Krios IV-Titan Krios IV at Diamond
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Mohammad W.
Bahar
,
Veronica
Nasta
,
Helen
Fox
,
Lee
Sherry
,
Keith
Grehan
,
Claudine
Porta
,
Andrew J.
Macadam
,
Nicola J.
Stonehouse
,
David J.
Rowlands
,
Elizabeth E.
Fry
,
David I.
Stuart
Diamond Proposal Number(s):
[20223]
Open Access
Abstract: Strategies to prevent the recurrence of poliovirus (PV) after eradication may utilise non-infectious, recombinant virus-like particle (VLP) vaccines. Despite clear advantages over inactivated or attenuated virus vaccines, instability of VLPs can compromise their immunogenicity. Glutathione (GSH), an important cellular reducing agent, is a crucial co-factor for the morphogenesis of enteroviruses, including PV. We report cryo-EM structures of GSH bound to PV serotype 3 VLPs showing that it can enhance particle stability. GSH binds the positively charged pocket at the interprotomer interface shown recently to bind GSH in enterovirus F3 and putative antiviral benzene sulphonamide compounds in other enteroviruses. We show, using high-resolution cryo-EM, the binding of a benzene sulphonamide compound with a PV serotype 2 VLP, consistent with antiviral activity through over-stabilizing the interprotomer pocket, preventing the capsid rearrangements necessary for viral infection. Collectively, these results suggest GSH or an analogous tight-binding antiviral offers the potential for stabilizing VLP vaccines.
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Nov 2022
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587, 18598, 14435, 15580, 15897, 15836, 21777]
Open Access
Abstract: Meiosis protein TEX12 is an essential component of the synaptonemal complex (SC), which mediates homologous chromosome synapsis. It is also recruited to centrosomes in meiosis, and aberrantly in certain cancers, leading to centrosome dysfunction. Within the SC, TEX12 forms an intertwined complex with SYCE2 that undergoes fibrous assembly, driven by TEX12’s C-terminal tip. However, we hitherto lack structural information regarding SYCE2-independent functions of TEX12. Here, we report X-ray crystal structures of TEX12 mutants in three distinct conformations, and utilise solution light and X-ray scattering to determine its wild-type dimeric four-helical coiled-coil structure. TEX12 undergoes conformational change upon C-terminal tip mutations, indicating that the sequence responsible for driving SYCE2-TEX12 assembly within the SC also controls the oligomeric state and conformation of isolated TEX12. Our findings provide the structural basis for SYCE2-independent roles of TEX12, including the possible regulation of SC assembly, and its known functions in meiotic centrosomes and cancer.
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Sep 2022
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Vasundara
Srinivasan
,
Hévila
Brognaro
,
Prince R.
Prabhu
,
Edmarcia Elisa
De Souza
,
Sebastian
Günther
,
Patrick Y. A.
Reinke
,
Thomas J.
Lane
,
Helen
Ginn
,
Huijong
Han
,
Wiebke
Ewert
,
Janina
Sprenger
,
Faisal H. M.
Koua
,
Sven
Falke
,
Nadine
Werner
,
Hina
Andaleeb
,
Najeeb
Ullah
,
Bruno Alves
Franca
,
Mengying
Wang
,
Angélica Luana C.
Barra
,
Markus
Perbandt
,
Martin
Schwinzer
,
Christina
Schmidt
,
Lea
Brings
,
Kristina
Lorenzen
,
Robin
Schubert
,
Rafael Rahal Guaragna
Machado
,
Erika Donizette
Candido
,
Danielle Bruna Leal
Oliveira
,
Edison Luiz
Durigon
,
Stephan
Niebling
,
Angelica
Struve Garcia
,
Oleksandr
Yefanov
,
Julia
Lieske
,
Luca
Gelisio
,
Martin
Domaracky
,
Philipp
Middendorf
,
Michael
Groessler
,
Fabian
Trost
,
Marina
Galchenkova
,
Aida Rahmani
Mashhour
,
Sofiane
Saouane
,
Johanna
Hakanpää
,
Markus
Wolf
,
Maria
Garcia Alai
,
Dusan
Turk
,
Arwen R.
Pearson
,
Henry N.
Chapman
,
Winfried
Hinrichs
,
Carsten
Wrenger
,
Alke
Meents
,
Christian
Betzel
Open Access
Abstract: SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host’s innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.
