Double-stranded DNA minicircles have been observed in a variety of biological settings and are also widely employed in biotechnology, therapeutic applications, and basic research. Here, we report a cryo-EM structure of a 95-basepair minicircle (dsMC95) at a 5.3 Å resolution. dsMC95 forms a closed ring as designed and no local deformation is observed. The two DNA strands are fully resolved, with the major and minor grooves clearly distinguishable. Analysis reveals a nine-fold periodicity in the helical twist, which corresponds to approximately 10.56 base pairs per turn. Together with groove width analysis, the data indicate that dsMC95 maintains a B-DNA configuration. The dsMC95 ring exhibits an in-plane ellipticity of 1.13 and an out-of-plane displacement of 15°, with differences in out-of-plane displacements observed between the two half-segments. The dsMC95 structure, which is the only free DNA cryo-EM structure with a resolution better than 6 Å to date, allows comparison to other structures to better understand DNA physical features such as bending. The findings advance our understanding of DNA structure under topological constraints and may inform studies of naturally occurring small circular DNA as well as the manipulation of DNA in nanotechnology applications.

Abstract The functional complexity of G protein-coupled receptors (GPCRs) arises from their structural dynamics, spanning timescales from nanoseconds to minutes. Single-molecule Förster Resonance Energy Transfer (smFRET) enables direct observation of these dynamics in individual receptors, either freely diffusing in solution, using confocal microscopy, or immobilized on surfaces, using Total Internal Reflection Fluorescence (TIRF) camera-based microscopy. However, these modalities are limited to distinct timescales – faster than milliseconds or slower than hundreds of milliseconds, respectively. To overcome these limitations, we employed smFRET with Anti-Brownian Electrokinetic (ABEL) trapping to extend the observation time of untethered human A2A adenosine receptors (A2AAR) reconstituted in lipid nanodiscs from milliseconds to seconds. We characterized conformational heterogeneity in apo and ligand-bound A2AAR and updated previous estimates of dwell times for long-lived receptor states from milliseconds to hundreds of milliseconds. Our results highlight the power of ABEL-FRET to probe GPCRs dynamics and offer valuable insights into GPCR conformational landscapes.

The kinin receptors B1R and B2R act as key regulators within the kallikrein-kinin system, mediating vasodilation, inflammation, and pain. Antagonists targeting these receptors have shown therapeutic potential in the treatment of angioedema, pancreatitis, and asthma. However, the molecular mechanisms underlying subtype selectivity of kinin receptors' antagonists remain unclear. Here we report three cryo-electron microscopy (cryo-EM) structures of B1R in complex with the peptide antagonist R715 and small-molecule antagonist ELN441958, as well as B2R in complex with small-molecule antagonist Win64338. Together with functional assays and molecular docking, these structures unveil the molecular basis of antagonist binding modes in kinin receptors. Three non-conserved residues at the bottom of the ligand binding pocket are identified to confer the antagonist selectivity to B1R and B2R. Furthermore, our results reveal a shared mechanism for antagonism in both B1R and B2R, wherein the antagonist binding restricts the conformational changes of the toggle switch tryptophan residue. Collectively, these insights lay the foundation for the structure-based design of selective antagonists targeting B1R and B2R.

CRISPR-Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA at specific sites and has been adapted as a powerful tool for genome manipulation. Cas9 recognizes its target through multiple conformational transitions coordinated between the Cas9 ribonucleoprotein and the DNA duplex. Such transitions, and consequently Cas9 targeting specificity, are expected to be significantly influenced by the collective duplex physical properties referred to as DNA shape. To advance our currently limited understanding of the interplay between DNA shape and Cas9 target interrogation, we solved two cryo-EM structures of SpyCas9 bound to a cognate target embedded in a relaxed 95-base-pair DNA double-stranded minicircle. The Cas9-bound DNA segment engages in similar interactions involved in PAM-binding and R-loop initiation as those observed in Cas9-bound linear DNA. However, R-loop is limited to less than three base-pairs, thus interfering with Cas9 cleavage. The minicircle DNA, which is fully resolved, retains its global shape. As Cas9 locally unwinds the protospacer, the closed-ring topology constrains the movement of the paired PAM-distal DNA duplex, thus interfering with R-loop propagation. These data provide detailed insight into the interplay between DNA shape and Cas9 structure and function, and may shed light on genome-editing and manipulation in environments with varied DNA topologies.

