The F-actin binding cytoskeletal protein α-catenin interacts with
β-catenin-cadherin complexes and stabilizes cell-cell junctions. The
β-catenin–α-catenin complex cannot bind to F-actin,
whereas interactions of α-catenin with the cytoskeletal protein vinculin
appear necessary to stabilize adherens junctions. Here we report the crystal
structure of nearly full-length human α-catenin at 3.7 Å
resolution. α-Catenin forms an asymmetric dimer, where the four-helix
bundle domains of each subunit engage in distinct intermolecular interactions.
This results in a left handshake-like dimer, where the two subunits have
remarkably different conformations. The crystal structure explains why dimeric
α-catenin has a higher affinity for F-actin than monomeric
α-catenin, why the β-catenin–α-catenin complex
does not bind to F-actin, how activated vinculin links the cadherin-catenin
complex to the cytoskeleton, and why α-catenin but not inactive vinculin
can bind to F-actin.
Actin has well-established functions in the cytoplasm, but its roles in the nucleus remain poorly defined. Here, by studying the nuclear actin-containing yeast INO80 chromatin remodeling complex, we provide genetic and biochemical evidence for a role of monomeric actin in INO80 chromatin remodeling. In contrast to cytoplasmic actin, nuclear actin is present as a monomer in the INO80 complex and its barbed end is not accessible for polymerization. An actin mutation affecting in vivo nuclear functions is identified in subdomain 2, which reduces the chromatin remodeling activities of the INO80 complex in vitro. Importantly, the highly conserved subdomain 2 at the pointed end of actin contributes to INO80 interactions with chromatin. Our results establish an evolutionarily conserved function of nuclear actin in its monomeric form and suggest that nuclear actin can utilize a fundamentally distinct mechanism from cytoplasmic actin.
PTEN is a tumor suppressor gene that has been shown to be under the regulatory control of a PTEN pseudogene expressed noncoding RNA, PTENpg1. Here, we characterize a previously unidentified PTENpg1 encoded antisense RNA (asRNA), which regulates PTEN transcription and PTEN mRNA stability. We find two PTENpg1 asRNA isoforms, alpha and beta. The alpha isoform functions in trans, localizes to the PTEN promoter, and epigenetically modulates PTEN transcription by the recruitment of DNMT3a and EZH2. In contrast, the beta isoform interacts with PTENpg1 through an RNA:RNA pairing interaction, which affects PTEN protein output via changes of PTENpg1 stability and microRNA sponge activity. Disruption of this asRNA-regulated network induces cell cycle arrest and sensitizes cells to doxorubicin, suggesting a biological function for the respective PTENpg1 expressed asRNAs.
Pseudogene; PTEN; PTENp1; PTENpg1; antisense RNA; noncoding RNA; Epigenetics; Transcriptional regulation; DNMT3a; EZH2
G protein-coupled receptors (GPCRs) mediate transmembrane signaling. Before ligand binding, GPCRs exist in a basal state. Crystal structures of several GPCRs bound with antagonists or agonists have been solved. However, the crystal structure of the ligand-free basal state of a GPCR, the starting point of GPCR activation and function, has not been determined. Here we report the X-ray crystal structure of the first ligand-free basal state of a GPCR in a lipid membrane-like environment. Oligomeric turkey β1-adrenergic receptors display two alternating dimer interfaces. One interface involves the transmembrane domain (TM) 1, TM2, the C-terminal H8, and the extracellular loop 1. The other interface engages residues from TM4, TM5, the intracellular loop 2 and the extracellular loop 2. Structural comparisons show that this ligand-free state is in an inactive conformation. This provides the structural information regarding GPCR dimerization and oligomerization.
