Ligand recognition and allosteric regulation of DRD1-Gs signaling complexes.

Dopamine receptors, including D1- and D2-like receptors, are important therapeutic targets in a variety of neurological syndromes, as well as cardiovascular and kidney diseases. Here, we present five cryoelectron microscopy (cryo-EM) structures of the dopamine D1 receptor (DRD1) coupled to Gs heterotrimer in complex with three catechol-based agonists, a non-catechol agonist, and a positive allosteric modulator for endogenous dopamine. These structures revealed that a polar interaction network is essential for catecholamine-like agonist recognition, whereas specific motifs in the extended binding pocket were responsible for discriminating D1- from D2-like receptors. Moreover, allosteric binding at a distinct inner surface pocket improved the activity of DRD1 by stabilizing endogenous dopamine interaction at the orthosteric site. DRD1-Gs interface revealed key features that serve as determinants for G protein coupling. Together, our study provides a structural understanding of the ligand recognition, allosteric regulation, and G protein coupling mechanisms of DRD1.

Tethered peptide activation mechanism of the adhesion GPCRs ADGRG2 and ADGRG4.

Adhesion G protein-coupled receptors (aGPCRs) constitute an evolutionarily ancient family of receptors that often undergo autoproteolysis to produce α and ß subunits1-3. A tethered agonism mediated by the 'Stachel sequence' of the ß subunit has been proposed to have central roles in aGPCR activation4-6. Here we present three cryo-electron microscopy structures of aGPCRs coupled to the Gs heterotrimer. Two of these aGPCRs are activated by tethered Stachel sequences-the ADGRG2-ß-Gs complex and the ADGRG4-ß-Gs complex (in which ß indicates the ß subunit of the aGPCR)-and the other is the full-length ADGRG2 in complex with the exogenous ADGRG2 Stachel-sequence-derived peptide agonist IP15 (ADGRG2(FL)-IP15-Gs). The Stachel sequences of both ADGRG2-ß and ADGRG4-ß assume a U shape and insert deeply into the seven-transmembrane bundles. Constituting the FXφφφXφ motif (in which φ represents a hydrophobic residue), five residues of ADGRG2-ß or ADGRG4-ß extend like fingers to mediate binding to the seven-transmembrane domain and activation of the receptor. The structure of the ADGRG2(FL)-IP15-Gs complex reveals the structural basis for the improved binding affinity of IP15 compared with VPM-p15 and indicates that rational design of peptidic agonists could be achieved by exploiting aGPCR-ß structures. By converting the 'finger residues' to acidic residues, we develop a method to generate peptidic antagonists towards several aGPCRs. Collectively, our study provides structural and biochemical insights into the tethered activation mechanism of aGPCRs.

Plasma-activated water: An alternative disinfectant for S protein inactivation to prevent SARS-CoV-2 infection.

SARS-CoV-2 is a highly contagious virus and is causing a global pandemic. SARS-CoV-2 infection depends on the recognition of and binding to the cellular receptor human angiotensin-converting enzyme 2 (hACE2) through the receptor-binding domain (RBD) of the spike protein, and disruption of this process can effectively inhibit SARS-CoV-2 invasion. Plasma-activated water efficiently inactivates bacteria and bacteriophages by causing damage to biological macromolecules, but its effect on coronavirus has not been reported. In this study, pseudoviruses with the SARS-CoV-2 S protein were used as a model, and plasma-activated water (PAW) effectively inhibited pseudovirus infection through S protein inactivation. The RBD was used to study the molecular details, and the RBD binding activity was inactivated by plasma-activated water through the RBD modification. The short-lived reactive species in the PAW, such as ONOO-, played crucial roles in this inactivation. Plasma-activated water after room-temperature storage of 30 days remained capable of significantly reducing the RBD binding with hACE2. Together, our findings provide evidence of a potent disinfection strategy to combat the epidemic caused by SARS-CoV-2.

Chemically Controlled Helical Polymorphism in Protein Tubes by Selective Modulation of Supramolecular Interactions.

