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Mechanotransduction at cell–cell adhesions is crucial for the structural integrity, organization, and morphogenesis of epithelia. At cell–cell junctions, ternary E-cadherin/β-catenin/αE-catenin complexes sense and transmit mechanical load by binding to F-actin. The interaction with F-actin, described as a two-state catch bond, is weak in solution but is strengthened by applied force due to force-dependent transitions between weak and strong actin-binding states. Here, we provide direct evidence from optical trapping experiments that the catch bond property principally resides in the αE-catenin actin-binding domain (ABD). Consistent with our previously proposed model, the deletion of the first helix of the five-helix ABD bundle enables stable interactions with F-actin under minimal load that are well described by a single-state slip bond, even when αE-catenin is complexed with β-catenin and E-cadherin. Our data argue for a conserved catch bond mechanism for adhesion proteins with structurally similar ABDs. We also demonstrate that a stably bound ABD strengthens load-dependent binding interactions between a neighboring complex and F-actin, but the presence of the other αE-catenin domains weakens this effect. These results provide mechanistic insight to the cooperative binding of the cadherin–catenin complex to F-actin, which regulate dynamic cytoskeletal linkages in epithelial tissues.

Tension transmitted between neighboring cells can exert profound effects on cell proliferation, differentiation, and tissue organization. Exactly how intercellular mechanical tension is sensed at the molecular level is unknown. One attractive hypothesis is that a linkage between the cell-cell adhesion molecule E-cadherin, its binding partners α- and β-catenin, and actin filaments may act as a tension sensor. However, how this linkage is established at the molecular level is not known. Buckley et al. used optical tweezers to determine how mechanical load influences interactions of the cadherin/catenin complex with single actin filaments. The data support a model in which force shifts the interaction from a force-independent, weakly bound state to a highly force-sensitive, strongly bound state. The findings may explain how cells maintain tissue integrity while still being able to move and change shape. Science , this issue p. 10.1126/science.1254211 A protein complex involved in cell adhesion forms a two-state catch bond with the cytoskeleton under mechanical load. Linkage between the adherens junction (AJ) and the actin cytoskeleton is required for tissue development and homeostasis. In vivo findings indicated that the AJ proteins E-cadherin, β-catenin, and the filamentous (F)–actin binding protein αE-catenin form a minimal cadherin-catenin complex that binds directly to F-actin. Biochemical studies challenged this model because the purified cadherin-catenin complex does not bind F-actin in solution. Here, we reconciled this difference. Using an optical trap–based assay, we showed that the minimal cadherin-catenin complex formed stable bonds with an actin filament under force. Bond dissociation kinetics can be explained by a catch-bond model in which force shifts the bond from a weakly to a strongly bound state. These results may explain how the cadherin-catenin complex transduces mechanical forces at cell-cell junctions.

Classical cadherins are transmembrane proteins at the core of intercellular adhesion complexes in cohesive metazoan tissues. The extracellular domain of classical cadherins forms intercellular bonds with cadherins on neighboring cells, whereas the cytoplasmic domain recruits catenins, which in turn associate with additional cytoskeleton binding and regulatory proteins. Cadherin/catenin complexes are hypothesized to play a role in the transduction of mechanical forces that shape cells and tissues during development, regeneration, and disease. Whether mechanical forces are transduced directly through cadherins is unknown. To address this question, we used a Förster resonance energy transfer (FRET)-based molecular tension sensor to test the origin and magnitude of tensile forces transmitted through the cytoplasmic domain of E-cadherin in epithelial cells. We show that the actomyosin cytoskeleton exerts pN-tensile force on E-cadherin, and that this tension requires the catenin-binding domain of E-cadherin and αE-catenin. Surprisingly, the actomyosin cytoskeleton constitutively exerts tension on E-cadherin at the plasma membrane regardless of whether or not E-cadherin is recruited to cell–cell contacts, although tension is further increased at cell–cell contacts when adhering cells are stretched. Our findings thus point to a constitutive role of E-cadherin in transducing mechanical forces between the actomyosin cytoskeleton and the plasma membrane, not only at cell–cell junctions but throughout the cell surface.

Here, by developing a high-affinity camelid antibody fragment that stabilizes the active state of the β 2 -adrenoceptor, the X-ray crystal structures of the receptor in complex with three agonists, including adrenaline, were determined. Structure of the activated β 2 -adrenoceptor This study reports three structures of fully active human β 2 adrenergic receptor (β 2 AR) in complex with diverse agonists: BI167107, hydroxybenzyl isoproterenol, and the endogenous agonist adrenaline. β 2 AR is a G-protein-coupled receptor (GPCR), ubiquitous membrane proteins that are targeted by many clinically used drugs. The molecular processes by which they bind to their endogenous agonists and activate effector proteins remain poorly understood. Despite the chemical diversity of the three agonists examined, all three stabilize highly similar active states in the receptor. Subtle structural differences provide insight into how a single GPCR is activated by multiple agonists, a phenomenon that is critically important to drug development. 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 1 , 2 or receptor stabilization by mutagenesis 3 , 4 , 5 . Many natural agonists such as adrenaline, which activates the β 2 -adrenoceptor (β 2 AR), 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 β 2 AR, and used this to obtain crystal structures of the activated receptor bound to multiple ligands. Here we present structures of the active-state human β 2 AR 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 β 2 AR and related GPCRs.

