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RNA helicases are molecular motors that are involved in virtually all aspects of RNA metabolism. Eukaryotic initiation factor (eIF) 4A is the prototypical member of the DEAD-box family of RNA helicases. It is thought to use energy from ATP hydrolysis to unwind mRNA structure and, in conjunction with other translation factors, it prepares mRNA templates for ribosome recruitment during translation initiation. In screening marine extracts for new eukaryotic translation initiation inhibitors, we identified the natural product hippuristanol. We show here that this compound is a selective and potent inhibitor of eIF4A RNA-binding activity that can be used to distinguish between eIF4A-dependent and -independent modes of translation initiation in vitro and in vivo . We also show that poliovirus replication is delayed when infected cells are exposed to hippuristanol. Our study demonstrates the feasibility of selectively targeting members of the DEAD-box helicase family with small-molecule inhibitors.

p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo–electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)–bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5′-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.

To spatially organize chromosomes, ring-shaped protein complexes including condensin and cohesin have been hypothesized to extrude DNA loops. Condensin has been shown to exhibit a DNA-translocating motor function, but extrusion has not been observed directly. Using single-molecule imaging, Ganji et al. visualized in real time a condensin-mediated, adenosine triphosphate-dependent, fast DNA loop extrusion process. Loop extrusion occurred asymmetrically, with condensin reeling in only one end of the DNA. These data provide unambiguous evidence of a loop extrusion mechanism for chromosome organization. Science , this issue p. 102 Single-molecule imaging supports a loop extrusion mechanism for the spatial organization of chromosomes. It has been hypothesized that SMC protein complexes such as condensin and cohesin spatially organize chromosomes by extruding DNA into large loops. We directly visualized the formation and processive extension of DNA loops by yeast condensin in real time. Our findings constitute unambiguous evidence for loop extrusion. We observed that a single condensin complex is able to extrude tens of kilobase pairs of DNA at a force-dependent speed of up to 1500 base pairs per second, using the energy of adenosine triphosphate hydrolysis. Condensin-induced loop extrusion was strictly asymmetric, which demonstrates that condensin anchors onto DNA and reels it in from only one side. Active DNA loop extrusion by SMC complexes may provide the universal unifying principle for genome organization.

Active coacervate droplets are liquid condensates coupled to a chemical reaction that turns over their components, keeping the droplets out of equilibrium. This turnover can be used to drive active processes such as growth, and provide an insight into the chemical requirements underlying (proto)cellular behaviour. Moreover, controlled growth is a key requirement to achieve population fitness and survival. Here we present a minimal, nucleotide-based coacervate model for active droplets, and report three key findings that make these droplets into evolvable protocells. First, we show that coacervate droplets form and grow by the fuel-driven synthesis of new coacervate material. Second, we find that these droplets do not undergo Ostwald ripening, which we attribute to the attractive electrostatic interactions and translational entropy within complex coacervates, active or passive. Finally, we show that the droplet growth rate reflects experimental conditions such as substrate, enzyme and protein concentration, and that a different droplet composition (addition of RNA) leads to altered growth rates and droplet fitness. These findings together make active coacervate droplets a powerful platform to mimic cellular growth at a single-droplet level, and to study fitness at a population level. Active coacervate droplets are droplets coupled to a chemical reaction that maintains them out of equilibrium, which can be used to drive active processes, but coacervates are still subject to passive processes that compete with or mask growth. Here, the authors present a nucleotide-based model for active coacervate droplets that form and grow by fuel-driven synthesis of ATP, and, importantly, do not undergo Ostwald ripening.