Polymorphism has been the subject of investigation across different research disciplines. In biology, polymorphism could be interpreted in such a way that discrete biomacromolecules can adopt diversiform specific conformations/packing arrangement, and this polymorph-dependent property is essential for many biochemical processes. For example, bacterial flagellar filament, composed of flagellin, switches between different supercoiled state allowing the bacteria to swim and tumble. However, in artificial supramolecular systems, it is often challenging to achieve polymorph control and prediction, and in most cases, two or more concomitant polymorphs of similar formation energies coexist. Here, we show that a tetrameric protein with properly oriented binding sites on its surface can arrange into diverse protein tubes with distinct helical parameters by adding specifically designed inducing ligands. We examined several parameters of the ligand that would influence the protein tube formation and found that the flexibility of the ligand linker and the dimerization pose of the ligand complex is critical for the successful production of the tubes and eventually influence the specific helical polymorphs of the formed tubes. A surface lattice accommodation model was further developed to rationalize the geometrical relationship between each helical tube type. Molecular simulation was used to elucidate the interactions between ligands and SBA and molecular basis for polymorphic switching of the protein tubes. Moreover, the kinetics of structural formation was studied and the ligand design was found that can affect the kinetics of the protein polymerization pathway. In short, our designed protein tubes serves as an enlightening system for understanding how a protein polymer composed of a single protein switches among different helical states.

Mechanism of Virus Inactivation by Cold Atmospheric-Pressure Plasma and Plasma-Activated Water.

Viruses cause serious pathogenic contamination that severely affects the environment and human health. Cold atmospheric-pressure plasma efficiently inactivates pathogenic bacteria; however, the mechanism of virus inactivation by plasma is not fully understood. In this study, surface plasma in argon mixed with 1% air and plasma-activated water was used to treat water containing bacteriophages. Both agents efficiently inactivated bacteriophages T4, Φ174, and MS2 in a time-dependent manner. Prolonged storage had marginal effects on the antiviral activity of plasma-activated water. DNA and protein analysis revealed that the reactive species generated by plasma damaged both nucleic acids and proteins, consistent with the morphological examination showing that plasma treatment caused the aggregation of bacteriophages. The inactivation of bacteriophages was alleviated by the singlet oxygen scavengers, demonstrating that singlet oxygen played a primary role in this process. Our findings provide a potentially effective disinfecting strategy to combat the environmental viruses using cold atmospheric-pressure plasma and plasma-activated water.IMPORTANCE Contamination with pathogenic and infectious viruses severely threatens human health and animal husbandry. Current methods for disinfection have different disadvantages, such as inconvenience and contamination of disinfection by-products (e.g., chlorine disinfection). In this study, atmospheric surface plasma in argon mixed with air and plasma-activated water was found to efficiently inactivate bacteriophages, and plasma-activated water still had strong antiviral activity after prolonged storage. Furthermore, it was shown that bacteriophage inactivation was associated with damage to nucleic acids and proteins by singlet oxygen. An understanding of the biological effects of plasma-based treatment is useful to inform the development of plasma into a novel disinfecting strategy with convenience and no by-product.

Four Parallel Pathways in T4 Ligase-Catalyzed Repair of Nicked DNA with Diverse Bending Angles.

The structural diversity of biological macromolecules in different environments contributes complexity to enzymological processes vital for cellular functions. Fluorescence resonance energy transfer and electron microscopy are used to investigate the enzymatic reaction of T4 DNA ligase catalyzing the ligation of nicked DNA. The data show that both the ligase-AMP complex and the ligase-AMP-DNA complex can have four conformations. This finding suggests the parallel occurrence of four ligation reaction pathways, each characterized by specific conformations of the ligase-AMP complex that persist in the ligase-AMP-DNA complex. Notably, these complexes have DNA bending angles of ≈0°, 20°, 60°, or 100°. The mechanism of parallel reactions challenges the conventional notion of simple sequential reaction steps occurring among multiple conformations. The results provide insights into the dynamic conformational changes and the versatile attributes of T4 DNA ligase and suggest that the parallel multiple reaction pathways may correspond to diverse T4 DNA ligase functions. This mechanism may potentially have evolved as an adaptive strategy across evolutionary history to navigate complex environments.

