Structural insights into vesicular monoamine storage and drug interactions.

Biogenic monoamines-vital transmitters orchestrating neurological, endocrinal and immunological functions1-5-are stored in secretory vesicles by vesicular monoamine transporters (VMATs) for controlled quantal release6,7. Harnessing proton antiport, VMATs enrich monoamines around 10,000-fold and sequester neurotoxicants to protect neurons8-10. VMATs are targeted by an arsenal of therapeutic drugs and imaging agents to treat and monitor neurodegenerative disorders, hypertension and drug addiction1,8,11-16. However, the structural mechanisms underlying these actions remain unclear. Here we report eight cryo-electron microscopy structures of human VMAT1 in unbound form and in complex with four monoamines (dopamine, noradrenaline, serotonin and histamine), the Parkinsonism-inducing MPP+, the psychostimulant amphetamine and the antihypertensive drug reserpine. Reserpine binding captures a cytoplasmic-open conformation, whereas the other structures show a lumenal-open conformation stabilized by extensive gating interactions. The favoured transition to this lumenal-open state contributes to monoamine accumulation, while protonation facilitates the cytoplasmic-open transition and concurrently prevents monoamine binding to avoid unintended depletion. Monoamines and neurotoxicants share a binding pocket that possesses polar sites for specificity and a wrist-and-fist shape for versatility. Variations in this pocket explain substrate preferences across the SLC18 family. Overall, these structural insights and supporting functional studies elucidate the mechanism of vesicular monoamine transport and provide the basis to develop therapeutics for neurodegenerative diseases and substance abuse.

The hepatitis C virus envelope protein complex is a dimer of heterodimers.

Fifty-eight million individuals worldwide are affected by chronic hepatitis C virus (HCV) infection, a primary driver of liver cancer for which no vaccine is available1. The HCV envelope proteins E1 and E2 form a heterodimer (E1/E2), which is the target for neutralizing antibodies2. However, the higher-order organization of these E1/E2 heterodimers, as well as that of any Hepacivirus envelope protein complex, remains unknown. Here we determined the cryo-electron microscopy structure of two E1/E2 heterodimers in a homodimeric arrangement. We reveal how the homodimer is established at the molecular level and provide insights into neutralizing antibody evasion and membrane fusion by HCV, as orchestrated by E2 motifs such as hypervariable region 1 and antigenic site 412, as well as the organization of the transmembrane helices, including two internal to E1. This study addresses long-standing questions on the higher-order oligomeric arrangement of Hepacivirus envelope proteins and provides a critical framework in the design of novel HCV vaccine antigens.

Calmodulin Triggers Activity-Dependent rRNA Biogenesis via Interaction with DDX21.

Protein synthesis in response to neuronal activity, known as activity-dependent translation, is critical for synaptic plasticity and memory formation. However, the signaling cascades that couple neuronal activity to the translational events remain elusive. In this study, we identified the role of calmodulin (CaM), a conserved Ca2+-binding protein, in ribosomal RNA (rRNA) biogenesis in neurons. We found the CaM-regulated rRNA synthesis is Ca2+-dependent and necessary for nascent protein synthesis and axon growth in hippocampal neurons. Mechanistically, CaM interacts with nucleolar DEAD (Asp-Glu-Ala-Asp) box RNA helicase (DDX21) in a Ca2+-dependent manner to regulate nascent rRNA transcription within nucleoli. We further found CaM alters the conformation of DDX21 to liberate the DDX21-sequestered RPA194, the catalytic subunit of RNA polymerase I, to facilitate transcription of ribosomal DNA. Using high-throughput screening, we identified the small molecules batefenterol and indacaterol that attenuate the CaM-DDX21 interaction and suppress nascent rRNA synthesis and axon growth in hippocampal neurons. These results unveiled the previously unrecognized role of CaM as a messenger to link the activity-induced Ca2+ influx to the nucleolar events essential for protein synthesis. We thus identified the ability of CaM to transmit information to the nucleoli of neurons in response to stimulation.

Cryo-EM reveals the conformational epitope of human monoclonal antibody PAM1.4 broadly reacting with polymorphic malarial protein VAR2CSA.

