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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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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[6386]
Open Access
Abstract: Incoherent neutron spectroscopy, in combination with dynamic light scattering, was used to investigate the effect of ligand binding on the center-of-mass self-diffusion and internal diffusive dynamics of Escherichia coli aspartate α-decarboxylase (ADC). The X-ray crystal structure of ADC in complex with the D-Serine inhibitor was also determined, and molecular dynamics simulations were used to further probe the structural rearrangements that occur as a result of ligand binding. These experiments reveal that D-Serine forms hydrogen bonds with some of the active site residues, that higher order oligomers of the ADC tetramer exist on ns-ms time-scales, and also show that ligand binding both affects the ADC internal diffusive dynamics and appears to further increase the size of the higher order oligomers.
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Aug 2022
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I03-Macromolecular Crystallography
I23-Long wavelength MX
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Christian M.
Orr
,
Hayden
Fisher
,
Xiaojie
Yu
,
Claude H.-T.
Chan
,
Yunyun
Gao
,
Patrick J.
Duriez
,
Steven G.
Booth
,
Isabel
Elliott
,
Tatyana
Inzhelevskaya
,
Ian
Mockridge
,
Christine A.
Penfold
,
Armin
Wagner
,
Martin J.
Glennie
,
Ann L.
White
,
Jonathan W.
Essex
,
Arwen R.
Pearson
,
Mark S.
Cragg
,
Ivo
Tews
Diamond Proposal Number(s):
[22563]
Open Access
Abstract: Antibodies protect from infection, underpin successful vaccines and elicit therapeutic responses in otherwise untreatable cancers and autoimmune conditions. The human IgG2 isotype displays a unique capacity to undergo disulfide shuffling in the hinge region, leading to modulation of its ability to drive target receptor signaling (agonism) in a variety of important immune receptors, through hitherto unexplained molecular mechanisms. To address the underlying process and reveal how hinge disulfide orientation affects agonistic activity, we generated a series of cysteine to serine exchange variants in the hinge region of the clinically relevant monoclonal antibody ChiLob7/4, directed against the key immune receptor CD40. We report how agonistic activity varies with disulfide pattern and is afforded by the presence of a disulfide crossover between F(ab) arms in the agonistic forms, independently of epitope, as observed in the determined crystallographic structures. This structural “switch” affects directly on antibody conformation and flexibility. Small-angle x-ray scattering and ensemble modeling demonstrated that the least flexible variants adopt the fewest conformations and evoke the highest levels of receptor agonism. This covalent change may be amenable for broad implementation to modulate receptor signaling in an epitope-independent manner in future therapeutics.
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Jul 2022
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Sebastian
Gunther
,
Patrick Y. A.
Reinke
,
Yaiza
Fernández-García
,
Julia
Lieske
,
Thomas J.
Lane
,
Helen M.
Ginn
,
Faisal H. M.
Koua
,
Christiane
Ehrt
,
Wiebke
Ewert
,
Dominik
Oberthuer
,
Oleksandr
Yefanov
,
Susanne
Meier
,
Kristina
Lorenzen
,
Boris
Krichel
,
Janine-Denise
Kopicki
,
Luca
Gelisio
,
Wolfgang
Brehm
,
Ilona
Dunkel
,
Brandon
Seychell
,
Henry
Gieseler
,
Brenna
Norton-Baker
,
Beatriz
Escudero-Pérez
,
Martin
Domaracky
,
Sofiane
Saouane
,
Alexandra
Tolstikova
,
Thomas A.
White
,
Anna
Hänle
,
Michael
Groessler
,
Holger
Fleckenstein
,
Fabian
Trost
,
Marina
Galchenkova
,
Yaroslav
Gevorkov
,
Chufeng
Li
,
Salah
Awel
,
Ariana
Peck
,
Miriam
Barthelmess
,
Frank
Schluenzen
,
Paulraj
Lourdu Xavier
,
Nadine
Werner
,
Hina
Andaleeb
,
Najeeb
Ullah
,
Sven
Falke
,
Vasundara
Srinivasan
,
Bruno Alves
França
,
Martin
Schwinzer
,
Hévila
Brognaro
,
Cromarte
Rogers
,
Diogo
Melo
,
Joanna J.
Zaitseva-Doyle
,
Juraj
Knoska
,
Gisel E.
