Head-to-tail polymerization by VEL proteins underpins cold-induced Polycomb silencing in flowering control.

Transcriptional silencing through the Polycomb silencing machinery utilizes a "read-write" mechanism involving histone tail modifications. However, nucleation of silencing and long-term stable transmission of the silenced state also requires P-olycomb Repressive Complex 2 (PRC2) accessory proteins, whose molecular role is poorly understood. The Arabidopsis VEL proteins are accessory proteins that interact with PRC2 to nucleate and propagate silencing at the FLOWERING LOCUS C (FLC) locus, enabling early flowering in spring. Here, we report that VEL proteins contain a domain related to an atypical four-helix bundle that engages in spontaneous concentration-dependent head-to-tail polymerization to assemble dynamic biomolecular condensates. Mutations blocking polymerization of this VEL domain prevent Polycomb silencing at FLC. Plant VEL proteins thus facilitate assembly of dynamic multivalent Polycomb complexes required for inheritance of the silenced state.

Plant vernalization proteins contain unusual PHD superdomains without histone H3 binding activity.

PHD fingers are modular domains in chromatin-associated proteins that decode the methylation status of histone H3 tails. A PHD finger signature is found in plant vernalization (VEL) proteins, which function as accessory factors of the Polycomb system to control flowering in Arabidopsis through an epigenetic silencing mechanism. It has been proposed that VEL PHD fingers bind to methylated histone H3 tails to facilitate association of the Polycomb silencing machinery with target genes. Here, we use structural analysis by X-ray crystallography to show that the VEL PHD finger forms the central module of a larger compact tripartite superdomain that also contains a zinc finger and a four-helix bundle. This PHD superdomain fold is only found in one other family, the OBERON proteins, which have multiple functions in Arabidopsis meristems to control plant growth. The putative histone-binding surface of OBERON proteins exhibits the characteristic three-pronged pocket of histone-binding PHD fingers and binds to methylated histone H3 tails. However, that of VEL PHD fingers lacks this architecture and exhibits unusually high positive surface charge. This VEL PHD superdomain neither binds to unmodified nor variously modified histone H3 tails, as demonstrated by isothermal calorimetry and NMR spectroscopy. Instead, the VEL PHD superdomain interacts with negatively charged polymers. Our evidence argues for evolution of a divergent function for the PHD superdomain in vernalization that does not involve histone tail decoding.

Selective function of the PDZ domain of Dishevelled in noncanonical Wnt signalling.

Dishevelled is a cytoplasmic hub that transduces Wnt signals to cytoplasmic effectors, which can be broadly characterised as canonical (ß-catenin dependent) and noncanonical, to specify cell fates and behaviours during development. To transduce canonical Wnt signals, Dishevelled binds to the intracellular face of Frizzled through its DEP domain and polymerises through its DIX domain to assemble dynamic signalosomes. Dishevelled also contains a PDZ domain, whose function remains controversial. Here, we use genome editing to delete the PDZ domain-encoding region from Drosophila dishevelled. Canonical Wingless signalling is entirely normal in these deletion mutants; however, they show defects in multiple contexts controlled by noncanonical Wnt signalling, such as planar polarity. We use nuclear magnetic resonance spectroscopy to identify bona fide PDZ-binding motifs at the C termini of different polarity proteins. Although deletions of these motifs proved aphenotypic in adults, we detected changes in the proximodistal distribution of the polarity protein Flamingo (also known as Starry night) in pupal wings that suggest a modulatory role of these motifs in polarity signalling. We also provide new genetic evidence that planar polarity relies on the DEP-dependent recruitment of Dishevelled to the plasma membrane by Frizzled.

Molecular mechanisms underlying the role of the centriolar CEP164-TTBK2 complex in ciliopathies.

Cilia formation is essential for human life. One of the earliest events in the ciliogenesis program is the recruitment of tau-tubulin kinase 2 (TTBK2) by the centriole distal appendage component CEP164. Due to the lack of high-resolution structural information on this complex, it is unclear how it is affected in human ciliopathies such as nephronophthisis. Furthermore, it is poorly understood if binding to CEP164 influences TTBK2 activities. Here, we present a detailed biochemical, structural, and functional analysis of the CEP164-TTBK2 complex and demonstrate how it is compromised by two ciliopathic mutations in CEP164. Moreover, we also provide insights into how binding to CEP164 is coordinated with TTBK2 activities. Together, our data deepen our understanding of a crucial step in cilia formation and will inform future studies aimed at restoring CEP164 functionality in a debilitating human ciliopathy.

