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Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter1,2 that activates the largest subtype family of G-protein-coupled receptors3. Drugs that target 5-HT1A, 5-HT1D, 5-HT1E and other 5-HT receptors are used to treat numerous disorders4. 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor–G-protein complexes: 5-HT1A in the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT1D bound to 5-HT; and 5-HT1E in complex with a 5-HT1E- and 5-HT1F-selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein–5-HT1A interface, and is able to increase 5-HT1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules—particularly in the case of 5-HT1A, in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT1A are structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors. Cryo-electron microscopy structures of three different serotonin receptors in complex with serotonin and other agonists provide insights into the role of lipids in regulating these receptors and the structural basis of ligand recognition.

CpG methylation by de novo DNA methyltransferases (DNMTs) 3A and 3B is essential for mammalian development and differentiation and is frequently dysregulated in cancer1. These two DNMTs preferentially bind to nucleosomes, yet cannot methylate the DNA wrapped around the nucleosome core2, and they favour the methylation of linker DNA at positioned nucleosomes3,4. Here we present the cryo-electron microscopy structure of a ternary complex of catalytically competent DNMT3A2, the catalytically inactive accessory subunit DNMT3B3 and a nucleosome core particle flanked by linker DNA. The catalytic-like domain of the accessory DNMT3B3 binds to the acidic patch of the nucleosome core, which orients the binding of DNMT3A2 to the linker DNA. The steric constraints of this arrangement suggest that nucleosomal DNA must be moved relative to the nucleosome core for de novo methylation to occur. Catalytically inactive DNMT3B3 is crucial in de novo CpG methylation of DNA, interacting with the nucleosome core to orient catalytically active DNMT3A2 so that it can bind to nearby linker DNA.

Vasoactive intestinal polypeptide receptor (VIP1R) is a widely expressed class B G protein-coupled receptor and a drug target for the treatment of neuronal, metabolic, and inflammatory diseases. However, our understanding of its mechanism of action and the potential of drug discovery targeting this receptor is limited by the lack of structural information of VIP1R. Here we report a cryo-electron microscopy structure of human VIP1R bound to PACAP27 and Gs heterotrimer, whose complex assembly is stabilized by a NanoBiT tethering strategy. Comparison with other class B GPCR structures reveals that PACAP27 engages VIP1R with its N-terminus inserting into the ligand binding pocket at the transmembrane bundle of the receptor, which subsequently couples to the G protein in a receptor-specific manner. This structure has provided insights into the molecular basis of PACAP27 binding and VIP receptor activation. The methodology of the NanoBiT tethering may help to provide structural information of unstable complexes., Vasoactive intestinal polypeptide receptor (VIP1R) is a widely expressed class B G protein-coupled receptor and a drug target for the treatment of inflammatory diseases. Here authors report a cryoelectron microscopy structure of human VIP1R bound to PACAP27 and Gs heterotrimer, which provides insights into PACAP27 binding and VIP receptor activation.

Long-chain fatty acids (LCFAs) play important roles in cellular energy metabolism, acting as both an important energy source and signalling molecules1. LCFA-CoA esters promote their own oxidation by acting as allosteric inhibitors of acetyl-CoA carboxylase, which reduces the production of malonyl-CoA and relieves inhibition of carnitine palmitoyl-transferase 1, thereby promoting LCFA-CoA transport into the mitochondria for β-oxidation2,3,4,5,6. Here we report a new level of regulation wherein LCFA-CoA esters per se allosterically activate AMP-activated protein kinase (AMPK) β1–containing isoforms to increase fatty acid oxidation through phosphorylation of acetyl-CoA carboxylase. Activation of AMPK by LCFA-CoA esters requires the allosteric drug and metabolite site formed between the α-subunit kinase domain and the β-subunit. β1 subunit mutations that inhibit AMPK activation by the small-molecule activator A769662, which binds to the allosteric drug and metabolite site, also inhibit activation by LCFA-CoAs. Thus, LCFA-CoA metabolites act as direct endogenous AMPK β1–selective activators and promote LCFA oxidation.

Formylpeptide receptors (FPRs) as G protein-coupled receptors (GPCRs) can recognize formylpeptides derived from pathogens or host cells to function in host defense and cell clearance. In addition, FPRs, especially FPR2, can also recognize other ligands with a large chemical diversity generated at different stages of inflammation to either promote or resolve inflammation in order to maintain a balanced inflammatory response. The mechanism underlying promiscuous ligand recognition and activation of FPRs is not clear. Here we report a cryo-EM structure of FPR2-Gi signaling complex with a peptide agonist. The structure reveals a widely open extracellular region with an amphiphilic environment for ligand binding. Together with computational docking and simulation, the structure suggests a molecular basis for the recognition of formylpeptides and a potential mechanism of receptor activation, and reveals conserved and divergent features in Gi coupling. Our results provide a basis for understanding the molecular mechanism of the functional promiscuity of FPRs., Formylpeptide receptors (FPRs) are a class of chemotactic G protein-coupled receptors (GPCRs) that recognize pathogen- and host-derived formylpeptides. Here the authors report the 3.17 Å cryo-EM structure of the human FPR2-Gi signaling complex with a bound peptide agonist and in combination with computational docking and MD simulations provide mechanistic insights into formylpeptide recognition by FPRs.

Peroxisome proliferator-activated receptor γ (PPARγ) is a key regulator of glucose homeostasis and lipid metabolism, and an important target for the development of modern anti-diabetic drugs. However, current PPARγ-targeting anti-diabetic drugs such as classical thiazolidinediones (TZDs) are associated with undesirable side effects. To address this concern, we here describe the structure-based design, synthesis, identification and detailed in vitro and in vivo characterization of a novel, decanoic acid (DA)-based and selective PPARγ modulator (SPPARγM), VSP-77, especially (S)-VSP-77, as the potential “hit” for the development of improved and safer anti-diabetic therapeutics. We have also determined the co-crystal structure of the PPARγ ligand-binding domain (LBD) in complex with two molecules of (S)-VSP-77, which reveal a previously undisclosed allosteric binding mode. Overall, these findings not only demonstrate the therapeutic advantage of (S)-VSP-77 over current TZD drugs and representative partial agonist INT131, but also provide a rational basis for the development of future SPPARγMs as safe and highly efficacious anti-diabetic drugs.

