Dual-role transcription factors stabilize intermediate expression levels.

Precise control of gene expression levels is essential for normal cell functions, yet how they are defined and tightly maintained, particularly at intermediate levels, remains elusive. Here, using a series of newly developed sequencing, imaging, and functional assays, we uncover a class of transcription factors with dual roles as activators and repressors, referred to as condensate-forming level-regulating dual-action transcription factors (TFs). They reduce high expression but increase low expression to achieve stable intermediate levels. Dual-action TFs directly exert activating and repressing functions via condensate-forming domains that compartmentalize core transcriptional unit selectively. Clinically relevant mutations in these domains, which are linked to a range of developmental disorders, impair condensate selectivity and dual-action TF activity. These results collectively address a fundamental question in expression regulation and demonstrate the potential of level-regulating dual-action TFs as powerful effectors for engineering controlled expression levels.

Structure-based discovery of conformationally selective inhibitors of the serotonin transporter.

The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has unusual anti-depressant and substance-withdrawal effects, and stabilizes the inward-open conformation. Unfortunately, ibogaine's promiscuity and cardiotoxicity limit the understanding of inward-open state ligands. We docked over 200 million small molecules against the inward-open state of the SERT. Thirty-six top-ranking compounds were synthesized, and thirteen inhibited; further structure-based optimization led to the selection of two potent (low nanomolar) inhibitors. These stabilized an outward-closed state of the SERT with little activity against common off-targets. A cryo-EM structure of one of these bound to the SERT confirmed the predicted geometry. In mouse behavioral assays, both compounds had anxiolytic- and anti-depressant-like activity, with potencies up to 200-fold better than fluoxetine (Prozac), and one substantially reversed morphine withdrawal effects.

Structures of the entire human opioid receptor family.

Opioids are effective analgesics, but their use is beset by serious side effects, including addiction and respiratory depression, which contribute to the ongoing opioid crisis. The human opioid system contains four opioid receptors (µOR, δOR, κOR, and NOPR) and a set of related endogenous opioid peptides (EOPs), which show distinct selectivity toward their respective opioid receptors (ORs). Despite being key to the development of safer analgesics, the mechanisms of molecular recognition and selectivity of EOPs to ORs remain unclear. Here, we systematically characterize the binding of EOPs to ORs and present five structures of EOP-OR-Gi complexes, including ß-endorphin- and endomorphin-bound µOR, deltorphin-bound δOR, dynorphin-bound κOR, and nociceptin-bound NOPR. These structures, supported by biochemical results, uncover the specific recognition and selectivity of opioid peptides and the conserved mechanism of opioid receptor activation. These results provide a structural framework to facilitate rational design of safer opioid drugs for pain relief.

Structural insights into the human D1 and D2 dopamine receptor signaling complexes.

The D1- and D2-dopamine receptors (D1R and D2R), which signal through Gs and Gi, 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-Gs and D2R-Gi 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.

Structure of the Cardiac Sodium Channel.

Voltage-gated sodium channel Nav1.5 generates cardiac action potentials and initiates the heartbeat. Here, we report structures of NaV1.5 at 3.2-3.5 Å resolution. NaV1.5 is distinguished from other sodium channels by a unique glycosyl moiety and loss of disulfide-bonding capability at the NaVß subunit-interaction sites. The antiarrhythmic drug flecainide specifically targets the central cavity of the pore. The voltage sensors are partially activated, and the fast-inactivation gate is partially closed. Activation of the voltage sensor of Domain III allows binding of the isoleucine-phenylalanine-methionine (IFM) motif to the inactivation-gate receptor. Asp and Ala, in the selectivity motif DEKA, line the walls of the ion-selectivity filter, whereas Glu and Lys are in positions to accept and release Na+ ions via a charge-delocalization network. Arrhythmia mutation sites undergo large translocations during gating, providing a potential mechanism for pathogenic effects. Our results provide detailed insights into Nav1.5 structure, pharmacology, activation, inactivation, ion selectivity, and arrhythmias.

How GPCR Phosphorylation Patterns Orchestrate Arrestin-Mediated Signaling.

Binding of arrestin to phosphorylated G-protein-coupled receptors (GPCRs) controls many aspects of cell signaling. The number and arrangement of phosphates may vary substantially for a given GPCR, and different phosphorylation patterns trigger different arrestin-mediated effects. Here, we determine how GPCR phosphorylation influences arrestin behavior by using atomic-level simulations and site-directed spectroscopy to reveal the effects of phosphorylation patterns on arrestin binding and conformation. We find that patterns favoring binding differ from those favoring activation-associated conformational change. Both binding and conformation depend more on arrangement of phosphates than on their total number, with phosphorylation at different positions sometimes exerting opposite effects. Phosphorylation patterns selectively favor a wide variety of arrestin conformations, differently affecting arrestin sites implicated in scaffolding distinct signaling proteins. We also reveal molecular mechanisms of these phenomena. Our work reveals the structural basis for the long-standing "barcode" hypothesis and has important implications for design of functionally selective GPCR-targeted drugs.

The Geometry of Abstraction in the Hippocampus and Prefrontal Cortex.