The inhibitory protein SOCS3 plays a key role in the immune and hematopoietic systems by regulating signaling induced by specific cytokines. SOCS3 functions by inhibiting the catalytic activity of Janus Kinases (JAKs) that initiate signaling within the cell. We determined the crystal structure of a ternary complex between murine SOCS3, JAK2 (kinase domain) and a fragment of the IL-6 receptor β-chain. The structure shows that SOCS3 binds JAK2 and receptor simultaneously, using two opposing surfaces. Whilst the phosphotyrosine-binding groove on the SOCS3 SH2 domain is occupied by receptor, JAK2 binds in a phospho-independent manner to a non-canonical surface. The kinase inhibitory region of SOCS3 occludes the substrate-binding groove on JAK2 and biochemical studies show it blocks substrate association. These studies reveal that SOCS3 targets specific JAK-cytokine receptor pairs and explains the mechanism and specificity of SOCS action.
SIX1 interacts with EYA to form a bipartite transcription factor essential for development. Loss of function of this complex causes branchio-oto-renal syndrome (BOR), while re-expression of SIX1 or EYA promotes metastasis. Here we describe the 2.0 Å structure of SIX1 bound to EYA2, which suggests a novel DNA binding mechanism for SIX1 and provides a rationale for the effect of BOR syndrome mutations. The structure also reveals that SIX1 uses predominantly a single helix to interact with EYA. Substitution of a single amino acid in this helix is sufficient to disrupt the SIX1–EYA interaction, SIX1-mediated epithelial-mesenchymal transition and metastasis in mouse models. Given that SIX1 and EYA are co-overexpressed in many tumor types, our data indicate that targeting the SIX1–EYA complex may be a potent approach to inhibit tumor progression in multiple cancer types.
Telomerase contains a large RNA subunit TER and a protein catalytic subunit TERT. Whether telomerase functions as monomer or dimer has been a matter of debate. Here we report biochemical and labeling data that show that in vivo assembled human telomerase contains two TERT subunits and binds two telomeric DNA substrates. Importantly, catalytic activity requires both TERT active sites to be functional, demonstrating that human telomerase functions as a dimer. We also present the three-dimensional structure of active, full-length human telomerase dimer, determined by single-particle electron microscopy in negative stain. Telomerase has a bilobal architecture, with the two monomers linked by a flexible interface. The monomer reconstruction at 23Å resolution, and fitting of the atomic structure of the beetle TERT subunit reveals the spatial relationship between RNA and protein subunits, providing insights into the telomerase architecture.
The 70kD heat shock proteins (Hsp70s) are ubiquitous molecular chaperones essential for cellular protein folding and proteostasis. Each Hsp70 has two functional domains: a nucleotide-binding domain (NBD) that binds and hydrolyzes ATP, and a substrate-binding domain (SBD) that binds extended polypeptides. NBD and SBD interact little when in ADP; however, ATP binding allosterically couples the polypeptide- and ATP-binding sites. ATP binding promotes polypeptide release; polypeptide rebinding stimulates ATP hydrolysis. This allosteric coupling is poorly understood. Here we present the crystal structure of an intact Hsp70 from Escherichia coli in an ATP-bound state at 1.96 Å resolution. NBD-ATP adopts a unique conformation, forming extensive interfaces with a radically changed SBD that has its α-helical lid displaced and the polypeptide-binding channel of its β-subdomain restructured. These conformational changes together with our biochemical tests provide a long-sought structural explanation for allosteric coupling in Hsp70 activity.
We report the early conformation of the E. coli signal recognition particle (SRP) and its receptor FtsY bound to the translating ribosome by cryo-electron microscopy. FtsY binds to the tetraloop of the SRP RNA whereas the NG-domains of the SRP protein and FtsY interact weakly in this conformation. Our results suggest that optimal positioning of the SRP RNA tetraloop and the Ffh NG-domain leads to FtsY recruitment.
Naïve pluripotent embryonic stem (ESCs) cells and embryonic germ (EGCs) cells are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however we find that EGCs exhibit equivalent levels of global DNA methylation to ESCs. Importantly, inhibition of MEK and Gsk3b by 2i conditions leads to a pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instils a naïve pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs.