Bone-cell regulation, fleshed out One of many medically relevant G protein–coupled receptors, parathyroid hormone receptor-1 (PTH1R) functions in the control of calcium homeostasis and bone physiology. Zhao et al. used cryo–election microscopy to observe the structure of PTH1R in a complex with a modified form of parathyroid hormone and stimulatory G protein. The structural model helps explain how parathyroid hormone interacts with its receptor and the molecular basis for receptor activation. Science , this issue p. 148

AMP-activated protein kinase (AMPK) is an attractive therapeutic target for managing metabolic diseases. A class of pharmacological activators, including Merck 991, binds the AMPK ADaM site, which forms the interaction surface between the kinase domain (KD) of the α-subunit and the carbohydrate-binding module (CBM) of the β-subunit. Here, we report the development of two new 991-derivative compounds, R734 and R739, which potently activate AMPK in a variety of cell types, including β(2)-specific skeletal muscle cells. Surprisingly, we found that they have only minor effects on direct kinase activity of the recombinant α(1)β(2)γ(1) isoform yet robustly enhance protection against activation loop dephosphorylation. This mode of activation is reminiscent of that of ADP, which activates AMPK by binding to the nucleotide-binding sites in the γ-subunit, more than 60 Å away from the ADaM site. To understand the mechanisms of full and partial AMPK activation, we determined the crystal structures of fully active phosphorylated AMPK α(1)β(1)γ(1) bound to AMP and R734/R739 as well as partially active nonphosphorylated AMPK bound to R734 and AMP and phosphorylated AMPK bound to R734 in the absence of added nucleotides at

How to catch a dynamic state AMP-activated protein kinase (AMPK) is a key sensor of energy status in eukaryotes. Its dynamic structure is regulated by allosteric factors including phosphorylation and binding of nucleotides and metabolites. Yan et al. developed conformation-specific antibodies that trap AMPK in a fully inactive state that has experienced a large, domain-level rotation. Biophysical experiments in cells and in vitro are consistent with the structural work and support a model in which the activation loop is fully exposed in the completely inactive, dephosphorylated state. These structures inform our understanding of the complex allosteric behavior in this crucial metabolic regulator. Science , abe7565, this issue p. 413

The development of novel analgesics with improved safety profiles to combat the opioid epidemic represents a central question to G protein coupled receptor structural biology and pharmacology: What chemical features dictate G protein or β-arrestin signaling? Here we use adaptively biased molecular dynamics simulations to determine how fentanyl, a potent β-arrestin biased agonist, binds the μ-opioid receptor (μOR). The resulting fentanyl-bound pose provides rational insight into a wealth of historical structure-activity-relationship on its chemical scaffold. Following an in-silico derived hypothesis we found that fentanyl and the synthetic opioid peptide DAMGO require M153 to induce β-arrestin coupling, while M153 was dispensable for G protein coupling. We propose and validate an activation mechanism where the n-aniline ring of fentanyl mediates μOR β-arrestin through a novel M153 “microswitch” by synthesizing fentanyl-based derivatives that exhibit complete, clinically desirable, G protein biased coupling. Together, these results provide molecular insight into fentanyl mediated β-arrestin biased signaling and a rational framework for further optimization of fentanyl-based analgesics with improved safety profiles., Author summary The global opioid crisis has drawn significant attention to the risks associated with over-use of synthetic opioids. Despite the public attention, and perhaps in-line with the profit-based incentives of the pharmaceutical industry, there is no public structure of mu-opioid receptor bound to fentanyl or fentanyl derivatives. A publicly available structure of the complex would allow open-source development of safer painkillers and synthetic antagonists. Current overdose antidotes, antagonists, require natural products in their synthesis which persists a sizable barrier to market and develop better antidotes. In this work we use advance molecular dynamics techniques to obtain the bound geometry of mu-opioid receptor with fentanyl (and derivatives) and uncovered a novel molecular switch involved in receptor activation. Based on our in-silico structure, we synthesized and tested novel compounds to validate our predicted structure. Herein we report the bound state of several dangerous fentanyl derivatives and introduce new derivatives with signaling profiles that may lead to lower risk of respiratory depression.

Drugs selectively targeting CB2 hold promise for treating neurodegenerative disorders, inflammation, and pain while avoiding psychotropic side effects mediated by CB1. The mechanisms underlying CB2 activation and signaling are poorly understood but critical for drug design. Here we report the cryo-EM structure of the human CB2-Gi signaling complex bound to the agonist WIN 55,212-2. The 3D structure reveals the binding mode of WIN 55,212-2 and structural determinants for distinguishing CB2 agonists from antagonists, which are supported by a pair of rationally designed agonist and antagonist. Further structural analyses with computational docking results uncover the differences between CB2 and CB1 in receptor activation, ligand recognition, and Gi coupling. These findings are expected to facilitate rational structure-based discovery of drugs targeting the cannabinoid system.

Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms1,2. As central mediators in this complex signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand. The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed, and is distinct from all other GPCR structures reported so far. Within this unique transmembrane fold is an extremely narrow and highly hydrophilic pocket that is not amenable to the binding of traditional GPCR ligands. We show that such a pocket is conserved across all FZDs, which may explain the long-standing difficulties in the development of ligands for these receptors. Molecular dynamics simulations on the microsecond timescale and mutational analysis uncovered two coupled, dynamic kinks located at helix VII that are involved in FZD4 activation. The stability of the structure in its ligand-free form, an unfavourable pocket for ligand binding and the two unusual kinks on helix VII suggest that FZDs may have evolved a novel ligand-recognition and activation mechanism that is distinct from that of other GPCRs.