Forkhead homologue 1 (Fkh1) is a yeast transcription factor that plays essential roles in cell-cycle dynamics. Here, we report the co-crystal structure of the DNA-binding domain (DBD) of the yeast Fkh1 protein in complex with a 19-base pair oligonucleotide containing the core binding site and flanking regions. The three-dimensional structure of the Fkh1-DBD reveals a previously unknown protein fold among all known Forkhead proteins. The winged-helix fold forms base-specific contacts of α-helix H3 with the major groove of the core binding site. Wing 1 and Wing 2 form DNA shape-mediated contacts with the minor groove of the binding site flanking regions. The conformation of Wing 2 is distinct from all known Forkhead proteins, with α-helices H5 and H6 wrapping back onto the protein core, creating a stable Wing 2 loop. Backbone interactions with β-strands S1 and S2 reveal a structural mechanism for previously observed flanking region preferences in SELEX-seq experiments. In vivo yeast experiments on Fkh1 mutants demonstrate that wing residues interacting with flanking regions are important for Fkh1 function. Molecular dynamics simulations relate Fkh1 function to conformational flexibility of wing residues. The novel Forkhead fold enables Fkh1 function with implications, such as structure-based protein design, for other DNA-binding proteins.

The prostaglandin D2 receptor 1 (DP1), a member of the prostanoid G protein-coupled receptor (GPCR) family, plays critical roles in allergic responses, sleep regulation, immune modulation, and vasodilation. Here, we present five high-resolution cryo-electron microscopy (cryo-EM) structures of the human DP1 receptor, including an apo structure, two inactive state structures bound to two different inverse agonists developed by ONO Pharmaceutical, and two active state structures in complex with the Gs protein and bound to the endogenous agonist PGD2 and its selective derivative BW245C. Structural analysis, complemented by molecular dynamics simulations and site-directed mutagenesis, reveals key residues involved in ligand recognition and suggests a distinct activation mechanism for DP1, which lacks most of the conserved class A GPCR motifs. Notably, the unique residue K76 within the conserved sodium pocket acts as a major activation switch, while amphiphilic helix 8 adopts an unconventional orientation essential for receptor function. These findings offer valuable insights into the structure and function of prostanoid receptors and may facilitate the development of therapeutics targeting DP1.

G protein-coupled receptors (GPCRs) constitute the largest transmembrane protein superfamily, with over 800 representatives in the human genome. Recognized as pivotal targets in pharmacological research and drug discovery, these receptors play a crucial role in advancing therapeutics. Understanding the molecular mechanisms of receptor-ligand interactions is imperative for drug discovery applications. However, experimental procedures for measuring ligand binding are complicated by various factors, including the transmembrane nature of the receptors and the high cost associated with specialized instruments and consumables. Here we introduce an application of the thermal shift assay (TSA) to measuring ligand binding affinities for GPCRs. TSA is a cost-effective and user-friendly method that detects changes in protein stability induced by alterations in environmental conditions. Employing the human A2A adenosine receptor as a representative GPCR, we determined binding constants for four orthosteric ligands and allosteric sodium using three mathematical models for TSA data approximation and analysis. Models were additionally validated by two antagonists of cysteinyl leukotriene GPCR (CysLT1R), used as antiasthmatic drugs. Our results suggest that the TSA approach demonstrates a high degree of reproducibility and agreement with existing literature data, thereby affirming its suitability for investigating GPCR interactions with various types of ligands.

GPR6 is an orphan G protein-coupled receptor with high constitutive activity found in D2-type dopamine receptor-expressing medium spiny neurons of the striatopallidal pathway, which is aberrantly hyperactivated in Parkinson's disease. Here, we solved crystal structures of GPR6 without the addition of a ligand (a pseudo-apo state) and in complex with two inverse agonists, including CVN424, which improved motor symptoms in patients with Parkinson's disease in clinical trials. In addition, we obtained a cryo-electron microscopy structure of the signaling complex between GPR6 and its cognate Gs heterotrimer. The pseudo-apo structure revealed a strong density in the orthosteric pocket of GPR6 corresponding to a lipid-like endogenous ligand. A combination of site-directed mutagenesis, native mass spectrometry, and computer modeling suggested potential mechanisms for high constitutive activity and inverse agonism in GPR6 and identified a series of lipids and ions bound to the receptor. The structures and results obtained in this study could guide the rational design of drugs that modulate GPR6 signaling.

