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

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography	The zinc-binding motif in tankyrases is required for the structural integrity of the catalytic ADP-ribosyltransferase domain Sven T. Sowa , Lari Lehtio Open Biology 12 DOI: 10.1098/rsob.210365 Diamond Proposal Number(s): [19951] Open Access Show Abstract ▼ Abstract: Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and are considered promising drug targets for cancer and fibrotic diseases. The catalytic ADP-ribosyltransferase domain of tankyrases contains a unique zinc-binding motif of unknown function. Recently, this motif was suggested to be involved in the catalytic activity of tankyrases. In this work, we set out to study the effect of the zinc-binding motif on the activity, stability and structure of human tankyrases. We generated mutants of human tankyrase (TNKS) 1 and TNKS2, abolishing the zinc-binding capabilities, and characterized the proteins biochemically and biophysically in vitro. We further generated a crystal structure of TNKS2, in which the zinc ion was oxidatively removed. Our work shows that the zinc-binding motif in tankyrases is a crucial structural element which is particularly important for the structural integrity of the acceptor site. While mutation of the motif rendered TNKS1 inactive, probably due to introduction of major structural defects, the TNKS2 mutant remained active and displayed an altered activity profile compared to the wild-type.	Mar 2022
I03-Macromolecular Crystallography	Structure and in silico simulations of a cold-active esterase reveals its prime cold-adaptation mechanism Nehad Noby , Husam Sabah Auhim , Samuel Winter , Harley L. Worthy , Amira M. Embaby , Hesham Saeed , Ahmed Hussein , Christopher R. Pudney , Pierre J. Rizkallah , Stephen A. Wells , D. Dafydd Jones Open Biology 11 DOI: 10.1098/rsob.210182 Diamond Proposal Number(s): [18812] Open Access Show Abstract ▼ Abstract: Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7's closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7's cold adaptation rather than changes in dynamics.	Dec 2021
	Parakeet: a digital twin software pipeline to assess the impact of experimental parameters on tomographic reconstructions for cryo-electron tomography James M. Parkhurst , Maud Dumoux , Mark Basham , Daniel Clare , C. Alistair Siebert , Trond Varslot , Angus Kirkland , James H. Naismith , Gwyndaf Evans Open Biology 11 DOI: 10.1098/rsob.210160 Open Access Show Abstract ▼ Abstract: In cryo-electron tomography (cryo-ET) of biological samples, the quality of tomographic reconstructions can vary depending on the transmission electron microscope (TEM) instrument and data acquisition parameters. In this paper, we present Parakeet, a ‘digital twin’ software pipeline for the assessment of the impact of various TEM experiment parameters on the quality of three-dimensional tomographic reconstructions. The Parakeet digital twin is a digital model that can be used to optimize the performance and utilization of a physical instrument to enable in silico optimization of sample geometries, data acquisition schemes and instrument parameters. The digital twin performs virtual sample generation, TEM image simulation, and tilt series reconstruction and analysis within a convenient software framework. As well as being able to produce physically realistic simulated cryo-ET datasets to aid the development of tomographic reconstruction and subtomogram averaging programs, Parakeet aims to enable convenient assessment of the effects of different microscope parameters and data acquisition parameters on reconstruction quality. To illustrate the use of the software, we present the example of a quantitative analysis of missing wedge artefacts on simulated planar and cylindrical biological samples and discuss how data collection parameters can be modified for cylindrical samples where a full 180° tilt range might be measured.	Oct 2021
I04-Macromolecular Crystallography	Bdellovibrio bacteriovorus phosphoglucose isomerase structures reveal novel rigidity in the active site of a selected subset of enzymes upon substrate binding R. W. Meek , I. T. Cadby , A. L. Lovering Open Biology 11 DOI: 10.1098/rsob.210098 Diamond Proposal Number(s): [10369, 14692] Open Access Show Abstract ▼ Abstract: Glycolysis and gluconeogenesis are central pathways of metabolism across all domains of life. A prominent enzyme in these pathways is phosphoglucose isomerase (PGI), which mediates the interconversion of glucose-6-phosphate and fructose-6-phosphate. The predatory bacterium Bdellovibrio bacteriovorus leads a complex life cycle, switching between intraperiplasmic replicative and extracellular ‘hunter’ attack-phase stages. Passage through this complex life cycle involves different metabolic states. Here we present the unliganded and substrate-bound structures of the B. bacteriovorus PGI, solved to 1.74 Å and 1.67 Å, respectively. These structures reveal that an induced-fit conformational change within the active site is not a prerequisite for the binding of substrates in some PGIs. Crucially, we suggest a phenylalanine residue, conserved across most PGI enzymes but substituted for glycine in B. bacteriovorus and other select organisms, is central to the induced-fit mode of substrate recognition for PGIs. This enzyme also represents the smallest conventional PGI characterized to date and probably represents the minimal requirements for a functional PGI.	Aug 2021
