Summary Cytosolic DNA arising from intracellular bacteria or viral infections is a powerful pathogen-associated molecular pattern (PAMP) that leads to innate immune host defense by the production of type I interferon and inflammatory cytokines. Recognition of cytosolic DNA by the recently discovered cyclic-GMP-AMP (cGA) synthase (cGAS) induces the production of cGA to activate the stimulator of interferon genes (STING). Here we report the crystal structure of cGAS alone and in complex with DNA, ATP and GTP along with functional studies. Our results explain cGAS’ broad specificity DNA sensing, show how cGAS catalyzes di-nucleotide formation and indicate activation by a DNA-induced structural switch. cGAS possesses a remarkable structural similarity to the antiviral cytosolic dsRNA sensor 2’-5’oligoadenylate synthase (OAS1), but contains a unique zinc-thumb that recognizes B-form dsDNA. Our results mechanistically unify dsRNA and dsDNA innate immune sensing by OAS1 and cGAS nucleotidyl transferases.

DNA in the eukaryotic nucleus is packaged in the form of nucleosomes, ~147 base pairs of DNA wrapped around a histone protein octamer. The position and histone composition of nucleosomes is governed by ATP dependent chromatin remodelers1–3 such as the 15 subunit INO80 complex4. INO80 regulates gene expression, DNA repair and replication by sliding nucleosomes, exchanging histone H2A.Z with H2A, and positioning +1 and -1 nucleosomes at promoter DNA5–8. A structure and mechanism for these remodeling reactions is lacking. Here we report the cryo-electron microscopy structure at 4.3Å resolution, with parts at 3.7Å, of an evolutionary conserved core INO80 complex from Chaetomium thermophilum bound to a nucleosome. INO80core cradles one entire gyre of the nucleosome through multivalent DNA and histone contacts. A Rvb1/2 AAA+ ATPase hetero-hexamer is an assembly scaffold for the complex and acts as stator for the motor and nucleosome gripping subunits. The Swi2/Snf2 ATPase motor binds to SHL-6, unwraps ~15 base pairs, disrupts the H2A:DNA contacts and is poised to pump entry DNA into the nucleosome. Arp5-Ies6 grip SHL-2/-3 acting as counter grip for the motor on the other side of the H2A/H2B dimer. The Arp5 insertion domain forms a grappler element that binds the nucleosome dyad, connects the Arp5 core and entry DNA over a distance of ~90Å and packs against histone H2A/H2B near the acidic patch. Our structure together with biochemical data8 suggest a unified mechanism for nucleosome sliding and histone editing by INO80. The motor pumps entry DNA across H2A/H2B against Arp5 and the grappler, sliding nucleosomes as a ratchet. Transient exposure of H2A/H2B by the motor and differential recognition of H2A.Z and H2A may regulate histone exchange during translocation.

The retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) melanoma differentiation-associated protein 5 (MDA5) senses cytoplasmic viral RNA and activates antiviral innate immunity. To reveal how paramyxoviruses counteract this response, we determined the crystal structure of the MDA5 adenosine 5'-triphosphate (ATP)-hydrolysis domain in complex with the viral inhibitor V protein. The V protein unfolded the ATP-hydrolysis domain of MDA5 via a β-hairpin motif and recognized a structural motif of MDA5 that is normally buried in the conserved helicase fold. This leads to disruption of the MDA5 ATP-hydrolysis site and prevention of RNA-bound MDA5 filament formation. The structure explains why V proteins inactivate MDA5, but not RIG-I, and mutating only two amino acids in RIG-I induces robust V protein binding. Our results suggest an inhibition mechanism of RLR signalosome formation by unfolding of receptor and inhibitor.

Swi2/Snf2-type ATPases broadly regulate genome-associated processes such as transcription, replication and repair by catalyzing disruption, assembly, or remodeling of nucleosomes or other protein:DNA complexes1,2. ATP-driven motor activity along DNA has been suggested to disrupt target protein:DNA interactions in the remodeling reaction3–5. However, the complex and highly specific remodeling reactions are poorly understood, mostly because we lack high-resolution structural information on how remodelers bind their substrate proteins. Mot1 (modifier of transcription 1, denoted BTAF1 in humans) is a Swi2/Snf2 enzyme that specifically displaces TATA box binding protein (TBP) from promoter DNA and globally regulates transcription by generating a highly dynamic TBP pool in the cell6,7. As a Swi2/Snf2 enzyme that functions as a single polypeptide and interacts with a relatively simple substrate, Mot1 offers an ideal system for a better understanding of this important enzyme family. To reveal how Mot1 specifically disrupts TBP:DNA, we combined crystal and electron microscopy structures of Mot1:TBP complexes with biochemical studies. Here we show that Mot1 wraps around TBP and appears to act like a bottle opener: a spring-like array of 16 HEAT (huntingtin, elongation factor 3, PP2A and lipid kinase TOR) repeats grips the DNA distal side of TBP via loop insertions, while the Swi2/Snf2 domain binds upstream DNA, positioned to weaken TBPs DNA interaction by DNA translocation. A “latch” subsequently blocks TBP’s DNA binding groove, acting as a chaperone to prevent DNA re-association for efficient promoter clearance. This work shows how a remodeling enzyme can combine both motor and chaperone activities to achieve functional specificity using a conserved Swi2/Snf2 translocase.