5-hydroxytryptamine (5-HT, also known as serotonin) regulates many physiological processes through the 5-HT receptor family. Here we report the crystal structure of 5-HT1B subtype receptor (5-HT1BR) bound to the psychotropic serotonin receptor inverse agonist methiothepin (MT). Crystallization was facilitated by replacing ICL3 with a novel optimized variant of BRIL (OB1) that enhances the formation of intermolecular polar interactions, making OB1 a potential useful tool for structural studies of membrane proteins. Unlike the agonist ergotamine (ERG), MT occupies only the conserved orthosteric binding pocket, explaining the wide spectrum effect of MT on serotonin receptors. Compared with ERG, MT shifts toward TM6 and sterically pushes residues W3276.48, F3306.50 and F3316.51 from inside the orthosteric binding pocket, leading to an outward movement of the extracellular end and a corresponding inward shift of the intracellular end of TM6, a feature shared by other reported inactive G protein-coupled receptor (GPCR) structures. Together with the previous agonist-bound serotonin receptor structures, the inverse agonist-bound 5-HT1BR structure identifies a basis for the ligand-mediated switch of 5-HT1BR activity and provides a structural understanding of the inactivation mechanism of 5-HT1BR and some other class A GPCRs, characterized by ligand-induced outward movement of the extracellular end of TM6 that is coupled with inward movement of the cytoplasmic end of this helix.

Wnt/β-catenin signalling is initiated by a ternary Wnt-Frizzled (FZD)-LDL receptor-related protein (LRP) 5/6 binding event. The resulting conformational changes in the FZD and LRP5/6 receptors promote the assembly of an intracellular signalosome driven by Dishevelled and Axin co-polymerization. Recent evidence suggests that the FZD receptor and LRP5/6 participate in the assembly of this signalosome by forming regulatory scaffolds for stabilizing Dishevelled and Axin adapters. In this review, we focus on the contributions of Wnts and their receptors in the assembly of the signalosome. We present an emerging model, which unifies Wnt receptor oligomerization with intracellular signalosome formation, and then discuss how FZD receptors might be targeted to either disrupt or enhance their capacity as a dynamic sensor of Wnt binding. Linked Articles This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and are important drug targets for many human diseases. The determination of the 3-D structure of GPCRs and their signaling complexes has promoted our understanding of GPCR biology and provided templates for structure-based drug discovery. In this review, we focus on the recent structure work on GPCR signaling complexes, the β2-adrenoreceptor-Gs and the rhodopsin-arrestin complexes in particular, and highlight the structural features of GPCR complexes involved in G protein- and arrestin-mediated signal transduction. The crystal structures reveal distinct structural mechanisms by which GPCRs recruit a G protein and an arrestin. A comparison of the two complex structures provides insight into the molecular mechanism of functionally selective GPCR signaling, and a structural basis for the discovery of G protein- and arrestin-biased treatments of human diseases related to GPCR signal transduction.

The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical β2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation.

G protein-coupled receptor kinases (GRKs) play pivotal roles in desensitizing GPCR signaling but little is known about how GRKs recognize and phosphorylate GPCRs due to the technical difficulties in detecting the highly dynamic GPCR/GRK interaction. By combining a genetic approach with multiple biochemical assays, we identified the key determinants for the assembly of the prototypical GPCR rhodopsin with its kinase GRK1. Our work reveals that the regulatory G-protein signaling homology (RH) domain of GRKs is the primary binding site to GPCRs and an active conformation of the GRK1 kinase domain is required for efficient interaction with rhodopsin. In addition, we provide a mechanistic solution for the longstanding puzzle about the gain-of-function Q41L mutation in GRK5. This mutation is in the RH domain and increases the capacity of the GRK mutant to interact with and to desensitize GPCRs. Finally we present the principal architecture of a rhodopsin/GRK complex through negative stain electron microscopy reconstruction. Together, these data define the key components for the rhodopsin/GRK1 interaction and provide a framework for understanding GRK-mediated desensitization of GPCRs.

Wnt/β-catenin signaling is activated when extracellular Wnt ligands bind Frizzled (FZD) receptors at the cell membrane. Wnts bind FZD cysteine-rich domains (CRDs) with high affinity through a palmitoylated N-terminal “thumb” and a disulfide-stabilized C-terminal “index finger,” yet how these binding events trigger receptor activation and intracellular signaling remains unclear. Here we report the crystal structure of the Frizzled-4 (FZD4) CRD in complex with palmitoleic acid, which reveals a CRD tetramer consisting of two cross-braced CRD dimers. Each dimer is stabilized by interactions of one hydrophobic palmitoleic acid tail with two CRD palmitoleoyl-binding grooves oriented end to end, suggesting that the Wnt palmitoleoyl group stimulates CRD–CRD interaction. Using bioluminescence resonance energy transfer (BRET) in live cells, we show that WNT5A stimulates dimerization of membrane-anchored FZD4 CRDs and oligomerization of full-length FZD4, which requires the integrity of CRD palmitoleoyl-binding residues. These results suggest that FZD receptors may form signalosomes in response to Wnt binding through the CRDs and that the Wnt palmitoleoyl group is important in promoting these interactions. These results complement our understanding of lipoprotein receptor-related proteins 5 and 6 (LRP5/6), Dishevelled, and Axin signalosome assembly and provide a more complete model for Wnt signalosome assembly both intracellularly and at the membrane.

