NONE-No attached Diamond beamline
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Charles J.
Buchanan
,
Ben
Gaunt
,
Peter J.
Harrison
,
Yun
Yang
,
Jiwei
Liu
,
Aziz
Khan
,
Andrew M.
Giltrap
,
Audrey
Le Bas
,
Philip N.
Ward
,
Kapil
Gupta
,
Maud
Dumoux
,
Tiong Kit
Tan
,
Lisa
Schimaski
,
Sergio
Daga
,
Nicola
Picchiotti
,
Margherita
Baldassarri
,
Elisa
Benetti
,
Chiara
Fallerini
,
Francesca
Fava
,
Annarita
Giliberti
,
Panagiotis I.
Koukos
,
Matthew J.
Davy
,
Abirami
Lakshminarayanan
,
Xiaochao
Xue
,
Georgios
Papadakis
,
Lachlan P.
Deimel
,
Virgínia
Casablancas-Antràs
,
Timothy D. W.
Claridge
,
Alexandre M. J. J.
Bonvin
,
Quentin J.
Sattentau
,
Simone
Furini
,
Marco
Gori
,
Jiandong
Huo
,
Raymond J.
Owens
,
Christiane
Schaffitzel
,
Imre
Berger
,
Alessandra
Renieri
,
James H.
Naismith
,
Andrew J.
Baldwin
,
Benjamin G.
Davis
Open Access
Abstract: Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily-modified pathogen proteins can be confounded by overlapping sugar signals and/or compound with known experimental constraints. ‘Universal saturation transfer analysis’ (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin lineage SARS-CoV-2 spike trimer binds sialoside sugars in an ‘end-on’ manner. uSTA-guided modelling and a high-resolution cryo-electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar-binding in SARS CoV 2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins in deeper human lung as potentially relevant to virulence and/or zoonosis.
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Jun 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Patrick
Rabe
,
Jos J. A. G.
Kamps
,
Kyle D.
Sutherlin
,
James D. S.
Linyard
,
Pierre
Aller
,
Cindy C.
Pham
,
Mikako
Makita
,
Ian
Clifton
,
Michael A.
Mcdonough
,
Thomas M.
Leissing
,
Denis
Shutin
,
Pauline A.
Lang
,
Agata
Butryn
,
Jurgen
Brem
,
Sheraz
Gul
,
Franklin D.
Fuller
,
In-Sik
Kim
,
Mun Hon
Cheah
,
Thomas
Fransson
,
Asmit
Bhowmick
,
Iris D.
Young
,
Lee
O'Riordan
,
Aaron S.
Brewster
,
Ilaria
Pettinati
,
Margaret
Doyle
,
Yasumasa
Joti
,
Shigeki
Owada
,
Kensuke
Tono
,
Alexander
Batyuk
,
Mark S.
Hunter
,
Roberto
Alonso-Mori
,
Uwe
Bergmann
,
Robin L.
Owen
,
Nicholas K.
Sauter
,
Timothy D. W.
Claridge
,
Carol V.
Robinson
,
Vittal K.
Yachandra
,
Junko
Yano
,
Jan F.
Kern
,
Allen M.
Orville
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[23459, 19458]
Open Access
Abstract: Isopenicillin N synthase (IPNS) catalyzes the unique reaction of L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.
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Aug 2021
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|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[18069]
Open Access
Abstract: ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2,3,4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6,7,8,9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT–DarG toxin–antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)10. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP–HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT–DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication.
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Aug 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12346]
Open Access
Abstract: Bacterial production of β‐lactamases with carbapenemase activity is a global health threat. The active sites of class D carbapenemases such as OXA‐48, which is of major clinical importance, uniquely contain a carbamylated lysine residue which is essential for catalysis. Although there is significant interest in characterizing this post‐translational modification, and it is a promising inhibition target, protein carbamylation is challenging to monitor in solution. We report the use of 19F‐NMR spectroscopy to monitor the carbamylation state of 19F‐labelled OXA‐48. This method was used to investigate the interactions of OXA‐48 with clinically used serine β‐ lactamase inhibitors, including avibactam and vaborbactam. Crystallographic studies on 19F‐labelled OXA‐48 provide a structural rationale for the sensitivity of the 19F‐label to active site interactions. The overall results demonstrate the use of 19F‐NMR to monitor reversible covalent post‐translational modifications.
