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Taking PAR apart Proteins can be reversibly modified through the addition of repeating, polymerized ADP-ribose (PAR) subunits catalysed by poly(ADP-ribose) polymerase (PARP). Removal of PAR requires a glycohydrolase (PARG), which cleaves the ribose–ribose bond between subunits. Ivan Ahel and colleagues report that bacteria and fungi have a divergent PARG, which is unrelated to other enzymes that cleave PAR. Its structure, in complex with ADP-ribose and with a PARG inhibitor, and the results of mutational analysis suggest that the mechanism used in mammals and bacteria may be conserved. PARP inhibitors are being developed as pharmaceuticals for diseases including cancer, and this work suggests that small, cell-permeable PARG inhibitors might also be possible drug candidates. Post-translational modification of proteins by poly(ADP-ribosyl)ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis 1 . Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose–ribose bond, and is synthesized from NAD by PAR polymerases 1 , 2 . PAR glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose–ribose bonds present in PAR chains; its deficiency leads to cell death 3 , 4 . Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that has all the main characteristics of the human PARG enzyme. We present the first PARG crystal structure (derived from the bacterium Thermomonospora curvata ), which reveals that the PARG catalytic domain is a distant member of the ubiquitous ADP-ribose-binding macrodomain family 5 , 6 . High-resolution structures of T. curvata PARG in complexes with ADP-ribose and the PARG inhibitor ADP-HPD, complemented by biochemical studies, allow us to propose a model for PAR binding and catalysis by PARG. The insights into the PARG structure and catalytic mechanism should greatly improve our understanding of how PARG activity controls reversible protein poly(ADP-ribosyl)ation and potentially of how the defects in this regulation are linked to human disease.

Structural and biochemical approaches are used to show how RNF146 activity is allosterically regulated by the binding of poly(ADP-ribose) ligand, and how substrate specificity is achieved with protein poly(ADP-ribosyl)ation and ubiquitination occurring in the same protein complex. PARylation-dependent ubiquitination mechanism PARylation is a post-translational modification in which ADP-ribose polymers are covalently attached to protein targets. One of its many cellular functions is to control the ubiquitination and degradation of cell regulators such as Axin and PTEN. Wenqing Xu and colleagues use structural and biochemical approaches to show how the activity of RNF146, an E3 ligase responsible for PARylation-dependent ubiquitination, is regulated by the binding of PAR ligand and how substrate specificity is achieved with PARylation and ubiquitination occurring in the same protein complex. RNF146 represents a new class of RING E3 ligases, the activity of which can be regulated by ligand binding. Protein poly(ADP-ribosyl)ation (PARylation) has a role in diverse cellular processes such as DNA repair, transcription, Wnt signalling, and cell death 1 , 2 , 3 , 4 , 5 , 6 . Recent studies have shown that PARylation can serve as a signal for the polyubiquitination and degradation of several crucial regulatory proteins, including Axin and 3BP2 (refs 7 , 8 , 9 ). The RING-type E3 ubiquitin ligase RNF146 (also known as Iduna) is responsible for PARylation-dependent ubiquitination (PARdU) 10 , 11 , 12 . Here we provide a structural basis for RNF146-catalysed PARdU and how PARdU specificity is achieved. First, we show that iso -ADP-ribose ( iso -ADPr), the smallest internal poly(ADP-ribose) (PAR) structural unit, binds between the WWE and RING domains of RNF146 and functions as an allosteric signal that switches the RING domain from a catalytically inactive state to an active one. In the absence of PAR, the RING domain is unable to bind and activate a ubiquitin-conjugating enzyme (E2) efficiently. Binding of PAR or iso -ADPr induces a major conformational change that creates a functional RING structure. Thus, RNF146 represents a new mechanistic class of RING E3 ligases, the activities of which are regulated by non-covalent ligand binding, and that may provide a template for designing inducible protein-degradation systems. Second, we find that RNF146 directly interacts with the PAR polymerase tankyrase (TNKS). Disruption of the RNF146–TNKS interaction inhibits turnover of the substrate Axin in cells. Thus, both substrate PARylation and PARdU are catalysed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate–TNKS interaction. We propose that the maintenance of unliganded RNF146 in an inactive state may serve to maintain the stability of the RNF146–TNKS complex, which in turn regulates the homeostasis of PARdU activity in the cell.

