I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[27001]
Open Access
Abstract: Viral macrodomains possess the ability to counteract host ADP-ribosylation, a post-translational modification implicated in the creation of an antiviral environment via immune response regulation. This brought them into focus as promising therapeutic targets, albeit the close homology to some of the human macrodomains raised concerns regarding potential cross-reactivity and adverse effects for the host. Here, we evaluate the structure and function of the macrodomain of SARS-CoV-2, the causative agent of COVID-19. We show that it can antagonize ADP-ribosylation by PARP14, a cellular (ADP-ribosyl)transferase necessary for the restriction of coronaviral infections. Furthermore, our structural studies together with ligand modelling revealed the structural basis for poly(ADP-ribose) binding and hydrolysis, an emerging new aspect of viral macrodomain biology. These new insights were used in an extensive evolutionary analysis aimed at evaluating the druggability of viral macrodomains not only from the Coronaviridae but also Togaviridae and Iridoviridae genera (causing diseases such as Chikungunya and infectious spleen and kidney necrosis virus disease, respectively). We found that they contain conserved features, distinct from their human counterparts, which may be exploited during drug design.
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Nov 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Marcin J.
Suskiewicz
,
Florian
Zobel
,
Tom E. H.
Ogden
,
Pietro
Fontana
,
Antonio
Ariza
,
Ji-chun
Yang
,
Kang
Zhu
,
Lily
Bracken
,
William J.
Hawthorne
,
Dragana
Ahel
,
David
Neuhaus
,
Ivan
Ahel
Diamond Proposal Number(s):
[9306, 18069]
Abstract: The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2, are early responders to DNA damage in human cells1,2. Upon binding to genomic lesions, these enzymes utilise NAD+ to modify a plethora of proteins with mono- and poly(ADP-ribose) signals that are important for subsequent chromatin decompaction and repair factor recruitment3,4. These post-translational modification events are predominantly serine-linked and require HPF1, an accessory factor that is specific for the DNA damage response and switches the amino-acid specificity of PARP1/2 from aspartate/glutamate to serine residues5–10. Here, we report a co-structure of HPF1 bound to the catalytic domain of PARP2 that, in combination with NMR and biochemical data, reveals a composite active site formed by residues from both PARP1/2 and HPF1. We further show that the assembly of this new catalytic centre is essential for DNA damage-induced protein ADP-ribosylation in human cells. In response to DNA damage and NAD+ binding site occupancy, the HPF1–PARP1/2 interaction is enhanced via allosteric networks operating within PARP1/2, providing an additional level of regulation in DNA repair induction. As HPF1 forms a joint active site with PARP1/2, our data implicate HPF1 as an important determinant of the response to clinical PARP inhibitors.
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Feb 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18069]
Open Access
Abstract: Protein ADP-ribosylation is a highly dynamic post-translational modification. The rapid turnover is achieved, among others, by ADP-(ribosyl)hydrolases (ARHs), an ancient family of enzymes that reverses this modification. Recently ARHs came into focus due to their role as regulators of cellular stresses and tumor suppressors. Here we present a comprehensive structural analysis of the enzymatically active family members ARH1 and ARH3. These two enzymes have very distinct substrate requirements. Our data show that binding of the adenosine ribose moiety is highly diverged between the two enzymes, whereas the active sites harboring the distal ribose closely resemble each other. Despite this apparent similarity, we elucidate the structural basis for the selective inhibition of ARH3 by the ADP-ribose analogues ADP-HPD and arginine-ADP-ribose. Together, our biochemical and structural work provides important insights into the mode of enzyme-ligand interaction, helps to understand differences in their catalytic behavior, and provides useful tools for targeted drug design.