G protein-coupled receptors (GPCRs) are essential transmembrane proteins playing key roles in human health and disease. Understanding their atomic-level molecular structure and conformational states is imperative for advancing drug development. Recent breakthroughs in single-particle cryogenic electron microscopy (cryo-EM) have propelled the structural biology of GPCRs into a new era. Nevertheless, the preparation of suitable GPCR samples and their complexes for cryo-EM analysis remains challenging due to their poor stability and highly dynamic nature. Here, we present our online buffer exchange-native MS method combined with Direct Mass Technology (OBE-nMS+DMT) which facilitates high-throughput analysis and guides sample preparation. We applied this method to optimize the GPR119-Gs complex sample prior to cryo-EM analysis, leading to a 3.51 Å resolution structure from only 396 movies collected on a 200 kV Glacios. This study suggests that the OBE-nMS+DMT method emerges as a powerful tool for prescreening sample conditions in cryo-EM studies of GPCRs and other membrane protein complexes.

Insulin vesicle heterogeneity within the pancreatic β cell has been previously observed, but the extent of this heterogeneity has not been determined due to limitations in organelle separation techniques. There has previously been a lack of unbiased methods for studying differences in organelles, such as the insulin vesicle. Direct current insulator-based dielectrophoresis (DC-iDEP) separates particles based on various biophysical properties without requiring fluorescent labels, characterizing them by their electrokinetic mobility ratio (EKMr). This method has been applied to insulin vesicles isolated from mammalian insulin-secreting cell lines in order to investigate the degree of insulin vesicle heterogeneity, as well as how these subpopulations fluctuate under various cell conditions. There were significant differences in the distribution of insulin vesicles between conditions, suggesting variations in subpopulation distribution. Insulin vesicles with different EKMr values may represent variations in maturity, size, cargo, or other relevant characteristics differing between insulin vesicle subpopulations.

Dear Editor, Human G protein-coupled receptor 55(GPR55)is an orphan GPCR,termed an atypical cannabinoid receptor,CB3R.1 This classification was further supported by studies demonstrating that the endogenous ligands anandamide(AEA)and 2-arachidonoylglycerol(2-AG)of CB1R and CB2R,along with their synthetic agonist CP55940,could activate GPR55.2 Interestingly,CB1R antagonists such as rimonabant and AM251 were also reported to exhibit activity on GPR55,although reports on rimonabant's effect on GPR55 are inconsistent across different laboratories.2,3 Unlike CB1R or CB2R,which primarily couple with G)prtoein,4 GPR55 activation induces diverse cellular responses by coupling with G12/13 or Gq protein.2,3 However,recent studies suggest that lysophosphatidylinositol(LPI)and its 2-arachidonyl analogs,rather than endocannabinoids,may serve as endogenous agonists of GPR55.5,6 Therefore,the deorphanization of GPR55 still remains debatable.

Organelle heterogeneity and inter-organelle contacts within a single cell contribute to the limited sensitivity of current organelle separation techniques, thus hindering organelle subpopulation characterization. Here, we use direct current insulator-based dielectrophoresis (DC-iDEP) as an unbiased separation method and demonstrate its capability by identifying distinct distribution patterns of insulin vesicles from INS-1E insulinoma cells. A multiple voltage DC-iDEP strategy with increased range and sensitivity has been applied, and a differentiation factor (ratio of electrokinetic to dielectrophoretic mobility) has been used to characterize features of insulin vesicle distribution patterns. We observed a significant difference in the distribution pattern of insulin vesicles isolated from glucose-stimulated cells relative to unstimulated cells, in accordance with maturation of vesicles upon glucose stimulation. We interpret the difference in distribution pattern to be indicative of high-resolution separation of vesicle subpopulations. DC-iDEP provides a path for future characterization of subtle biochemical differences of organelle subpopulations within any biological system.

Nicotinamide adenine dinucleotide (NAD+) is required for a myriad of metabolic, signaling, and post-translational events in cells. Its levels in tissues and organs are closely associated with health conditions. The homeostasis of NAD+ is regulated by biosynthetic pathways and consuming enzymes. As a membrane-bound protein with robust NAD+ hydrolase activity, cluster of differentiation 38 (CD38) is a major degrader of NAD+. Deficiency or inhibition of CD38 enhances NAD+ levels in vivo, resulting in various therapeutic benefits. As a metabolic precursor of NAD+, nicotinamide mononucleotide can be rapidly hydrolyzed by CD38, whereas nicotinamide riboside (NR) lacks CD38 substrate activity. Given their structural similarities, we explored the inhibition potential of NR. To our surprise, NR exhibits marked inhibitory activity against CD38 by forming a stable ribosyl-ester bond with the glutamate residue 226 at the active site. Inspired by this discovery, we designed and synthesized a clickable NR featuring an azido substitution at the 5 '-OH position. This cell-permeable NR analogue enables covalent labeling and imaging of both extracellular and intracellular CD38 in live cells. Our work discovers an unrecognized molecular function of NR and generates a covalent probe for health-related CD38. These findings offer new insights into the role of NR in modulating NAD+ metabolism and CD38-mediated signaling as well as an innovative tool for in-depth studies of CD38 in physiology and pathophysiology.