Cytosolic DNA arising from intracellular bacterial or viral infections is a powerful pathogen-associated molecular pattern (PAMP) that leads to innate immune host defence by the production of type I interferon and inflammatory cytokines. Recognition of cytosolic DNA by the recently discovered cyclic-GMP-AMP (cGAMP) synthase (cGAS) induces the production of cGAMP 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 the broad DNA sensing specificity of cGAS, show how cGAS catalyses dinucleotide formation and indicate activation by a DNA-induced structural switch. cGAS possesses a remarkable structural similarity to the antiviral cytosolic double-stranded RNA sensor 2′-5′oligoadenylate synthase (OAS1), but contains a unique zinc thumb that recognizes B-form double-stranded DNA. Our results mechanistically unify dsRNA and dsDNA innate immune sensing by OAS1 and cGAS nucleotidyl transferases. Cytosolic DNA arising from intracellular bacterial or viral infections induces type I interferon through activation of the DNA sensor cGAS, which catalyses the synthesis of cyclic dinucleotide which in turn activates STING; here the crystal structures of a carboxy-terminal fragment of cGAS alone and in complex with UTP and DNA–ATP–GTP complex are determined. DNA sensing by cGAS The mechanism of sensing and signalling of cytosolic DNA by the innate immune system is a topic of intense research interest as it is the means by which invading bacteria and viruses are detected. Cytosolic DNA is known to induce type I interferon through activation of the DNA sensor cyclic-GMP-AMP synthetase (cGAS), which catalyses the synthesis of a cyclic dinucleotide which in turn activates a protein known as STING (stimulator of interferon genes). Karl-Peter Hopfner and co-workers present the crystal structures of a C-terminal fragment of cGAS alone, in complex with UTP, and as a DNA–ATP–GTP complex. In a complementary paper [in this issue], Veit Hornung and coworkers show that the product of cGAS is distinct from previously characterized cyclic dinucleotides. Rather it is an unorthodox cyclic dinucleotide with a 2′–5′ linkage between guanosine and adenosine. This two-step synthesis of cGAMP(2′–5′) could be a focus for the development of specific inhibitors for the treatment of autoimmune diseases that engage the cGAS–STING axis.

Adenosine 5′ triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F 0 F 1 -ATPase from Bacillus PS3 . On the cell surface and within the cytosol, iATPSnFR 1.0 responds to relevant ATP concentrations (30 μM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements. ATP has essential roles in cell signalling and energy homeostasis and biosensors to detect it have many potential applications. Here, the authors develop a new ATP sensor that can be targeted to the membrane or cytosol.

An X-ray structure of human P2Y 12 receptor, a clinical drug target for platelet aggregation inhibitors, is presented in complex with an agonist, providing insight into the δ-group of class A G-protein-coupled receptors. Key platelet aggregation GPCR structures Two papers in this issue of Nature present the crystal structures of the human P2Y 12 receptor, first in complex with the antithrombotic drug AZD1283, and second, bound to a full agonist (a close analogue of endogenous agonist ADP) and to a partial agonist. P2Y receptors are a family of purinergic G-protein-coupled receptors (GPCRs) that are activated by extracellular nucleotides. The P2Y 12 receptor is found mainly on the surface of platelets, where it regulates platelet activation and thrombus formation, and it is the target of several important antithrombotic drugs. In overall structure, P2Y 12 receptor is found to be similar to other GPCRs, although both the shape and location of the ligand-binding pocket are unusual. Comparisons of the three newly determined structures reveal that agonist binding induces a large-scale rearrangement of the extracellular domains of the GPCR. The P2Y 12 receptor (P2Y 12 R), one of eight members of the P2YR family expressed in humans, is one of the most prominent clinical drug targets for inhibition of platelet aggregation. Although mutagenesis and modelling studies of the P2Y 12 R provided useful insights into ligand binding 1 , 2 , 3 , 4 , the agonist and antagonist recognition and function at the P2Y 12 R remain poorly understood at the molecular level. Here we report the structures of the human P2Y 12 R in complex with the full agonist 2-methylthio-adenosine-5′-diphosphate (2MeSADP, a close analogue of endogenous agonist ADP) at 2.5 Å resolution, and the corresponding ATP derivative 2-methylthio-adenosine-5′-triphosphate (2MeSATP) at 3.1 Å resolution. These structures, together with the structure of the P2Y 12 R with antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283) 5 , reveal striking conformational changes between nucleotide and non-nucleotide ligand complexes in the extracellular regions. Further analysis of these changes provides insight into a distinct ligand binding landscape in the δ-group of class A G-protein-coupled receptors (GPCRs). Agonist and non-nucleotide antagonist adopt different orientations in the P2Y 12 R, with only partially overlapped binding pockets. The agonist-bound P2Y 12 R structure answers long-standing questions surrounding P2Y 12 R–agonist recognition, and reveals interactions with several residues that had not been reported to be involved in agonist binding. As a first example, to our knowledge, of a GPCR in which agonist access to the binding pocket requires large-scale rearrangements in the highly malleable extracellular region, the structural and docking studies will therefore provide invaluable insight into the pharmacology and mechanisms of action of agonists and different classes of antagonists for the P2Y 12 R and potentially for other closely related P2YRs.