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 β 2 adrenergic receptor (β 2 AR) 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 β 2 AR 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. Very little is known about how a G-protein-coupled receptor (GPCR) transitions from an inactive to an active state, but this study has solved the X-ray crystal structures of the human M2 muscarinic acetylcholine receptor bound to a high-affinity agonist in an active state and to a high-affinity agonist and a small-molecule allosteric modulator in an active state; the structures provide insights into the activation mechanism and allosteric modulation of muscarinic receptors. Allostery in the M2 muscarinic acetylcholine receptor The structures of many G-protein-coupled receptors (GPCRs), including members of the class B and class F families, are now available but little is known about the transitions from the inactive to active states. In this study the authors solve the X-ray crystal structures of the human M2 muscarinic acetylcholine receptor in the active state bound to the agonist iperoxo alone and in combination with LY2119620, a positive allosteric modulator. The structures reveal that the activated M2 receptor has an extremely small orthosteric binding site, with LY2119620 'sitting' right on top of the agonist. The authors also note that the region that makes up the allosteric site in the inactive conformation of the M2 receptor is too large to bind to LY2119620; this means that the extracellular region needs to contract (by binding to the high-affinity agonist) before LY2119620 can bind to the allosteric site. This GPCR is essential for the physiological control of cardiovascular function, cognition, and pain perception, and since allosteric sites are less conserved in sequence and structure than the orthosteric binding site, the hope is that ligands that bind to allosteric sites could be turned into drugs that selectively interact with only one of the five muscarinic receptor subtypes.

A highly crystallizable T4 lysozyme (T4L) was fused to the N-terminus of the β(2) adrenergic receptor (β(2)AR), a G-protein coupled receptor (GPCR) for catecholamines. We demonstrate that the N-terminal fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner.

Stabilization of an active and inactive conformation of the β 2 -adrenergic receptor by allosteric nanobodies reveals differential ligand-dependent regulation of receptor states to control G-protein-coupled receptor activation. Agonist binding to the β2-adrenergic receptor In this manuscript, the authors studied how a positive allosteric nanobody (Nb80) and a newly discovered negative allosteric nanobody (Nb60) alter the structure of the β2-adrenergic receptor (β 2 AR). Their data support a three-state model for receptor activation in this important G-protein-coupled receptor, rather than a simple inactive–active two-state model. They also find that full agonists primarily stabilize the active Nb80-stabilized receptor state (while having negligible effects on the inactive Nb60-bound state), but partial agonists appear to regulate multiple receptor states to control receptor activation. 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’) 1 . Furthermore, increasing biophysical evidence, primarily using the β 2 -adrenergic receptor (β 2 AR) as a model system, supports the existence of multiple active and inactive conformational states 2 , 3 , 4 , 5 . 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 β 2 AR conformations using single domain camelid antibodies (nanobodies)—a previously described positive allosteric nanobody (Nb80) 6 , 7 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 β 2 AR in the presence of Nb80 compared to the affinity of isoprenaline for β 2 AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β 2 AR 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 (R 80 ). In contrast, for a number of partial agonists, both stabilization of R 80 and destabilization of the inactive, Nb60-bound state (R 60 ) 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.

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 M1 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 M1 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. X-ray crystal structures of the M1 and M4 muscarinic acetylcholine receptors, revealing differences in the orthosteric and allosteric binding sites that help to explain the subtype selectivity of drugs targeting this family of receptors. M1 and M4 muscarinic acetylcholine receptor structures Arthur Christopoulos and colleagues present the first X-ray crystal structures of the M1 and M4 muscarinic acetylcholine receptors, G-protein-coupled receptors (GPCRs) that regulate many vital functions of the central and peripheral nervous systems. The structures reveal differences in the orthosteric and allosteric binding sites that help to explain the subtype selectivity of drugs targeting this family of receptors. The M1 and M4 receptor subtypes are potential drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia.

The X-ray crystal structure of the M3 muscarinic acetylcholine receptor bound to the bronchodilator drug tiotropium is reported; comparison of this structure with that of the M2 muscarinic acetylcholine receptor reveals key differences that could potentially be exploited to develop subtype-selective drugs. Muscrarinic receptor structures compared The muscarinic acetylcholine receptors (mAChRs) constitute a family of G-protein-coupled receptors. These membrane proteins are targets for treatment of a broad range of conditions, including Alzheimer's disease, schizophrenia and chronic obstructive pulmonary disease. The five mAChR subtypes (M1–M5) share a high degree of sequence homology, but show marked differences in G-protein-coupling preference and physiological function. This pair of papers from Brian Kobilka's group presents the structures of two of the five subtypes. Haga et al . report the X-ray crystal structure of the M2 receptor, which is essential for the physiological control of cardiovascular function; Kruse et al . determine the structure of the M3 receptor, active in the bronchial airways and elsewhere. Comparison of the two structures reveals key differences that could potentially be exploited to develop subtype-selective drugs. 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 , 3 , 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 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 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 route to 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.

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 2 adrenergic receptor (β 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 2 AR and nucleotide-free Gs heterotrimer. The principal interactions between the β 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 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. X-ray structure of a GPCR complex G-protein-coupled receptors (GPCRs) mediate the majority of a cell's responses to hormones and neurotransmitters, and to the senses of sight, olfaction and taste. This makes GPCRs potentially the most important group of drug targets in the human body. GPCRs are deeply embedded in the cell membrane, crossing it seven times, so structure determination for these complexes is particularly challenging — as recounted in a recent News Feature (see http://go.nature.com/ftqnx4 ). The eagerly-awaited X-ray crystal structure of a GPCR transmembrane signalling complex has now been determined by Brian Kobilka's group. The structure presented is of an agonist-occupied monomer of the β 2 adrenergic receptor in complex with G s , the stimulatory G protein for adenylyl cyclase. An accompanying paper reports the use of peptide amide hydrogen-deuterium exchange mass spectrometry to probe the protein dynamics of this signalling complex.