To survive bacteriophage (phage) infections, bacteria developed numerous anti-phage defence systems 1 – 7 . Some of them (for example, type III CRISPR–Cas, CBASS, Pycsar and Thoeris) consist of two modules: a sensor responsible for infection recognition and an effector that stops viral replication by destroying key cellular components 8 – 12 . In the Thoeris system, a Toll/interleukin-1 receptor (TIR)-domain protein, ThsB, acts as a sensor that synthesizes an isomer of cyclic ADP ribose, 1′′−3′ glycocyclic ADP ribose (gcADPR), which is bound in the Smf/DprA-LOG (SLOG) domain of the ThsA effector and activates the silent information regulator 2 (SIR2)-domain-mediated hydrolysis of a key cell metabolite, NAD + (refs.  12 – 14 ). Although the structure of ThsA has been solved 15 , the ThsA activation mechanism remained incompletely understood. Here we show that 1′′−3′ gcADPR, synthesized in vitro by the dimeric ThsB′ protein, binds to the ThsA SLOG domain, thereby activating ThsA by triggering helical filament assembly of ThsA tetramers. The cryogenic electron microscopy (cryo-EM) structure of activated ThsA revealed that filament assembly stabilizes the active conformation of the ThsA SIR2 domain, enabling rapid NAD + depletion. Furthermore, we demonstrate that filament formation enables a switch-like response of ThsA to the 1′′−3′ gcADPR signal. A study reports that the Theoris anti-phage defence system is activated through helical filament assembly of the ThsA effector and details the activation mechanism.

Nucleotide-binding, leucine-rich repeat receptors (NLRs) initiate immune responses when they sense a pathogen-associated effector. In animals, oligomerization of NLRs upon binding their effectors is key to downstream activity, but plant systems differ in many ways and their activation mechanisms have been less clear. In two papers, Wang et al. studied the composition and structure of an NLR called ZAR1 in the small mustard plant Arabidopsis (see the Perspective by Dangl and Jones). They determined cryo–electron microscopy structures that illustrate differences between inactive and intermediate states. The active, intermediate state of ZAR1 forms a wheel-like pentamer, called the resistosome. In this activated complex, a set of helices come together to form a funnel-shaped structure required for immune responsiveness and association with the plasma membrane. Science , this issue p. eaav5868 , p. eaav5870 ; see also p. 31 Structural, biochemical, and functional studies show how a plant immune resistosome complex mediates cell death and disease resistance. Pathogen recognition by nucleotide-binding (NB), leucine-rich repeat (LRR) receptors (NLRs) plays roles in plant immunity. The Xanthomonas campestris pv. campestris effector AvrAC uridylylates the Arabidopsis PBL2 kinase, and the latter (PBL2 UMP ) acts as a ligand to activate the NLR ZAR1 precomplexed with the RKS1 pseudokinase. Here we report the cryo–electron microscopy structures of ZAR1-RKS1 and ZAR1-RKS1-PBL2 UMP in an inactive and intermediate state, respectively. The ZAR1 LRR domain, compared with animal NLR LRR domains, is differently positioned to sequester ZAR1 in an inactive state. Recognition of PBL2 UMP is exclusively through RKS1, which interacts with ZAR1 LRR . PBL2 UMP binding stabilizes the RKS1 activation segment, which sterically blocks ZAR1 adenosine diphosphate (ADP) binding. This engenders a more flexible NB domain without conformational changes in the other ZAR1 domains. Our study provides a structural template for understanding plant NLRs.

Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable, non-selective cation channel that has an essential role in diverse physiological processes such as core body temperature regulation, immune response and apoptosis 1 – 4 . TRPM2 is polymodal and can be activated by a wide range of stimuli 1 – 7 , including temperature, oxidative stress and NAD + -related metabolites such as ADP-ribose (ADPR). Its activation results in both Ca 2+ entry across the plasma membrane and Ca 2+ release from lysosomes 8 , and has been linked to diseases such as ischaemia-reperfusion injury, bipolar disorder and Alzheimer’s disease 9 – 11 . Here we report the cryo-electron microscopy structures of the zebrafish TRPM2 in the apo resting (closed) state and in the ADPR/Ca 2+ -bound active (open) state, in which the characteristic NUDT9-H domains hang underneath the MHR1/2 domain. We identify an ADPR-binding site located in the bi-lobed structure of the MHR1/2 domain. Our results provide an insight into the mechanism of activation of the TRPM channel family and define a framework for the development of therapeutic agents to treat neurodegenerative diseases and temperature-related pathological conditions. Structures of the transient receptor potential melastatin 2 channel in the apo resting (closed) state and in the ADP-ribose/Ca 2+ -bound active (open) state are determined by cryo-electron microscopy.