Malaria during pregnancy is a major global health problem caused by infection with Plasmodium falciparum parasites. Severe effects arise from the accumulation of infected erythrocytes in the placenta. Here, erythrocytes infected by late blood-stage parasites adhere to placental chondroitin sulphate A (CS) via VAR2CSA-type P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. Immunity to placental malaria is acquired through exposure and mediated through antibodies to VAR2CSA. Through evolution, the VAR2CSA proteins have diversified in sequence to escape immune recognition but retained their overall macromolecular structure to maintain CS binding affinity. This structural conservation may also have allowed development of broadly reactive antibodies to VAR2CSA in immune women. Here we show the negative stain and cryo-EM structure of the only known broadly reactive human monoclonal antibody, PAM1.4, in complex with VAR2CSA. The data shows how PAM1.4's broad VAR2CSA reactivity is achieved through interactions with multiple conserved residues of different sub-domains forming conformational epitope distant from the CS binding site on the VAR2CSA core structure. Thus, while PAM1.4 may represent a class of antibodies mediating placental malaria immunity by inducing phagocytosis or NK cell-mediated cytotoxicity, it is likely that broadly CS binding-inhibitory antibodies target other epitopes at the CS binding site. Insights on both types of broadly reactive monoclonal antibodies may aid the development of a vaccine against placental malaria.

Identifying Heterozipper ß-Sheet in Twisted Amyloid Aggregation.

Amyloid peptide (AP) self-assembly is a hierarchical process. However, the mechanistic rule of guiding peptides to organize well-ordered nanostructure in a clear and precise manner remains poorly understood. Herein we explored the molecular insight of AP motif aggregates underlying hierarchical process with helical fibrillar structure by atomic force microscope, cryo-electron microscopy (cryo-EM), and molecular dynamics simulation. AP assembly encompasses well-ordered twisted fibrils with uniform morphology, size, and periodicity. More importantly, a heterozipper ß-sheet was identified in a protofilament of AP assembly determined by cryo-EM with a high resolution of 3.5 Å. Each peptide heterozipper was further composed of two antiparallel ß strands and arranged by an alternative manner in a protofilament. The hydrophobic core and hydrophilic area in each zipper played the significant role for peptide assembling. This work proposed and verified the rule facilitating the basic building unit to form twisted fibrils and gave the explanation of peptide hierarchical assembling.

MADS2 regulates priming defence in postharvest peach through combined salicylic acid and abscisic acid signaling.

MADS-box genes play well-documented roles in plant development, but relatively little is known regarding their involvement in defence responses. In this study, pre-treatment of peach (Prunus persica) fruit with ß-aminobutyric acid (BABA) activated resistance against Rhizopus stolonifer, leading to a significant delay in the symptomatic appearance of disease. This was associated with an integrated defence response that included a H2O2 burst, ABA accumulation, and callose deposition. cDNA library screening identified nucleus-localized MADS2 as an interacting partner with NPR1, and this was further confirmed by yeast two-hybrid, luciferase complementation imaging, and co-immunoprecipitation assays. The DNA-binding activity of NPR1 conferred by the NPR1-MADS2 complex was required for the transcription of SA-dependent pathogenesis-related (PR) and ABA-inducible CalS genes in order to gain the BABA-induced resistance, in which MAPK1-induced post-translational modification of MADS2 was also involved. In accordance with this, overexpression of PpMADS2 in Arabidopsis potentiated the transcription of a group of PR genes and conferred fungal resistance in the transgenic plants. Conversely, Arabidopsis mads2-knockout lines showed high sensitivity to the fungal pathogen. Our results indicate that MADS2 positively participates in BABA-elicited defence in peach through a combination of SA-dependent NPR1 activation and ABA signaling-induced callose accumulation, and that this defence is also related to the post-translational modification of MADS2 by MAPK1 for signal amplification.

Structure of the human ClC-1 chloride channel.

ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes, thereby orchestrating skeletal muscle excitability. Malfunction of ClC-1 is associated with myotonia congenita, a disease impairing muscle relaxation. Here, we present the cryo-electron microscopy (cryo-EM) structure of human ClC-1, uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters. The chloride conducting pathway exhibits distinct features, including a central glutamate residue ("fast gate") known to confer voltage-dependence (a mechanistic feature not present in ClC-K), linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule. These characteristics agree with the lower chloride flux of ClC-1 compared with ClC-K and enable us to propose a model for chloride passage in voltage-dependent CLC channels. Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-ß-synthase (CBS) domains and the intracellular vestibule ("slow gating"). The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating, opening avenues for rational design of therapies against ClC-1-related diseases.

Composition and processing activity of a semi-recombinant holo U7 snRNP.