γ-Secretase is an intramembrane aspartyl protease that cleaves the C99 fragment of amyloid precursor protein to generate extracellular Aβ peptides. These peptides can oligomerize and aggregate to form amyloid plaques, processes that are widely believed to be causal for Alzheimer's disease. In spite of this critical function, it remains unknown how γ-secretase recognizes C99 and its other substrates, including Notch. In this study we determined E22-K55 as the minimal C99 fragment that was sufficient and required for initial cleavage. Within this fragment, we identified four determinants: (i) a transferable extracellular determinant that differed between C99 and Notch, and which included negative charge in the case of C99, (ii) the amino acid sequence of the C-terminal half of the transmembrane helix, (iii) an invariant lysine or arginine at the intracellular membrane border, and (iv) a positive charge cluster that included the invariant lysine/arginine. We demonstrated that the charge clusters of C99 and Notch receptors could directly bind phosphatidylinositol 4,5-bisphosphate (PIP2). The PIP2-binding cluster was required for γ-secretase cleavage, and modulation of membrane PIP2 levels strongly affected γ-secretase cleavage levels and the Aβ40/Aβ42 ratio, providing support for the importance of the PIP2 interaction in cells. Together, these studies provide critically needed insight into substrate recognition by γ-secretase.

The polycomb repressive complexes 2 (PRC2) complex catalyzes tri-methylation of histone H3 lysine 27 (H3K27), a repressive chromatin marker associated with gene silencing. Overexpression and mutations of PRC2 are found in a wide variety of cancers, making the catalytic activity of PRC2 an important target of cancer therapy. This review highlights recent structural breakthroughs of the human PRC2 complex bound to the H3K27 peptide and a small molecule inhibitor, which provide critically needed insight into PRC2-targeted drug discovery.

Jasmonate ZIM-domain (JAZ) transcriptional repressors play a key role in regulating jasmonate (JA) signaling in plants. Below a threshold concentration of jasmonoyl isoleucine (JA-Ile), the active form of JA, the C-terminal Jas motif of JAZ proteins binds MYC transcription factors to repress JA signaling. With increasing JA-Ile concentration, the Jas motif binds to JA-Ile and the COI1 subunit of the SCFCOI1 E3 ligase, which mediates ubiquitination and proteasomal degradation of JAZ repressors, resulting in derepression of MYC transcription factors. JA signaling subsequently becomes desensitized, in part by feedback induction of JAZ splice variants that lack the C-terminal Jas motif but include an N-terminal cryptic MYC-interaction domain (CMID). The CMID sequence is dissimilar to the Jas motif and is incapable of recruiting SCFCOI1, allowing CMID-containing JAZ splice variants to accumulate in the presence of JA and to re-repress MYC transcription factors as an integral part of reestablishing signal homeostasis. The mechanism by which the CMID represses MYC transcription factors remains elusive. Here we describe the crystal structure of the MYC3-CMIDJAZ10 complex. In contrast to the Jas motif, which forms a single continuous helix when bound to MYC3, the CMID adopts a loop-helix-loop-helix architecture with modular interactions with both the Jas-binding groove and the backside of the Jas-interaction domain of MYC3. This clamp-like interaction allows the CMID to bind MYC3 tightly and block access of MED25 (a subunit of the Mediator coactivator complex) to the MYC3 transcriptional activation domain, shedding light on the enigmatic mechanism by which JAZ splice variants desensitize JA signaling.

In the jasmonate signaling pathway, a region of 17 amino acids within the Jas motif of JAZ proteins and a conserved region within the N-terminus of MYC proteins are sufficient for JAZ-MYC interactions. Crystal structures of Jas-MYC complexes have revealed the structural basis of this important interaction. Here, we describe methods of cloning, expression, and purification of MYC N-terminal proteins and their co-crystallization with Jas motif peptides.

Arrestins comprise a family of signal regulators of G-protein-coupled receptors (GPCRs), which include arrestins 1 to 4. While arrestins 1 and 4 are visual arrestins dedicated to rhodopsin, arrestins 2 and 3 (Arr2 and Arr3) are β-arrestins known to regulate many nonvisual GPCRs. The dynamic and promiscuous coupling of Arr2 to nonvisual GPCRs has posed technical challenges to tackle the basis of arrestin binding to GPCRs. Here we report the structure of Arr2 in complex with neurotensin receptor 1 (NTSR1), which reveals an overall assembly that is strikingly different from the visual arrestin-rhodopsin complex by a 90° rotation of Arr2 relative to the receptor. In this new configuration, intracellular loop 3 (ICL3) and transmembrane helix 6 (TM6) of the receptor are oriented toward the N-terminal domain of the arrestin, making it possible for GPCRs that lack the C-terminal tail to couple Arr2 through their ICL3. Molecular dynamics simulation and crosslinking data further support the assembly of the Arr2–NTSR1 complex. Sequence analysis and homology modeling suggest that the Arr2–NTSR1 complex structure may provide an alternative template for modeling arrestin-GPCR interactions.

Fusarium is a genus of filamentous fungi that includes species that cause devastating diseases in major staple crops, such as wheat, maize, rice, and barley, resulting in severe yield losses and mycotoxin contamination of infected grains. Phenamacril is a novel fungicide that is considered environmentally benign due to its exceptional specificity; it inhibits the ATPase activity of the sole class I myosin of only a subset of Fusarium species including the major plant pathogens F. graminearum, F. asiaticum and F. fujikuroi. To understand the underlying mechanisms of inhibition, species specificity, and resistance mutations, we have determined the crystal structure of phenamacril-bound F. graminearum myosin I. Phenamacril binds in the actin-binding cleft in a new allosteric pocket that contains the central residue of the regulatory Switch 2 loop and that is collapsed in the structure of a myosin with closed actin-binding cleft, suggesting that pocket occupancy blocks cleft closure. We have further identified a single, transferable phenamacril-binding residue found exclusively in phenamacril-sensitive myosins to confer phenamacril selectivity., Author summary Phenamacril is a recently identified myosin I inhibitor that is a potent and highly species-specific and myosin subtype-selective fungicide. We report the high-resolution structure of the phenamacril-bound myosin I motor domain of the major crop pathogen Fusarium graminearum, providing insight into the molecular mechanism of phenamacril action and resistance. These results are of broad significance for understanding the mode of actions of myosin-based fungicides and for designing novel myosin I inhibitors for crop protection and for treatment of human myosin dysfunction diseases.

