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In the early stage of transcription, eukaryotic RNA polymerase II (Pol II) exchanges initiation factors with elongation factors to form an elongation complex for processive transcription. Here we report the structure of the Pol II elongation complex bound with the basal elongation factors Spt4/5, Elf1, and TFIIS. Spt4/5 (the Spt4/Spt5 complex) and Elf1 modify a wide area of the Pol II surface. Elf1 bridges the Pol II central cleft, completing a "DNA entry tunnel" for downstream DNA. Spt4 and the Spt5 NGN and KOW1 domains encircle the upstream DNA, constituting a "DNA exit tunnel." The Spt5 KOW4 and KOW5 domains augment the "RNA exit tunnel," directing the exiting nascent RNA. Thus, the elongation complex establishes a completely different transcription and regulation platform from that of the initiation complexes.

Maintenance of cell volume against osmotic change is crucial for proper cell functions. Leucine-rich repeat-containing 8 proteins are anion-selective channels that extrude anions to decrease the cell volume on cellular swelling. Here, we present the structure of human leucine-rich repeat-containing 8A, determined by single-particle cryo-electron microscopy. The structure shows a hexameric assembly, and the transmembrane region features a topology similar to gap junction channels. The LRR region, with 15 leucine-rich repeats, forms a long, twisted arc. The channel pore is located along the central axis and constricted on the extracellular side, where highly conserved polar and charged residues at the tip of the extracellular helix contribute to permeability to anions and other osmolytes. Two structural populations were identified, corresponding to compact and relaxed conformations. Comparing the two conformations suggests that the LRR region is flexible and mobile, with rigid-body motions, which might be implicated in structural transitions on pore opening.

Tetraspanins play critical roles in various physiological processes, ranging from cell adhesion to virus infection. The members of the tetraspanin family have four membrane-spanning domains and short and large extracellular loops, and associate with a broad range of other functional proteins to exert cellular functions. Here we report the crystal structure of CD9 and the cryo-electron microscopic structure of CD9 in complex with its single membranespanning partner protein, EWI-2. The reversed cone-like molecular shape of CD9 generates membrane curvature in the crystalline lipid layers, which explains the CD9 localization in regions with high membrane curvature and its implications in membrane remodeling. The molecular interaction between CD9 and EWI-2 is mainly mediated through the small residues in the transmembrane region and protein/lipid interactions, whereas the fertilization assay revealed the critical involvement of the LEL region in the sperm-egg fusion, indicating the different dependency of each binding domain for other partner proteins.

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No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Purification of human eIF2The α-, β-, and γ-subunits of human eIF2, and the eIF2-specific chaperone protein human Cdc123 (33) were co-expressed in FreeStyle 293-F cells, using the four pEBMulti-Neo plasmid vectors (Wako), and eIF2γ was expressed with C-terminal FLAG and His8 tags. The cells were lysed in buffer A [20 mM MES-KOH buffer (pH 6.0), containing 150 mM KCl, 1 mM MgCl2, 10%(v/v) glycerol, and 5 mM 2-mercaptoethanol] supplemented with 20 mM imidazole, 0.5 mM EDTA, 0.1%(v/v) Triton X-100 and protease inhibitors. After 30 min on ice and centrifugation, the supernatant was applied to a HisTrap (GE Healthcare) column equilibrated with buffer A supplemented with 20 mM imidazole, and eluted with a linear gradient of 20-500 mM imidazole. The fraction containing eIF2 was collected and applied to a HiTrap SP (GE Healthcare) column equilibrated with buffer A, and eluted with a linear gradient of 200-640 mM KCl. After three-fold dilution with buffer B [20 mM HEPES-KOH buffer (pH 7.5) containing 100 mM KCl, 0.1 mM MgCl2, 10%(v/v) glycerol, and 1 mM DTT], the sample was applied to a HiTrap Heparin (GE Healthcare) column equilibrated with buffer B, and eluted with a linear gradient of 0.2-1 M KCl. The fraction containing eIF2 was further purified on a Superdex200 (GE Healthcare) column equilibrated with buffer B. eIF2 was phosphorylated by PKR, as described for Komagataella pastoris eIF2 (14). Purification of human eIF2BHuman eIF2Bα and eIF2Bβγδε were purified separately.The fragment encoding human eIF2Bα was cloned into pET-28c (Novagen), in which the thrombin cleavage site was replaced by the HRV 3C protease cleavage site. The T7 Express

Members of the leucine-rich repeat-containing 8 (LRRC8) protein family, composed of the five LRRC8A-E isoforms, are pore-forming components of the volume-regulated anion channel (VRAC). LRRC8A and at least one of the other LRRC8 isoforms assemble into heteromers to generate VRAC transport activities. Despite the availability of the LRRC8A structures, the structural basis of how LRRC8 isoforms other than LRRC8A contribute to the functional diversity of VRAC has remained elusive. Here, we present the structure of the human LRRC8D isoform, which enables the permeation of organic substrates through VRAC. The LRRC8D homo-hexamer structure displays a twofold symmetric arrangement, and together with a structure-based electrophysiological analysis, revealed two key features. The pore constriction on the extracellular side is wider than that in the LRRC8A structures, which may explain the increased permeability of organic substrates. Furthermore, an N-terminal helix protrudes into the pore from the intracellular side and may be critical for gating.

Death-associated protein kinase 1 (DAPK1) is a 160 kDa serine/threonine protein kinase that belongs to the Ca(2+)/calmodulin-dependent protein kinase subfamily. DAPK1 is a possible target for the treatment of acute ischemic stroke and endometrial adenocarcinomas. In the present study, we investigated the binding characteristics of 17 natural flavonoids to DAPK1 using a 1-anilinonaphthalene-8-sulfonic acid competitive binding assay and revealed that morin was the strongest binder among the selected compounds. The crystallographic analysis of DAPK1 and 7 selected flavonoid complexes revealed the structure-binding affinity relationship in atomic-level detail. It was suggested that the high affinity of morin could be accounted for by the ionic interaction between 2'-OH and K42 and that such an interaction would not take place with either cyclin-dependent protein kinases or PIM kinases because of their broader entrance regions. Thus, morin would be a more selective inhibitor of DAPK1 than either of these other types of kinases. In addition, we found that the binding of kaempferol to DAPK1 was associated with a chloride ion. The present study provides a better understanding of the molecular properties of the ATP site of DAPK1 and may be useful for the design of specific DAPK1 inhibitors.