I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18069]
Open Access
Abstract: The Aurora B chromosomal passenger complex (CPC) is a conserved regulator of mitosis. Its functions require localization first to the chromosome arms and then centromeres in mitosis and subsequently the central spindle in anaphase. Here, we analyze the requirements for core CPC subunits, survivin and INCENP, and the mitotic kinesin-like protein 2 (MKLP2) in targeting to these distinct localizations. Centromere recruitment of the CPC requires interaction of survivin with histone H3 phosphorylated at threonine 3, and we provide a complete structure of this assembly. Furthermore, we show that the INCENP RRKKRR-motif is required for both centromeric localization of the CPC in metaphase and MKLP2-dependent transport in anaphase. MKLP2 and DNA bind competitively to this motif, and INCENP T59 phosphorylation acts as a switch preventing MKLP2 binding in metaphase. In anaphase, CPC binding promotes the microtubule-dependent ATPase activity of MKLP2. These results explain how centromere targeting of the CPC in mitosis is coupled to its movement to the central spindle in anaphase.
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Jul 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[20221]
Abstract: Guanylate cyclase activating protein 1 (GCAP1) is a neuronal calcium sensor (NCS) involved in the early biochemical steps underlying the phototransduction cascade. By switching from a Ca2+-bound form in the dark to a Mg2+-bound state following light activation of the cascade, GCAP1 triggers the activation of the retinal guanylate cyclase (GC), thus replenishing the levels of 3′,5′-cyclic monophosphate (cGMP) necessary to re-open CNG channels. Here, we investigated the structural and functional effects of three missense mutations in GCAP1 associated with cone-rod dystrophy, which severely perturb the homeostasis of cGMP and Ca2+. Substitutions affect residues directly involved in Ca2+ coordination in either EF3 (D100G) or EF4 (E155A and E155G) Ca2+ binding motifs. We found that all GCAP1 variants form relatively stable dimers showing decreased apparent affinity for Ca2+ and blocking the enzyme in a constitutively active state at physiological levels of Ca2+. Interestingly, by corroborating spectroscopic experiments with molecular dynamics simulations we show that beside local structural effects, mutation of the bidentate glutamate in an EF-hand calcium binding motif can profoundly perturb the flexibility of the adjacent EF-hand as well, ultimately destabilizing the whole domain. Therefore, while Ca2+-binding to GCAP1 per se occurs sequentially, allosteric effects may connect EF hand motifs, which appear to be essential for the integrity of the structural switch mechanism in GCAP1, and perhaps in other NCS proteins.
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Jul 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Tessa
Keenan
,
Fabio
Parmeggiani
,
Julien
Malassis
,
Clement Q.
Fontenelle
,
Jean-baptiste
Vendeville
,
Wendy
Offen
,
Peter
Both
,
Kun
Huang
,
Andrea
Marchesi
,
Alex
Heyam
,
Carl
Young
,
Simon J.
Charnock
,
Gideon J.
Davies
,
Bruno
Linclau
,
Sabine L.
Flitsch
,
Martin A.
Fascione
Diamond Proposal Number(s):
[13587, 18598]
Abstract: Fluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluorogalactose, giving insight into changes in the active site that may account for the specificity of the enzyme toward certain substrate analogs.
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Jul 2020
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I03-Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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Jiandong
Huo
,
Yuguang
Zhao
,
Jingshan
Ren
,
Daming
Zhou
,
Helen M. E.
Duyvesteyn
,
Helen M.
Ginn
,
Loic
Carrique
,
Tomas
Malinauskas
,
Reinis R.
Ruza
,
Pranav N. M.
Shah
,
Tiong Kit
Tan
,
Pramila
Rijal
,
Naomi
Coombes
,
Kevin R.
Bewley
,
Julia A.
Tree
,
Julika
Radecke
,
Neil
Paterson
,
Piyasa
Supasa
,
Juthathip
Mongkolsapaya
,
Gavin R.