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Jul 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
|
Dong
Zhang
,
Marios S.
Markoulides
,
Dmitrijs
Stepanovs
,
Anna M.
Rydzik
,
Ahmed
El-Hussein
,
Corentin A. M.
Bon
,
Jos J. A. G.
Kamps
,
Klaus-Daniel
Umland
,
Patrick M.
Collins
,
Samuel T.
Cahill
,
David Y.
Wang
,
Timothy D. W.
Claridge
,
Jurgen
Brem
,
Michael
Mcdonough
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[9306, 12346, 16949]
Open Access
Abstract: Metallo-β-lactamases (MBLs) enable bacterial resistance to almost all classes of β-lactam antibiotics. We report studies on enethiol containing MBL inhibitors, which were prepared by rhodanine hydrolysis. The enethiols inhibit MBLs from different subclasses. Crystallographic analyses reveal that the enethiol sulphur displaces the di-Zn(II) ion bridging ‘hydrolytic’ water. In some, but not all, cases biophysical analyses provide evidence that rhodanine/enethiol inhibition involves formation of a ternary MBL enethiol rhodanine complex. The results demonstrate how low molecular weight active site Zn(II) chelating compounds can inhibit a range of clinically relevant MBLs and provide additional evidence for the potential of rhodanines to be hydrolysed to potent inhibitors of MBL protein fold and, maybe, other metallo-enzymes, perhaps contributing to the complex biological effects of rhodanines. The results imply that any medicinal chemistry studies employing rhodanines (and related scaffolds) as inhibitors should as a matter of course include testing of their hydrolysis products.
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Feb 2018
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I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[313]
Abstract: The most important resistance mechanism to β-lactam antibiotics involves hydrolysis by two β-lactamase categories: the nucleophilic serine (SBL) and the metallo- (MBL) β-lactamases. Cyclobutanones are hydrolytically stable β-lactam analogues with potential to inhibit both SBLs and MBLs. We describe solution and crystallographic studies on the interaction of a cyclobutanone penem analogue with the clinically important MBL SPM-1. NMR experiments using 19F-labeled SPM-1 imply the cyclobutanone binds to SPM-1 with micromolar affinity. A crystal structure of the SPM-1:cyclobutanone complex reveals binding of the hydrated cyclobutanone via interactions with one of the zinc ions, stabilisation of the hydrate by hydrogen bonding to zinc-bound water, and hydrophobic contacts with aromatic residues. NMR analyses using a 13C-labeled cyclobutanone support assignment of the bound species as the hydrated ketone. The results inform on how MBLs bind substrates and stabilize tetrahedral intermediates. They support further investigations on the use of transition state and/or intermediate analogues as inhibitors of all β-lactamase classes.
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Dec 2017
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Tzu-Lan
Yeh
,
Thomas m.
Leissing
,
Martine I.
Abboud
,
Cyrille C.
Thinnes
,
Onur
Atasoylu
,
James P.
Holt-Martyn
,
Dong
Zhang
,
Anthony
Tumber
,
Kerstin
Lippl
,
Christopher T.
Lohans
,
Ivanhoe K. H.
Leung
,
Helen
Morcrette
,
Ian J.
Clifton
,
Timothy D. W.
Claridge
,
Akane
Kawamura
,
Emily
Flashman
,
Xin
Lu
,
Peter J.
Ratcliffe
,
Rasheduzzaman
Chowdhury
,
Christopher W.
Pugh
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[12346, 9306]
Open Access
Abstract: Inhibition of the human 2-oxoglutarate (2OG) dependent hypoxia inducible factor (HIF) prolyl hydroxylases (human PHD1–3) causes upregulation of HIF, thus promoting erythropoiesis and is therefore of therapeutic interest. We describe cellular, biophysical, and biochemical studies comparing four PHD inhibitors currently in clinical trials for anaemia treatment, that describe their mechanisms of action, potency against isolated enzymes and in cells, and selectivities versus representatives of other human 2OG oxygenase subfamilies. The ‘clinical’ PHD inhibitors are potent inhibitors of PHD catalyzed hydroxylation of the HIF-α oxygen dependent degradation domains (ODDs), and selective against most, but not all, representatives of other human 2OG dependent dioxygenase subfamilies. Crystallographic and NMR studies provide insights into the different active site binding modes of the inhibitors. Cell-based results reveal the inhibitors have similar effects on the upregulation of HIF target genes, but differ in the kinetics of their effects and in extent of inhibition of hydroxylation of the N- and C-terminal ODDs; the latter differences correlate with the biophysical observations.