B16-Test Beamline B21-High Throughput SAXS	Structural basis for the coiled-coil architecture of human CtIP C. R. Morton , N. J. Rzechorzek , J. D. Maman , M. Kuramochi , H. Sekiguchi , R. Rambo , Y. C. Sasaki , O. R. Davies , Luca Pellegrini Open Biology 11 DOI: 10.1098/rsob.210060 Open Access Show Abstract ▼ Abstract: The DNA repair factor CtIP has a critical function in double-strand break (DSB) repair by homologous recombination, promoting the assembly of the repair apparatus at DNA ends and participating in DNA-end resection. However, the molecular mechanisms of CtIP function in DSB repair remain unclear. Here, we present an atomic model for the three-dimensional architecture of human CtIP, derived from a multi-disciplinary approach that includes X-ray crystallography, small-angle X-ray scattering (SAXS) and diffracted X-ray tracking (DXT). Our data show that CtIP adopts an extended dimer-of-dimers structure, in agreement with a role in bridging distant sites on chromosomal DNA during the recombinational repair. The zinc-binding motif in the CtIP N-terminus alters dynamically the coiled-coil structure, with functional implications for the long-range interactions of CtIP with DNA. Our results provide a structural basis for the three-dimensional arrangement of chains in the CtIP tetramer, a key aspect of CtIP function in DNA DSB repair.	Jun 2021
I03-Macromolecular Crystallography	Viral macrodomains: a structural and evolutionary assessment of the pharmacological potential Johannes Gregor Matthias Rack , Valentina Zorzini , Zihan Zhu , Marion Schuller , Dragana Ahel , Ivan Ahel Open Biology 10 DOI: 10.1098/rsob.200237 Diamond Proposal Number(s): [27001] Open Access Show Abstract ▼ Abstract: Viral macrodomains possess the ability to counteract host ADP-ribosylation, a post-translational modification implicated in the creation of an antiviral environment via immune response regulation. This brought them into focus as promising therapeutic targets, albeit the close homology to some of the human macrodomains raised concerns regarding potential cross-reactivity and adverse effects for the host. Here, we evaluate the structure and function of the macrodomain of SARS-CoV-2, the causative agent of COVID-19. We show that it can antagonize ADP-ribosylation by PARP14, a cellular (ADP-ribosyl)transferase necessary for the restriction of coronaviral infections. Furthermore, our structural studies together with ligand modelling revealed the structural basis for poly(ADP-ribose) binding and hydrolysis, an emerging new aspect of viral macrodomain biology. These new insights were used in an extensive evolutionary analysis aimed at evaluating the druggability of viral macrodomains not only from the Coronaviridae but also Togaviridae and Iridoviridae genera (causing diseases such as Chikungunya and infectious spleen and kidney necrosis virus disease, respectively). We found that they contain conserved features, distinct from their human counterparts, which may be exploited during drug design.	Nov 2020
I02-Macromolecular Crystallography	The efficiency of insulin production and its content in insulin-expressing model β-cells correlate with their Zn 2+ levels Petra Dzianová , Seiya Asai , Martina Chrudinová , Lucie Kosinová , Pavlo Potalitsyn , Pavel Šácha , Romana Hadravová , Irena Selicharová , Jan Kříž , Johan P. Turkenburg , Andrzej Marek Brzozowski , Jiří Jiráček , Lenka Žáková Open Biology 10 DOI: 10.1098/rsob.200137 Diamond Proposal Number(s): [13587] Open Access Show Abstract ▼ Abstract: Insulin is produced and stored inside the pancreatic β-cell secretory granules, where it is assumed to form Zn2+-stabilized oligomers. However, the actual storage forms of this hormone and the impact of zinc ions on insulin production in vivo are not known. Our initial X-ray fluorescence experiment on granules from native Langerhans islets and insulinoma-derived INS-1E cells revealed a considerable difference in the zinc content. This led our further investigation to evaluate the impact of the intra-granular Zn2+ levels on the production and storage of insulin in different model β-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 β-cells and in the native rat pancreatic islets by flow cytometry, confocal microscopy, immunoblotting, specific messenger RNA (mRNA) and total insulin analysis. These studies revealed an impaired insulin production in the permanent β-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn2+ levels was paralleled by a lower expression of ZnT8 zinc transporter mRNA and hampered proinsulin processing/folding in both permanent cell lines. To summarize, we showed that the disruption of zinc homeostasis in the model β-cells correlated with their impaired insulin and ZnT8 production. This indicates a need for in-depth fundamental research about the role of zinc in insulin production and storage.	Oct 2020