The dopamine system, including five dopamine receptors (D1R-D5R), plays essential roles in the central nervous system (CNS), and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders. Here, we report two cryo-EM structures of human D3R in complex with an inhibitory G protein and bound to the D3R-selective agonists PD128907 and pramipexole, the latter of which is used to treat patients with Parkinson's disease. The structures reveal agonist binding modes distinct from the antagonist-bound D3R structure and conformational signatures for ligand-induced receptor activation. Mutagenesis and homology modeling illuminate determinants of ligand specificity across dopamine receptors and the mechanisms for Gi protein coupling. Collectively our work reveals the basis of agonist binding and ligand-induced receptor activation and provides structural templates for designing specific ligands to treat CNS diseases targeting the dopaminergic system.

The D1- and D2-dopamine receptors (D1R and D2R), which signal through G(s) and G(i), respectively, represent the principal stimulatory and inhibitory dopamine receptors in the central nervous system. D1R and D2R also represent the main therapeutic targets for Parkinson’s disease, schizophrenia, and many other neuropsychiatric disorders, and insight into their signaling is essential for understanding both therapeutic and side effects of dopaminergic drugs. Here, we report four cryoelectron microscopy (cryo-EM) structures of D1R-G(s) and D2R-G(i) signaling complexes with selective and non-selective dopamine agonists, including two currently used anti-Parkinson’s disease drugs, apomorphine and bromocriptine. These structures, together with mutagenesis studies, reveal the conserved binding mode of dopamine agonists, the unique pocket topology underlying ligand selectivity, the conformational changes in receptor activation, and potential structural determinants for G protein-coupling selectivity. These results provide both a molecular understanding of dopamine signaling and multiple structural templates for drug design targeting the dopaminergic system.

G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation.

Nuclear receptors are defined as a family of ligand regulated transcription factors [1] , [2] , [3] , [4] , [5] , [6] . While this definition reflects that ligand binding is a key property of nuclear receptors, it is still a heated subject of debate if all the nuclear receptors (48 human members) can bind ligands (ligands referred here to both physiological and synthetic ligands). Recent studies in nuclear receptor structure biology and pharmacology have undoubtedly increased our knowledge of nuclear receptor functions and their regulation. As a result, they point to new avenues for the discovery and development of nuclear receptor regulators, including nuclear receptor ligands. Here we review the recent literature on orphan nuclear receptor structural analysis and ligand identification, particularly on the orphan nuclear receptors that do not heterodimerize with retinoid X receptors, which we term as non-X orphan receptors. We also propose a speculative “retinoid hypothesis” for a subset of non-X orphan nuclear receptors, which we hope to help shed light on orphan nuclear receptor biology and drug discovery. This article is part of a Special Issue entitled ‘Orphan Nuclear Receptors’.

Corticotropin-releasing factor (CRF) and the three related peptides urocortins 1-3 (UCN1-UCN3) are endocrine hormones that control the stress responses by activating CRF1R and CRF2R, two members of class B G-protein-coupled receptors (GPCRs). Here, we present two cryoelectron microscopy (cryo-EM) structures of UCN1-bound CRF1R and CRF2R with the stimulatory G protein. In both structures, UCN1 adopts a single straight helix with its N terminus dipped into the receptor transmembrane bundle. Although the peptide-binding residues in CRF1R and CRF2R are different from other members of class B GPCRs, the residues involved in receptor activation and G protein coupling are conserved. In addition, both structures reveal bound cholesterol molecules to the receptor transmembrane helices. Our structures define the basis of ligand-binding specificity in the CRF receptor-hormone system, establish a common mechanism of class B GPCR activation and G protein coupling, and provide a paradigm for studying membrane protein-lipid interactions for class B GPCRs.

G protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors that mediate numerous cell signaling pathways, and are targets of more than one-third of clinical drugs. Thanks to the advancement of novel structural biology technologies, high-resolution structures of GPCRs in complex with their signaling transducers, including G-protein and arrestin, have been determined. These 3D complex structures have significantly improved our understanding of the molecular mechanism of GPCR signaling and provided a structural basis for signaling-biased drug discovery targeting GPCRs. Here we summarize structural studies of GPCR signaling complexes with G protein and arrestin using rhodopsin as a model system, and highlight the key features of GPCR conformational states in biased signaling including the sequence motifs of receptor TM6 that determine selective coupling of G proteins, and the phosphorylation codes of GPCRs for arrestin recruitment. We envision the future of GPCR structural biology not only to solve more high-resolution complex structures but also to show stepwise GPCR signaling complex assembly and disassembly and dynamic process of GPCR signal transduction.

“Universal” synthetic antibody (sAB)-based fiducial marks have been generated by customized phage display selections to facilitate the rapid structure determination of G-protein coupled receptor (GPCR) signaling complexes by single particle cryo-electron microscopy (SP cryo-EM). sABs were generated to the two major G-protein subclasses: trimeric G(i) and G(s), as well as mini-G(s) and were tested to ensure binding in the context of their cognate GPCRs. Epitope binning revealed that multiple distinct epitopes exist for each G(αβγ)-protein. Several Gβγ-specific sABs, cross-reactive between trimeric G(i) and G(s), were identified suggesting they could be used across all subclasses in a “plug and play” fashion. sABs were also generated to a representative of another class of GPCR signaling partner, G-protein receptor kinase 1 (GRK1) and evaluated further, supporting the generalizability of the approach. EM data indicated that the subclass specific sABs provide effective single and dual fiducials for multiple GPCR signaling complexes.

