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The μ-opioid receptor (μOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by μOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Here we present the 3.5 Å resolution cryo-electron microscopy structure of the μOR bound to the agonist peptide DAMGO and nucleotide-free Gi. DAMGO occupies the morphinan ligand pocket, with its N terminus interacting with conserved receptor residues and its C terminus engaging regions important for opioid-ligand selectivity. Comparison of the μOR-Gi complex to previously determined structures of other GPCRs bound to the stimulatory G protein Gs reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein α-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the Gi protein-coupling specificity of the µOR.

Nuclear pore complexes (NPCs) create large conduits for cargo transport between the nucleus and cytoplasm across the nuclear envelope (NE).sup.1-3. These multi-megadalton structures are composed of about thirty different nucleoporins that are distributed in three main substructures (the inner, cytoplasmic and nucleoplasmic rings) around the central transport channel.sup.4-6. Here we use cryo-electron tomography on DLD-1 cells that were prepared using cryo-focused-ion-beam milling to generate a structural model for the human NPC in its native environment. We show that--compared with previous human NPC models obtained from purified NEs--the inner ring in our model is substantially wider; the volume of the central channel is increased by 75% and the nucleoplasmic and cytoplasmic rings are reorganized. Moreover, the NPC membrane exhibits asymmetry around the inner-ring complex. Using targeted degradation of Nup96, a scaffold nucleoporin of the cytoplasmic and nucleoplasmic rings, we observe the interdependence of each ring in modulating the central channel and maintaining membrane asymmetry. Our findings highlight the inherent flexibility of the NPC and suggest that the cellular environment has a considerable influence on NPC dimensions and architecture. Structure of human nuclear pore complex in its cellular environment reveals a substantially dilated central channel and shows that its nucleoplasmic and cytoplasmic rings restrict channel dimensions and create membrane asymmetry at the inner ring., Author(s): Anthony P. Schuller [sup.1] , Matthias Wojtynek [sup.2] [sup.3] , David Mankus [sup.4] , Meltem Tatli [sup.2] , Rafael Kronenberg-Tenga [sup.2] , Saroj G. Regmi [sup.5] , Phat V. [...]

Droughts and climate-change-driven warming are leading to more frequent and intense wildfires.sup.1-3, arguably contributing to the severe 2019-2020 Australian wildfires.sup.4. The environmental and ecological impacts of the fires include loss of habitats and the emission of substantial amounts of atmospheric aerosols.sup.5-7. Aerosol emissions from wildfires can lead to the atmospheric transport of macronutrients and bio-essential trace metals such as nitrogen and iron, respectively.sup.8-10. It has been suggested that the oceanic deposition of wildfire aerosols can relieve nutrient limitations and, consequently, enhance marine productivity.sup.11,12, but direct observations are lacking. Here we use satellite and autonomous biogeochemical Argo float data to evaluate the effect of 2019-2020 Australian wildfire aerosol deposition on phytoplankton productivity. We find anomalously widespread phytoplankton blooms from December 2019 to March 2020 in the Southern Ocean downwind of Australia. Aerosol samples originating from the Australian wildfires contained a high iron content and atmospheric trajectories show that these aerosols were likely to be transported to the bloom regions, suggesting that the blooms resulted from the fertilization of the iron-limited waters of the Southern Ocean. Climate models project more frequent and severe wildfires in many regions.sup.1-3. A greater appreciation of the links between wildfires, pyrogenic aerosols.sup.13, nutrient cycling and marine photosynthesis could improve our understanding of the contemporary and glacial-interglacial cycling of atmospheric CO.sub.2 and the global climate system. Oceanic deposition of wildfire aerosols can enhance marine productivity, as supported here by satellite and in situ profiling floats data showing that emissions from the 2019-2020 Australian wildfires fuelled phytoplankton blooms in the Southern Ocean., Author(s): Weiyi Tang [sup.1] [sup.11] , Joan Llort [sup.2] [sup.3] , Jakob Weis [sup.2] [sup.4] , Morgane M. G. Perron [sup.2] , Sara Basart [sup.3] , Zuchuan Li [sup.1] [sup.5] [...]

Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.

G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and β-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’). Furthermore, increasing biophysical evidence, primarily using the β2-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies)—a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for β2AR in the presence of Nb80 compared to the affinity of isoprenaline for β2AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states.

Spontaneous emission of radiation is one of the fundamental mechanisms by which an excited quantum system returns to equilibrium. For spins, however, spontaneous emission is generally negligible compared to other non-radiative relaxation processes because of the weak coupling between the magnetic dipole and the electromagnetic field. In 1946, Purcell realized that the rate of spontaneous emission can be greatly enhanced by placing the quantum system in a resonant cavity. This effect has since been used extensively to control the lifetime of atoms and semiconducting heterostructures coupled to microwave or optical cavities, and is essential for the realization of high-efficiency single-photon sources. Here we report the application of this idea to spins in solids. By coupling donor spins in silicon to a superconducting microwave cavity with a high quality factor and a small mode volume, we reach the regime in which spontaneous emission constitutes the dominant mechanism of spin relaxation. The relaxation rate is increased by three orders of magnitude as the spins are tuned to the cavity resonance, demonstrating that energy relaxation can be controlled on demand. Our results provide a general way to initialize spin systems into their ground state and therefore have applications in magnetic resonance and quantum information processing. They also demonstrate that the coupling between the magnetic dipole of a spin and the electromagnetic field can be enhanced up to the point at which quantum fluctuations have a marked effect on the spin dynamics; as such, they represent an important step towards the coherent magnetic coupling of individual spins to microwave photons.

Synaptotagmin-1 and neuronal SNARE proteins have central roles in evoked synchronous neurotransmitter release; however, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca(2+)- and Mg(2+)-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free-electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many side chains. The structures reveal several interfaces, including a large, specific, Ca(2+)-independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca(2+)-triggered neurotransmitter release in mouse hippocampal neuronal synapses and for Ca(2+)-triggered vesicle fusion in a reconstituted system. We propose that this interface forms before Ca(2+) triggering, moves en bloc as Ca(2+) influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.

Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 Å X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

Natural iron fertilization of the Southern Ocean by windblown dust has been suggested to enhance biological productivity and modulate the climate1–3. Yet, this process has never been quantified across the Southern Ocean and at annual timescales4,5. Here we combined 11 years of nitrate observations from autonomous biogeochemical ocean profiling floats with a Southern Hemisphere dust simulation to empirically derive the relationship between dust-iron deposition and annual net community production (ANCP) in the iron-limited Southern Ocean. Using this relationship, we determined the biological response to dust-iron in the pelagic perennially ice-free Southern Ocean at present and during the last glacial maximum (LGM). We estimate that dust-iron now supports 33% ± 15% of Southern Ocean ANCP. During the LGM, when dust deposition was 5–40-fold higher than today, the contribution of dust to Southern Ocean ANCP was much greater, estimated at 64% ± 13%. We provide quantitative evidence of basin-wide dust-iron fertilization of the Southern Ocean and the potential magnitude of its impact on glacial–interglacial timescales, supporting the idea of the important role of dust in the global carbon cycle and climate6–8.Nitrate observations over 11 years from autonomous biogeochemical ocean profiling combined with a Southern Hemisphere dust simulation find that iron supplied by dust supports about 30% of Southern Ocean productivity. [ABSTRACT FROM AUTHOR]

Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors.