Regulation of Dishevelled DEP domain swapping by conserved phosphorylation sites.

Wnt signals bind to Frizzled receptors to trigger canonical and noncanonical signaling responses that control cell fates during animal development and tissue homeostasis. All Wnt signals are relayed by the hub protein Dishevelled. During canonical (ß-catenin-dependent) signaling, Dishevelled assembles signalosomes via dynamic head-to-tail polymerization of its Dishevelled and Axin (DIX) domain, which are cross-linked by its Dishevelled, Egl-10, and Pleckstrin (DEP) domain through a conformational switch from monomer to domain-swapped dimer. The domain-swapped conformation of DEP masks the site through which Dishevelled binds to Frizzled, implying that DEP domain swapping results in the detachment of Dishevelled from Frizzled. This would be incompatible with noncanonical Wnt signaling, which relies on long-term association between Dishevelled and Frizzled. It is therefore likely that DEP domain swapping is differentially regulated during canonical and noncanonical Wnt signaling. Here, we use NMR spectroscopy and cell-based assays to uncover intermolecular contacts in the DEP dimer that are essential for its stability and for Dishevelled function in relaying canonical Wnt signals. These contacts are mediated by an intrinsically structured sequence spanning a conserved phosphorylation site upstream of the DEP domain that serves to clamp down the swapped N-terminal α-helix onto the structural core of a reciprocal DEP molecule in the domain-swapped configuration. Mutations of this phosphorylation site and its cognate surface on the reciprocal DEP core attenuate DEP-dependent dimerization of Dishevelled and its canonical signaling activity in cells without impeding its binding to Frizzled. We propose that phosphorylation of this crucial residue could be employed to switch off canonical Wnt signaling.

Analysis of ligand binding and resulting conformational changes in pyrophosphatase NUDT9.

Nudix hydrolase 9 (NUDT9) is a member of the nucleoside linked to another moiety X (NUDIX) protein superfamily, which hydrolyses a broad spectrum of organic pyrophosphates from metabolic processes. ADP-ribose (ADPR) has been the only known endogenous substrate accepted by NUDT9 so far. The Ca2+ -permeable transient receptor potential melastatin subfamily 2 (TRPM2) channel contains a homologous NUDT9-homology (NUDT9H) domain and is activated by ADPR. Sustained Ca2+ influx via ADPR-activated TRPM2 triggers apoptotic mechanisms. Thus, a precise regulation of cellular ADPR levels by NUDT9 is essential. A detailed characterization of the enzyme-substrate interaction would help to understand the high substrate specificity of NUDT9. Here, we analysed ligand binding to NUDT9 using a variety of biophysical techniques. We identified 2'-deoxy-ADPR as an additional substrate for NUDT9. Similar enzyme kinetics and binding affinities were determined for the two ligands. The high-affinity binding was preserved in NUDT9 containing the mutated NUDIX box derived from the human NUDT9H domain. NMR spectroscopy indicated that ADPR and 2'-deoxy-ADPR bind to the same binding site of NUDT9. Backbone resonance assignment and subsequent molecular docking allowed further characterization of the binding pocket. Substantial conformational changes of NUDT9 upon ligand binding were observed which might allow for the development of NUDT9-based ADPR fluorescence resonance energy transfer sensors that may help with the analysis of ADPR signalling processes in cells in the future.

Controlled Ligand Exchange Between Ruthenium Organometallic Cofactor Precursors and a Naïve Protein Scaffold Generates Artificial Metalloenzymes Catalysing Transfer Hydrogenation.

Many natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII (η6 -arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non-biological ligand. Tandem mass spectrometry and 19 F NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein-derived ligands. By introduction of ruthenium cofactors into a 4-helical bundle, transfer hydrogenation catalysts were generated that displayed a 35-fold rate increase when compared to the respective small molecule reaction in solution.

FtsK in motion reveals its mechanism for double-stranded DNA translocation.

FtsK protein contains a fast DNA motor that is involved in bacterial chromosome dimer resolution. During cell division, FtsK translocates double-stranded DNA until both dif recombination sites are placed at mid cell for subsequent dimer resolution. Here, we solved the 3.6-Å resolution electron cryo-microscopy structure of the motor domain of FtsK while translocating on its DNA substrate. Each subunit of the homo-hexameric ring adopts a unique conformation and one of three nucleotide states. Two DNA-binding loops within four subunits form a pair of spiral staircases within the ring, interacting with the two DNA strands. This suggests that simultaneous conformational changes in all ATPase domains at each catalytic step generate movement through a mechanism related to filament treadmilling. While the ring is only rotating around the DNA slowly, it is instead the conformational states that rotate around the ring as the DNA substrate is pushed through.