G-protein-coupled receptors comprise the largest family of mammalian transmembrane receptors. They mediate numerous cellular pathways by coupling with downstream signalling transducers, including the hetrotrimeric G proteins G(s) (stimulatory) and G(i) (inhibitory) and several arrestin proteins. The structural mechanisms that define how G-protein-coupled receptors selectively couple to a specific type of G protein or arrestin remain unknown. Here, using cryo-electron microscopy, we show that the major interactions between activated rhodopsin and G(i) are mediated by the C-terminal helix of the G(i) α-subunit, which is wedged into the cytoplasmic cavity of the transmembrane helix bundle and directly contacts the amino terminus of helix 8 of rhodopsin. Structural comparisons of inactive, G(i)-bound and arrestin-bound forms of rhodopsin with inactive and G(s)-bound forms of the β(2)-adrenergic receptor provide a foundation to understand the unique structural signatures that are associated with the recognition of G(s), G(i) and arrestin by activated G-protein-coupled receptors.

AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis and a promising drug target for managing metabolic diseases such as type 2 diabetes. Many pharmacological AMPK activators, and possibly unidentified physiological metabolites, bind to the allosteric drug and metabolite (ADaM) site at the interface between the kinase domain (KD) in the α-subunit and the carbohydrate-binding module (CBM) in the β-subunit. Here, using double electron-electron resonance (DEER) spectroscopy, we demonstrate that the CBM-KD interaction is partially dissociated and the interface highly disordered in the absence of pharmacological ADaM site activators as inferred from a low depth of modulation and broad DEER distance distributions. ADaM site ligands such as 991, and to a lesser degree phosphorylation, stabilize the KD-CBM association and strikingly reduce conformational heterogeneity in the ADaM site. Our findings that the ADaM site, formed by the KD-CBM interaction, can be modulated by diverse ligands and by phosphorylation suggest that it may function as a hub for integrating regulatory signals.

G-protein-coupled receptors comprise the largest family of mammalian transmembrane receptors. They mediate numerous cellular pathways by coupling with downstream signalling transducers, including the hetrotrimeric G proteins Gs (stimulatory) and Gi (inhibitory) and several arrestin proteins. The structural mechanisms that define how G-protein-coupled receptors selectively couple to a specific type of G protein or arrestin remain unknown. Here, using cryo-electron microscopy, we show that the major interactions between activated rhodopsin and Gi are mediated by the C-terminal helix of the Gi α-subunit, which is wedged into the cytoplasmic cavity of the transmembrane helix bundle and directly contacts the amino terminus of helix 8 of rhodopsin. Structural comparisons of inactive, Gi-bound and arrestin-bound forms of rhodopsin with inactive and Gs-bound forms of the β2-adrenergic receptor provide a foundation to understand the unique structural signatures that are associated with the recognition of Gs, Gi and arrestin by activated G-protein-coupled receptors.

Summary G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor's C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular β sheet with the N-terminal β strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to β-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs.

Rice D53 repression motif links two sites of TPL corepressors to mediate TPL oligomerization and nucleosome association., TOPLESS are tetrameric plant corepressors of the conserved Tup1/Groucho/TLE (transducin-like enhancer of split) family. We show that they interact through their TOPLESS domains (TPDs) with two functionally important ethylene response factor–associated amphiphilic repression (EAR) motifs of the rice strigolactone signaling repressor D53: the universally conserved EAR-3 and the monocot-specific EAR-2. We present the crystal structure of the monocot-specific EAR-2 peptide in complex with the TOPLESS-related protein 2 (TPR2) TPD, in which the EAR-2 motif binds the same TPD groove as jasmonate and auxin signaling repressors but makes additional contacts with a second TPD site to mediate TPD tetramer-tetramer interaction. We validated the functional relevance of the two TPD binding sites in reporter gene assays and in transgenic rice and demonstrate that EAR-2 binding induces TPD oligomerization. Moreover, we demonstrate that the TPD directly binds nucleosomes and the tails of histones H3 and H4. Higher-order assembly of TPD complexes induced by EAR-2 binding markedly stabilizes the nucleosome-TPD interaction. These results establish a new TPD-repressor binding mode that promotes TPD oligomerization and TPD-nucleosome interaction, thus illustrating the initial assembly of a repressor-corepressor-nucleosome complex.

AMP-activated protein kinase (AMPK) is a central cellular energy sensor that adapts metabolism and growth to the energy state of the cell. AMPK senses the ratio of adenine nucleotides (adenylate energy charge) by competitive binding of AMP, ADP, and ATP to three sites (CBS1, CBS3, and CBS4) in its γ-subunit. Because these three binding sites are functionally interconnected, it remains unclear how nucleotides bind to individual sites, which nucleotides occupy each site under physiological conditions, and how binding to one site affects binding to the other sites. Here, we comprehensively analyze nucleotide binding to wild-type and mutant AMPK protein complexes by quantitative competition assays and by hydrogen-deuterium exchange MS. We also demonstrate that NADPH, in addition to the known AMPK ligand NADH, directly and competitively binds AMPK at the AMP-sensing CBS3 site. Our findings reveal how AMP binding to one site affects the conformation and adenine nucleotide binding at the other two sites and establish CBS3, and not CBS1, as the high affinity exchangeable AMP/ADP/ATP-binding site. We further show that AMP binding at CBS4 increases AMP binding at CBS3 by 2 orders of magnitude and reverses the AMP/ATP preference of CBS3. Together, these results illustrate how the three CBS sites collaborate to enable highly sensitive detection of cellular energy states to maintain the tight ATP homeostastis required for cellular metabolism.