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Aug 2022
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[15916]
Open Access
Abstract: Obtaining the high-resolution structures of proteins and their complexes is a crucial aspect of understanding the mechanisms of life. Experimental structure determination methods are time-consuming, expensive and cannot keep pace with the growing number of protein sequences available through genomic DNA sequencing. Thus, the ability to accurately predict the structure of proteins from their sequence is a holy grail of structural and computational biology that would remove a bottleneck in our efforts to understand as well as rationally engineer living systems. Recent advances in protein structure prediction, in particular the breakthrough with the AI-based tool AlphaFold2 (AF2), hold promise for achieving this goal, but the practical utility of AF2 remains to be explored. Focusing on proteins with essential roles in centrosome and centriole biogenesis, we demonstrate the quality and usability of the AF2 prediction models and we show that they can provide important insights into the modular organization of two key players in this process, CEP192 and CEP44. Furthermore, we used the AF2 algorithm to elucidate and then experimentally validate previously unknown prime features in the structure of TTBK2 bound to CEP164, as well as the Chibby1-FAM92A complex for which no structural information was available to date. These findings have important implications in understanding the regulation and function of these complexes. Finally, we also discuss some practical limitations of AF2 and anticipate the implications for future research approaches in the centriole/centrosome field.
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Apr 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Chloe R.
Koulouris
,
Sian E.
Gardiner
,
Tessa K.
Harris
,
Karen T.
Elvers
,
S. Mark
Roe
,
Jason A.
Gillespie
,
Simon E.
Ward
,
Olivera
Grubisha
,
Robert A.
Nicholls
,
John R.
Atack
,
Benjamin D.
Bax
Diamond Proposal Number(s):
[19990]
Open Access
Abstract: Human serine racemase (hSR) catalyses racemisation of L-serine to D-serine, the latter of which is a co-agonist of the NMDA subtype of glutamate receptors that are important in synaptic plasticity, learning and memory. In a ‘closed’ hSR structure containing the allosteric activator ATP, the inhibitor malonate is enclosed between the large and small domains while ATP is distal to the active site, residing at the dimer interface with the Tyr121 hydroxyl group contacting the α-phosphate of ATP. In contrast, in ‘open’ hSR structures, Tyr121 sits in the core of the small domain with its hydroxyl contacting the key catalytic residue Ser84. The ability to regulate SR activity by flipping Tyr121 from the core of the small domain to the dimer interface appears to have evolved in animals with a CNS. Multiple X-ray crystallographic enzyme-fragment structures show Tyr121 flipped out of its pocket in the core of the small domain. Data suggest that this ligandable pocket could be targeted by molecules that inhibit enzyme activity.
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Apr 2022
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I03-Macromolecular Crystallography
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Dapeng
Li
,
Simon
Brackenridge
,
Lucy C.
Walters
,
Olivia
Swanson
,
Karl
Harlos
,
Daniel
Rozbesky
,
Derek W.
Cain
,
Kevin
Wiehe
,
Richard M.
Scearce
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Maggie
Barr
,
Zekun
Mu
,
Robert
Parks
,
Max
Quastel
,
Robert J.
Edwards
,
Yunfei
Wang
,
Wes
Rountree
,
Kevin O.
Saunders
,
Guido
Ferrari
,
Persephone
Borrow
,
E. Yvonne
Jones
,
S. Munir
Alam
,
Mihai L.
Azoitei
,
Geraldine M.
Gillespie
,
Andrew J.
Mcmichael
,
Barton F.
Haynes
Open Access
Abstract: The non-classical class Ib molecule human leukocyte antigen E (HLA-E) has limited polymorphism and can bind HLA class Ia leader peptides (VL9). HLA-E-VL9 complexes interact with the natural killer (NK) cell receptors NKG2A-C/CD94 and regulate NK cell-mediated cytotoxicity. Here we report the isolation of 3H4, a murine HLA-E-VL9-specific IgM antibody that enhances killing of HLA-E-VL9-expressing cells by an NKG2A+ NK cell line. Structural analysis reveal that 3H4 acts by preventing CD94/NKG2A docking on HLA-E-VL9. Upon in vitro maturation, an affinity-optimized IgG form of 3H4 showes enhanced NK killing of HLA-E-VL9-expressing cells. HLA-E-VL9-specific IgM antibodies similar in function to 3H4 are also isolated from naïve B cells of cytomegalovirus (CMV)-negative, healthy humans. Thus, HLA-E-VL9-targeting mouse and human antibodies isolated from the naïve B cell antibody pool have the capacity to enhance NK cell cytotoxicity.
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Mar 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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George W.
Mobbs
,
Adli A.
Aziz
,
Samuel R.
Dix
,
G. Michael
Blackburn
,
Sveta E.
Sedelnikova
,
Thomas C.
Minshull
,
Mark J.