Screaton
,
Miles
Carroll
,
Alain
Townsend
,
Elizabeth E.
Fry
,
Raymond J.
Owens
,
David I.
Stuart
Diamond Proposal Number(s):
[19946, 26983]
Open Access
Abstract: There are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognises angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that the monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding facilitates conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment.
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Jun 2020
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B24-Cryo Soft X-ray Tomography
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Ilias
Kounatidis
,
Megan L.
Stanifer
,
Michael A.
Phillips
,
Perrine
Paul-gilloteaux
,
Xavier
Heiligenstein
,
Hongchang
Wang
,
Chidinma
Okolo
,
Thomas M.
Fish
,
Matthew C.
Spink
,
David I.
Stuart
,
Ilan
Davis
,
Steeveh
Boulant
,
Jonathan M.
Grimes
,
Ian M.
Dobbie
,
Maria
Harkiolaki
Diamond Proposal Number(s):
[21046, 18314]
Open Access
Abstract: Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.
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Jun 2020
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I03-Macromolecular Crystallography
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Matteo
Marchesini
,
Andrea
Gherli
,
Anna
Montanaro
,
Laura
Patrizi
,
Claudia
Sorrentino
,
Luca
Pagliaro
,
Chiara
Rompietti
,
Samuel
Kitara
,
Sabine
Heit
,
Claus E.
Olesen
,
Jesper V.
Møller
,
Monia
Savi
,
Leonardo
Bocchi
,
Rocchina
Vilella
,
Federica
Rizzi
,
Marilena
Baglione
,
Giorgia
Rastelli
,
Caterina
Loiacono
,
Roberta
La Starza
,
Cristina
Mecucci
,
Kimberly
Stegmaier
,
Franco
Aversa
,
Donatella
Stilli
,
Anne-marie
Lund Winther
,
Paolo
Sportoletti
,
Maike
Bublitz
,
William
Dalby-brown
,
Giovanni
Roti
Diamond Proposal Number(s):
[18069]
Abstract: The identification of SERCA (sarco/endoplasmic reticulum calcium ATPase) as a target for modulating gain-of-function NOTCH1 mutations in Notch-dependent cancers has spurred the development of this compound class for cancer therapeutics. Despite the innate toxicity challenge associated with SERCA inhibition, we identified CAD204520, a small molecule with better drug-like properties and reduced off-target Ca2+ toxicity compared with the SERCA inhibitor thapsigargin. In this work, we describe the properties and complex structure of CAD204520 and show that CAD204520 preferentially targets mutated over wild-type NOTCH1 proteins in T cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL). Uniquely among SERCA inhibitors, CAD204520 suppresses NOTCH1-mutated leukemic cells in a T-ALL xenografted model without causing cardiac toxicity. This study supports the development of SERCA inhibitors for Notch-dependent cancers and extends their application to cases with isolated mutations in the PEST degradation domain of NOTCH1, such as MCL or chronic lymphocytic leukemia (CLL).
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May 2020
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B21-High Throughput SAXS
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Xiaojie
Yu
,
H.t. Claude
Chan
,
Hayden
Fisher
,
Christine A.
Penfold
,
Jinny
Kim
,
Tatyana
Inzhelevskaya
,
C. Ian
Mockridge
,
Ruth R.
French
,
Patrick J.
Duriez
,
Leon R.
Douglas
,
Vikki
English
,
J. Sjef
Verbeek
,
Ann L.
White
,
Ivo
Tews
,
Martin J.
Glennie
,
Mark S.
Cragg
Diamond Proposal Number(s):
[21035]
Open Access
Abstract: Anti-CD40 monoclonal antibodies (mAbs) comprise agonists and antagonists, which display promising therapeutic activities in cancer and autoimmunity, respectively. We previously showed that epitope and isotype interact to deliver optimal agonistic anti-CD40 mAbs. The impact of Fc engineering on antagonists, however, remains largely unexplored. Here, we show that clinically relevant antagonists used for treating autoimmune conditions can be converted into potent FcγR-independent agonists with remarkable antitumor activity by isotype switching to hIgG2. One antagonist is converted to a super-agonist with greater potency than previously reported highly agonistic anti-CD40 mAbs. Such conversion is dependent on the unique disulfide bonding properties of the hIgG2 hinge. This investigation highlights the transformative capacity of the hIgG2 isotype for converting antagonists to agonists to treat cancer.