One of the hallmarks of Alzheimer's disease is the formation of extracellular amyloid plaques that consist mainly of abnormally aggregated forms of amyloid β (Aβ) peptides. These peptides are generated by γ-secretase-catalyzed cleavage of a dimeric membrane-bound C-terminal fragment (C99) of the amyloid precursor protein. Although C99 homodimerization has been linked to Aβ production and changes in the aggregation-determining Aβ42/Aβ40 ratio, the motif through which C99 dimerizes has remained controversial. Here, we have used two independent assays to gain insight into C99 homodimerization in the context of the membrane of live cells: bioluminescence resonance energy transfer and Tango membrane protein-protein interaction assays, which were further confirmed by traditional pull-down assays. Our results indicate a four-amino acid region within the C99 transmembrane helix (43TVIV46) as well as its local secondary structure as critical determinants for homodimerization. These four amino acids are also a hot spot of familial Alzheimer's disease-linked mutations that both decrease C99 homodimerization and γ-secretase cleavage and alter the initial cleavage site to increase the Aβ42/40 ratio.

Thiazolidinediones (TZD) function as potent anti-diabetic drugs through their direct action on the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ), but their therapeutic benefits are compromised by severe side effects. To address this concern, here we developed a potent “hit” compound, VSP-51, which is a novel selective PPARγ-modulating ligand with improved therapeutic profiles in vitro compared to the multi-billion dollar TZD drug rosiglitazone (Rosi). Unlike Rosi, VSP-51 is a partial agonist of PPARγ with improved insulin sensitivity due to its ability to bind PPARγ with high affinity without stimulating adipocyte differentiation and the expression of adipogenesis-related genes. We have determined the crystal structure of the PPARγ ligand-binding domain (LBD) in complex with VSP-51, which revealed a unique mode of binding for VSP-51 and provides the molecular basis for the discrimination between VSP-51 from TZDs and other ligands such as telmisartan, SR1663 and SR1664. Taken together, our findings demonstrate that: a) VSP-51 can serve as a promising candidate for anti-diabetic drug discovery; and b) provide a rational basis for the development of future pharmacological agents targeting PPARγ with advantages over current TZD drugs.

The nuclear pore complex subunit TPR is found in at least five different oncogenic fusion kinases, including TPR-MET, yet how TPR fusions promote activation of kinases and their oncogenic activities remains poorly understood. Here we report the crystal structure of TPR(2-142), the MET fusion partner of oncogenic TPR-MET. TPR(2-142) contains a continuous 124-residue α helix that forms an antiparallel tetramer from two leucine zipper-containing parallel coiled coils. Remarkably, single mutations cause strikingly different conformations of the coiled coil, indicating its highly dynamic nature. We further show that fusion of TPR(2-142) to the MET intracellular domain strongly and selectively stabilizes the αG helix of the MET kinase domain, and mutations of only the TPR leucine zipper residues at the junction to MET, but not other leucine zipper residues, abolish kinase activation. Together, these results provide critical insight into the TPR structure and its ability to induce dimerization and activation of fusion kinases.

Crystal structures of two phytohormone signal-transducing α/β hydrolases: karrikin-signaling KAI2 and strigolactone-signaling DWARF14

The glucagon-like peptide-1 receptor is a class B G protein coupled receptor (GPCR) that plays key roles in glucose metabolism and is a major therapeutic target for diabetes. The classic two-domain model for class B GPCR activation proposes that the apo-state receptor is auto-inhibited by its extracellular domain, which physically interacts with the transmembrane domain. The binding of the C-terminus of the peptide hormone to the extracellular domain allows the N-terminus of the hormone to insert into the transmembrane domain to induce receptor activation. In contrast to this model, here we demonstrate that glucagon-like peptide-1 receptor can be activated by N-terminally truncated glucagon-like peptide-1 or exendin-4 when fused to the receptor, raising the question regarding the role of N-terminal residues of peptide hormone in glucagon-like peptide-1 receptor activation. Mutations of cysteine 347 to lysine or arginine in intracellular loop 3 transform the receptor into a G protein-biased receptor and allow it to be activated by a nonspecific five-residue linker that is completely devoid of exendin-4 or glucagon-like peptide-1 sequence but still requires the presence of an intact extracellular domain. Moreover, the extracellular domain can activate the receptor in trans in the presence of an intact peptide hormone, and specific mutations in three extracellular loops abolished this extracellular domain trans-activation. Together, our data reveal a dominant role of the extracellular domain in glucagon-like peptide-1 receptor activation and support an intrinsic agonist model of the extracellular domain, in which peptide binding switches the receptor from the auto-inhibited state to the auto-activated state by releasing the intrinsic agonist activity of the extracellular domain.

Amyloid beta peptide (Aβ) is produced through the proteolytic processing of a transmembrane protein, amyloid precursor protein (APP), by β- and γ-secretases. Aβ accumulation in the brain is proposed to be an early toxic event in the pathogenesis of Alzheimer's disease, which is the most common form of dementia associated with plaques and tangles in the brain. Currently, it is unclear what the physiological and pathological forms of Aβ are and by what mechanism Aβ causes dementia. Moreover, there are no efficient drugs to stop or reverse the progression of Alzheimer's disease. In this paper, we review the structures, biological functions, and neurotoxicity role of Aβ. We also discuss the potential receptors that interact with Aβ and mediate Aβ intake, clearance, and metabolism. Additionally, we summarize the therapeutic developments and recent advances of different strategies for treating Alzheimer's disease. Finally, we will report on the progress in searching for novel, potentially effective agents as well as selected promising strategies for the treatment of Alzheimer's disease. These prospects include agents acting on Aβ, its receptors and tau protein, such as small molecules, vaccines and antibodies against Aβ; inhibitors or modulators of β- and γ-secretase; Aβ-degrading proteases; tau protein inhibitors and vaccines; amyloid dyes and microRNAs.