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Nov 2018
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B23-Circular Dichroism
I04-Macromolecular Crystallography
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Dominika
Kwasna
,
Syed Arif
Abdul Rehman
,
Jayaprakash
Natarajan
,
Stephen
Matthews
,
Ross
Madden
,
Virginia
De Cesare
,
Simone
Weidlich
,
Satpal
Virdee
,
Ivan
Ahel
,
Ian
Gibbs-seymour
,
Yogesh
Kulathu
Diamond Proposal Number(s):
[16778]
Open Access
Abstract: Deubiquitinating enzymes (DUBs) are important regulators of ubiquitin signaling. Here, we report the discovery of deubiquitinating activity in ZUFSP/C6orf113. High-resolution crystal structures of ZUFSP in complex with ubiquitin reveal several distinctive features of ubiquitin recognition and catalysis. Our analyses reveal that ZUFSP is a novel DUB with no homology to any known DUBs, leading us to classify ZUFSP as the seventh DUB family. Intriguingly, the minimal catalytic domain does not cleave polyubiquitin. We identify two ubiquitin binding domains in ZUFSP: a ZHA (ZUFSP helical arm) that binds to the distal ubiquitin and an atypical UBZ domain in ZUFSP that binds to polyubiquitin. Importantly, both domains are essential for ZUFSP to selectively cleave K63-linked polyubiquitin. We show that ZUFSP localizes to DNA lesions, where it plays an important role in genome stability pathways, functioning to prevent spontaneous DNA damage and also promote cellular survival in response to exogenous DNA damage.
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Mar 2018
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I02-Macromolecular Crystallography
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Marion
Schuller
,
Kerstin
Riedel
,
Ian
Gibbs-seymour
,
Kristin
Uth
,
Christian
Sieg
,
André P
Gehring
,
Ivan
Ahel
,
Franz
Bracher
,
Benedikt M.
Kessler
,
Jonathan M.
Elkins
,
Stefan
Knapp
Abstract: Macrodomains are conserved protein interaction modules that can be found in all domains of life as well as in certain viruses. Macrodomains mediate recognition of sequence motifs harbouring adenosine diphosphate ribose (ADPR) modifications, thereby regulating a variety of cellular processes. Due to their role in cancer or viral pathogenesis, macrodomains have emerged as potential therapeutic targets, but the unavailability of small molecule inhibitors has hampered target validation studies so far. Here, we describe an efficient screening strategy for identification of small molecule inhibitors that displace ADPR from macrodomains. We report the discovery and characterisation of a macrodomain inhibitor, GeA-69, selectively targeting macrodomain 2 (MD2) of PARP14 with low micromolar affinity. Co-crystallisation of a GeA-69 analogue with PARP14 MD2 revealed an allosteric binding mechanism explaining its selectivity over other human macrodomains. We show that GeA-69 engages PARP14 MD2 in intact cells and prevents its localisation to sites of DNA damage.
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Oct 2017
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9306, 12346]
Open Access
Abstract: The discovery and study of toxin-antitoxin (TA) systems helps us advance our understanding of the strategies prokaryotes employ to regulate cellular processes related to the general stress response, such as defense against phages, growth control, biofilm formation, persistence, and programmed cell death. Here we identify and characterize a TA system found in various bacteria, including the global pathogen Mycobacterium tuberculosis. The toxin of the system (DarT) is a domain of unknown function (DUF) 4433, and the antitoxin (DarG) a macrodomain protein. We demonstrate that DarT is an enzyme that specifically modifies thymidines on single-stranded DNA in a sequence-specific manner by a nucleotide-type modification called ADP-ribosylation. We also show that this modification can be removed by DarG. Our results provide an example of reversible DNA ADP-ribosylation, and we anticipate potential therapeutic benefits by targeting this enzyme-enzyme TA system in bacterial pathogens such as M. tuberculosis.
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Dec 2016
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I04-Macromolecular Crystallography
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Alasdair R.
Gunn
,
Benito
Banos-pinero
,
Peggy
Paschke
,
Luis
Sanchez-pulido
,
Antonio
Ariza
,
Joseph
Day
,
Mehera
Emrich
,
David
Leys
,
Chris P.
Ponting
,
Ivan
Ahel
,
Nicholas D.