Transcription has the capacity to modify mechanically DNA topology, DNA structure, and nucleosome arrangement. Resulting from ongoing transcription, these modifications in turn, may provide instant feedback to the transcription machinery. To substantiate the connection between transcription and DNA dynamics, we charted an ENCODE map of transcription-dependent dynamic supercoiling in human Burkitt lymphoma cells using psoralen photobinding to probe DNA topology in vivo. Dynamic supercoils spread ~1.5 kb upstream of the start sites of active genes. Low and high output promoters handle this torsional stress differently as shown using inhibitors of transcription and topoisomerases, and by chromatin immunoprecipation of RNA polymerase and topoisomerases I and II. Whereas lower outputs are managed adequately by topoisomerase I, high output promoters additionally require topoisomerase II. The genome-wide coupling between transcription and DNA topology emphasizes the importance of dynamic supercoiling for gene regulation.
The pathogenic sequelae of BRCA1 mutation in human and mouse cells are mitigated by concomitant deletion of 53BP1, which binds histone H4 dimethylated at Lys20 (H4K20me2) to promote nonhomologous end-joining, suggesting a balance between BRCA1 and 53BP1 regulates DNA double-strand break (DSB) repair mechanism choice. Here, we document that acetylation is a key determinant of this balance. TIP60 acetyltransferase deficiency reduced BRCA1 at DSB chromatin with commensurate increases in 53BP1, while HDAC inhibition yielded the opposite effect. TIP60 -dependent H4 acetylation diminished 53BP1 binding to H4K20me2 in part through disruption of a salt bridge between H4K16 and Glu1551 in the 53BP1 Tudor domain. Moreover, TIP60 deficiency impaired HR and conferred sensitivity to PARP inhibition in a 53BP1-dependent manner. These findings demonstrate that acetylation in cis to H4K20me2 regulates relative BRCA1 and 53BP1 DSB chromatin occupancy to direct DNA repair mechanism.
TIP60; Acetylation; BRCA1; 53BP1; Homologous recombination; PARP inhibitors
Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM 3D reconstructions of human full-length PLCβ3 in complex with murine Gαq. The distal CTD forms an extended, monomeric helical bundle consisting of three anti-parallel segments with structural similarity to membrane-binding bin–amphiphysin–Rvs (BAR) domains. Sequence conservation of the distal CTD identifies putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gαq in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests the distal CTD plays roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis.
Influenza virus hemagglutinin (HA) mediates receptor binding and viral entry during influenza infection. The development of receptor analogs as viral entry blockers has not been successful, suggesting that sialic acid may not be an ideal scaffold to obtain broad and potent HA inhibitors. Here we report crystal structures of Fab fragments from three human antibodies that neutralize the 1957 pandemic H2N2 influenza virus in complex with H2 HA. All three antibodies use an aromatic residue to plug a conserved cavity in the HA receptor-binding site. Each antibody interacts with the absolutely conserved HA1 Trp153 at the cavity base through π-π stacking with the signature Phe54 of two VH1-69 antibodies or a tyrosine from HCDR3 in the other antibody. This remarkably conserved interaction can be used as a starting point to design inhibitors targeting this conserved hydrophobic pocket in influenza viruses.
Oligomeric complexes of Trax and Translin proteins, known as C3POs, participate in a variety of eukaryotic nucleic acid metabolism pathways including RNAi and tRNA processing. In RNAi in humans and Drosophila, C3PO activates pre-RISC by removing the passenger strand of the siRNA precursor duplex using nuclease activity present in Trax. It is not known how C3POs engage with nucleic acid substrates. Here we identify a single protein from Archaeoglobus fulgidus that assembles into an octamer with striking similarity to human C3PO. The structure in complex with duplex RNA reveals that the octamer entirely encapsulates a single thirteen base-pair RNA duplex inside a large inner cavity. Trax-like subunit catalytic sites target opposite strands of the duplex for cleavage, separated by seven base pairs. The structure provides insight into the mechanism of RNA recognition and cleavage by an archaeal C3PO-like complex.
DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling we used biotinylated-trimethylpsoralen as a DNA structure probe to show the genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF binding sites. Under-wound domains are transcriptionally active, enriched in topoisomerase I, “open” chromatin fibers and DNaseI sites, but are depleted of topoisomerase II. Furthermore DNA supercoiling impacts on additional levels of chromatin compaction as under-wound domains are cytologically decondensed, topologically constrained, and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation providing an evolutionary purpose for clustering genes along chromosomes.
The toxin colicin E3 targets the 30S subunit of bacterial ribosomes and cleaves a phosphodiester bond in the decoding center. We present the crystal structure of the 70S ribosome in complex with the cytotoxic domain of colicin E3 (E3-rRNase). The structure reveals how the rRNase domain of colicin binds to the A site of the decoding center in the 70S ribosome and cleaves 16S rRNA between A1493 and G1494. The cleavage mechanism involves the concerted action of conserved residues Glu62 and His58 of the cytotoxic domain of colicin E3 that activate the 16S rRNA for 2′ OH induced hydrolysis. Conformational changes observed for E3-rRNase, 16S rRNA and Helix 69 of 23S rRNA suggest that a dynamic binding platform is required for colicin E3 binding and function.
Most human genes produce multiple splicing isoforms with distinct functions. To systematically understand splicing regulation, we conducted an unbiased screen and identified >100 intronic splicing enhancers (ISEs) that were clustered by sequence similarity into six groups. All ISEs functioned in another cell type and heterologous introns, and their distribution and conservation patterns in different pre-mRNA regions are similar to exonic splicing silencers. Consistently all ISEs inhibited use of splice sites from exonic locations. The putative trans-factors of each ISE group were identified and validated. Five distinct ISE motifs were recognized by hnRNP H and F whose C-terminal domains were sufficient to render context-dependent activities of ISEs. The sixth group was controlled by factors that either activate or suppress splicing. This work provided a comprehensive picture of general ISE activities and provided new models of how a single element can function oppositely depending on its locations and binding factors.
splicing regulation; splicing factors; RNA binding protein; context dependent activity
Promoter-proximal pausing by RNA polymerase II (Pol II) ensures both gene-specific regulation and RNA quality control. Structural considerations suggested initiation factor eviction would be required for elongation factor engagement and pausing of transcription complexes. Here we show that selective inhibition of Cdk7—part of TFIIH—increases TFIIE retention, prevents DRB-sensitivity inducing factor (DSIF) recruitment and attenuates pausing in human cells. Pause release depends on Cdk9—cyclin T1 (P-TEFb); Cdk7 is also required for Cdk9-activating phosphorylation and Cdk9-dependent downstream events—Pol II carboxyl-terminal domain Ser2 phosphorylation and histone H2B ubiquitylation—in vivo. Cdk7 inhibition, moreover, impairs Pol II transcript 3′-end formation. Cdk7 thus acts through TFIIE and DSIF to establish and through P-TEFb to relieve barriers to elongation: incoherent feedforward that might create a window to recruit RNA-processing machinery. Therefore, cyclin-dependent kinases govern Pol II handoff from initiation to elongation factors and co-transcriptional RNA maturation.
RIG-I is a cytosolic helicase that senses 5’-ppp-RNA contained in negative strand RNA viruses and triggers innate antiviral immune responses. Calorimetric binding studies establish that the RIG-I C-terminal regulatory domain (CTD) binds to blunt-end double-stranded 5’-ppp-RNA a factor of 17 more tightly than to its single-stranded counterpart. Here we report on the crystal structure of RIG-I CTD domain bound to both blunt-ends of a self-complementary 5’-ppp-dsRNA 12-mer, with interactions involving 5’-pp clearly visible in the complex. The structure, supported by mutation studies, defines how a lysine-rich basic cleft within the RIG-I CTD domain sequesters the observable 5’-pp of the bound RNA, with a stacked Phe capping the terminal base pair. Key intermolecular interactions observed in the crystalline state are retained in the complex of 5’-ppp-dsRNA 24-mer and full-length RIG-I under in vivo conditions, as evaluated from the impact of binding pocket RIG-I mutations and 2’-OCH3 RNA modifications on the interferon response.