In eukaryotic membranes, type IV P-type adenosine triphosphatases (P4-ATPases) mediate the translocation of phospholipids from the outer to the inner leaflet and maintain lipid asymmetry, which is critical for membrane trafficking and signaling pathways. Here, we report the cryoelectron microscopy structures of six distinct intermediates of the human ATP8A1-CDC50a heterocomplex at resolutions of 2.6 to 3.3 angstroms, elucidating the lipid translocation cycle of this P4-ATPase. ATP-dependent phosphorylation induces a large rotational movement of the actuator domain around the phosphorylation site in the phosphorylation domain, accompanied by lateral shifts of the first and second transmembrane helices, thereby allowing phosphatidylserine binding. The phospholipid head group passes through the hydrophilic cleft, while the acyl chain is exposed toward the lipid environment. These findings advance our understanding of the flippase mechanism and the disease-associated mutants of P4-ATPases.

Adenosine triphosphate (ATP) plays fundamental roles in cellular biochemistry and was recently discovered to function as a biological hydrotrope. Here, we use mass spectrometry to interrogate ATP-mediated regulation of protein thermal stability and protein solubility on a proteome-wide scale. Thermal proteome profiling reveals high affinity interactions of ATP as a substrate and as an allosteric modulator that has widespread influence on protein complexes and their stability. Further, we develop a strategy for proteome-wide solubility profiling, and discover ATP-dependent solubilization of at least 25% of the insoluble proteome. ATP increases the solubility of positively charged, intrinsically disordered proteins, and their susceptibility for solubilization varies depending on their localization to different membrane-less organelles. Moreover, a few proteins, exhibit an ATP-dependent decrease in solubility, likely reflecting polymer formation. Our data provides a proteome-wide, quantitative insight into how ATP influences protein structure and solubility across the spectrum of physiologically relevant concentrations. ATP can function as a biological hydrotrope, but its global effects on protein solubility have not yet been characterized. Here, the authors quantify the effect of ATP on the thermal stability and solubility of the cellular proteome, providing insights into protein solubility regulation by ATP.

Permeability glycoprotein (PgP) uses the energy from adenosine triphosphate (ATP) hydrolysis to transport substrates out of the cell. Many of its substrates are drugs, so it plays an important role in drug resistance. Structures in the inward-facing conformation have been determined for mouse, yeast, and algal PgP. Kim and Chen present the cryo–electron microscopy structure of human PgP in an outward-facing conformation. Two ATP molecules are bound between two nucleotide-binding domains. The substrate-binding site, located in the transmembrane domain, is open to the outside of the cell, but compressed, and no substrate is bound. This suggests that ATP binding, rather than ATP hydrolysis, promotes the transition to the outward-facing conformation and substrate release. Science , this issue p. 915 The structure of a key drug-efflux membrane protein reveals features that enable substrate translocation and release. The multidrug transporter permeability (P)–glycoprotein is an adenosine triphosphate (ATP)–binding cassette exporter responsible for clinical resistance to chemotherapy. P-glycoprotein extrudes toxic molecules and drugs from cells through ATP-powered conformational changes. Despite decades of effort, only the structures of the inward-facing conformation of P-glycoprotein are available. Here we present the structure of human P-glycoprotein in the outward-facing conformation, determined by cryo–electron microscopy at 3.4-angstrom resolution. The two nucleotide-binding domains form a closed dimer occluding two ATP molecules. The drug-binding cavity observed in the inward-facing structures is reorientated toward the extracellular space and compressed to preclude substrate binding. This observation indicates that ATP binding, not hydrolysis, promotes substrate release. The structure evokes a model in which the dynamic nature of P-glycoprotein enables translocation of a large variety of substrates.