Inflammasomes are cytosolic innate immune complexes that activate caspase-1 following detection of pathogenic and endogenous dangers 1 – 5 , and NACHT-, leucine-rich repeat (LRR)- and pyrin domain (PYD)-containing protein  3 (NLRP3) is an inflammasome sensor of membrane damage highly important in regard to the induction of inflammation 2 , 6 , 7 . Here we report cryogenic electron microscopy structures of disc-shaped active NLRP3 oligomers in complex with adenosine 5′-O-(3-thio)triphosphate, the centrosomal NIMA-related kinase 7 (NEK7) and the adaptor protein ASC, which recruits caspase-1. In these NLRP3–NEK7–ASC complexes, the central NACHT domain of NLRP3 assumes an ATP-bound conformation in which two of its subdomains rotate by about 85° relative to the ADP-bound inactive conformation 8 – 12 . The fish-specific NACHT-associated domain conserved in NLRP3 but absent in most NLRPs 13 becomes ordered in its key regions to stabilize the active NACHT conformation and mediate most interactions in the disc. Mutations on these interactions compromise NLRP3-mediated caspase-1 activation. The N-terminal PYDs from all NLRP3 subunits combine to form a PYD filament that recruits ASC PYD to elicit downstream signalling. Surprisingly, the C-terminal LRR domain and the LRR-bound NEK7 do not participate in disc interfaces. Together with previous structures of an inactive NLRP3 cage in which LRR–LRR interactions play an important role 8 – 11 , we propose that the role of NEK7 is to break the inactive cage to transform NLRP3 into the active NLRP3 inflammasome disc. We report cryogenic electron microscopy structures of disc-shaped active NLRP3 oligomers in complex with NEK7 and ASC, and propose that the role of NEK7 is to transform NLRP3 into the active NLRP3 inflammasome disc.

ATP-hydrolysis-coupled actin polymerization is a fundamental mechanism of cellular force generation 1 – 3 . In turn, force 4 , 5 and actin filament (F-actin) nucleotide state 6 regulate actin dynamics by tuning F-actin’s engagement of actin-binding proteins through mechanisms that are unclear. Here we show that the nucleotide state of actin modulates F-actin structural transitions evoked by bending forces. Cryo-electron microscopy structures of ADP–F-actin and ADP-P i –F-actin with sufficient resolution to visualize bound solvent reveal intersubunit interfaces bridged by water molecules that could mediate filament lattice flexibility. Despite extensive ordered solvent differences in the nucleotide cleft, these structures feature nearly identical lattices and essentially indistinguishable protein backbone conformations that are unlikely to be discriminable by actin-binding proteins. We next introduce a machine-learning-enabled pipeline for reconstructing bent filaments, enabling us to visualize both continuous structural variability and side-chain-level detail. Bent F-actin structures reveal rearrangements at intersubunit interfaces characterized by substantial alterations of helical twist and deformations in individual protomers, transitions that are distinct in ADP–F-actin and ADP-P i –F-actin. This suggests that phosphate rigidifies actin subunits to alter the bending structural landscape of F-actin. As bending forces evoke nucleotide-state dependent conformational transitions of sufficient magnitude to be detected by actin-binding proteins, we propose that actin nucleotide state can serve as a co-regulator of F-actin mechanical regulation. The nucleotide state of actin modulates F-actin structural transitions evoked by bending forces.