Directional growth of quasi-2D Cu2O monocrystals on rGO membranes in aqueous environments.

The preparation technology of unconventional low-dimensional Cu2O monocrystals, which exhibit specific crystal planes and present significantly unique interfacial and physicochemical properties, is attracting increasing attention and interest. Herein, by integrating a high-temperature oxidation process under vacuum and a pure-water incubation process under ambient conditions, we propose the self-assembled growth and synthesis of quasi-two-dimensional Cu2O monocrystals on reduced graphene oxide (rGO) membranes. The prepared Cu2O crystals have a single (110) crystal plane, regular rectangular morphology, and potentially well conductivity. Experimental and theoretical results suggest that this assembly is attributed to the pre-nucleation clusters aggregation and directional attachment of Cu and O on the rGO membranes in aqueous environment and cation-π interactions between the (110) crystal plane of Cu2O and rGO surface. Our findings offer a potential avenue for the discovery and design of advanced low-dimensional single-crystal materials with specific interfacial properties in a pure aqueous environment.

Cold Atmospheric-Pressure Plasma Caused Protein Damage in Methicillin-Resistant Staphylococcus aureus Cells in Biofilms.

Biofilms formed by multidrug-resistant bacteria are a major cause of hospital-acquired infections. Cold atmospheric-pressure plasma (CAP) is attractive for sterilization, especially to disrupt biofilms formed by multidrug-resistant bacteria. However, the underlying molecular mechanism is not clear. In this study, CAP effectively reduced the living cells in the biofilms formed by methicillin-resistant Staphylococcus aureus, and 6 min treatment with CAP reduced the S. aureus cells in biofilms by 3.5 log10. The treatment with CAP caused the polymerization of SaFtsZ and SaClpP proteins in the S. aureus cells of the biofilms. In vitro analysis demonstrated that recombinant SaFtsZ lost its self-assembly capability, and recombinant SaClpP lost its peptidase activity after 2 min of treatment with CAP. Mass spectrometry showed oxidative modifications of a cluster of peaks differing by 16 Da, 31 Da, 32 Da, 47 Da, 48 Da, 62 Da, and 78 Da, induced by reactive species of CAP. It is speculated that the oxidative damage to proteins in S. aureus cells was induced by CAP, which contributed to the reduction of biofilms. This study elucidates the biological effect of CAP on the proteins in bacterial cells of biofilms and provides a basis for the application of CAP in the disinfection of biofilms.

Novel 2D CaCl crystals with metallicity, room-temperature ferromagnetism, heterojunction, piezoelectricity-like property and monovalent calcium ions.

Under ambient conditions, the only known valence state of calcium ions is +2, and the corresponding crystals with calcium ions are insulating and nonferromagnetic. Here, using cryo-electron microscopy, we report direct observation of two-dimensional (2D) CaCl crystals on reduced graphene oxide (rGO) membranes, in which the calcium ions are only monovalent (i.e. +1). Remarkably, metallic rather than insulating properties are displayed by those CaCl crystals. More interestingly, room-temperature ferromagnetism, graphene-CaCl heterojunction, coexistence of piezoelectricity-like property and metallicity, as well as the distinct hydrogen storage and release capability of the CaCl crystals in rGO membranes are experimentally demonstrated. We note that such CaCl crystals are obtained by simply incubating rGO membranes in salt solutions below the saturated concentration, under ambient conditions. Theoretical studies suggest that the formation of those abnormal crystals is attributed to the strong cation-π interactions of the Ca cations with the aromatic rings in the graphene surfaces. The findings highlight the realistic potential applications of such abnormal CaCl material with unusual electronic properties in designing novel transistors and magnetic devices, hydrogen storage, catalyzers, high-performance conducting electrodes and sensors, with a size down to atomic scale.

Aberrant brain structural network and altered topological organization in minimal hepatic encephalopathy.

