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The structure-specific endonuclease XPF-ERCC1 participates in multiple DNA damage repair pathways including nucleotide excision repair (NER) and inter-strand crosslink repair (ICLR). How XPF-ERCC1 is catalytically activated by DNA junction substrates is not currently understood. Here we report cryo-electron microscopy structures of both DNA-free and DNA-bound human XPF-ERCC1. DNA-free XPF-ERCC1 adopts an auto-inhibited conformation in which the XPF helical domain masks the ERCC1 (HhH)2 domain and restricts access to the XPF catalytic site. DNA junction engagement releases the ERCC1 (HhH)2 domain to couple with the XPF-ERCC1 nuclease/nuclease-like domains. Structure-function data indicate xeroderma pigmentosum patient mutations frequently compromise the structural integrity of XPF-ERCC1. Fanconi anaemia patient mutations in XPF often display substantial in-vitro activity but are resistant to activation by ICLR recruitment factor SLX4. Our data provide insights into XPF-ERCC1 architecture and catalytic activation., The endonuclease XPF-ERCC1 is a key component of the repair machinery to process both intra-strand and inter-strand DNA crosslinks. Here the authors present the cryo-EM structures of DNA-free and DNA-bound human XPF-ERCC1 and find that DNA-free XPF-ERCC1 adopts an auto-inhibited conformation, which is opened up upon DNA binding and they also characterise the biochemical properties of patient-derived XPF-ERCC1 mutations.

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of “pseudoschizonts,” which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition., Understanding the essential factors needed for malaria parasite development could help us find new therapeutic targets. This study reveals that N-myristoylation is a posttranslational modification of proteins essential for the parasites’ growth and their invasion of red blood cells.

ADP-ribosylation is integral to a diverse range of cellular processes such as DNA repair, chromatin regulation and RNA processing. However, proteome-wide investigation of its cellular functions has been limited due to numerous technical challenges including the complexity of the poly(ADP-ribose) (PAR) chains, low abundance of the modification and lack of sensitive enrichment methods. We herein show that an adenosine analogue with a terminal alkyne functionality at position 2 of the adenine (2-alkyne adenosine or 2YnAd) is suitable for selective enrichment, fluorescence detection and mass spectrometry proteomics analysis of the candidate ADP-ribosylome in mammalian cells. Although similar labelling profiles were observed via fluorescence imaging for 2YnAd and 6YnAd, a previously reported clickable NAD+ precursor, quantitative mass spectrometry analysis of the two probes in MDA-MB-231 breast cancer cells revealed a significant increase in protein coverage of the 2YnAd probe. To facilitate global enrichment of ADP-ribosylated proteins, we developed a dual metabolic labelling approach that involves simultaneous treatment of live cells with both 2YnAd and 6YnAd. By combining this dual metabolic labelling strategy with highly sensitive tandem mass tag (TMT) isobaric mass spectrometry and hierarchical Bayesian analysis, we have quantified the responses of thousands of endogenous proteins to clinical PARP inhibitors Olaparib and Rucaparib.

Summary RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RETECD-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands., Graphical abstract, Highlights • X-ray structure of zebrafish RETCLD1-4 module reveals conformational flexibility • Conformational differences between RETCLD1-4 and a liganded RETECD cryo-EM structure • Spatial separation of RETECD CRD dimer C termini imposed by each ligand dimer • Differences in GDNF and GDF15 co-receptor engagement of RET and multimerization evidence, Adams et al. use X-ray crystallography and cryo-electron microscopy to probe conformational changes in RET due to binding of GDNF ligand-GFRα1 co-receptor. The study shows a two-site clamping mechanism with flexible adaptations near the RET calcium sites but near identical spacings of the RET cysteine-rich domains established by GDNF/GDF15 ligand dimers.

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last ten to fifteen hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified sixteen NMT substrates for which myristoylation was significantly reduced by NMT inhibitor treatment, and of these, six proteins were substantially reduced in abundance. In a viability screen, we showed that for four of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least three mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of ‘pseudoschizonts’, which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.

RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognise and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2-CLD3. Comparison with a cryo-EM structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1 complex indicates conformational changes within a clade-specific CLD3 loop adjacent to co-receptor. Our observations indicate RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognises a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualise linear arrays of RETECD-GDNF-GFRα1 suggesting a conserved contact stabilises higher-order species. Our study reveals ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.HighlightsCrystal structure of zebrafish RET cadherin-like module reveals conformational flexibility at the calcium-dependent CLD2-CLD3 interfaceComparison of X-ray and cryo-EM structures indicate conformational differences between unliganded and liganded RET involving a clade-specific CLD3 loopStrict spatial separation of RETECD C-termini is imposed by each cysteine-rich domain interaction with GFL dimerDifferences in co-receptor engagement and higher-order ligand-bound RET complexes indicate potentially divergent signalling mechanisms

IFN-stimulated gene (ISG) 15 is a ubiquitin-like protein induced after type I IFN stimulation. There is a dearth of in vivo models to study free unconjugated ISG15 function. We found that free ISG15 enhances the production of IFN-γ and IL-1β during murine infection with Toxoplasma gondii. In our model, ISG15 is induced in a type I IFN–dependent fashion and released into the serum. Increased ISG15 levels are dependent on an actively invading and replicating parasite. Two cysteine residues in the hinge domain are necessary determinants for ISG15 to induce increased cytokine levels during infection. Increased ISG15 is concurrent with an influx of IL-1β–producing CD8α+ dendritic cells to the site of infection. In this article, we present Toxoplasma infection as a novel in vivo murine model to study the immunomodulatory properties of free ISG15 and uniquely link it to IL-1β production by CD8α+ dendritic cells driven by two cysteines in the hinge region of the protein.

Summary Activation of the main DNA interstrand crosslink repair pathway in higher eukaryotes requires mono-ubiquitination of FANCI and FANCD2 by FANCL, the E3 ligase subunit of the Fanconi anemia core complex. FANCI and FANCD2 form a stable complex; however, the molecular basis of their ubiquitination is ill defined. FANCD2 mono-ubiquitination by FANCL is stimulated by the presence of the FANCB and FAAP100 core complex components, through an unknown mechanism. How FANCI mono-ubiquitination is achieved remains unclear. Here, we use structural electron microscopy, combined with crosslink-coupled mass spectrometry, to find that FANCB, FANCL, and FAAP100 form a dimer of trimers, containing two FANCL molecules that are ideally poised to target both FANCI and FANCD2 for mono-ubiquitination. The FANCC-FANCE-FANCF subunits bridge between FANCB-FANCL-FAAP100 and the FANCI-FANCD2 substrate. A transient interaction with FANCC-FANCE-FANCF alters the FANCI-FANCD2 configuration, stabilizing the dimerization interface. Our data provide a model to explain how equivalent mono-ubiquitination of FANCI and FANCD2 occurs., Graphical Abstract, Highlights • FANCB, FANCL, and FAAP100 form a symmetric dimer of trimers • FANCL is ideally poised for the symmetric mono-ubiquitination of FANCI-FANCD2 • Two separate FANCC-FANCE-FANCF complexes bind to the opposing poles of FANCB-FANCL-FAAP100 • FANCC-FANCE-FANCF stabilizes FANCI-FANCD2 for efficient mono-ubiquitination, Mono-ubiquitination of FANCI-FANCD2 by the Fanconi anemia core complex activates a major DNA interstrand-crosslink repair pathway important for genome stability maintenance. Here, Swuec et al. reveal the structural basis of this reaction by showing that the core complex exists as a dimeric catalytic module for the symmetric mono-ubiquitination of FANCI-FANCD2.

We investigate the environmental stability of fullerene solutions by static and dynamic light scattering, FTIR, NMR and mass spectroscopies, and quantum chemical calculations. We find that visible light exposure of fullerene solutions in toluene, a good solvent, under ambient laboratory conditions results in C 60 oxidation to form fullerene epoxides, and subsequently causes fullerene clustering in solution. The clusters grow with time, even in absence of further illumination, and can reach dimensions from ≈ 100 nm to the μ m scale over ≈ 1 day. Static light scattering suggests that resulting aggregates are fractal, with a characteristic power law ( d f ) that increases from approximately 1.3 to 2.0 during light exposure. The clusters are bound by weak Coulombic interactions and are found to be reversible, disintegrating by mechanical agitation and thermal stress, and reforming over time. Our findings are relevant to the solution processing of composites and organic photovoltaics, whose reproducibility and performance requires control of fullerene solution stability under storage conditions.

The transcription factor E4bp4/Nfil3 has been shown to have a critical role in the development of all innate lymphoid cell types including NK cells. In this study, we show that posttranslational modifications of E4bp4 by either SUMOylation or phosphorylation have profound effects on both E4bp4 function and NK cell development. We examined the activity of E4bp4 mutants lacking posttranslational modifications and found that Notch1 was a novel E4bp4 target gene. We observed that abrogation of Notch signaling impeded NK cell production and the total lack of NK cell development from E4bp4−/− progenitors was completely rescued by short exposure to Notch peptide ligands. This work reveals both novel mechanisms in NK cell development by a transcriptional network including E4bp4 with Notch, and that E4bp4 is a central hub to process extrinsic stimuli.