Rotational symmetry of the structured Chip/LDB-SSDP core module of the Wnt enhanceosome.

The Chip/LIM-domain binding protein (LDB)-single-stranded DNA-binding protein (SSDP) (ChiLS) complex controls numerous cell-fate decisions in animal cells, by mediating transcription of developmental control genes via remote enhancers. ChiLS is recruited to these enhancers by lineage-specific LIM-domain proteins that bind to its Chip/LDB subunit. ChiLS recently emerged as the core module of the Wnt enhanceosome, a multiprotein complex that primes developmental control genes for timely Wnt responses. ChiLS binds to NPFxD motifs within Pygopus (Pygo) and the Osa/ARID1A subunit of the BAF chromatin remodeling complex, which could synergize with LIM proteins in tethering ChiLS to enhancers. Chip/LDB and SSDP both contain N-terminal dimerization domains that constitute the bulk of their structured cores. Here, we report the crystal structures of these dimerization domains, in part aided by DARPin chaperones. We conducted systematic surface scanning by structure-designed mutations, followed by in vitro and in vivo binding assays, to determine conserved surface residues required for binding between Chip/LDB, SSDP, and Pygo-NPFxD. Based on this, and on the 4:2 (SSDP-Chip/LDB) stoichiometry of ChiLS, we derive a highly constrained structural model for this complex, which adopts a rotationally symmetrical SSDP2-LDB2-SSDP2 architecture. Integrity of ChiLS is essential for Pygo binding, and our mutational analysis places the NPFxD pockets on either side of the Chip/LDB dimer, each flanked by an SSDP dimer. The symmetry and multivalency of ChiLS underpin its function as an enhancer module integrating Wnt signals with lineage-specific factors to operate context-dependent transcriptional switches that are pivotal for normal development and cancer.

Novel CaM-binding motif in its NudT9H domain contributes to temperature sensitivity of TRPM2.

TRPM2 is a non-selective, Ca2+-permeable cation channel, which plays a role in cell death but also contributes to diverse immune cell functions. In addition, TRPM2 contributes to the control of body temperature and is involved in perception of non-noxious heat and thermotaxis. TRPM2 is regulated by many factors including Ca2+, ADPR, 2'-deoxy-ADPR, Ca2+-CaM, and temperature. However, the molecular basis for the temperature sensitivity of TRPM2 as well as the interplay between the regulatory factors is still not understood. Here we identify a novel CaM-binding site in the unique NudT9H domain of TRPM2. Using a multipronged biophysical approach we show that binding of Ca2+-CaM to this site occurs upon partial unfolding at temperatures >35 °C and prevents further thermal destabilization. In combination with patch-clamp measurements of full-length TRPM2 our results suggest a role of this CaM-binding site in the temperature sensitivity of TRPM2. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Constitutive scaffolding of multiple Wnt enhanceosome components by Legless/BCL9.

Wnt/ß-catenin signaling elicits context-dependent transcription switches that determine normal development and oncogenesis. These are mediated by the Wnt enhanceosome, a multiprotein complex binding to the Pygo chromatin reader and acting through TCF/LEF-responsive enhancers. Pygo renders this complex Wnt-responsive, by capturing ß-catenin via the Legless/BCL9 adaptor. We used CRISPR/Cas9 genome engineering of Drosophila legless (lgs) and human BCL9 and B9L to show that the C-terminus downstream of their adaptor elements is crucial for Wnt responses. BioID proximity labeling revealed that BCL9 and B9L, like PYGO2, are constitutive components of the Wnt enhanceosome. Wnt-dependent docking of ß-catenin to the enhanceosome apparently causes a rearrangement that apposes the BCL9/B9L C-terminus to TCF. This C-terminus binds to the Groucho/TLE co-repressor, and also to the Chip/LDB1-SSDP enhanceosome core complex via an evolutionary conserved element. An unexpected link between BCL9/B9L, PYGO2 and nuclear co-receptor complexes suggests that these ß-catenin co-factors may coordinate Wnt and nuclear hormone responses.