The curse of dimensionality plagues models of reinforcement learning and decision making. The process of abstraction solves this by constructing variables describing features shared by different instances, reducing dimensionality and enabling generalization in novel situations. Here, we characterized neural representations in monkeys performing a task described by different hidden and explicit variables. Abstraction was defined operationally using the generalization performance of neural decoders across task conditions not used for training, which requires a particular geometry of neural representations. Neural ensembles in prefrontal cortex, hippocampus, and simulated neural networks simultaneously represented multiple variables in a geometry reflecting abstraction but that still allowed a linear classifier to decode a large number of other variables (high shattering dimensionality). Furthermore, this geometry changed in relation to task events and performance. These findings elucidate how the brain and artificial systems represent variables in an abstract format while preserving the advantages conferred by high shattering dimensionality.

Activation and Signaling Mechanism Revealed by Cannabinoid Receptor-Gi Complex Structures.

Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.

Crystal Structure of the Human Cannabinoid Receptor CB2.

The cannabinoid receptor CB2 is predominately expressed in the immune system, and selective modulation of CB2 without the psychoactivity of CB1 has therapeutic potential in inflammatory, fibrotic, and neurodegenerative diseases. Here, we report the crystal structure of human CB2 in complex with a rationally designed antagonist, AM10257, at 2.8 Å resolution. The CB2-AM10257 structure reveals a distinctly different binding pose compared with CB1. However, the extracellular portion of the antagonist-bound CB2 shares a high degree of conformational similarity with the agonist-bound CB1, which led to the discovery of AM10257's unexpected opposing functional profile of CB2 antagonism versus CB1 agonism. Further structural analysis using mutagenesis studies and molecular docking revealed the molecular basis of their function and selectivity for CB2 and CB1. Additional analyses of our designed antagonist and agonist pairs provide important insight into the activation mechanism of CB2. The present findings should facilitate rational drug design toward precise modulation of the endocannabinoid system.

Angiotensin Analogs with Divergent Bias Stabilize Distinct Receptor Conformations.

"Biased" G protein-coupled receptor (GPCR) agonists preferentially activate pathways mediated by G proteins or ß-arrestins. Here, we use double electron-electron resonance spectroscopy to probe the changes that ligands induce in the conformational distribution of the angiotensin II type I receptor. Monitoring distances between 10 pairs of nitroxide labels distributed across the intracellular regions enabled mapping of four underlying sets of conformations. Ligands from different functional classes have distinct, characteristic effects on the conformational heterogeneity of the receptor. Compared to angiotensin II, the endogenous agonist, agonists with enhanced Gq coupling more strongly stabilize an "open" conformation with an accessible transducer-binding site. ß-arrestin-biased agonists deficient in Gq coupling do not stabilize this open conformation but instead favor two more occluded conformations. These data suggest a structural mechanism for biased ligand action at the angiotensin receptor that can be exploited to rationally design GPCR-targeting drugs with greater specificity of action.

5-HT2C Receptor Structures Reveal the Structural Basis of GPCR Polypharmacology.

Drugs frequently require interactions with multiple targets-via a process known as polypharmacology-to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT2C receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here, we solved two structures of the 5-HT2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT2A-C receptor-selective inverse agonist ritanserin at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs.

Functional Selectivity in Cytokine Signaling Revealed Through a Pathogenic EPO Mutation.

Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.

Bias Factor and Therapeutic Window Correlate to Predict Safer Opioid Analgesics.

Biased agonism has been proposed as a means to separate desirable and adverse drug responses downstream of G protein-coupled receptor (GPCR) targets. Herein, we describe structural features of a series of mu-opioid-receptor (MOR)-selective agonists that preferentially activate receptors to couple to G proteins or to recruit ßarrestin proteins. By comparing relative bias for MOR-mediated signaling in each pathway, we demonstrate a strong correlation between the respiratory suppression/antinociception therapeutic window in a series of compounds spanning a wide range of signaling bias. We find that ßarrestin-biased compounds, such as fentanyl, are more likely to induce respiratory suppression at weak analgesic doses, while G protein signaling bias broadens the therapeutic window, allowing for antinociception in the absence of respiratory suppression.

Structures of the Human HCN1 Hyperpolarization-Activated Channel.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the control of rhythmic activity in cardiac and neuronal pacemaker cells. In HCN, the polarity of voltage dependence is uniquely reversed. Intracellular cyclic adenosine monophosphate (cAMP) levels tune the voltage response, enabling sympathetic nerve stimulation to increase the heart rate. We present cryo-electron microscopy structures of the human HCN channel in the absence and presence of cAMP at 3.5 Å resolution. HCN channels contain a K+ channel selectivity filter-forming sequence from which the amino acids create a unique structure that explains Na+ and K+ permeability. The voltage sensor adopts a depolarized conformation, and the pore is closed. An S4 helix of unprecedented length extends into the cytoplasm, contacts the C-linker, and twists the inner helical gate shut. cAMP binding rotates cytoplasmic domains to favor opening of the inner helical gate. These structures advance understanding of ion selectivity, reversed polarity gating, and cAMP regulation in HCN channels.