Lakin
Diamond Proposal Number(s):
[12346]
Open Access
Abstract: ADP-ribosylation by ADP-ribosyltransferases (ARTs) has a well-established role in DNA strand break repair by promoting enrichment of repair factors at damage sites through ADP-ribose interaction domains. Here we exploit the simple eukaryote Dictyostelium to uncover a role for ADP-ribosylation in regulating DNA interstrand crosslink repair and redundancy of this pathway with non-homologous end-joining (NHEJ). In silico searches identify a protein that contains a permutated macrodomain (Aprataxin/APLF-and-PNKP-Like protein; APL). Structural analysis reveals permutated macrodomains retain features associated with ADP-ribose interactions and APL is capable of binding poly-ADP-ribose through its macrodomain. APL is enriched in chromatin in response to cisplatin, an agent that induces DNA interstrand crosslinks (ICLs). This is dependent on the macrodomain of APL, and the ART Adprt2, indicating a role for ADP-ribosylation in the cellular response to cisplatin. Although adprt2(-) cells are sensitive to cisplatin, ADP-ribosylation is evident in these cells due to redundant signalling by the DSB-responsive ART Adprt1a, promoting NHEJ-mediated repair. These data implicate ADP-ribosylation in DNA ICL repair and identify NHEJ can function to resolve this form of DNA damage in the absence of Adprt2.
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Oct 2016
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Johannes Gregor Matthias
Rack
,
Rosa
Morra
,
Eva
Barkauskaite
,
Rolf
Kraehenbuehl
,
Antonio
Ariza
,
Yue
Qu
,
Mary
Ortmayer
,
Orsolya
Leidecker
,
David r
Cameron
,
Ivan
Matic
,
Anton y.
Peleg
,
David
Leys
,
Ana
Traven
,
Ivan
Ahel
Diamond Proposal Number(s):
[9306]
Open Access
Abstract: Sirtuins are an ancient family of NAD+-dependent deacylases connected with the regulation of fundamental cellular processes including metabolic homeostasis and genome integrity. We show the existence of a hitherto unrecognized class of sirtuins, found predominantly in microbial pathogens. In contrast to earlier described classes, these sirtuins exhibit robust protein ADP-ribosylation activity. In our model organisms, Staphylococcus aureus and Streptococcus pyogenes, the activity is dependent on prior lipoylation of the target protein and can be reversed by a sirtuin-associated macrodomain protein. Together, our data describe a sirtuin-dependent reversible protein ADP-ribosylation system and establish a crosstalk between lipoylation and mono- ADP-ribosylation. We propose that these posttranslational modifications modulate microbial virulence by regulating the response to host-derived reactive oxygen species.
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Jul 2015
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9306]
Open Access
Abstract: Poly(ADP-ribosyl)ation is a common post-translational modification that mediates a wide variety of cellular processes including DNA damage repair, chromatin regulation, transcription, and apoptosis. The difficulty associated with accessing poly(ADP-ribose) (PAR) in a homogeneous form has been an impediment to understanding the interactions of PAR with poly(ADP-ribose) glycohydrolase (PARG) and other binding proteins. Here we describe the chemical synthesis of the ADP-ribose dimer, and we use this compound to obtain the first human PARG substrate-enzyme cocrystal structure. Chemical synthesis of PAR is an attractive alternative to traditional enzymatic synthesis and fractionation, allowing access to products such as dimeric ADP-ribose, which has been detected but never isolated from natural sources. Additionally, we describe the synthesis of an alkynylated dimer and demonstrate that this compound can be used to synthesize PAR probes including biotin and fluorophore-labeled compounds. The fluorescently labeled ADP-ribose dimer was then utilized in a general fluorescence polarization-based PAR-protein binding assay. Finally, we use intermediates of our synthesis to access various PAR fragments, and evaluation of these compounds as substrates for PARG reveals the minimal features for substrate recognition and enzymatic cleavage. Homogeneous PAR oligomers and unnatural variants produced from chemical synthesis will allow for further detailed structural and biochemical studies on the interaction of PAR with its many protein binding partners.
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Mar 2015
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I03-Macromolecular Crystallography
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Eva
Barkauskaite
,
Amy
Brassington
,
Edwin S.
Tan
,
Jim
Warwicker
,
Mark S.
Dunstan
,
Benito
Banos
,
Pierre
Lafite
,
Marijan
Ahel
,
Timothy J.
Mitchison
,
Ivan
Ahel
,
David
Leys
Diamond Proposal Number(s):
[7146]
Abstract: Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios.
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Aug 2013
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