Essential role of the Dishevelled DEP domain in a Wnt-dependent human-cell-based complementation assay.

Dishevelled (DVL) assembles Wnt signalosomes through dynamic head-to-tail polymerisation by means of its DIX domain. It thus transduces Wnt signals to cytoplasmic effectors including ß-catenin, to control cell fates during normal development, tissue homeostasis and also in cancer. To date, most functional studies of Dishevelled relied on its Wnt-independent signalling activity resulting from overexpression, which is sufficient to trigger polymerisation, bypassing the requirement for Wnt signals. Here, we generate a human cell line devoid of endogenous Dishevelled (DVL1- DVL3), which lacks Wnt signal transduction to ß-catenin. However, Wnt responses can be restored by DVL2 stably re-expressed at near-endogenous levels. Using this assay to test mutant DVL2, we show that its DEP domain is essential, whereas its PDZ domain is dispensable, for signalling to ß-catenin. Our results imply two mutually exclusive functions of the DEP domain in Wnt signal transduction - binding to Frizzled to recruit Dishevelled to the receptor complex, and dimerising to cross-link DIX domain polymers for signalosome assembly. Our assay avoids the caveats associated with overexpressing Dishevelled, and provides a powerful tool for rigorous functional tests of this pivotal human signalling protein.

Wnt Signalosome Assembly by DEP Domain Swapping of Dishevelled.

Extracellular signals are often transduced by dynamic signaling complexes ("signalosomes") assembled by oligomerizing hub proteins following their recruitment to signal-activated transmembrane receptors. A paradigm is the Wnt signalosome, which is assembled by Dishevelled via reversible head-to-tail polymerization by its DIX domain. Its activity causes stabilization of ß-catenin, a Wnt effector with pivotal roles in animal development and cancer. How Wnt triggers signalosome assembly is unknown. Here, we use structural analysis, as well as biophysical and cell-based assays, to show that the DEP domain of Dishevelled undergoes a conformational switch, from monomeric to swapped dimer, to trigger DIX-dependent polymerization and signaling to ß-catenin. This occurs in two steps: binding of monomeric DEP to Frizzled followed by DEP domain swapping triggered by its high local concentration upon Wnt-induced recruitment into clathrin-coated pits. DEP domain swapping confers directional bias on signaling, and the dimerization provides cross-linking between Dishevelled polymers, illustrating a key principle underlying signalosome formation.

Experimental and Theoretical Evaluation of the Ethynyl Moiety as a Halogen Bioisostere.

Bioisosteric replacements are widely used in medicinal chemistry to improve physicochemical and ADME properties of molecules while retaining or improving affinity. Here, using the p53 cancer mutant Y220C as a test case, we investigate both computationally and experimentally whether an ethynyl moiety is a suitable bioisostere to replace iodine in ligands that form halogen bonds with the protein backbone. This bioisosteric transformation is synthetically feasible via Sonogashira cross-coupling. In our test case of a particularly strong halogen bond, replacement of the iodine with an ethynyl group resulted in a 13-fold affinity loss. High-resolution crystal structures of the two analogues in complex with the p53-Y220C mutant enabled us to correlate the different affinities with particular features of the binding site and subtle changes in ligand binding mode. In addition, using QM calculations and analyzing the PDB, we provide general guidelines for identifying cases where such a transformation is likely to improve ligand recognition.

An ancient Pygo-dependent Wnt enhanceosome integrated by Chip/LDB-SSDP.

TCF/LEF factors are ancient context-dependent enhancer-binding proteins that are activated by ß-catenin following Wnt signaling. They control embryonic development and adult stem cell compartments, and their dysregulation often causes cancer. ß-catenin-dependent transcription relies on the NPF motif of Pygo proteins. Here, we use a proteomics approach to discover the Chip/LDB-SSDP (ChiLS) complex as the ligand specifically binding to NPF. ChiLS also recognizes NPF motifs in other nuclear factors including Runt/RUNX2 and Drosophila ARID1, and binds to Groucho/TLE. Studies of Wnt-responsive dTCF enhancers in the Drosophila embryonic midgut indicate how these factors interact to form the Wnt enhanceosome, primed for Wnt responses by Pygo. Together with previous evidence, our study indicates that ChiLS confers context-dependence on TCF/LEF by integrating multiple inputs from lineage and signal-responsive factors, including enhanceosome switch-off by Notch. Its pivotal function in embryos and stem cells explain why its integrity is crucial in the avoidance of cancer.