Dickman
,
Patrick
Baker
,
Sheila
Nathan
,
Mohd Firdaus
Raih
,
David W.
Rice
Diamond Proposal Number(s):
[8987, 17773]
Open Access
Abstract: Burkholderia pseudomallei lethal factor 1 (BLF1) exhibits site-specific glutamine deamidase activity against the eukaryotic RNA helicase, eIF4A, thereby blocking mammalian protein synthesis. The structure of a complex between BLF1 C94S and human eIF4A shows that the toxin binds in the cleft between the two RecA-like eIF4A domains forming interactions with residues from both and with the scissile amide of the target glutamine, Gln339, adjacent to the toxin active site. The RecA-like domains adopt a radically twisted orientation compared to other eIF4A structures and the nature and position of conserved residues suggests this may represent a conformation associated with RNA binding. Comparison of the catalytic site of BLF1 with other deamidases and cysteine proteases reveals that they fall into two classes, related by pseudosymmetry, that present either the re or si faces of the target amide/peptide to the nucleophilic sulfur, highlighting constraints in the convergent evolution of their Cys-His active sites.
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Mar 2022
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[15836]
Open Access
Abstract: The synaptonemal complex (SC) is a supramolecular protein scaffold that mediates chromosome synapsis and facilitates crossing over during meiosis. In mammals, SC proteins are generally assumed to have no other function. Here, we show that SC protein TEX12 also localises to centrosomes during meiosis independently of chromosome synapsis. In somatic cells, ectopically expressed TEX12 similarly localises to centrosomes, where it is associated with centrosome amplification, a pathology correlated with cancer development. Indeed, TEX12 is identified as a cancer-testis antigen and proliferation of some cancer cells is TEX12-dependent. Moreover, somatic expression of TEX12 is aberrantly activated via retinoic acid signalling, which is commonly disregulated in cancer. Structure-function analysis reveals that phosphorylation of TEX12 on tyrosine 48 is important for centrosome amplification but not for recruitment of TEX12 to centrosomes. We conclude that TEX12 normally localises to meiotic centrosomes, but its misexpression in somatic cells can contribute to pathological amplification and dysfunction of centrosomes in cancers.
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Dec 2021
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[23722]
Open Access
Abstract: It has been proposed that adaptation to high temperature involved the synthesis of monolayer-forming ether phospholipids. Recently, a novel membrane architecture was proposed to explain the membrane stability in polyextremophiles unable to synthesize such lipids, in which apolar polyisoprenoids populate the bilayer midplane and modify its physico-chemistry, extending its stability domain. Here, we have studied the effect of the apolar polyisoprenoid squalane on a model membrane analogue using neutron diffraction, SAXS and fluorescence spectroscopy. We show that squalane resides inside the bilayer midplane, extends its stability domain, reduces its permeability to protons but increases that of water, and induces a negative curvature in the membrane, allowing the transition to novel non-lamellar phases. This membrane architecture can be transposed to early membranes and could help explain their emergence and temperature tolerance if life originated near hydrothermal vents. Transposed to the archaeal bilayer, this membrane architecture could explain the tolerance to high temperature in hyperthermophiles which grow at temperatures over 100 °C while having a membrane bilayer. The induction of a negative curvature to the membrane could also facilitate crucial cell functions that require high bending membranes.
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Jun 2021
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Krios II-Titan Krios II at Diamond
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Melisa
Lazaro
,
Roberto
Melero
,
Charlotte
Huet
,
Jorge
Lopez-Alonso
,
Sandra
Delgado
,
Alexandra
Dodu
,
Eduardo M.
Bruch
,
Luciano A.
Abriata
,
Pedro M.
Alzari
,
Mikel
Valle
,
María-Natalia
Lisa
Diamond Proposal Number(s):
[14743]
Open Access
Abstract: Glutamate dehydrogenases (GDHs) are widespread metabolic enzymes that play key roles in nitrogen homeostasis. Large glutamate dehydrogenases composed of 180 kDa subunits (L-GDHs180) contain long N- and C-terminal segments flanking the catalytic core. Despite the relevance of L-GDHs180 in bacterial physiology, the lack of structural data for these enzymes has limited the progress of functional studies. Here we show that the mycobacterial L-GDH180 (mL-GDH180) adopts a quaternary structure that is radically different from that of related low molecular weight enzymes. Intersubunit contacts in mL-GDH180 involve a C-terminal domain that we propose as a new fold and a flexible N-terminal segment comprising ACT-like and PAS-type domains that could act as metabolic sensors for allosteric regulation. These findings uncover unique aspects of the structure-function relationship in the subfamily of L-GDHs.
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Jun 2021
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