The ability of proteins and nucleic acids to undergo liquid-liquid phase separation has recently emerged as an important molecular principle of how cells rapidly and reversibly compartmentalize their components into membrane-less organelles such as the nucleolus, processing bodies or stress granules.sup.1,2. How the assembly and turnover of these organelles are controlled, and how these biological condensates selectively recruit or release components are poorly understood. Here we show that members of the large and highly abundant family of RNA-dependent DEAD-box ATPases (DDXs).sup.3 are regulators of RNA-containing phase-separated organelles in prokaryotes and eukaryotes. Using in vitro reconstitution and in vivo experiments, we demonstrate that DDXs promote phase separation in their ATP-bound form, whereas ATP hydrolysis induces compartment turnover and release of RNA. This mechanism of membrane-less organelle regulation reveals a principle of cellular organization that is conserved from bacteria to humans. Furthermore, we show that DDXs control RNA flux into and out of phase-separated organelles, and thus propose that a cellular network of dynamic, DDX-controlled compartments establishes biochemical reaction centres that provide cells with spatial and temporal control of various RNA-processing steps, which could regulate the composition and fate of ribonucleoprotein particles. RNA-dependent DEAD-box ATPases (DDXs) regulate the dynamics of phase-separated organelles, with ATP-bound DDXs promoting phase separation, and ATP hydrolysis inducing compartment disassembly and RNA release., Author(s): Maria Hondele [sup.1] , Ruchika Sachdev [sup.1] , Stephanie Heinrich [sup.1] , Juan Wang [sup.2] , Pascal Vallotton [sup.1] , Beatriz M. A. Fontoura [sup.2] , Karsten Weis [sup.1] [...]

G-protein-coupled receptors (GPCRs) are integral membrane proteins that have an essential role in human physiology, yet the molecular processes through which they bind to their endogenous agonists and activate effector proteins remain poorly understood. So far, it has not been possible to capture an active-state GPCR bound to its native neurotransmitter. Crystal structures of agonist-bound GPCRs have relied on the use of either exceptionally high-affinity agonists or receptor stabilization by mutagenesis. Many natural agonists such as adrenaline, which activates the β2-adrenoceptor (β2AR), bind with relatively low affinity, and they are often chemically unstable. Using directed evolution, we engineered a high-affinity camelid antibody fragment that stabilizes the active state of the β2AR, and used this to obtain crystal structures of the activated receptor bound to multiple ligands. Here we present structures of the active-state human β2AR bound to three chemically distinct agonists: the ultrahigh-affinity agonist BI167107, the high-affinity catecholamine agonist hydroxybenzyl isoproterenol, and the low-affinity endogenous agonist adrenaline. The crystal structures reveal a highly conserved overall ligand recognition and activation mode despite diverse ligand chemical structures and affinities that range from 100 nM to ∼80 pM. Overall, the adrenaline-bound receptor structure is similar to the others, but it has substantial rearrangements in extracellular loop three and the extracellular tip of transmembrane helix 6. These structures also reveal a water-mediated hydrogen bond between two conserved tyrosines, which appears to stabilize the active state of the β2AR and related GPCRs.

The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of β-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of β-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate β-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of β-arrestin-1. The structure of the β-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in β-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of β-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on β-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

Muscarinic M1-M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the Ml and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the Ml and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains., The M1-M5 muscarinic acetylcholine receptors constitute an important family of class A G-protein-coupled receptor (GPCRs) activated by the neurotransmitter, acetylcholine (1). Both the M1 and M4 receptors have been associated [...]

Protease-activated receptor 1 (PAR1) is the prototypical member of a family of G-protein-coupled receptors that mediate cellular responses to thrombin and related proteases. Thrombin irreversibly activates PAR1 by cleaving the amino-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the heptahelical bundle of the receptor to affect G-protein activation. Here we report the 2.2 A resolution crystal structure of human PAR1 bound to vorapaxar, a PAR1 antagonist. The structure reveals an unusual mode of drug binding that explains how a small molecule binds virtually irreversibly to inhibit receptor activation by the tethered ligand of PAR1. In contrast to deep, solvent-exposed binding pockets observed in other peptide-activated G- protein-coupled receptors, the vorapaxar-binding pocket is superficial but has little surface exposed to the aqueous solvent. Protease-activated receptors are important targets for drug development. The structure reported here will aid the development of improved PAR1 antagonists and the discovery of antagonists to other members of this receptor family., Protease-activated receptors (PARs) are G-protein-coupled receptors (GPCRs) that mediate cellular responses to specific proteases (1,2). The coagulation protease thrombin activates the prototypical PAR, PAR1, by specific cleavage of the N-terminal [...]

