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Instruments / Technical Area	Publication Details	Published
Krios I-Titan Krios I at Diamond	Structural basis of tankyrase activation by polymerization Nisha Pillay , Laura Mariotti , Mariola Zaleska , Oviya Inian , Matthew Jessop , Sam Hibbs , Ambroise Desfosses , Paul C. R. Hopkins , Catherine M. Templeton , Fabienne Beuron , Edward P. Morris , Sebastian Guettler Nature 20 DOI: 10.1038/s41586-022-05449-8 Diamond Proposal Number(s): [28549] Open Access Show Abstract ▼ Abstract: The poly-ADP-ribosyltransferase tankyrase (TNKS, TNKS2) controls a wide range of disease-relevant cellular processes, including WNT–β-catenin signalling, telomere length maintenance, Hippo signalling, DNA damage repair and glucose homeostasis1,2. This has incentivized the development of tankyrase inhibitors. Notwithstanding, our knowledge of the mechanisms that control tankyrase activity has remained limited. Both catalytic and non-catalytic functions of tankyrase depend on its filamentous polymerization3,4,5. Here we report the cryo-electron microscopy reconstruction of a filament formed by a minimal active unit of tankyrase, comprising the polymerizing sterile alpha motif (SAM) domain and its adjacent catalytic domain. The SAM domain forms a novel antiparallel double helix, positioning the protruding catalytic domains for recurring head-to-head and tail-to-tail interactions. The head interactions are highly conserved among tankyrases and induce an allosteric switch in the active site within the catalytic domain to promote catalysis. Although the tail interactions have a limited effect on catalysis, they are essential to tankyrase function in WNT–β-catenin signalling. This work reveals a novel SAM domain polymerization mode, illustrates how supramolecular assembly controls catalytic and non-catalytic functions, provides important structural insights into the regulation of a non-DNA-dependent poly-ADP-ribosyltransferase and will guide future efforts to modulate tankyrase and decipher its contribution to disease mechanisms.	Nov 2022
Krios II-Titan Krios II at Diamond	Structure of a nucleosome-bound MuvB transcription factor complex reveals DNA remodelling Marios G. Koliopoulos , Reyhan Muhammad , Theodoros I. Roumeliotis , Fabienne Beuron , Jyoti S. Choudhary , Claudio Alfieri Nature Communications 13 DOI: 10.1038/s41467-022-32798-9 Diamond Proposal Number(s): [21809] Open Access Show Abstract ▼ Abstract: Genes encoding the core cell cycle machinery are transcriptionally regulated by the MuvB family of protein complexes in a cell cycle-specific manner. Complexes of MuvB with the transcription factors B-MYB and FOXM1 activate mitotic genes during cell proliferation. The mechanisms of transcriptional regulation by these complexes are still poorly characterised. Here, we combine biochemical analysis and in vitro reconstitution, with structural analysis by cryo-electron microscopy and cross-linking mass spectrometry, to functionally examine these complexes. We find that the MuvB:B-MYB complex binds and remodels nucleosomes, thereby exposing nucleosomal DNA. This remodelling activity is supported by B-MYB which directly binds the remodelled DNA. Given the remodelling activity on the nucleosome, we propose that the MuvB:B-MYB complex functions as a pioneer transcription factor complex. In this work, we rationalise prior biochemical and cellular studies and provide a molecular framework of interactions on a protein complex that is key for cell cycle regulation.	Aug 2022
Krios I-Titan Krios I at Diamond Krios IV-Titan Krios IV at Diamond	Structural basis of Ty3 retrotransposon integration at RNA Polymerase III-transcribed genes Guillermo Abascal-Palacios , Laura Jochem , Carlos Pla-Prats , Fabienne Beuron , Alessandro Vannini Nature Communications 12 DOI: 10.1038/s41467-021-27338-w Diamond Proposal Number(s): [21809] Open Access Show Abstract ▼ Abstract: Retrotransposons are endogenous elements that have the ability to mobilise their DNA between different locations in the host genome. The Ty3 retrotransposon integrates with an exquisite specificity in a narrow window upstream of RNA Polymerase (Pol) III-transcribed genes, representing a paradigm for harmless targeted integration. Here we present the cryo-EM reconstruction at 4.0 Å of an active Ty3 strand transfer complex bound to TFIIIB transcription factor and a tRNA gene. The structure unravels the molecular mechanisms underlying Ty3 targeting specificity at Pol III-transcribed genes and sheds light into the architecture of retrotransposon machinery during integration. Ty3 intasome contacts a region of TBP, a subunit of TFIIIB, which is blocked by NC2 transcription regulator in RNA Pol II-transcribed genes. A newly-identified chromodomain on Ty3 integrase interacts with TFIIIB and the tRNA gene, defining with extreme precision the integration site position.	Dec 2021
