I24-Microfocus Macromolecular Crystallography
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Jim
Baggen
,
Maarten
Jacquemyn
,
Leentje
Persoons
,
Els
Vanstreels
,
Valerie E.
Pye
,
Antoni G.
Wrobel
,
Valeria
Calvaresi
,
Stephen R.
Martin
,
Chloe
Roustan
,
Nora B.
Cronin
,
Eamonn
Reading
,
Hendrik Jan
Thibaut
,
Thomas
Vercruysse
,
Piet
Maes
,
Frederik
De Smet
,
Angie
Yee
,
Toey
Nivitchanyong
,
Marina
Roell
,
Natalia
Franco-Hernandez
,
Herve
Rhinn
,
Alusha Andre
Mamchak
,
Maxime
Ah Young-Chapon
,
Eric
Brown
,
Peter
Cherepanov
,
Dirk
Daelemans
Diamond Proposal Number(s):
[25587]
Open Access
Abstract: SARS-CoV-2 is associated with broad tissue tropism, a characteristic often determined by the availability of entry receptors on host cells. Here, we show that TMEM106B, a lysosomal transmembrane protein, can serve as an alternative receptor for SARS-CoV-2 entry into angiotensin-converting enzyme 2 (ACE2)-negative cells. Spike substitution E484D increased TMEM106B binding, thereby enhancing TMEM106B-mediated entry. TMEM106B-specific monoclonal antibodies blocked SARS-CoV-2 infection, demonstrating a role of TMEM106B in viral entry. Using X-ray crystallography, cryogenic electron microscopy (cryo-EM), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we show that the luminal domain (LD) of TMEM106B engages the receptor-binding motif of SARS-CoV-2 spike. Finally, we show that TMEM106B promotes spike-mediated syncytium formation, suggesting a role of TMEM106B in viral fusion. Together, our findings identify an ACE2-independent SARS-CoV-2 infection mechanism that involves cooperative interactions with the receptors heparan sulfate and TMEM106B.
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Jul 2023
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[25832]
Open Access
Abstract: Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one for each replisome. However, the molecular mechanisms underpinning helicase loading at bacterial chromosome origins (oriC) are unclear. Here we investigated the essential DNA replication initiation protein DnaD in the model organism Bacillus subtilis. A set of DnaD residues required for ssDNA binding was identified, and photo-crosslinking revealed that this ssDNA binding region interacts preferentially with one strand of oriC. Biochemical and genetic data support the model that DnaD recognizes a new single-stranded DNA (ssDNA) motif located in oriC, the DnaD Recognition Element (DRE). Considered with single particle cryo-electron microscopy (cryo-EM) imaging of DnaD, we propose that the location of the DRE within oriC orchestrates strand-specific recruitment of helicase during DNA replication initiation. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin.
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May 2023
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Krios I-Titan Krios I at Diamond
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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
Diamond Proposal Number(s):
[15621, 16023]
Open Access
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.
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Sep 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Hannah L.
Woodward
,
Paolo
Innocenti
,
Kwai-Ming J.
Cheung
,
Angela
Hayes
,
Jennie
Roberts
,
Alan T.
Henley
,
Amir
Faisal
,
Grace Wing-Yan
Mak
,
Gary
Box
,
Isaac M.
Westwood
,
Nora
Cronin
,
Michael
Carter
,
Melanie
Valenti
,
Alexis
De Haven Brandon
,
Lisa
O’fee
,
Harry
Saville
,
Jessica
Schmitt
,
Rosemary
Burke
,
Fabio
Broccatelli
,
Rob L. M.
Van Montfort
,
Florence I.
Raynaud
,
Suzanne A.
Eccles
,
Spiros
Linardopoulos
,
Julian
Blagg
,
Swen
Hoelder
Diamond Proposal Number(s):
[10088]
Open Access
Abstract: Monopolar spindle 1 (MPS1) occupies a central role in mitosis and is one of the main components of the spindle assembly checkpoint. The MPS1 kinase is an attractive cancer target, and herein, we report the discovery of the clinical candidate BOS172722. The starting point for our work was a series of pyrido[3,4-d]pyrimidine inhibitors that demonstrated excellent potency and kinase selectivity but suffered from rapid turnover in human liver microsomes (HLM). Optimizing HLM stability proved challenging since it was not possible to identify a consistent site of metabolism and lowering lipophilicity proved unsuccessful. Key to overcoming this problem was the finding that introduction of a methyl group at the 6-position of the pyrido[3,4-d]pyrimidine core significantly improved HLM stability. Met ID studies suggested that the methyl group suppressed metabolism at the distant aniline portion of the molecule, likely by blocking the preferred pharmacophore through which P450 recognized the compound. This work ultimately led to the discovery of BOS172722 as a Phase 1 clinical candidate.
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Sep 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8015, 10088]
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.
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Aug 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Paolo
Innocenti
,
Hannah L.
Woodward
,
Savade
Solanki
,
Sébastien
Naud
,
Isaac
Westwood
,
Nora
Cronin
,
Angela
Hayes
,
Jennie
Roberts
,
Alan T.
Henley
,
Ross
Baker
,
Amir
Faisal
,
Grace Wing-Yan
Mak
,
Gary
Box
,
Melanie
Valenti
,
Alexis
De Haven Brandon
,
Lisa
O’ Fee
,
Harry
Saville
,
Jessica
Schmitt
,
Berry
Matijssen
,
Rosemary
Burke
,
Rob
Van Montfort
,
Florence I.
Raynaud
,
Suzanne A.