The opioid receptor family comprises three members, the μ-, δ- and κ-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-proteincoupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The δ-opioid receptor (δ-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood (1). The structures of the μ-OR and κ-OR have recently been solved (2,3). Here we report the crystal structure of the mouse δ-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the μ-OR and κ-OR, the δ-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology (4,5), in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the o-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well., Opioid receptors have an important role in the central nervous system, regulating pain perception, hedonic homeostasis, mood and wellbeing (1). Thus, they have long been the focus of physiological and [...]

Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled μ-opioid receptor (μ-OR) in the central nervous system. Here we describe the 2.8 Å crystal structure of the mouse μ-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the μ-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction., Opium extracts from the plant Papaver somniferum have been used for therapeutic and recreational purposes for thousands of years. Opioid alkaloids and related pharmaceuticals are the most effective analgesics for [...]

Acetylcholine, the first neurotransmitter to be identified (1), exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate (2-4). Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences (5,6). We describe here the structure of the [G.sub.q/11]-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the [G.sub.i/o]-coupled M2 receptor (7), offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en routeto the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors., The mAChR family consists of five subtypes, M1-M5, which can be subdivided into two major classes (Fig. 1a). The M1, M3 and M5 receptors show selectivity for G proteins of [...]

The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves (1-5). Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation., Muscarinic receptors constitute a family with five subtypes, M1-M5 (ref. 1). M1, M3 and M5 subtypes couple with the [G.sub.q] family of G proteins, and M2 and M4 subtypes with [...]

Optical laser fields have been widely used to achieve quantum control over the motional and internal degrees of freedom of atoms and ions (1,2), molecules and atomic gases. A route to controlling the quantum states of macroscopic mechanical oscillators in a similar fashion is to exploit the parametric coupling between optical and mechanical degrees of freedom through radiation pressure in suitably engineered optical cavities (3-6). If the optomechanical coupling is 'quantum coherent'--that is, if the coherent coupling rate exceeds both the optical and the mechanical decoherence rate--quantum states are transferred from the optical field to the mechanical oscillator and vice versa. This transfer allows control of the mechanical oscillator state using the wide range of available quantum optical techniques. So far, however, quantum-coherent coupling of micromechanical oscillators has only been achieved using microwave fields at millikelvin temperatures (7,8). Optical experiments have not attained this regime owing to the large mechanical decoherence rates (9) and the difficulty of overcoming optical dissipation (10). Here we achieve quantum-coherent coupling between optical photons and a micromechanical oscillator. Simultaneously, coupling to the cold photon bath cools the mechanical oscillator to an average occupancy of 1.7 ± 0.1 motional quanta. Excitation with weak classical light pulses reveals the exchange of energy between the optical light field and the micromechanical oscillator in the time domain at the level of less than one quantum on average. This optomechanical system establishes an efficient quantum interface between mechanical oscillators and optical photons, which can provide decoherence-free transport of quantum states through optical fibres. Our results offer a route towards the use of mechanical oscillators as quantum transducers or in microwave-to-optical quantum links (11-15)., Mechanical oscillators are at the heart of many precision experiments and can exhibit exceptionally low dissipation. The possibility of controlling the quantum states of engineered micro- or nano-mechanical oscillators has [...]

G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The [β.sub.2] adrenergic receptor ([β.sub.2]AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric [β.sub.2]AR and nucleotide-free Gs heterotrimer. The principal interactions between the [β.sub.2]AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the [β.sub.2]AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR., Introduction The [β.sub.2] adrenergic receptor ([β.sub.2]AR) has been a model system for the large and diverse family of G protein-coupled receptors (GPCRs) for over 40 years. It was one of [...]

Superfamily 1 and superfamily 2 RNA helicases are ubiquitous messenger-RNA-protein complex (mRNP) remodelling enzymes that have critical roles in all aspects of RNA metabolism (1,2). The superfamily 2 DEAD-box ATPase [...]