I24-Microfocus Macromolecular Crystallography	Structure of human RNA polymerase III Ewan Phillip Ramsay , Guillermo Abascal-Palacios , Julia L. Daiß , Helen King , Jerome Gouge , Michael Pilsl , Fabienne Beuron , Edward Morris , Philip Gunkel , Christoph Engel , Alessandro Vannini Nature Communications 11 DOI: 10.1038/s41467-020-20262-5 Diamond Proposal Number(s): [8015, 10088] Open Access Show Abstract ▼ Abstract: In eukaryotes, RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs. Pol III is a determinant of cellular growth and lifespan across eukaryotes. Upregulation of Pol III transcription is observed in cancer and causative Pol III mutations have been described in neurodevelopmental disorders and hypersensitivity to viral infection. Here, we report a cryo-EM reconstruction at 4.0 Å of human Pol III, allowing mapping and rationalization of reported genetic mutations. Mutations causing neurodevelopmental defects cluster in hotspots affecting Pol III stability and/or biogenesis, whereas mutations affecting viral sensing are located in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing. Integrating x-ray crystallography and SAXS, we also describe the structure of the higher eukaryote specific RPC5 C-terminal extension. Surprisingly, experiments in living cells highlight a role for this module in the assembly and stability of human Pol III.	Dec 2020
Krios I-Titan Krios I at Diamond	Structural basis of Cullin 2 RING E3 ligase regulation by the COP9 signalosome Sarah V. Faull , Andy M. C. Lau , Chloe Martens , Zainab Ahdash , Kjetil Hansen , Hugo Yebenes , Carla Schmidt , Fabienne Beuron , Nora B. Cronin , Edward Morris , Argyris Politis Nature Communications 10 DOI: 10.1038/s41467-019-11772-y Diamond Proposal Number(s): [15621, 16023] Open Access Show Abstract ▼ Abstract: Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 from activated Cullins. Here we present structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single-particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (XL-MS). These structures suggest a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen-deuterium exchange (HDX)-MS we show that CRL2 activates CSN5/CSN6 in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identify sensory regions of the CSN that mediate its stepwise activation and provide a framework for understanding the regulatory mechanism of other Cullin family members.	Sep 2019
Krios II-Titan Krios II at Diamond	Structural basis of RNA polymerase III transcription initiation Guillermo Abascal-Palacios , Ewan Phillip Ramsay , Fabienne Beuron , Edward Morris , Alessandro Vannini Nature 553, 301 - 306 DOI: 10.1038/nature25441 Diamond Proposal Number(s): [15629, 16599, 166601] Show Abstract ▼ Abstract: RNA polymerase (Pol) III transcribes essential non-coding RNAs, including the entire pool of transfer RNAs, the 5S ribosomal RNA and the U6 spliceosomal RNA, and is often deregulated in cancer cells. The initiation of gene transcription by Pol III requires the activity of the transcription factor TFIIIB to form a transcriptionally active Pol III preinitiation complex (PIC). Here we present electron microscopy reconstructions of Pol III PICs at 3.4–4.0 Å and a reconstruction of unbound apo-Pol III at 3.1 Å. TFIIIB fully encircles the DNA and restructures Pol III. In particular, binding of the TFIIIB subunit Bdp1 rearranges the Pol III-specific subunits C37 and C34, thereby promoting DNA opening. The unwound DNA directly contacts both sides of the Pol III cleft. Topologically, the Pol III PIC resembles the Pol II PIC, whereas the Pol I PIC is more divergent. The structures presented unravel the molecular mechanisms underlying the first steps of Pol III transcription and also the general conserved mechanisms of gene transcription initiation.	Jan 2018
I03-Macromolecular Crystallography	Tankyrase requires SAM domain-dependent polymerization to support Wnt-β-catenin signaling Laura Mariotti , Catherine m. Templeton , Michael Ranes , Patricia Paracuellos Torrecilla , Nora Cronin , Fabienne Beuron , Edward Morris , Sebastian Guettler Molecular Cell 63, 498 - 513 DOI: 10.1016/j.molcel.2016.06.019 Diamond Proposal Number(s): [8015, 10088] Show Abstract ▼ Abstract: The poly(ADP-ribose) polymerase (PARP) Tankyrase (TNKS and TNKS2) is paramount to Wnt-β-catenin signaling and a promising therapeutic target in Wnt-dependent cancers. The pool of active β-catenin is normally limited by destruction complexes, whose assembly depends on the polymeric master scaffolding protein AXIN. Tankyrase, which poly(ADP-ribosyl)ates and thereby destabilizes AXIN, also can polymerize, but the relevance of these polymers has remained unclear. We report crystal structures of the polymerizing TNKS and TNKS2 sterile alpha motif (SAM) domains, revealing versatile head-to-tail interactions. Biochemical studies informed by these structures demonstrate that polymerization is required for Tankyrase to drive β-catenin-dependent transcription. We show that the polymeric state supports PARP activity and allows Tankyrase to effectively access destruction complexes through enabling avidity-dependent AXIN binding. This study provides an example for regulated signal transduction in non-membrane-enclosed compartments (signalosomes), and it points to novel potential strategies to inhibit Tankyrase function in oncogenic Wnt signaling.	Aug 2016

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