I02-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Identification of a PGXPP degron motif in dishevelled and structural basis for its binding to the E3 ligase KLHL12 Zhuoyao Chen , Gregory A. Wasney , Sarah Picaud , Panagis Filippakopoulos , Masoud Vedadi , Vincenzo D'Angiolella , Alex N. Bullock Open Biology 10 DOI: 10.1098/rsob.200041 Diamond Proposal Number(s): [15433] Open Access Show Abstract ▼ Abstract: Wnt signalling is dependent on dishevelled proteins (DVL1-3), which assemble an intracellular Wnt signalosome at the plasma membrane. The levels of DVL1-3 are regulated by multiple Cullin-RING E3 ligases that mediate their ubiquitination and degradation. The BTB-Kelch protein KLHL12 was the first E3 ubiquitin ligase to be identified for DVL1-3, but the molecular mechanisms determining its substrate interactions have remained unknown. Here, we mapped the interaction of DVL1-3 to a ‘PGXPP' motif that is conserved in other known partners and substrates of KLHL12, including PLEKHA4, PEF1, SEC31 and DRD4. To determine the binding mechanism, we solved a 2.4 Å crystal structure of the Kelch domain of KLHL12 in complex with a DVL1 peptide that bound with low micromolar affinity. The DVL1 substrate adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades of the Kelch domain β-propeller. In cells, the mutation or deletion of this motif reduced the binding and ubiquitination of DVL1 and increased its stability confirming this sequence as a degron motif for KLHL12 recruitment. These results define the molecular mechanisms determining DVL regulation by KLHL12 and establish the KLHL12 Kelch domain as a new protein interaction module for a novel proline-rich motif.	Jun 2020
I02-Macromolecular Crystallography	Crystal structure of human RIOK2 bound to a specific inhibitor Jing Wang , Thibault Varin , Michal Vieth , Jonathan M. Elkins Open Biology 9 DOI: 10.1098/rsob.190037 Open Access Show Abstract ▼ Abstract: The RIO kinases (RIOKs) are a universal family of atypical kinases that are essential for assembly of the pre-40S ribosome complex. Here, we present the crystal structure of human RIO kinase 2 (RIOK2) bound to a specific inhibitor. This first crystal structure of an inhibitor-bound RIO kinase reveals the binding mode of the inhibitor and explains the structure–activity relationship of the inhibitor series. The inhibitor binds in the ATP-binding site and forms extensive hydrophobic interactions with residues at the entrance to the ATP-binding site. Analysis of the conservation of active site residues reveals the reasons for the specificity of the inhibitor for RIOK2 over RIOK1 and RIOK3, and it provides a template for inhibitor design against the human RIOK family.	Apr 2019
I03-Macromolecular Crystallography	The human CTF4-orthologue AND-1 interacts with DNA polymerase α/primase via its unique C-terminal HMG box Mairi L. Kilkenny , Aline C. Simon , Jack Mainwaring , David Wirthensohn , Sandro Holzer , Luca Pellegrini Open Biology 7 DOI: 10.1098/rsob.170217 Open Access Show Abstract ▼ Abstract: A dynamic multi-protein assembly known as the replisome is responsible for DNA synthesis in eukaryotic cells. In yeast, the hub protein Ctf4 bridges DNA helicase and DNA polymerase and recruits factors with roles in metabolic processes coupled to DNA replication. An important question in DNA replication is the extent to which the molecular architecture of the replisome is conserved between yeast and higher eukaryotes. Here, we describe the biochemical basis for the interaction of the human CTF4-orthologue AND-1 with DNA polymerase α (Pol α)/primase, the replicative polymerase that initiates DNA synthesis. AND-1 has maintained the trimeric structure of yeast Ctf4, driven by its conserved SepB domain. However, the primary interaction of AND-1 with Pol α/primase is mediated by its C-terminal HMG box, unique to mammalian AND-1, which binds the B subunit, at the same site targeted by the SV40 T-antigen for viral replication. In addition, we report a novel DNA-binding activity in AND-1, which might promote the correct positioning of Pol α/primase on the lagging-strand template at the replication fork. Our findings provide a biochemical basis for the specific interaction between two critical components of the human replisome, and indicate that important principles of replisome architecture have changed significantly in evolution.	Nov 2017

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