G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs (1), the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human [β.sub.2] adrenergic receptor ([β.sub.2]AR) as a guide, we designed a [β.sub.2]AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent [β.sub.2]AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound [β.sub.2]AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method (2), and determined its structure at 3.5 Å resolution. A comparison to the inactive structure and an antibodystabilized active structure (companion paper (3)) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30 µs) molecular dynamics simulations showing that an agonistbound active conformation spontaneously relaxes to an inactivelike conformation in the absence of a G protein or stabilizing antibody., The relationship between agonist binding to GPCRs and the conformational changes that facilitate G protein binding and activation remains largely unknown. Characterization of GPCR activation at a molecular level has [...]

G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human [β.sub.2] adrenergic receptor ([β.sub.2]AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive [β.sub.2]AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation., GPCRs activated by diffusible ligands have a spectrum of functional states (1). A GPCR may activate more than one G protein isoform or a G-protein-independent pathway such as arrestin. In [...]

The nuclear pore complex (NPC) mediates all exchange between the cytoplasm and the nucleus. Small molecules can passively diffuse through the NPC, whereas larger cargos require transport receptors to translocate1. How the NPC facilitates the translocation of transport receptor/cargo complexes remains unclear. To investigate this process, we tracked single protein-functionalized quantum dot cargos as they moved through human NPCs. Here we show that import proceeds by successive substeps comprising cargo capture, filtering and translocation, and release into the nucleus. Most quantum dots are rejected at one of these steps and return to the cytoplasm, including very large cargos that abort at a size-selective barrier. Cargo movement in the central channel is subdiffusive and cargos that can bind more transport receptors diffuse more freely. Without Ran GTPase, a critical regulator of transport directionality (1), cargos still explore the entire NPC, but have a markedly reduced probability of exit into the nucleus, suggesting that NPC entry and exit steps are not equivalent and that the pore is functionally asymmetric to importing cargos. The overall selectivity of the NPC seems to arise from the cumulative action of multiple reversible substeps and a final irreversible exit step., Receptors of the importin-β/karyopherin family use a steep RanGTP gradient across the nuclear envelope to carry cargos directionally across the NPC2. Unstructured phenylalanine-glycine repeat (FG-repeat) proteins in the NPC are [...]

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs (1-5) have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the [β.sub.2] adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures., In the ligand-free basal state, GPCRs exist in an equilibrium of conformations (6). Ligand binding modulates receptor function by stabilizing different intramolecular interactions and establishing a new conformational equilibrium. Activating [...]

Author(s): Xiangyu Liu [1]; Seungkirl Ahn [2]; Alem W. Kahsai [2]; Kai-Cheng Meng [3]; Naomi R. Latorraca [4, 5]; Biswaranjan Pani [2]; A. J. Venkatakrishnan [4, 5, 6]; Ali Masoudi [...]

Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a [...]

Author(s): Douglas A. Hattendorf [1]; Anna Andreeva [2]; Akanksha Gangar [2]; Patrick J. Brennwald (corresponding author) [2]; William I. Weis (corresponding author) [1] Polarized exocytosis requires coordination between the actin [...]

Author(s): Petr Kaláb [1]; Arnd Pralle [1]; Ehud Y. Isacoff [1]; Rebecca Heald (corresponding author) [1]; Karsten Weis (corresponding author) [1] The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating [...]

Author(s): Sara M. Weis [1]; David A. Cheresh (corresponding author) [1] The VEGF family includes VEGF-A, placenta growth factor, VEGF-B, VEGF-C and VEGF-D. VEGF-A (hereafter referred to as VEGF) is [...]

Author(s): Kira M. S. Misura [1]; Richard H. Scheller [2]; William I. Weis (corresponding author) [1] The organization of the membrane compartments of eukaryotic cells and intercellular communication depend on [...]

The crystal structure of human importin-Beta bound to the IBB domain of human importin-alpha has been solve. The association occurs via combined specific-sequence recognition and gross electrostatic complementarity. The structure provides an indication as to how the binding of Ran-GTP to importin-Beta could result in IBB dissociation, and provides support for the hypothesis that importins alpha and beta have a common ancestor.