Peña-Murillo
,
Aida Rahmani
Mashhour
,
Vincent
Hennicke
,
Pontus
Fischer
,
Johanna
Hakanpää
,
Jan
Meyer
,
Philip
Gribbon
,
Bernhard
Ellinger
,
Maria
Kuzikov
,
Markus
Wolf
,
Andrea R.
Beccari
,
Gleb
Bourenkov
,
David
Von Stetten
,
Guillaume
Pompidor
,
Isabel
Bento
,
Saravanan
Panneerselvam
,
Ivars
Karpics
,
Thomas R.
Schneider
,
Maria Marta
Garcia-Alai
,
Stephan
Niebling
,
Christian
Günther
,
Christina
Schmidt
,
Robin
Schubert
,
Huijong
Han
,
Juliane
Boger
,
Diana C. F.
Monteiro
,
Linlin
Zhang
,
Xinyuanyuan
Sun
,
Jonathan
Pletzer-Zelgert
,
Jan
Wollenhaupt
,
Christian G.
Feiler
,
Manfred S.
Weiss
,
Eike-Christian
Schulz
,
Pedram
Mehrabi
,
Katarina
Karničar
,
Aleksandra
Usenik
,
Jure
Loboda
,
Henning
Tidow
,
Ashwin
Chari
,
Rolf
Hilgenfeld
,
Charlotte
Uetrecht
,
Russell
Cox
,
Andrea
Zaliani
,
Tobias
Beck
,
Matthias
Rarey
,
Stephan
Günther
,
Dusan
Turk
,
Winfried
Hinrichs
,
Henry N.
Chapman
,
Arwen R.
Pearson
,
Christian
Betzel
,
Alke
Meents
Open Access
Abstract: The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.
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Apr 2021
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Pedram
Mehrabi
,
Henrike
Mueller-Werkmeister
,
Jan-Philipp
Leimkohl
,
Hendrik
Schikora
,
Jelena
Ninkovic
,
Silvia
Krivokuca
,
Ladislav
Andriček
,
Sascha W.
Epp
,
Darren A.
Sherrell
,
Robin L.
Owen
,
Arwen R.
Pearson
,
Friedjof
Tellkamp
,
Eike
Schulz
,
R. J. Dwayne
Miller
Open Access
Abstract: Serial synchrotron crystallography (SSX) is an emerging technique for static and time-resolved protein structure determination. Using specifically patterned silicon chips for sample delivery, the `hit-and-return' (HARE) protocol allows for efficient time-resolved data collection. The specific pattern of the crystal wells in the HARE chip provides direct access to many discrete time points. HARE chips allow for optical excitation as well as on-chip mixing for reaction initiation, making a large number of protein systems amenable to time-resolved studies. Loading of protein microcrystals onto the HARE chip is streamlined by a novel vacuum loading platform that allows fine-tuning of suction strength while maintaining a humid environment to prevent crystal dehydration. To enable the widespread use of time-resolved serial synchrotron crystallography (TR-SSX), detailed technical descriptions of a set of accessories that facilitate TR-SSX workflows are provided.
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Mar 2020
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I24-Microfocus Macromolecular Crystallography
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James F
Ross
,
Gemma C.
Wildsmith
,
Michael
Johnson
,
Daniel
Hurdiss
,
Kristian
Hollingsworth
,
Rebecca F.
Thompson
,
Majid
Mosayebi
,
Chi H.
Trinh
,
Emanuele
Paci
,
Arwen R.
Pearson
,
Michael E.
Webb
,
W. Bruce
Turnbull
Open Access
Abstract: The self-assembly of proteins into higher order structures is ubiquitous in living systems. It is also an essential process for the bottom-up creation of novel molecular architectures and devices for synthetic biology. However, the complexity of protein-protein interaction surfaces makes it challenging to mimic natural assembly processes in artificial systems. Indeed, many successful computationally designed protein assemblies are pre-screened for ‘designability’, limiting the choice of components. Here, we report a simple and pragmatic strategy to assemble chosen multi-subunit proteins into more complex structures. A coiled-coil domain appended to one face of the pentameric cholera toxin B-subunit (CTB) enabled the ordered assembly of tubular supra-molecular complexes. X-ray crystallography and analysis of a tubular structure has revealed a hierarchical assembly process that displays features reminiscent of the polymorphic assembly of polyomavirus proteins. The approach provides a simple and straightforward method to direct the assembly of protein building blocks which present either termini on a single face of an oligomer. This scaffolding approach can be used to generate bespoke supramolecular assemblies of functional proteins. Additionally, structural resolution of the scaffolded assemblies highlight ‘native-state’ forced protein-protein interfaces, which may prove useful as starting conformations for future computational design.