Targeted antifolates with heteroatom replacements of the carbon vicinal to the phenyl ring in 1 by N (4), O (8), or S (9), or with N-substituted formyl (5), acetyl (6), or trifluoroacetyl (7) moieties, were synthesized and tested for selective cellular uptake by folate receptor (FR) α and β or the proton-coupled folate transporter. Results show increased in vitro antiproliferative activity toward engineered Chinese hamster ovary cells expressing FRs by 4-9 over the CH2 analogue 1. Compounds 4-9 inhibited de novo purine biosynthesis and glycinamide ribonucleotide formyltransferase (GARFTase). X-ray crystal structures for 4 with FRα and GARFTase showed that the bound conformations of 4 required flexibility for attachment to both FRα and GARFTase. In mice bearing IGROV1 ovarian tumor xenografts, 4 was highly efficacious. Our results establish that heteroatom substitutions in the 3-atom bridge region of 6-substituted pyrrolo[2,3-d]pyrimidines related to 1 provide targeted antifolates that warrant further evaluation as anticancer agents.

Mutations in the amyloid precursor protein (APP) gene and the aberrant cleavage of APP by γ-secretase are associated with Alzheimer’s disease (AD). Here we have developed a simple and sensitive cell-based assay to detect APP cleavage by γ-secretase. Unexpectedly, most familial AD (FAD)-linked APP mutations make APP partially resistant to γ-secretase. Mutations that alter residues N terminal to the γ-secretase cleavage site Aβ42 have subtle effects on cleavage efficiency and cleavage-site selectivity. In contrast, mutations that alter residues C terminal to the Aβ42 site reduce cleavage efficiency and dramatically shift cleavage-site specificity toward the aggregation-prone Aβ42. Moreover, mutations that remove positive charge at residue 53 greatly reduce the APP cleavage by γ-secretase. These results suggest a model of γ-secretase substrate recognition, in which the APP region C terminal to the Aβ42 site and the positively charged residue at position 53 are the primary determinants for substrate binding and cleavage-site selectivity. We further demonstrate that this model can be extended to γ-secretase processing of notch receptors, a family of highly conserved cell-surface signaling proteins.

Aim: Dominant negative mutant G proteins have provided critical insight into the mechanisms of G protein-coupled receptor (GPCR) signaling, but the mechanisms underlying the dominant negative characteristics are not completely understood. The aim of this study was to determine the structure of the dominant negative Gαi1β1γ2 G203A/A326S complex (Gi-DN) and to reveal the structural basis of the mutation-induced phenotype of Gαi1β1γ2. Methods: The three subunits of the Gi-DN complex were co-expressed with a baculovirus expression system. The Gi-DN heterotrimer was purified, and the structure of its complex with GDP was determined through X-ray crystallography. Results: The Gi-DN heterotrimer structure revealed a dual mechanism underlying the dominant negative characteristics. The mutations weakened the hydrogen bonding network between GDP/GTP and the binding pocket residues, and increased the interactions in the Gα-Gβγ interface. Concomitantly, the Gi-DN heterotrimer adopted a conformation, in which the C-terminus of Gαi and the N-termini of both the Gβ and Gγ subunits were more similar to the GPCR-bound state compared with the wild type complex. From these structural observations, two additional mutations (T48F and D272F) were designed that completely abolish the GDP binding of the Gi-DN heterotrimer. Conclusion: Overall, the results suggest that the mutations impede guanine nucleotide binding and Gα-Gβγ protein dissociation and favor the formation of the G protein/GPCR complex, thus blocking signal propagation. In addition, the structure provides a rationale for the design of other mutations that cause dominant negative effects in the G protein, as exemplified by the T48F and D272F mutations.

Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a heterotrimeric αβγ complex that functions as a central regulator of energy homeostasis. Energy stress manifests as a drop in the ratio of adenosine triphosphate (ATP) to AMP/ADP, which activates AMPK’s kinase activity, allowing it to upregulate ATP-generating catabolic pathways and to reduce energy-consuming catabolic pathways and cellular programs. AMPK senses the cellular energy state by competitive binding of the three adenine nucleotides AMP, ADP, and ATP to three sites in its γ subunit, each, which in turn modulates the activity of AMPK’s kinase domain in its α subunit. Our current understanding of adenine nucleotide binding and the mechanisms by which differential adenine nucleotide occupancies activate or inhibit AMPK activity has been largely informed by crystal structures of AMPK in different activity states. Here we provide an overview of AMPK structures, and how these structures, in combination with biochemical, biophysical, and mutational analyses provide insights into the mechanisms of adenine nucleotide binding and AMPK activity modulation.

The crystal structure of the rhodopsin–arrestin complex provides important insights into how G protein–coupled receptor signaling is terminated by arrestin and a structural basis for understanding the mechanism of arrestin-biased signaling.

Groucho-related corepressors in plants bind a peptide motif found in numerous repressors through a novel peptide recognition fold., TOPLESS (TPL) and TOPLESS-related (TPR) proteins comprise a conserved family of plant transcriptional corepressors that are related to Tup1, Groucho, and TLE (transducin-like enhancer of split) corepressors in yeast, insects, and mammals. In plants, TPL/TPR corepressors regulate development, stress responses, and hormone signaling through interaction with small ethylene response factor–associated amphiphilic repression (EAR) motifs found in diverse transcriptional repressors. How EAR motifs can interact with TPL/TPR proteins is unknown. We confirm the amino-terminal domain of the TPL family of corepressors, which we term TOPLESS domain (TPD), as the EAR motif–binding domain. To understand the structural basis of this interaction, we determined the crystal structures of the TPD of rice (Os) TPR2 in apo (apo protein) state and in complexes with the EAR motifs from Arabidopsis NINJA (novel interactor of JAZ), IAA1 (auxin-responsive protein 1), and IAA10, key transcriptional repressors involved in jasmonate and auxin signaling. The OsTPR2 TPD adopts a new fold of nine helices, followed by a zinc finger, which are arranged into a disc-like tetramer. The EAR motifs in the three different complexes adopt a similar extended conformation with the hydrophobic residues fitting into the same surface groove of each OsTPR2 monomer. Sequence alignments and structure-based mutagenesis indicate that this mode of corepressor binding is highly conserved in a large set of transcriptional repressors, thus providing a general mechanism for gene repression mediated by the TPL family of corepressors.