Many proteins cannot fold without the assistance of chaperonin machines like GroEL and GroES. The nature of this assistance, however, remains poorly understood. Here we demonstrate that unfolding of a substrate protein by GroEL enhances protein folding. We first show that capture of a protein on the open ring of a GroEL–ADP–GroES complex, GroEL’s physiological acceptor state for non-native proteins in vivo, leaves the substrate protein in an unexpectedly compact state. Subsequent binding of ATP to the same GroEL ring causes rapid, forced unfolding of the substrate protein. Notably, the fraction of the substrate protein that commits to the native state following GroES binding and protein release into the GroEL–GroES cavity is proportional to the extent of substrate-protein unfolding. Forced protein unfolding is thus a central component of the multilayered stimulatory mechanism used by GroEL to drive protein folding.
While liganded nuclear receptors are established to regulate Pol II-dependent transcription units, their role in regulating Pol III-transcribed DNA repeats remains largely unknown. Here, we report that ~2–3% of the ~100,000–200,000 human DR2 Alu repeats in proximity to activated Pol II transcription units are activated by retinoic acid receptor in human embryonic stem cells to generate Pol III-dependent RNAs. These transcripts are processed, initially in a DICER-dependent fashion, into small RNAs (~28–65nt.), referred to as riRNAs, that cause degradation of a subset of critical stem cell mRNAs, including Nanog, modulating exit from the proliferative stem cell state. This regulation requires AGO3-dependent accumulation of processed DR2 Alu transcripts and subsequent recruitment of AGO3-associated decapping complexes to the target mRNA. In this way, the RAR and Pol III-dependent DR2 Alu transcriptional events in stem cells functionally complement the Pol II-dependent neuronal transcriptional program.
Nucleosome positioning is critical for gene expression and most DNA-related processes. Here, we review the dominant patterns of nucleosome positioning that have been observed, and summarize current understanding of their underlying determinants. The genome-wide pattern of nucleosome positioning is determined by the combination of DNA sequence, ATP-dependent nucleosome remodeling enzymes, and transcription factors including activators, components of the preinitiation complex, and elongating RNA polymerase II. These determinants influence each other such that the resulting nucleosome positioning patterns are likely to differ among genes and among cells within a population, with consequent effects on gene expression.
The core mechanism of intracellular vesicle fusion consists of SNAREpin zippering between vesicular and target membranes. Recent studies indicate that the same SNARE-binding protein, Complexin (CPX), can act either as a facilitator or as an inhibitor of membrane fusion, giving rise to a major controversy. Here, we employ energetic measurements using the Surface Forces Apparatus which reveal that CPX acts sequentially on assembling SNAREpins, first facilitating zippering by nearly doubling the distance at which v- and t-SNAREs can engage, and then by clamping them into a half-zippered fusion-incompetent state. Specifically, we find that the central helix of CPX allows SNAREs to form this intermediate energetic state at 9–15 nm, but not when the bilayers are closer than 9 nm. Stabilizing the activated-clamped state at separations < 9 nm requires the accessory helix of CPX, which prevents membrane-proximal assembly of SNAREpins.
Dosage compensation in mammals occurs at two levels. In addition to balancing X-chromosome dosage between males and females via X-inactivation, mammals also balance dosage of Xs and autosomes. It has been proposed that X-autosome equalization occurs by upregulation of Xa (active X). To investigate mechanism, we perform allele-specific ChIP-seq for chromatin epitopes and analyze RNA-seq data. The hypertranscribed Xa demonstrates enrichment of active chromatin marks relative to autosomes. We derive predictive models for relationships among POL-II, active mark densities, and gene expression, and suggest that Xa upregulation involves increased transcription initiation and elongation. Enrichment of active marks on Xa does not scale proportionally with transcription output, a disparity explained by nonlinear quantitative dependencies among active histone marks, POL-II occupancy, and transcription. Significantly, the trend of nonlinear upregulation also occurs on autosomes. Thus, Xa upregulation involves combined increases of active histone marks and POL-II occupancy, without invoking X-specific dependencies between chromatin states and transcription.