Explore the vast range of titles available.

Gap junctions establish direct pathways for cells to transfer metabolic and electrical messages. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions; however, the molecular basis for these effects remains unknown. Here, we incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid nanodisc system, mimicking a native cell-to-cell junction. Structural characterization by CryoEM reveals a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 1.9 Å resolution. Together with all-atom molecular dynamics simulations, it is shown that Cx46/50 in turn imparts long-range stabilization to the dynamic local lipid environment that is specific to the extracellular lipid leaflet. In addition, ~400 water molecules are resolved in the CryoEM map, localized throughout the intercellular permeation pathway and contributing to the channel architecture. These results illustrate how the aqueous-lipid environment is integrated with the architectural stability, structure and function of gap junction communication channels. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions, however, the molecular basis for these effects remains unknown. Here authors report the CryoEM structure of Cx46/50 lipid-embedded channels, by which they reveal a lipid-induced stabilization to the channel.

The Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) assembles into large 12-meric holoenzymes, which is thought to enable regulatory processes required for synaptic plasticity underlying learning, memory and cognition. Here we used single particle electron microscopy (EM) to determine a pseudoatomic model of the CaMKIIα holoenzyme in an extended and activation-competent conformation. The holoenzyme is organized by a rigid central hub complex, while positioning of the kinase domains is highly flexible, revealing dynamic holoenzymes ranging from 15–35 nm in diameter. While most kinase domains are ordered independently, ∼20% appear to form dimers and <3% are consistent with a compact conformation. An additional level of plasticity is revealed by a small fraction of bona-fide 14-mers (<4%) that may enable subunit exchange. Biochemical and cellular FRET studies confirm that the extended state of CaMKIIα resolved by EM is the predominant form of the holoenzyme, even under molecular crowding conditions. Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) forms a 12 subunit holoenzyme central to synaptic plasticity. Here the authors report a 3D structure of the CaMKII holoenzyme in an activation-competent state obtained by single particle EM, and suggest a role for the intrinsically disordered linker domain in facilitating cooperative activation.

The transcription factor ASCIZ (ATMIN, ZNF822) has an unusually high number of recognition motifs for the product of its main target gene, the hub protein LC8 (DYNLL1). Using a combination of biophysical methods, structural analysis by NMR and electron microscopy, and cellular transcription assays, we developed a model that proposes a concerted role of intrinsic disorder and multiple LC8 binding events in regulating LC8 transcription. We demonstrate that the long intrinsically disordered C-terminal domain of ASCIZ binds LC8 to form a dynamic ensemble of complexes with a gradient of transcriptional activity that is inversely proportional to LC8 occupancy. The preference for low occupancy complexes at saturating LC8 concentrations with both human and Drosophila ASCIZ indicates that negative cooperativity is an important feature of ASCIZ-LC8 interactions. The prevalence of intrinsic disorder and multivalency among transcription factors suggests that formation of heterogeneous, dynamic complexes is a widespread mechanism for tuning transcriptional regulation. Proteins help to regulate almost every process in the body, and come in various forms, sizes and purposes. Cells contain thousands of different proteins, but not every protein is needed at all times. To create new proteins, the information on a gene first needs to be transcribed into RNA (template molecules of the DNA) in a process known as transcription. A complex machinery inside the cell then uses the copy as a template to assemble the protein. So-called transcription factors (also proteins) can switch the copying process on or off by binding to the start point of a gene. They can act alone or in complex with other proteins. The transcription factor called ASCIZ, for example, helps to regulate the production of a protein called LC8. LC8 attaches to more than 100 different proteins and plays an important role in many cell processes. Therefore, fine-tuning its production is essential. The shape of a protein is critical to its purpose. Like most proteins, transcription factors are made up of chains of amino acids that fold into a specific three-dimensional (3D) structurewith a region that recognizes and binds to a specific DNA sequence. But many transcription factors also contain flexible, ‘disordered’ regions that do not fold into a rigid 3D shape. These may help to control the activity of genes, but their exact role is unclear. ASCIZ contains an exceptionally long, disordered region that has multiple positions for binding LC8 along its chain. Previous research has shown that ASCIZ binds to the LC8 gene and increases transcription to produce more LC8 proteins. Once the protein levels are high enough, LC8 is thought to bind to the disordered region of ASCIZ and switch off transcription. Human ASCIZ proteins have 11 binding sites for LC8 molecules, while fruit flies have seven. Until now it was not clear why so many different binding sites exist. To address this question, Clark et al. combined biophysical, structural and molecular biology techniques to analyze proteins from humans and fruit flies and to test their role in human cells. This revealed that LC8 and ASCIZ form a dynamic mixture of complexes, instead of a single fully-occupied complex. As the number of LC8 molecules bound to ASCIZ increased, the rate of transcription dropped. However, all of the binding sites were rarely fully occupied. Instead, three to four attached LC8 molecules seemed to be sufficient to ensure that LC8 levels remain balanced. When the number of LC8 molecules exceeded this value, the attachment rate for additional LC8 slowed down. So, even when there was an excess of LC8, most of the human ASCIZ binding sites were only partially filled. This way, the production of LC8 proteins was slowed, rather than fully shut down. As a result, the cells were able to fine-tune the transcription rate of LC8 and maintain a stable and balanced pool of these proteins. This work suggests that disordered regions on transcription factors could help to keep cellular systems steady in the face of changing conditions. In the future, the combination of methods used here could reveal new information about other proteins with disordered regions.