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Sep 2017
|
|
I24-Microfocus Macromolecular Crystallography
|
Christos
Pliotas
,
Samuel C.
Grayer
,
Silvia
Ekkerman
,
Anthony K. N.
Chan
,
Jess
Healy
,
Phedra
Marius
,
Wendy
Bartlett
,
Amjad
Khan
,
Wilian A.
Cortopassi
,
Shane A.
Chandler
,
Tim
Rasmussen
,
Justin L. P.
Benesch
,
Robert S.
Paton
,
Timothy D. W.
Claridge
,
Samantha
Miller
,
Ian R.
Booth
,
James
Naismith
,
Stuart J.
Conway
Open Access
Abstract: Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme–substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.
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Aug 2017
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I04-Macromolecular Crystallography
|
Christopher T.
Lohans
,
David Y.
Wang
,
Christian
Jorgensen
,
Samuel T.
Cahill
,
Ian J.
Clifton
,
Michael A.
Mcdonough
,
Henry P.
Oswin
,
James
Spencer
,
Carmen
Domene
,
Timothy D. W.
Claridge
,
Jurgen
Brem
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[12346]
Open Access
Abstract: The class D (OXA) serine β-lactamases are a major cause of resistance to β-lactam antibiotics. The class D enzymes are unique amongst β-lactamases because they have a carbamylated lysine that acts as a general acid/base in catalysis. Previous crystallographic studies led to the proposal that β-lactamase inhibitor avibactam targets OXA enzymes in part by promoting decarbamylation. Similarly, halide ions are proposed to inhibit OXA enzymes via decarbamylation. NMR analyses, in which the carbamylated lysines of OXA-10, -23 and -48 were 13C-labelled, indicate that reaction with avibactam does not ablate lysine carbamylation in solution. While halide ions did not decarbamylate the 13C-labelled OXA enzymes in the absence of substrate or inhibitor, avibactam-treated OXA enzymes were susceptible to decarbamylation mediated by halide ions, suggesting halide ions may inhibit OXA enzymes by promoting decarbamylation of acyl-enzyme complex. Crystal structures of the OXA-10 avibactam complex were obtained with bromide, iodide, and sodium ions bound between Trp-154 and Lys-70. Structures were also obtained wherein bromide and iodide ions occupy the position expected for the ‘hydrolytic water’ molecule. In contrast with some solution studies, Lys-70 was decarbamylated in these structures. These results reveal clear differences between crystallographic and solution studies on the interaction of class D β-lactamases with avibactam and halides, and demonstrate the utility of 13C-NMR for studying lysine carbamylation in solution.
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Jun 2017
|
|
I24-Microfocus Macromolecular Crystallography
|
Martine I.
Abboud
,
Philip
Hinchliffe
,
Jurgen
Brem
,
Robert
Macsics
,
Inga
Pfeffer
,
Anne
Makena
,
Klaus-Daniel
Umland
,
Anna M.
Rydzik
,
Guo-Bo
Li
,
James
Spencer
,
Timothy D. W.
Claridge
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[8922]
Open Access
Abstract: Resistance to β-lactam antibiotics mediated by metallo-β-lactamases (MBLs) is a growing problem. We describe the use of protein-observe 19F-NMR (PrOF NMR) to study the dynamics of the São Paulo MBL (SPM-1) from β-lactam-resistant Pseudomonas aeruginosa. Cysteinyl variants on the α3 and L3 regions, which flank the di-ZnII active site, were selectively 19F-labeled using 3-bromo-1,1,1-trifluoroacetone. The PrOF NMR results reveal roles for the mobile α3 and L3 regions in the binding of both inhibitors and hydrolyzed β-lactam products to SPM-1. These results have implications for the mechanisms and inhibition of MBLs by β-lactams and non-β-lactams and illustrate the utility of PrOF NMR for efficiently analyzing metal chelation, identifying new binding modes, and studying protein binding from a mixture of equilibrating isomers.
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Mar 2017
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