G protein-coupled receptors (GPCRs) represent some of themost important drug targets in the human genome, however,our understanding of these receptors has primarily beenconfined to systems that require the overexpression of targetreceptors. In a recent study published inCell, Janicot et al.introduced a universal biosensor platform capable of detect-ing GPCR signaling at endogenous levels, which facilitates itsapplication across various cell lines, including primary cells,thereby improving our understanding of context-dependentGPCR activity and system bias

Solvatochromic compounds have emerged as valuable environment-sensitive probes for biological research. Here we used thiol-reactive solvatochromic analogs of the green fluorescent protein (GFP) chromophore to track conformational changes in two proteins, recoverin and the A2A adenosine receptor (A2AAR). Two dyes showed Ca2+-induced fluorescence changes when attached to recoverin. Our best-performing dye, DyeC, exhibited agonist-induced changes in both intensity and shape of its fluorescence spectrum when attached to A2AAR; none of these effects were observed with other common environment-sensitive dyes. Molecular dynamics simulations showed that activation of the A2AAR led to a more confined and hydrophilic environment for DyeC. Additionally, an allosteric modulator of A2AAR induced distinct fluorescence changes in the DyeC spectrum, indicating a unique receptor conformation. Our study demonstrated that GFP-inspired dyes are effective for detecting structural changes in G protein-coupled receptors (GPCRs), offering advantages such as intensity-based and ratiometric tracking, redshifted fluorescence spectra, and sensitivity to allosteric modulation.

GPR20 is a class-A orphan G protein-coupled receptor (GPCR) and a potential therapeutic target for gastrointestinal stromal tumors (GIST) owing to its differentially high expression. An antibody-drug conjugate (ADC) containing a GPR20-binding antibody (Ab046) was recently developed in clinical trials for GIST treatment. GPR20 constitutively activates Gi proteins in the absence of any known ligand, but it remains obscure how this high basal activity is achieved. Here we report three cryo-EM structures of human GPR20 complexes including Gi-coupled GPR20 in the absence or presence of the Fab fragment of Ab046 and Gi-free GPR20. Remarkably, the structures demonstrate a uniquely folded N-terminal helix capping onto the transmembrane domain and our mutagenesis study suggests a key role of this cap region in stimulating the basal activity of GPR20. We also uncover the molecular interactions between GPR20 and Ab046, which may enable the design of tool antibodies with enhanced affinity or new functionality for GPR20. Furthermore, we report the orthosteric pocket occupied by an unassigned density which might be essential for exploring opportunities for deorphanization.

γ-Aminobutyric acid (GABA) transporter 1 (GAT1)1 regulates neuronal excitation of the central nervous system by clearing the synaptic cleft of the inhibitory neurotransmitter GABA upon its release from synaptic vesicles. Elevating the levels of GABA in the synaptic cleft, by inhibiting GABA reuptake transporters, is an established strategy to treat neurological disorders, such as epilepsy2. Here we determined the cryo-electron microscopy structure of full-length, wild-type human GAT1 in complex with its clinically used inhibitor tiagabine3, with an ordered part of only 60 kDa. Our structure reveals that tiagabine locks GAT1 in the inward-open conformation, by blocking the intracellular gate of the GABA release pathway, and thus suppresses neurotransmitter uptake. Our results provide insights into the mixed-type inhibition of GAT1 by tiagabine, which is an important anticonvulsant medication. Its pharmacodynamic profile, confirmed by our experimental data, suggests initial binding of tiagabine to the substrate-binding site in the outward-open conformation, whereas our structure presents the drug stalling the transporter in the inward-open conformation, consistent with a two-step mechanism of inhibition4. The presented structure of GAT1 gives crucial insights into the biology and pharmacology of this important neurotransmitter transporter and provides blueprints for the rational design of neuromodulators, as well as moving the boundaries of what is considered possible in single-particle cryo-electron microscopy of challenging membrane proteins. Structural determination of GAT1 using cryo-electron microscopy provides insights into the biology and pharmacology of this GABA transporter.