geurink , Anthony Aimon , Gabriel Amitai , Dom Bellini , James Bennett , Michael Fairhead , Oleg Fedorov , Ronen Gabizon , Jin Gan , Jingxu Guo , Alexander Plotnikov , Nava Reznik , Gian Filippo Ruda , Laura Diaz-saez , Verena M. Straub , Tamas Szommer , Srikannathasan Velupillai , Daniel Zaidman , Yanling Zhang , Alun R. Coker , Christopher G. Dowson , Haim Barr , Chu Wang , Kilian V. M. Huber , Paul E. Brennan , Huib Ovaa , Frank Von Delft , Nir London Journal Of The American Chemical Society DOI: 10.1021/jacs.9b02822 Show Abstract ▼ Abstract: Covalent probes can display unmatched potency, selectivity and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered non-selective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against ten cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. By contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.	May 2019
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Molecular determinants regulating selective binding of autophagy adapters and receptors to ATG8 proteins Martina Wirth , Wenxin Zhang , Minoo Razi , Lynet Nyoni , Dhira Joshi , Nicola O’reilly , Terje Johansen , Sharon A. Tooze , Stephane Mouilleron Nature Communications 10 DOI: 10.1038/s41467-019-10059-6 Diamond Proposal Number(s): [13775] Open Access Show Abstract ▼ Abstract: Autophagy is an essential recycling and quality control pathway. Mammalian ATG8 proteins drive autophagosome formation and selective removal of protein aggregates and organelles by recruiting autophagy receptors and adaptors that contain a LC3-interacting region (LIR) motif. LIR motifs can be highly selective for ATG8 subfamily proteins (LC3s/GABARAPs), however the molecular determinants regulating these selective interactions remain elusive. Here we show that residues within the core LIR motif and adjacent C-terminal region as well as ATG8 subfamily-specific residues in the LIR docking site are critical for binding of receptors and adaptors to GABARAPs. Moreover, rendering GABARAP more LC3B-like impairs autophagy receptor degradation. Modulating LIR binding specificity of the centriolar satellite protein PCM1, implicated in autophagy and centrosomal function, alters its dynamics in cells. Our data provides new mechanistic insight into how selective binding of LIR motifs to GABARAPs is achieved, and elucidate the overlapping and distinct functions of ATG8 subfamily proteins.	May 2019
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Design and evolution of an enzyme with a non-canonical organocatalytic mechanism Ashleigh J. Burke , Sarah L. Lovelock , Amina Frese , Rebecca Crawshaw , Mary Ortmayer , Mark Dunstan , Colin Levy , Anthony P. Green Nature 453 DOI: 10.1038/s41586-019-1262-8 Diamond Proposal Number(s): [12788, 17773] Show Abstract ▼ Abstract: The combination of computational design and laboratory evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions. However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-molecule organocatalysis, here we report the generation of a hydrolytic enzyme that uses Nδ-methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design. Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free Nδ-methylhistidine in solution. Crystallographic snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. Nδ-methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chemical transformations.	May 2019
I04-1-Macromolecular Crystallography (fixed wavelength)	Crystal structure of phycocyanin from heterocyst-forming filamentous cyanobacterium Nostoc sp. WR13 Hiral M. Patel , Aleksander W. Roszak , Datta Madamwar , Richard J. Cogdell International Journal Of Biological Macromolecules DOI: 10.1016/j.ijbiomac.2019.05.099 Diamond Proposal Number(s): [16258] Show Abstract ▼ Abstract: Phycocyanin (PC) is the principal pigment protein in the light-harvesting antenna of cyanobacteria. Here the biochemical characterization and the 1.51 Å crystal structure of PC from cyanobacterium Nostoc sp. WR13 (Nst-PC) is reported. The P63 crystal lattice is composed of the minimal biological entities of Nst-PC, the (αβ)3 trimeric rings. The structure has been refined to R factor 11.5% (Rfree 15.4%) using anisotropic atomic B factors. A phylogenetic study shows that the α and β chains of Nst-PC are significantly clustered in a distinct clade with Acaryochloris marina. The structure was examined to look for any significant differences between Nst-PC and PC from non-desert species. Only minor differences were found in the chromophore microenvironments. The tentative energy transfer pathways in Nst-PC were modeled based on simple structural considerations.	May 2019
I04-1-Macromolecular Crystallography (fixed wavelength)	Design and characterization of PE-PilA, a candidate fusion antigen for non-typeable Haemophilus influenzae vaccine Normand Blais , Don Somers , Denis Faubert , Steve Labbé , Cindy Castado , Carine Ysebaert , Louis-patrick Gagnon , Josée Champagne , Martin Gagné , Denis Martin Infection And Immunity DOI: 10.1128/IAI.00022-19 Open Access Show Abstract ▼ Abstract: Non-typeable Haemophilus influenzae (NTHi) is a pathogen known for being a frequent cause of acute otitis media in children and respiratory tract infections in adults with chronic obstructive pulmonary disease. In the present study, a vaccine antigen has been developed based on the fusion of two known NTHi adhesive proteins, Protein E (PE) and pilin subunit (PilA). The quality of the combined antigen was investigated through functional, biophysical and structural analyses. It was shown that PE and PilA individual structures are not modified in the PE-PilA fusion and that PE-PilA assembles as a dimer in solution, reflecting PE dimerization. PE-PilA was found to bind vitronectin in ELISA, as isolated PE does. Disulfide bridges were conserved and homogeneous, which was determined by peptide mapping and Top-down analysis on PE, PilA and PE-PilA molecules. Finally, the PE-PilA crystal showed a PE entity with a 3D-structure similar to that of recently published isolated PE, while the structure of the PilA entity was similar to that of a 3D-model elaborated from two other type 4 pilin subunits. Taken together, our observations suggest that the two tethered proteins behave independently within the chimeric molecule and display structures similar to the respective isolated antigens, which are important characteristics for eliciting an optimal antibody-mediated immunity. PE and PilA can thus be further developed as a single fusion protein in a vaccine perspective, in the knowledge that tethering the two antigens does not perceptibly compromise the structural attributes offered by the individual antigens.	May 2019
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-ones: Studies towards the identification of potent, cell penetrant Jumonji C domain containing histone lysine demethylase 4 subfamily (KDM4) inhibitors, compound profiling in cell-based target engagement assays Yann-vai Le Bihan , Rachel M. Lanigan , Butrus Atrash , Mark G. Mclaughlin , Srikannathasan Velupillai , Andrew G. Malcolm , Katherine S. England , Gian Filippo Ruda , N. Yi Mok , Anthony Tumber , Kathy Tomlin , Harry Saville , Erald Shehu , Craig Mcandrew , Leanne Carmichael , James M. Bennett , Fiona Jeganathan , Paul Eve , Adam Donovan , Angela Hayes , Francesca Wood , Florence I. Raynaud , Oleg Fedorov , Paul Brennan , Rosemary Burke , Rob Van Montfort , Olivia W. Rossanese , Julian Blagg , Vassilios Bavetsias European Journal Of Medicinal Chemistry DOI: 10.1016/j.ejmech.2019.05.041 Diamond Proposal Number(s): [20145] Open Access Show Abstract ▼ Abstract: Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between the histone substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were targeted. Additionally, the conformational restriction of the flexible pyridopyrimidinone C8-substituent was investigated. These approaches yielded potent and cell-penetrant dual KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Ki = 0.004 and 0.007 μM, respectively). Compound cellular profiling in two orthogonal target engagement assays revealed a significant reduction from biochemical to cell-based activity across multiple analogues; this decrease was shown to be consistent with 2OG competition, and suggest that sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells.	May 2019

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