We investigated Brucella melitensis methionyl-tRNA-synthetase (BmMetRS) with molecular, structural and phenotypic methods to learn if BmMetRS is a promising target for brucellosis drug development. Recombinant BmMetRS was expressed, purified from wild type Brucella melitensis biovar Abortus 2308 strain ATCC/CRP #DD-156 and screened by a thermal melt assay against a focused library of one hundred previously classified methionyl-tRNA-synthetase inhibitors of the blood stage form of Trypanosoma brucei. Three compounds showed appreciable shift of denaturation temperature and were selected for further studies on inhibition of the recombinant enzyme activity and cell viability against wild type B. melitensis strain 16M. BmMetRS protein complexed with these three inhibitors resolved into three-dimensional crystal structures and was analyzed. All three selected methionyl-tRNA-synthetase compounds inhibit recombinant BmMetRS enzymatic functions in an aminoacylation assay at varying concentrations. Furthermore, growth inhibition of B. melitensis strain 16M by the compounds was shown. Inhibitor-BmMetRS crystal structure models were used to illustrate the molecular basis of the enzyme inhibition. Our current data suggests that BmMetRS is a promising target for brucellosis drug development. However, further studies are needed to optimize lead compound potency, efficacy and safety as well as determine the pharmacokinetics, optimal dosage, and duration for effective treatment.

Gap junctions establish direct pathways for cell-to-cell communication through the assembly of twelve connexin subunits that form intercellular channels connecting neighbouring cells. Co-assembly of different connexin isoforms produces channels with unique properties and enables communication across cell types. Here we used single-particle cryo-electron microscopy to investigate the structural basis of connexin co-assembly in native lens gap junction channels composed of connexin 46 and connexin 50 (Cx46/50). We provide the first comparative analysis to connexin 26 (Cx26), which—together with computational studies—elucidates key energetic features governing gap junction permselectivity. Cx46/50 adopts an open-state conformation that is distinct from the Cx26 crystal structure, yet it appears to be stabilized by a conserved set of hydrophobic anchoring residues. ‘Hot spots’ of genetic mutations linked to hereditary cataract formation map to the core structural–functional elements identified in Cx46/50, suggesting explanations for many of the disease-causing effects. Cryo-electron microscopy structures of connexin channels composed of connexin 46 and connexin 50 in an open-state reveal features that govern permselectivity and the location of mutated residues linked to herediatry cataracts.

[This corrects the article DOI: 10.1371/journal.pone.0160350.].

Nature Communications 8: Article number: 15742 (2017); Published 7 June 2017; Updated 25 May 2018 The previously published version of this Article contained an error in Figure 7. In panel b, the % Compact subunits was shown as ‘<30%’ but should have instead been ‘<3%’. This error has been corrected in both the PDF and HTML versions of the Article.

This corrects the article DOI: 10.1038/ncomms15742.