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May 2020
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Krios I-Titan Krios I at Diamond
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Open Access
Abstract: Gap junction channels (GJCs) mediate intercellular communication and are gated by numerous conditions such as pH. The electron cryomicroscopy (cryo-EM) structure of Cx26 GJC at physiological pH recapitulates previous GJC structures in lipid bilayers. At pH 6.4, we identify two conformational states, one resembling the open physiological-pH structure and a closed conformation that displays six threads of density, that join to form a pore-occluding density. Crosslinking and hydrogen-deuterium exchange mass spectrometry reveal closer association between the N-terminal (NT) domains and the cytoplasmic loops (CL) at acidic pH. Previous electrophysiologic studies suggest an association between NT residue N14 and H100 near M2, which may trigger the observed movement of M2 toward M1 in our cryo-EM maps, thereby accounting for additional NT-CL crosslinks at acidic pH. We propose that these pH-induced interactions and conformational changes result in extension, ordering, and association of the acetylated NT domains to form a hexameric “ball-and-chain” gating particle.
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Apr 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I23-Long wavelength MX
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Diamond Proposal Number(s):
[18565]
Open Access
Abstract: A growing number of bacteria are recognized to conduct electrons across their cell envelope, and yet molecular details of the mechanisms supporting this process remain unknown. Here, we report the atomic structure of an outer membrane spanning protein complex, MtrAB, that is representative of a protein family known to transport electrons between the interior and exterior environments of phylogenetically and metabolically diverse microorganisms. The structure is revealed as a naturally insulated biomolecular wire possessing a 10-heme cytochrome, MtrA, insulated from the membrane lipidic environment by embedding within a 26 strand β-barrel formed by MtrB. MtrAB forms an intimate connection with an extracellular 10-heme cytochrome, MtrC, which presents its hemes across a large surface area for electrical contact with extracellular redox partners, including transition metals and electrodes.
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Apr 2020
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I03-Macromolecular Crystallography
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Jerome
Fortin
,
Ruxiao
Tian
,
Ida
Zarrabi
,
Graham
Hill
,
Eleanor
Williams
,
Gonzalo
Sanchez-duffhues
,
Midory
Thorikay
,
Parameswaran
Ramachandran
,
Robert
Siddaway
,
Jong Fu
Wong
,
Annette
Wu
,
Lorraine N.
Apuzzo
,
Jillian
Haight
,
Annick
You-ten
,
Bryan E.
Snow
,
Andrew
Wakeham
,
David J.
Goldhamer
,
Daniel
Schramek
,
Alex N.
Bullock
,
Peter Ten
Dijke
,
Cynthia
Hawkins
,
Tak W.
Mak
Diamond Proposal Number(s):
[15433]
Abstract: Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors for which there is currently no effective treatment. Some of these tumors combine gain-of-function mutations in ACVR1, PIK3CA, and histone H3-encoding genes. The oncogenic mechanisms of action of ACVR1 mutations are currently unknown. Using mouse models, we demonstrate that Acvr1 G328V arrests the differentiation of oligodendroglial lineage cells, and cooperates with Hist1h3b K27M and Pik3ca H1047R to generate high-grade diffuse gliomas. Mechanistically, Acvr1 G328V upregulates transcription factors which control differentiation and DIPG cell fitness. Furthermore, we characterize E6201 as a dual inhibitor of ACVR1 and MEK1/2, and demonstrate its efficacy toward tumor cells in vivo. Collectively, our results describe an oncogenic mechanism of action for ACVR1 mutations, and suggest therapeutic strategies for DIPGs.
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Mar 2020
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