Key Points Poly(ADP-ribose) (PAR) is synthesized from NAD + by PAR polymerases (PARPs) and regulates many physiological processes such as the maintenance of DNA integrity, gene expression and cell division. PARPs form a superfamily of 17 members in humans, and display diverse subcellular distributions and functions. Some members might function together and possess overlapping properties. PAR that is synthesized in response to DNA-strand breaks is a DNA-damage signalling molecule that allows a rapid and efficient cellular evaluation of the damage range. It is also an essential recruiting molecule that, in a few seconds, concentrates key factors of the single-strand break repair pathway at the site of the lesion. The poly(ADP-ribosyl)ation of histones that are associated with open chromatin conformation at the DNA-damage site provided the first clue to the roles of PAR as an epigenetic modification. Recent evidence revealed an important role of PAR in the epigenetic regulation of chromatin structure and in gene expression under physiological conditions in which the integrity of the DNA is not affected. The dogma that the DNA-damage-dependent PARP-1 is activated by DNA-strand breaks has to be reconsidered now due to recent studies that showed the activation of PARP-1 in the absence of DNA interruptions. Elucidating the triggers is currently one of our most exciting challenges. PARP-1 and PAR play key roles in various acute and chronic inflammatory disorders as well as in a number of degenerative diseases by contributing to the caspase-independent, apoptosis-inducing factor (AIF)-dependent cell death. PARP inhibition confers protection to these pathologies. PARP inhibitors have promising pharmacological applications in potentializing the effect of antitumour drugs in cancer therapy as well as in the treatment of inflammatory, neurological and cardiac disorders. Emerging evidence indicates a possible functional interplay between the PAR metabolic pathway and the SIRT1-mediated deacetylation pathway in the regulation of chromatin structure and function that is associated with broad biological activities. The transfer of poly(ADP-ribose) (PAR) to proteins is mediated by the growing family of PAR polymerases. This post-translational modification regulates many important cellular processes, including maintenance of genome integrity, gene expression and cell division, and is emerging as an important epigenetic mark. The addition to proteins of the negatively charged polymer of ADP-ribose (PAR), which is synthesized by PAR polymerases (PARPs) from NAD + , is a unique post-translational modification. It regulates not only cell survival and cell-death programmes, but also an increasing number of other biological functions with which novel members of the PARP family have been associated. These functions include transcriptional regulation, telomere cohesion and mitotic spindle formation during cell division, intracellular trafficking and energy metabolism.

A proteomic method to identify human proteins post-translationally modified by poly(ADP-ribosyl)ation is reported, which will help yield further insights into the biological role of this modification. Poly(ADP-ribosyl)ation is catalyzed by a family of enzymes known as PARPs. We describe a method to characterize the human aspartic acid– and glutamic acid–ADP-ribosylated proteome. We identified 1,048 ADP-ribosylation sites on 340 proteins involved in a wide array of nuclear functions; among these were many previously unknown PARP downstream targets whose ADP-ribosylation was sensitive to PARP inhibitor treatment. We also confirmed that iniparib had a negligible effect on PARP activity in intact cells.

In vitro experiments, using purified proteins and an assay that detects DNA unwinding, reveal the mechanism of activation of eukaryotic DNA replication. Unravelling DNA replication DNA replication in eukaryotes begins with the loading of a double hexamer of minichromosome maintenance (MCM) proteins onto the origin. Replication is then activated by separating the double hexamer into single-hexamer MCM rings that, together with Cdc45 and GINS, make up the CMG helicase, which is required for DNA unwinding. John Diffley and colleagues describe the role of ATP hydrolysis in regulating double-hexamer assembly and then CMG formation. Notably, there is an inactive CMG state that precedes the helicase-active CMG form that can translocate along the unwound DNA strand. The active CMG moves unidirectionally so that the two helicases pass by each other to establish bidirectional replication. The initiation of eukaryotic DNA replication occurs in two discrete stages 1 : first, the minichromosome maintenance (MCM) complex assembles as a head-to-head double hexamer that encircles duplex replication origin DNA during G1 phase; then, ‘firing factors’ convert each double hexamer into two active Cdc45–MCM–GINS helicases (CMG) during S phase. This second stage requires separation of the two origin DNA strands and remodelling of the double hexamer so that each MCM hexamer encircles a single DNA strand. Here we show that the MCM complex, which hydrolyses ATP during double-hexamer formation 2 , 3 , remains stably bound to ADP in the double hexamer. Firing factors trigger ADP release, and subsequent ATP binding promotes stable CMG assembly. CMG assembly is accompanied by initial DNA untwisting and separation of the double hexamer into two discrete but inactive CMG helicases. Mcm10, together with ATP hydrolysis, then triggers further DNA untwisting and helicase activation. After activation, the two CMG helicases translocate in an ‘N terminus-first’ direction, and in doing so pass each other within the origin; this requires that each helicase is bound entirely to single-stranded DNA. Our experiments elucidate the mechanism of eukaryotic replicative helicase activation, which we propose provides a fail-safe mechanism for bidirectional replisome establishment.