Competitive binding of a benzimidazole to the histone-binding pocket of the Pygo PHD finger.

The Pygo-BCL9 complex is a chromatin reader, facilitating ß-catenin-mediated oncogenesis, and is thus emerging as a potential therapeutic target for cancer. Its function relies on two ligand-binding surfaces of Pygo's PHD finger that anchor the histone H3 tail methylated at lysine 4 (H3K4me) with assistance from the BCL9 HD1 domain. Here, we report the first use of fragment-based screening by NMR to identify small molecules that block protein-protein interactions by a PHD finger. This led to the discovery of a set of benzothiazoles that bind to a cleft emanating from the PHD-HD1 interface, as defined by X-ray crystallography. Furthermore, we discovered a benzimidazole that docks into the H3K4me specificity pocket and displaces the native H3K4me peptide from the PHD finger. Our study demonstrates the ligandability of the Pygo-BCL9 complex and uncovers a privileged scaffold as a template for future development of lead inhibitors of oncogenesis.

Structure of the SAS-6 cartwheel hub from Leishmania major.

Centrioles are cylindrical cell organelles with a ninefold symmetric peripheral microtubule array that is essential to template cilia and flagella. They are built around a central cartwheel assembly that is organized through homo-oligomerization of the centriolar protein SAS-6, but whether SAS-6 self-assembly can dictate cartwheel and thereby centriole symmetry is unclear. Here we show that Leishmania major SAS-6 crystallizes as a 9-fold symmetric cartwheel and provide the X-ray structure of this assembly at a resolution of 3.5 Å. We furthermore demonstrate that oligomerization of Leishmania SAS-6 can be inhibited by a small molecule in vitro and provide indications for its binding site. Our results firmly establish that SAS-6 can impose cartwheel symmetry on its own and indicate how this process might occur mechanistically in vivo. Importantly, our data also provide a proof-of-principle that inhibition of SAS-6 oligomerization by small molecules is feasible. DOI: http://dx.doi.org/10.7554/eLife.01812.001.

Evolutionary adaptation of the fly Pygo PHD finger toward recognizing histone H3 tail methylated at arginine 2.

Pygo proteins promote Armadillo- and ß-catenin-dependent transcription, by relieving Groucho-dependent repression of Wnt targets. Their PHD fingers bind histone H3 tail methylated at lysine 4, and to the HD1 domain of their Legless/BCL9 cofactors, linking Pygo to Armadillo/ß-catenin. Intriguingly, fly Pygo orthologs exhibit a tryptophan > phenylalanine substitution in their histone pocket-divider which reduces their affinity for histones. Here, we use X-ray crystallography and NMR, to discover a conspicuous groove bordering this phenylalanine in the Drosophila PHD-HD1 complex--a semi-aromatic cage recognizing asymmetrically methylated arginine 2 (R2me2a), a chromatin mark of silenced genes. Our structural model of the ternary complex reveals a distinct mode of dimethylarginine recognition, involving a polar interaction between R2me2a and its groove, the structural integrity of which is crucial for normal tissue patterning. Notably, humanized fly Pygo derepresses Notch targets, implying an inherent Notch-related function of classical Pygo orthologs, disabled in fly Pygo, which thus appears dedicated to Wnt signaling.

An essential role for the ATG8 ortholog LC3C in antibacterial autophagy.

Autophagy defends the mammalian cytosol against bacterial invasion. Efficient bacterial engulfment by autophagy requires cargo receptors that bind (a) homolog(s) of the ubiquitin-like protein Atg8 on the phagophore membrane. The existence of multiple ATG8 orthologs in higher eukaryotes suggests that they may perform distinct functions. However, no specific role has been assigned to any mammalian ATG8 ortholog. We recently discovered that the autophagy receptor CALCOCO2/NDP52, which detects cytosol-invading Salmonella enterica serovar Typhimurium (S. Typhimurium), preferentially binds LC3C. The CALCOCO2/NDP52-LC3C interaction is essential for cell-autonomous immunity against cytosol-exposed S. Typhimurium, because cells lacking either protein fail to target bacteria into the autophagy pathway. The selectivity of CALCOCO2/NDP52 for LC3C is determined by a novel LC3C interacting region (CLIR), in which the lack of the key aromatic residue of canonical LIRs is compensated by LC3C-specific interactions. Our findings provide a new layer of regulation to selective autophagy, suggesting that specific interactions between autophagy receptors and the ATG8 orthologs are of biological importance.