Rotaviruses (RVs) are highly important pathogens that cause severe diarrhea among infants and young children worldwide. The understanding of the molecular mechanisms underlying RV replication and pathogenesis has been hampered by the lack of an entirely plasmid-based reverse genetics system. In this study, we describe the recovery of recombinant RVs entirely from cloned cDNAs. The strategy requires coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. We used this system to obtain insights into the process by which RV nonstructural protein NSP1 subverts host innate immune responses. By insertion into the NSP1 gene segment, we recovered recombinant viruses that encode split-green fluorescent protein-tagged NSP1 and NanoLuc luciferase. This technology will provide opportunities for studying RV biology and foster development of RV vaccines and therapeutics.

Previously, we determined the crystal structures of the dimeric ligand binding region of the metabotropic glutamate receptor subtype 1. Each protomer binds Lglutamate within the crevice between the LB1 and LB2 domains. We proposed that the two different conformations of the dimer interface between the two LB1 domains define the activated and resting states of the receptor protein. In this study, the residues in the ligandbinding site and the dimer interface were mutated, and the effects were analyzed in the full-length and truncated soluble receptor forms. The variations in the ligand binding activities of the purified truncated receptors are comparable with those of the full-length form. The mutated full-length receptors were also analyzed by inositol phosphate production and Ca 2؉ response. , and Gly 293 residues, which interact with the ␥-carboxyl group of glutamate, lost their responsiveness to glutamate but not to quisqualate. Furthermore, a mutant receptor containing alanine instead of isoleucine at position 120 located within an ␣ helix constituting the dimer interface showed no intracellular response to ligand stimulation. The results demonstrate the crucial role of the dimer interface in receptor activation.Glutamate is a major neurotransmitter in excitatory neurons in the central nervous system. Glutamate released into the synaptic space is recognized by two distinct receptors, glutamate-gated ion channels and metabotropic glutamate receptors (mGluRs) 1 (1, 2). The mGluRs consist of eight subtypes (mGluR1 to -8), which couple with a variety of effector systems, including inositol phosphate pathway, adenylyl cyclase, ion channels, etc. The mGluRs are considered to modulate synaptic neurotransmission and thus to play roles in memory, learning, and brain disorders such as epilepsy and neurodegenerative diseases.The mGluR consists of three regions: a large extracellular region, a seven-transmembrane-spanning region, and an intracellular region. Previously, we determined the crystal structures of the extracellular ligand-binding region (LBR) of mGluR1 (3). In combination with biochemical studies (4, 5), the mGluR1-LBR (m1-LBR) was found to be a homodimer consisting of two protomers. Each protomer consists of an LB1 domain and an LB2 domain. The glutamate-binding structure is a dimer composed of closed and open protomers, which differ in the relative orientation of the LB1 and LB2 domains. Without glutamate, two crystal forms of m1-LBR were obtained; one form exists as an open-open dimer, and the other is an openclosed form. The two main functioning sites were then elucidated: the ligand-recognition site and the LB1 dimer interface. In the ligand binding site, glutamate interacts mainly with 13 amino acid residues from the LB1 and LB2 domains of the protomer. We proposed that the ligand-binding domain of mGluR1 is in dynamic equilibrium between the activated state and the resting state, which are defined mainly by the different dimer interface conformations of the three crystal forms. An antagonist binding c...

The structures of the ligand-binding domains (LBDs) of human peroxisome proliferator-activated receptors (PPARα, PPARγ and PPARδ) in complexes with a pan agonist, an α/δ dual agonist and a PPARδ-specific agonist were determined. The results explain how each ligand is recognized by the PPAR LBDs at an atomic level.

BackgroundIn the early stage of eukaryotic DNA replication, the template DNA is unwound by the MCM helicase, which is activated by forming a complex with the Cdc45 and GINS proteins. The eukaryotic GINS forms a heterotetramer, comprising four types of subunits. On the other hand, the archaeal GINS appears to be either a tetramer formed by two types of subunits in a 2:2 ratio (α2β2) or a homotetramer of a single subunit (α4). Due to the low sequence similarity between the archaeal and eukaryotic GINS subunits, the atomic structures of the archaeal GINS complexes are attracting interest for comparisons of their subunit architectures and organization.ResultsWe determined the crystal structure of the α2β2 GINS tetramer from Thermococcus kodakaraensis (TkoGINS), comprising Gins51 and Gins23, and compared it with the reported human GINS structures. The backbone structure of each subunit and the tetrameric assembly are similar to those of human GINS. However, the location of the C-terminal small domain of Gins51 is remarkably different between the archaeal and human GINS structures. In addition, TkoGINS exhibits different subunit contacts from those in human GINS, as a consequence of the different relative locations and orientations between the domains. Based on the GINS crystal structures, we built a homology model of the putative homotetrameric GINS from Thermoplasma acidophilum (TacGINS). Importantly, we propose that a long insertion loop allows the differential positioning of the C-terminal domains and, as a consequence, exclusively leads to the formation of an asymmetric homotetramer rather than a symmetrical one.ConclusionsThe DNA metabolizing proteins from archaea are similar to those from eukaryotes, and the archaeal multi-subunit complexes are occasionally simplified versions of the eukaryotic ones. The overall similarity in the architectures between the archaeal and eukaryotic GINS complexes suggests that the GINS function, directed through interactions with other protein components, is basically conserved. On the other hand, the different subunit contacts, including the locations and contributions of the C-terminal domains to the tetramer formation, imply the possibility that the archaeal and eukaryotic GINS complexes contribute to DNA unwinding reactions by significantly different mechanisms in terms of the atomic details.