In animal cells, replication-dependent histone pre-mRNAs are cleaved at the 3' end by U7 snRNP consisting of two core components: a ∼60-nucleotide U7 snRNA and a ring of seven proteins, with Lsm10 and Lsm11 replacing the spliceosomal SmD1 and SmD2. Lsm11 interacts with FLASH and together they recruit the endonuclease CPSF73 and other polyadenylation factors, forming catalytically active holo U7 snRNP. Here, we assembled core U7 snRNP bound to FLASH from recombinant components and analyzed its appearance by electron microscopy and ability to support histone pre-mRNA processing in the presence of polyadenylation factors from nuclear extracts. We demonstrate that semi-recombinant holo U7 snRNP reconstituted in this manner has the same composition and functional properties as endogenous U7 snRNP, and accurately cleaves histone pre-mRNAs in a reconstituted in vitro processing reaction. We also demonstrate that the U7-specific Sm ring assembles efficiently in vitro on a spliceosomal Sm site but the engineered U7 snRNP is functionally impaired. This approach offers a unique opportunity to study the importance of various regions in the Sm proteins and U7 snRNA in 3' end processing of histone pre-mRNAs.

Alterations in Sucrose and Phenylpropanoid Metabolism Affected by BABA-Primed Defense in Postharvest Grapes and the Associated Transcriptional Mechanism.

Defense elicitors can induce fruit disease resistance to control postharvest decay but may incur quality impairment. Our present work aimed to investigate the resistance against Botrytis cinerea induced by the elicitor ß-aminobutyric acid (BABA) and to elucidate the specific transcriptional mechanism implicated in defense-related metabolic regulations. The functional dissection results demonstrated that, after inoculation with the fungal necrotroph B. cinerea, a suite of critical genes encoding enzymes related to the sucrose metabolism and phenylpropanoid pathway in priming defense in grapes were transcriptionally induced by treatment with 10 mM BABA. In contrast, more UDP-glucose, a shared precursor of phenylpropanoid and sucrose metabolism, may be redirected to the phenylpropanoid pathway for the synthesis of phytoalexins, including trans-resveratrol and ɛ-viniferin, in 100 mM BABA-treated grapes, resulting in direct resistance but compromised soluble sugar contents. An R2R3-type MYB protein from Vitis vinifera, VvMYB44, was isolated and characterized. VvMYB44 expression was significantly induced upon the grapes expressed defensive reaction. Subcellular localization, yeast two-hybrid, and coimmunoprecipitation assays revealed that the nuclear-localized VvMYB44 physically interacted with the salicylic acid-responsive transcription coactivator NPR1 in vivo for defense expression. In addition, VvMYB44 directly bound to the promoter regions of sucrose and phenylpropanoid metabolism-related genes and transactivated their expression, thus tipping the balance of antifungal compound accumulation and soluble sugar maintenance. Hence, these results suggest that 2R-type VvMYB44 might be a potential positive participant in BABA-induced priming defense in grape berries that contributes to avoiding the excessive consumption of soluble sugars during the postharvest storage.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Dual function of VvWRKY18 transcription factor in the ß-aminobutyric acid-activated priming defense in grapes.

Induction of phytoalexin production after invading pathogens is recognized as an essential aspect of the plant-induced resistance. The WRKY family includes plant-specific transcriptional factors associated with plant defense responses, but the comprehensive mechanisms are poorly understood. Here, we attempted to elaborate the regulatory function of VvWRKY18 from the group IIa of WRKY transcription factor (TF) from Vitis vinifera, in the regulation of ß-aminobutyric acid (BABA)-activated stilbene phytoalexins biosynthesis and PATHOGENESIS-RELATED (PR) genes expressions in grapes. BABA at 10 mmol L-1 triggered a priming protection in grapes and conferred a potentiation of the expression levels of VvWRKY18, VvNPR1, and several salicylic acid (SA)-responsive genes, which was accompanied by enhanced stilbene production upon Botrytis cinerea infection. In addition, a physical interaction between VvWRKY18 and the regulatory protein VvNPR1 was detected in vivo and in vitro by yeast-2-hybrid (Y2H), pull-down and co-immunoprecipitation assay (Co-IP) assays. Furthermore, yeast-1-hybrid (Y1H) and dual-luciferase reporter (DLR) assays indicated that VvWRKY18 activated the transcription of STILBENE SYNTHASE (STS) genes, including VvSTS1 and VvSTS2, by directly binding the W-box elements within the specific promoters and resultantly enhancing stilbene phytoalexins biosynthesis. Further investigation demonstrated that heterologous expression of VvWRKY18 elevated the transcriptions of STS and PR genes, thus contributing to potentiating the defense of transgenic Arabidopsis thaliana plants and resultantly inhibiting B. cinerea invasion. Hence, VvWRKY18 serves as a singular effector involved in the synthesis of stilbene phytoalexins in grapes and its interaction with VvNPR1 provided DNA binding ability required for VvNPR1 to initiate systemic acquired resistance (SAR) defense.