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Mar 2019
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[6386]
Open Access
Abstract: The catalase from Scytalidium thermophilum is a homotetramer containing a heme d in each active site. Although the enzyme has a classical monofunctional catalase fold, it also possesses oxidase activity towards a number of small organics, including catechol and phenol. In order to further investigate this, the crystal structure of the complex of the catalase with the classical catalase inhibitor 3-amino-1,2,4-triazole (3TR) was determined at 1.95 Å resolution. Surprisingly, no binding to the heme site was observed; instead, 3TR occupies a binding site corresponding to the NADPH-binding pocket in mammalian catalases at the entrance to a lateral channel leading to the heme. Kinetic analysis of site-directed mutants supports the assignment of this pocket as the binding site for oxidase substrates.
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Oct 2018
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[302]
Open Access
Abstract: Twinning is a crystal growth anomaly, which has posed a challenge in macromolecular crystallography (MX) since the earliest days. Many approaches have been used to treat twinned data in order to extract structural information. However, in most cases it is usually simpler to rescreen for new crystallization conditions that yield an untwinned crystal form or, if possible, collect data from non-twinned parts of the crystal. Here, we report 11 structures of engineered variants of the E. coli enzyme N-acetyl-neuraminic lyase which, despite twinning and incommensurate modulation, have been successfully indexed, solved and deposited. These structures span a resolution range of 1.45–2.30 Å, which is unusually high for datasets presenting such lattice disorders in MX and therefore these data provide an excellent test set for improving and challenging MX data processing programs.
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Oct 2018
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[6386]
Open Access
Abstract: Copper amine oxidases (CuAOs) are metalloenzymes that reduce molecular oxygen to hydrogen peroxide during catalytic turnover of primary amines. In addition to Cu2+ in the active site, two peripheral calcium sites, ca. 32 Å from the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Ca2+ (Asp533, Leu534, Asp535, Asp678, Ala 679) is essential for full-length protein production, while the surface Ca2+ (Glu573, Tyr667, Asp670, Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The mutation E573Q at the surface-site prevents calcium binding and TPQ biogenesis. However, TPQ biogenesis can be restored by a suppressor mutation (I342F) in the proposed oxygen delivery channel to the active site. While supporting TPQ biogenesis (ca. 60 % WTECAO TPQ), I342F/E573Q has almost no amine oxidase activity (ca. 4.6 % WTECAO activity). To understand how these long range mutations result in major effects on TPQ biogenesis and catalysis we employed UV-vis spectroscopy, steady-state kinetics, inhibition assays and X-ray crystallography. We show that the surface metal site controls the equilibrium (disproportionation) of the Cu2+-substrate reduced TPQ (TPQAMQ) Cu1+-TPQ semiquinone (TPQSQ) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to the Cu2+-TPQAMQ or destabilizes the Cu1+-TPQSQ. Crystal structure analysis shows that TPQ biogenesis is stalled at deprotonation in the Cu2+-tyrosinate state. Our findings support WTECAO using the inner sphere electron transfer mechanism for oxygen reduction during catalysis, and whilst a Cu1+-tyrosyl radical intermediate is not essential for TPQ biogenesis, it is required for efficient biogenesis.
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Aug 2018
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I03-Macromolecular Crystallography
|
Open Access
Abstract: The antimetabolite pentyl pantothenamide has broad spectrum antibiotic activity but exhibits enhanced activity against Escherichia coli. The PanDZ complex has been proposed to regulate the pantothenate biosynthetic pathway in E. coli by limiting the supply of β-alanine in response to coenzyme A concentration. We show that formation of this complex between activated aspartate decarboxylase (PanD) and PanZ leads to sequestration of the pyruvoyl cofactor as a ketone hydrate and demonstrate that both PanZ overexpression-linked β-alanine auxotrophy and pentyl pantothenamide toxicity are due to formation of this complex. This both demonstrates that the PanDZ complex regulates pantothenate biosynthesis in a cellular context and validates the complex as a target for antibiotic development.
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Aug 2017
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