B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9306, 12346, 15613]
Open Access
Abstract: Dynamic ADP-ribosylation signalling is a crucial pathway that controls fundamental cellular processes, in particular, the response to cellular stresses such as DNA damage, reactive oxygen species and infection. In some pathogenic microbes the response to oxidative stress is controlled by a SirTM/zinc-containing macrodomain (Zn-Macro) pair responsible for establishment and removal of the modification, respectively. Targeting this defence mechanism against the host’s innate immune response may lead to novel approaches to support the fight against emerging antimicrobial resistance. Earlier studies suggested that Zn-Macros play a key role in the activation of this defence. Therefore, we used phylogenetic, biochemical, and structural approaches to elucidate the functional properties of these enzymes. Using the substrate mimetic asparagine-ADP-ribose as well as the ADP-ribose product, we characterise the catalytic role of the zinc ion in the removal of the ADP-ribosyl modification. Furthermore, we determined structural properties that contribute to substrate selectivity within the different Zn-Macro branches. Together, our data not only give new insights into the Zn-Macro family but also highlight their distinct features that may be exploited for the development of future therapies.
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Sep 2024
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459]
Open Access
Abstract: Modification of nucleic acids by ADP-ribosylation is catalyzed by various ADP-ribosyltransferases, including the DarT enzyme. The latter is part of the bacterial toxin-antitoxin (TA) system DarTG, which was shown to provide control of DNA replication and bacterial growth as well as protection against bacteriophages. Two subfamilies have been identified, DarTG1 and DarTG2, which are distinguished by their associated antitoxins. While DarTG2 catalyzes reversible ADP-ribosylation of thymidine bases employing a macrodomain as antitoxin, the DNA ADP-ribosylation activity of DarTG1 and the biochemical function of its antitoxin, a NADAR domain, are as yet unknown. Using structural and biochemical approaches, we show that DarT1-NADAR is a TA system for reversible ADP-ribosylation of guanosine bases. DarT1 evolved the ability to link ADP-ribose to the guanine amino group, which is specifically hydrolyzed by NADAR. We show that guanine de-ADP-ribosylation is also conserved among eukaryotic and non-DarT-associated NADAR members, indicating a wide distribution of reversible guanine modifications beyond DarTG systems.
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Jun 2023
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346, 18069]
Open Access
Abstract: In the mammalian DNA damage response, ADP-ribosylation signalling is of crucial importance to mark sites of DNA damage as well as recruit and regulate repairs factors. Specifically, the PARP1:HPF1 complex recognises damaged DNA and catalyses the formation of serine-linked ADP-ribosylation marks (mono-Ser-ADPr), which are extended into ADP-ribose polymers (poly-Ser-ADPr) by PARP1 alone. Poly-Ser-ADPr is reversed by PARG, while the terminal mono-Ser-ADPr is removed by ARH3. Despite its significance and apparent evolutionary conservation, little is known about ADP-ribosylation signalling in non-mammalian Animalia. The presence of HPF1, but absence of ARH3, in some insect genomes, including Drosophila species, raises questions regarding the existence and reversal of serine-ADP-ribosylation in these species. Here we show by quantitative proteomics that Ser-ADPr is the major form of ADP-ribosylation in the DNA damage response of Drosophila melanogaster and is dependent on the dParp1:dHpf1 complex. Moreover, our structural and biochemical investigations uncover the mechanism of mono-Ser-ADPr removal by Drosophila Parg. Collectively, our data reveal PARP:HPF1-mediated Ser-ADPr as a defining feature of the DDR in Animalia. The striking conservation within this kingdom suggests that organisms that carry only a core set of ADP-ribosyl metabolising enzymes, such as Drosophila, are valuable model organisms to study the physiological role of Ser-ADPr signalling.