Eccles
,
Spiros
Linardopoulos
,
Julian
Blagg
,
Swen
Hoelder
Open Access
Abstract: Monopolar spindle 1 (MPS1) plays a central role in the transition of cells from metaphase to anaphase and is one of the main components of the spindle assembly checkpoint. Chromosomally unstable cancer cells rely heavily on MPS1 to cope with the stress arising from abnormal numbers of chromosomes and centrosomes and are thus more sensitive to MPS1 inhibition than normal cells. We report the discovery and optimization of a series of new pyrido[3,4-d]pyrimidine based inhibitors via a structure-based hybridization approach from our previously reported inhibitor CCT251455 and a modestly potent screening hit. Compounds in this novel series display excellent potency and selectivity for MPS1, which translates into biomarker modulation in an in vivo human tumor xenograft model.
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Apr 2016
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[8015]
Open Access
Abstract: Many essential biological processes are mediated by complex molecular machines comprising multiple subunits. Knowledge on the architecture of individual subunits and their positions within the overall multimeric complex is key to understanding the molecular mechanisms of macromolecular assemblies. The anaphase-promoting complex/cyclosome (APC/C) is a large multisubunit complex that regulates cell cycle progression by ubiquitinating cell cycle proteins for proteolysis by the proteasome. The holo-complex is composed of 15 different proteins that assemble to generate a complex of 20 subunits. Here, we describe the crystal structures of Apc4 and the N-terminal domain of Apc5 (Apc5N). Apc4 comprises a WD40 domain split by a long α-helical domain, whereas Apc5N has an α-helical fold. In a separate study, we had fitted these atomic models to a 3.6-Å-resolution cryo-electron microscopy map of the APC/C. We describe how, in the context of the APC/C, regions of Apc4 disordered in the crystal assume order through contacts to Apc5, whereas Apc5N shows small conformational changes relative to its crystal structure. We discuss the complementary approaches of high-resolution electron microscopy and protein crystallography to the structure determination of subunits of multimeric complexes.
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Oct 2015
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8015]
Abstract: In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence χ (Chi)1, 2 and is catalysed by either an AddAB- or RecBCD-type helicase–nuclease (reviewed in refs 3, 4). These enzyme complexes unwind and digest the DNA duplex from the broken end until they encounter a χ sequence5, whereupon they produce a 3′ single-stranded DNA tail onto which they initiate loading of the RecA protein6. Consequently, regulation of the AddAB/RecBCD complex by χ is a key control point in DNA repair and other processes involving genetic recombination. Here we report crystal structures of Bacillus subtilis AddAB in complex with different χ-containing DNA substrates either with or without a non-hydrolysable ATP analogue. Comparison of these structures suggests a mechanism for DNA translocation and unwinding, suggests how the enzyme binds specifically to χ sequences, and explains how χ recognition leads to the arrest of AddAB (and RecBCD) translocation that is observed in single-molecule experiments
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Mar 2014
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Ioannis
Manolaridis
,
Kiran
Kulkarni
,
Roger B.
Dodd
,
Satoshi
Ogasawara
,
Ziguo
Zhang
,
Ganka
Bineva
,
Nicola
O'Reilly
,
Sarah J.
Hanrahan
,
Andrew
Thompson
,
Nora
Cronin
,
So
Iwata
,
David
Barford
Diamond Proposal Number(s):
[8015]
Abstract: CAAX proteins have essential roles in multiple signalling pathways, controlling processes such as proliferation, differentiation and carcinogenesis1. The ~120 mammalian CAAX proteins function at cellular membranes and include the Ras superfamily of small GTPases, nuclear lamins, the γ-subunit of heterotrimeric GTPases, and several protein kinases and phosphatases2. The proper localization of CAAX proteins to cell membranes is orchestrated by a series of post-translational modifications of the carboxy-terminal CAAX motifs3 (
WHERE C is cysteine, A is an aliphatic amino acid and X is any amino acid). These reactions involve prenylation of the cysteine residue, cleavage at the AAX tripeptide and methylation of the carboxyl-prenylated cysteine residue. The major CAAX protease activity is mediated by Rce1 (Ras and a-factor converting enzyme 1), an intramembrane protease (IMP) of the endoplasmic reticulum4, 5. Information on the architecture and proteolytic mechanism of Rce1 has been lacking. Here we report the crystal structure of a Methanococcus maripaludis homologue of Rce1, whose endopeptidase specificity for farnesylated peptides mimics that of eukaryotic Rce1. Its structure, comprising eight transmembrane α-helices, and catalytic site are distinct from those of other IMPs. The catalytic residues are located ~10 Å into the membrane and are exposed to the cytoplasm and membrane through a conical cavity that accommodates the prenylated CAAX substrate. We propose that the farnesyl lipid binds to a site at the opening of two transmembrane α-helices, which results in the scissile bond being positioned adjacent to a glutamate-activated nucleophilic water molecule. This study suggests that Rce1 is the founding member of a novel IMP family, the glutamate IMPs.
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Dec 2013
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: The anaphase-promoting complex/cyclosome (APC/C) regulates sister chromatid segregation and the exit from mitosis. Selection of most APC/C substrates is controlled by coactivator subunits (either Cdc20 or Cdh1) that interact with substrate destruction motifs—predominantly the destruction (D) box and KEN box degrons. How coactivators recognize D box degrons and how this is inhibited by APC/C regulatory proteins is not defined at the atomic level. Here, from the crystal structure of S. cerevisiae Cdh1 in complex with its specific inhibitor Acm1, which incorporates D and KEN box pseudosubstrate motifs, we describe the molecular basis for D box recognition. Additional interactions between Acm1 and Cdh1 identify a third protein-binding site on Cdh1 that is likely to confer coactivator-specific protein functions including substrate association. We provide a structural rationalization for D box and KEN box recognition by coactivators and demonstrate that many noncanonical APC/C degrons bind APC/C coactivators at the D box coreceptor.
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May 2013
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