RIG-I detects cytosolic viral dsRNA with 50 triphosphates (5 0 -pppdsRNA), thereby initiating an antiviral innate immune response. Here we report the crystal structure of superfamily 2 (SF2) ATPase domain of RIG-I in complex with a nucleotide analogue. RIG-I SF2 comprises two RecA-like domains 1A and 2A and a helical insertion domain 2B, which together form a 'C'-shaped structure. Domains 1A and 2A are maintained in a 'signal-off' state with an inactive ATP hydrolysis site by an intriguing helical arm. By mutational analysis, we show surface motifs that are critical for dsRNA-stimulated ATPase activity, indicating that dsRNA induces a structural movement that brings domains 1A and 2A/B together to form an active ATPase site. The structure also indicates that the regulatory domain is close to the end of the helical arm, where it is well positioned to recruit 5 0 -ppp-dsRNA to the SF2 domain. Overall, our results indicate that the activation of RIG-I occurs through an RNA-and ATP-driven structural switch in the SF2 domain.

The cytosolic antiviral innate immune sensor RIG-I distinguishes 5′ tri- or diphosphate containing viral double-stranded (ds) RNA from self-RNA by an incompletely understood mechanism that involves ATP hydrolysis by RIG-I's RNA translocase domain. Recently discovered mutations in ATPase motifs can lead to the multi-system disorder Singleton-Merten Syndrome (SMS) and increased interferon levels, suggesting misregulated signaling by RIG-I. Here we report that SMS mutations phenocopy a mutation that allows ATP binding but prevents hydrolysis. ATPase deficient RIG-I constitutively signals through endogenous RNA and co-purifies with self-RNA even from virus infected cells. Biochemical studies and cryo-electron microscopy identify a 60S ribosomal expansion segment as a dominant self-RNA that is stably bound by ATPase deficient RIG-I. ATP hydrolysis displaces wild-type RIG-I from this self-RNA but not from 5' triphosphate dsRNA. Our results indicate that ATP-hydrolysis prevents recognition of self-RNA and suggest that SMS mutations lead to unintentional signaling through prolonged RNA binding.DOI: http://dx.doi.org/10.7554/eLife.10859.001

Arrays of regularly spaced nucleosomes dominate chromatin and are often phased by alignment to reference sites like active promoters. How the distances between nucleosomes (spacing), and between phasing sites and nucleosomes are determined remains unclear, and specifically, how ATP-dependent chromatin remodelers impact these features. Here, we used genome-wide reconstitution to probe how Saccharomyces cerevisiae ATP-dependent remodelers generate phased arrays of regularly spaced nucleosomes. We find that remodelers bear a functional element named the ‘ruler’ that determines spacing and phasing in a remodeler-specific way. We use structure-based mutagenesis to identify and tune the ruler element residing in the Nhp10 and Arp8 modules of the INO80 remodeler complex. Generally, we propose that a remodeler ruler regulates nucleosome sliding direction bias in response to (epi)genetic information. This finally conceptualizes how remodeler-mediated nucleosome dynamics determine stable steady-state nucleosome positioning relative to other nucleosomes, DNA bound factors, DNA ends and DNA sequence elements.

The fundamental molecular determinants by which ATP-dependent chromatin remodelers organize nucleosomes across eukaryotic genomes remain largely elusive. Here, chromatin reconstitutions on physiological, whole-genome templates reveal how remodelers read and translate genomic information into nucleosome positions. Using the yeast genome and the multi-subunit INO80 remodeler as a paradigm, we identify DNA shape/mechanics encoded signature motifs as sufficient for nucleosome positioning and distinct from known DNA sequence preferences of histones. INO80 processes such information through an allosteric interplay between its core- and Arp8-modules that probes mechanical properties of nucleosomal and linker DNA. At promoters, INO80 integrates this readout of DNA shape/mechanics with a readout of co-evolved sequence motifs via interaction with general regulatory factors bound to these motifs. Our findings establish a molecular mechanism for robust and yet adjustable +1 nucleosome positioning and, more generally, remodelers as information processing hubs that enable active organization and allosteric regulation of the first level of chromatin.