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Jun 2023
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9306, 12346]
Open Access
Abstract: Structurally complex genomic regions, such as centromeres, are inherently difficult to duplicate. The mechanism behind centromere inheritance is not well understood, and one of the key questions relates to the reassembly of centromeric chromatin following DNA replication. Here, we define ERCC6L2 as a key regulator of this process. ERCC6L2 accumulates at centromeres and promotes deposition of core centromeric factors. Interestingly, ERCC6L2−/− cells show unrestrained replication of centromeric DNA, likely caused by the erosion of centromeric chromatin. Beyond centromeres, ERCC6L2 facilitates replication at genomic repeats and non-canonical DNA structures. Notably, ERCC6L2 interacts with the DNA-clamp PCNA through an atypical peptide, presented here in a co-crystal structure. Finally, ERCC6L2 also restricts DNA end resection, acting independently of the 53BP1-REV7-Shieldin complex. We propose a mechanistic model, which reconciles seemingly distinct functions of ERCC6L2 in DNA repair and DNA replication. These findings provide a molecular context for studies linking ERCC6L2 to human disease.
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Apr 2023
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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: 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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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Johannes Gregor Matthias
Rack
,
Qiang
Liu
,
Valentina
Zorzini
,
Jim
Voorneveld
,
Antonio
Ariza
,
Kourosh
Honarmand Ebrahimi
,
Julia M.
Reber
,
Sarah C.
Krassnig
,
Dragana
Ahel
,
Gijsbert A.
Van Der Marel
,
Aswin
Mangerich
,
James S. O.
Mccullagh
,
Dmitri V.
Filippov
,
Ivan
Ahel
Diamond Proposal Number(s):
[18069, 23459]
Open Access
Abstract: Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry.
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Jul 2021
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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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I02-Macromolecular Crystallography
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Christian
Roth
,
Olga V.
Moroz
,
Johan P.
Turkenburg
,
Elena
Blagova
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Jitka
Waterman
,
Antonio
Ariza
,
Li
Ming
,
Sun
Tianqi
,
Carsten
Andersen
,
Gideon J.
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[1221, 9948]
Open Access
Abstract: Amylases are probably the best studied glycoside hydrolases and have a huge biotechnological value for industrial processes on starch. Multiple amylases from fungi and microbes are currently in use. Whereas bacterial amylases are well suited for many industrial processes due to their high stability, fungal amylases are recognized as safe and are preferred in the food industry, although they lack the pH tolerance and stability of their bacterial counterparts. Here, we describe three amylases, two of which have a broad pH spectrum extending to pH 8 and higher stability well suited for a broad set of industrial applications. These enzymes have the characteristic GH13 α-amylase fold with a central (β/α)8-domain, an insertion domain with the canonical calcium binding site and a C-terminal β-sandwich domain. The active site was identified based on the binding of the inhibitor acarbose in form of a transglycosylation product, in the amylases from Thamnidium elegans and Cordyceps farinosa. The three amylases have shortened loops flanking the nonreducing end of the substrate binding cleft, creating a more open crevice. Moreover, a potential novel binding site in the C-terminal domain of the Cordyceps enzyme was identified, which might be part of a starch interaction site. In addition, Cordyceps farinosa amylase presented a successful example of using the microseed matrix screening technique to significantly speed-up crystallization.
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Oct 2019
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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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[1221]
Abstract: Glucoamylases are one of the most important classes of enzymes in the industrial degradation of starch biomass. They consist of a catalytic domain and a carbohydrate-binding domain (CBM), with the latter being important for the interaction with the polymeric substrate. Whereas the catalytic mechanisms and structures of the individual domains are well known, the spatial arrangement of the domains with respect to each other and its influence on activity are not fully understood. Here, the structures of three industrially used fungal glucoamylases, two of which are full length, have been crystallized and determined. It is shown for the first time that the relative orientation between the CBM and the catalytic domain is flexible, as they can adopt different orientations independently of ligand binding, suggesting a role as an anchor to increase the contact time and the relative concentration of substrate near the active site. The flexibility in the orientations of the two domains presented a considerable challenge for the crystallization of the enzymes.
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May 2018
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