Genomic context sensitizes regulatory elements to genetic disruption.

Genomic context critically modulates regulatory function but is difficult to manipulate systematically. The murine insulin-like growth factor 2 (Igf2)/H19 locus is a paradigmatic model of enhancer selectivity, whereby CTCF occupancy at an imprinting control region directs downstream enhancers to activate either H19 or Igf2. We used synthetic regulatory genomics to repeatedly replace the native locus with 157-kb payloads, and we systematically dissected its architecture. Enhancer deletion and ectopic delivery revealed previously uncharacterized long-range regulatory dependencies at the native locus. Exchanging the H19 enhancer cluster with the Sox2 locus control region (LCR) showed that the H19 enhancers relied on their native surroundings while the Sox2 LCR functioned autonomously. Analysis of regulatory DNA actuation across cell types revealed that these enhancer clusters typify broader classes of context sensitivity genome wide. These results show that unexpected dependencies influence even well-studied loci, and our approach permits large-scale manipulation of complete loci to investigate the relationship between regulatory architecture and function.

How Flies See Motion.

How neurons detect the direction of motion is a prime example of neural computation: Motion vision is found in the visual systems of virtually all sighted animals, it is important for survival, and it requires interesting computations with well-defined linear and nonlinear processing steps-yet the whole process is of moderate complexity. The genetic methods available in the fruit fly Drosophila and the charting of a connectome of its visual system have led to rapid progress and unprecedented detail in our understanding of how neurons compute the direction of motion in this organism. The picture that emerged incorporates not only the identity, morphology, and synaptic connectivity of each neuron involved but also its neurotransmitters, its receptors, and their subcellular localization. Together with the neurons' membrane potential responses to visual stimulation, this information provides the basis for a biophysically realistic model of the circuit that computes the direction of visual motion.

RBM33 is a unique m6A RNA-binding protein that regulates ALKBH5 demethylase activity and substrate selectivity.

Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.

Structural insights into adhesion GPCR ADGRL3 activation and Gq, Gs, Gi, and G12 coupling.

Adhesion G-protein-coupled receptors (aGPCRs) play key roles in a diversity of physiologies. A hallmark of aGPCR activation is the removal of the inhibitory GAIN domain and the dipping of the cleaved stalk peptide into the ligand-binding pocket of receptors; however, the detailed mechanism remains obscure. Here, we present cryoelectron microscopy (cryo-EM) structures of ADGRL3 in complex with Gq, Gs, Gi, and G12. The structures reveal unique ligand-engaging mode, distinctive activation conformation, and key mechanisms of aGPCR activation. The structures also reveal the uncharted structural information of GPCR/G12 coupling. A comparison of Gq, Gs, Gi, and G12 engagements with ADGRL3 reveals the key determinant of G-protein coupling on the far end of αH5 of Gα. A detailed analysis of the engagements allows us to design mutations that specifically enhance one pathway over others. Taken together, our study lays the groundwork for understanding aGPCR activation and G-protein-coupling selectivity.

GPCRs steer Gi and Gs selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

Serotonin (or 5-hydroxytryptamine, 5-HT) is an important neurotransmitter that activates 12 different G protein-coupled receptors (GPCRs) through selective coupling of Gs, Gi, or Gq proteins. The structural basis for G protein subtype selectivity by these GPCRs remains elusive. Here, we report the structures of the serotonin receptors 5-HT4, 5-HT6, and 5-HT7 with Gs, and 5-HT4 with Gi1. The structures reveal that transmembrane helices TM5 and TM6 alternate lengths as a macro-switch to determine receptor's selectivity for Gs and Gi, respectively. We find that the macro-switch by the TM5-TM6 length is shared by class A GPCR-G protein structures. Furthermore, we discover specific residues within TM5 and TM6 that function as micro-switches to form specific interactions with Gs or Gi. Together, these results present a common mechanism of Gs versus Gi protein coupling selectivity or promiscuity by class A GPCRs and extend the basis of ligand recognition at serotonin receptors.

Selective chemical tracking of Dnmt1 catalytic activity in live cells.

Enzymatic methylation of cytosine to 5-methylcytosine in DNA is a fundamental epigenetic mechanism involved in mammalian development and disease. DNA methylation is brought about by collective action of three AdoMet-dependent DNA methyltransferases, whose catalytic interactions and temporal interplay are poorly understood. We used structure-guided engineering of the Dnmt1 methyltransferase to enable catalytic transfer of azide tags onto DNA from a synthetic cofactor analog, Ado-6-azide, in vitro. We then CRISPR-edited the Dnmt1 locus in mouse embryonic stem cells to install the engineered codon, which, following pulse internalization of the Ado-6-azide cofactor by electroporation, permitted selective azide tagging of Dnmt1-specific genomic targets in cellulo. The deposited covalent tags were exploited as "click" handles for reading adjoining sequences and precise genomic mapping of the methylation sites. The proposed approach, Dnmt-TOP-seq, enables high-resolution temporal tracking of the Dnmt1 catalysis in mammalian cells, paving the way to selective studies of other methylation pathways in eukaryotic systems.