ISG15 is strongly induced after type I IFN stimulation producing a protein comprised of two ubiquitin-like domains. Intracellularly, ISG15 can be covalently linked and modify the function of target proteins (ISGylation). In addition, free unconjugated ISG15 can be released from cells. We found that ISG15 is released in the serum of Toxoplasma gondii infected mice early after infection in a type-I IFN independent manner. Once in the extracellular space, free ISG15 forms dimers and enhances the release of key cytokines involved in the immune response to the parasite: IL-12, IFN-γ, and IL-1β. Its action is dependent on an actively invading and replicating live parasite. ISG15 induces an increase of IL-1β later during infection by leading to increased IL-1β producing CD8α+ dendritic cells at the site of infection. Here, we define for the first time the molecular determinants of active free ISG15 and link ISG15 to IL-1β production by CD8α+ dendritic cells. Thus we define ISG15 as a novel secreted modulator of the cytokine response during Toxoplasma infection.

Histone lysine methyltransferases (HKMTs) are an important class of targets for epigenetic therapy. 1 (chaetocin), an epidithiodiketopiperazine (ETP) natural product, has been reported to be a specific inhibitor of the SU(VAR)3-9 class of HKMTs. We have studied the inhibition of the HKMT G9a by 1 and functionally related analogues. Our results reveal that only the structurally unique ETP core is required for inhibition, and such inhibition is time-dependent and irreversible (in the absence of DTT), ultimately resulting in protein denaturation. Mass spectrometric data provide a molecular basis for this effect, demonstrating covalent adduct formation between 1 and the protein. This provides a potential rationale for the selectivity observed in the inhibition of a variety of HKMTs by 1 in vitro and has implications for the activity of ETPs against these important epigenetic targets.

The functions of cohesin are central to genome integrity, chromosome organization and transcription regulation through its prevention of premature sister-chromatid separation and the formation of DNA loops. The loading of cohesin onto chromatin depends on the Scc2–Scc4 complex; however, little is known about how it stimulates the cohesion-loading activity. Here we determine the large ‘hook' structure of Scc2 responsible for catalysing cohesin loading. We identify key Scc2 surfaces that are crucial for cohesin loading in vivo. With the aid of previously determined structures and homology modelling, we derive a pseudo-atomic structure of the full-length Scc2–Scc4 complex. Finally, using recombinantly purified Scc2–Scc4 and cohesin, we performed crosslinking mass spectrometry and interaction assays that suggest Scc2–Scc4 uses its modular structure to make multiple contacts with cohesin., The cohesin complex maintains genome integrity by ensuring correct sister-chromatid segregation during mitosis and meiosis. Here, Chao et al. present a pseudo-atomic model of the full-length Scc2–Scc4 cohesin loader complex and reveal key Scc2 surfaces crucial for cohesin loading.

In the version of this Letter originally published, Michele S. Y. Tan was incorrectly listed as Michele Y. S. Tan due to a technical error. This has now been amended in all online versions of the Letter.

Hexanucleotide repeat expansions in C9orf72 are a major cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Understanding the disease mechanisms and a method for clinical diagnostic genotyping have been hindered because of the difficulty in estimating the expansion size. We found 96 repeat-primed PCR expansions: 85/2,974 in six neurodegenerative diseases cohorts (FTLD, ALS, Alzheimer disease, sporadic Creutzfeldt-Jakob disease, Huntington disease-like syndrome, and other nonspecific neurodegenerative disease syndromes) and 11/7,579 (0.15%) in UK 1958 birth cohort (58BC) controls. With the use of a modified Southern blot method, the estimated expansion range (smear maxima) in cases was 800-4,400. Similarly, large expansions were detected in the population controls. Differences in expansion size and morphology were detected between DNA samples from tissue and cell lines. Of those in whom repeat-primed PCR detected expansions, 68/69 were confirmed by blotting, which was specific for greater than 275 repeats. We found that morphology in the expansion smear varied among different individuals and among different brain regions in the same individual. Expansion size correlated with age at clinical onset but did not differ between diagnostic groups. Evidence of instability of repeat size in control families, as well as neighboring SNP and microsatellite analyses, support multiple expansion events on the same haplotype background. Our method of estimating the size of large expansions has potential clinical utility. C9orf72-related disease might mimic several neurodegenerative disorders and, with potentially 90,000 carriers in the United Kingdom, is more common than previously realized.