PURPOSE: We aimed to investigate the multilevel impairments of brain structural network in patients with minimal hepatic encephalopathy (MHE). METHODS: Twenty-two patients with MHE and 22 well-matched healthy controls (HC) underwent structural magnetic resonance imaging (MRI) brain scans and neuropsychological evaluations. Individual brain structural networks were constructed using diffusion tensor imaging. Comparing with HC, we investigated the possible impairments of brain structural network in MHE, by applying graph-theory approaches to analyze the topological organization at global, modular, and local levels. The correlations between altered brain structural network and neuropsychological tests scores and venous ammonia levels were also examined in MHE patients. RESULTS: In the MHE group, small-worldness showed significant decrease and normalized characteristic path length showed increase at the global level. In the modular section, six modules were identified. The inter-modular connective strengths showed significant increase between modules 2 and 4 and between modules 4 and 5. The results of node analysis showed similar hub distributions in the MHE and HC groups except for the right postcentral gyrus, which was only found in the MHE group. No significant differences were found in connective strength of edges between MHE and HC groups using network-based statistics. CONCLUSION: The altered brain structural networks with reduced network integration and module segregation were demonstrated in patients with MHE. The dysconnectivity of brain structural network could provide an explanation for the brain dysfunctions of MHE.

Designing Melittin-Graphene Hybrid Complexes for Enhanced Antibacterial Activity.

Antimicrobial peptides (AMPs) promise a fundamental solution to the devastating threat of drug-resistant bacteria. However, drawbacks of AMPs (e.g., poor cell membrane penetration efficiency) seriously block their clinical use. In this work, rational design of a hybrid complex of melittin (as a representative AMP) and graphene or graphene oxide (Gra or GO) nanosheets for enhanced antibacterial ability is achieved, via combining in-silico prediction and in-tube test. In comparison to pristine melittin, the specifically designed AMP-Gra (/GO) complex exhibits remarkable efficiency in transmembrane perforation with an over tenfold decrease in the threshold working concentration of peptide; moreover, it has an up to 20-fold enhancement in antibacterial activity against both Gram-negative and Gram-positive bacteria. Such improvement is ascribed to the synergetic insertion of nanosheets and melittin due to similarity in antibacterial mechanism between them and is further regulated by the structural factors of the complex, including the intersheet spacing and surface functionalization of the Gra/GO sheets, etc. These results provide practical guidelines to engineer AMPs with nanotechnology for improved antimicrobial performances, especially based on targeted functionalization of the Gra/GO nanosheets.

Active DNA unwinding and transport by a membrane-adapted helicase nanopore.

Nanoscale transport through nanopores and live-cell membranes plays a vital role in both key biological processes as well as biosensing and DNA sequencing. Active translocation of DNA through these nanopores usually needs enzyme assistance. Here we present a nanopore derived from truncated helicase E1 of bovine papillomavirus (BPV) with a lumen diameter of c.a. 1.3 nm. Cryogenic electron microscopy (cryo-EM) imaging and single channel recording confirm its insertion into planar lipid bilayer (BLM). The helicase nanopore in BLM allows the passive single-stranded DNA (ssDNA) transport and retains the helicase activity in vitro. Furthermore, we incorporate this helicase nanopore into the live cell membrane of HEK293T cells, and monitor the ssDNA delivery into the cell real-time at single molecule level. This type of nanopore is expected to provide an interesting tool to study the biophysics of biomotors in vitro, with potential applications in biosensing, drug delivery and real-time single cell analysis.

Membrane Fusion Involved in Neurotransmission: Glimpse from Electron Microscope and Molecular Simulation.

Membrane fusion is one of the most fundamental physiological processes in eukaryotes for triggering the fusion of lipid and content, as well as the neurotransmission. However, the architecture features of neurotransmitter release machinery and interdependent mechanism of synaptic membrane fusion have not been extensively studied. This review article expounds the neuronal membrane fusion processes, discusses the fundamental steps in all fusion reactions (membrane aggregation, membrane association, lipid rearrangement and lipid and content mixing) and the probable mechanism coupling to the delivery of neurotransmitters. Subsequently, this work summarizes the research on the fusion process in synaptic transmission, using electron microscopy (EM) and molecular simulation approaches. Finally, we propose the future outlook for more exciting applications of membrane fusion involved in synaptic transmission, with the aid of stochastic optical reconstruction microscopy (STORM), cryo-EM (cryo-EM), and molecular simulations.