Dyneins are microtubule-based AAA(+) motor complexes that power ciliary beating, cell division, cell migration and intracellular transport. Here we report the most complete structure obtained so far, to our knowledge, of the 380-kDa motor domain of Dictyostelium discoideum cytoplasmic dynein at 2.8 Å resolution; the data are reliable enough to discuss the structure and mechanism at the level of individual amino acid residues. Features that can be clearly visualized at this resolution include the coordination of ADP in each of four distinct nucleotide-binding sites in the ring-shaped AAA(+) ATPase unit, a newly identified interaction interface between the ring and mechanical linker, and junctional structures between the ring and microtubule-binding stalk, all of which should be critical for the mechanism of dynein motility. We also identify a long-range allosteric communication pathway between the primary ATPase and the microtubule-binding sites. Our work provides a framework for understanding the mechanism of dynein-based motility.

Summary The multi-protein kinetochore complex must assemble at a specific site on each chromosome to achieve accurate chromosome segregation. Defining the nature of the DNA-protein interactions that specify the position of the kinetochore and provide a scaffold for kinetochore formation remain key goals. Here, we demonstrate that the centromeric histone-fold containing CENP-T-W and CENP-S-X complexes co-assemble to form a stable CENP-T-W-S-X heterotetramer. High-resolution structural analysis of the individual complexes and the heterotetramer reveals similarity to other histone fold-containing complexes including canonical histones within a nucleosome. The CENP-T-W-S-X heterotetramer binds to and supercoils DNA. Mutants designed to compromise heterotetramerization or the DNA-protein contacts around the heterotetramer strongly reduce the DNA binding and supercoiling activities in vitro and compromise kinetochore assembly in vivo. These data suggest that the CENP-T-W-S-X complex forms a unique nucleosome-like structure to generate contacts with DNA, extending the “histone code” beyond canonical nucleosome proteins.

Facilitates chromatin transcription (FACT) plays essential roles in chromatin remodeling during DNA transcription, replication, and repair. Our structural and biochemical studies of human FACT-histone interactions present precise views of nucleosome reorganization, conducted by the FACT-SPT16 (suppressor of Ty 16) Mid domain and its adjacent acidic AID segment. AID accesses the H2B N-terminal basic region exposed by partial unwrapping of the nucleosomal DNA, thereby triggering the invasion of FACT into the nucleosome. The crystal structure of the Mid domain complexed with an H3-H4 tetramer exhibits two separate contact sites; the Mid domain forms a novel intermolecular β structure with H4. At the other site, the Mid-H2A steric collision on the H2A-docking surface of the H3-H4 tetramer within the nucleosome induces H2A-H2B displacement. This integrated mechanism results in disrupting the H3 αN helix, which is essential for retaining the nucleosomal DNA ends, and hence facilitates DNA stripping from histone.

Lectin-like, oxidized low-density lipoprotein (LDL) receptor 1, LOX-1, is the major receptor for oxidized LDL (OxLDL) in endothelial cells. We have determined the crystal structure of the ligand binding domain of LOX-1, with a short stalk region connecting the domain to the membrane-spanning region, as a homodimer linked by an interchain disulfide bond. In vivo assays with LOX-1 mutants revealed that the "basic spine," consisting of linearly aligned arginine residues spanning over the dimer surface, is responsible for ligand binding. Single amino acid substitution in the dimer interface caused a severe reduction in LOX-1 binding activity, suggesting that the correct dimer arrangement is crucial for binding to OxLDL. Based on the LDL model structure, possible binding modes of LOX-1 to OxLDL are proposed.

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Ring-shaped sliding clamps and clamp loader ATPases are essential factors for rapid and accurate DNA replication. The clamp ring is opened and resealed at the primer-template junctions by the ATP-fueled clamp loader function. The processivity of the DNA polymerase is conferred by its attachment to the clamp loaded onto the DNA. In eukarya and archaea, the replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) play crucial roles as the clamp loader and the clamp, respectively. Here, we report the electron microscopic structure of an archaeal RFC-PCNA-DNA complex at 12-Å resolution. This complex exhibits excellent fitting of each atomic structure of RFC, PCNA, and the primed DNA. AAA ϩ ATPase ͉ clamp loader ͉ DNA replication ͉ electron microscopy ͉ single-particle analysis I n highly processive genomic DNA duplication, the DNA polymerase is tethered on the DNA strand through a direct interaction with the sliding clamp, which is topologically linked to the DNA by the action of the clamp loader (1). In this reaction, the clamp loader opens and reseals the clamp ring at the primer-template junctions in an ATP-dependent manner. Functional (2-8) and structural (9-12) analyses have indicated that the clamp-loading mechanism is conserved across the domains of life (13-15). All of the sliding clamps from phage to eukarya form similar planer rings, despite their distinct subunit compositions and lower sequence identities. Likewise, the clamp loader complexes from various organisms commonly exist as pentameric complexes with similar subunit configurations. The complexes have a unique oligomeric shape with the open ring in the N-terminal regions of each subunit, which folds into an architecture classified within the AAA ϩ ATPase superfamily (16), while the C-terminal regions form the closed ''collar'' structure. The crystal structure of the yeast clamp loader, replication factor C (RFC), in complex with the sliding clamp, proliferating cell nuclear antigen (PCNA), revealed their detailed contact mode and the elegant match of the spiral configuration of the Nterminal domains of RFC with that of the double-stranded (ds) DNA, and thus allowed the reasonable model building of the RFC-PCNA binary complex docked with a DNA duplex (12).We previously reported the 23-Å resolution EM structure of a clamp-loading RFC-PCNA-DNA ternary complex from Pyrococcus furiosus (Pfu), which was stabilized by introducing a nonhydrolyzable ATP analog, ATP␥S (17). The structure showed the two building blocks, a larger horseshoe and a smaller closed ring. It appeared the best interpretation based on the available data that the horseshoe and the closed ring correspond to RFC and PCNA, respectively. Although the atomic structures of the PCNA trimer (18) and RFC small subunits (RFCSs) (11) were available, along with the information about the 1:4 stoichiometry for RFC large subunit (RFCL) and RFCS in the RFC hetero-pentamer (5), the fitting of the atomic model into the EM map was not completely satisfactory, and some ambiguity remain...

The nuclear receptor, peroxisome proliferator-activated receptor c (PPARc), recognizes various synthetic and endogenous ligands by the ligand-binding domain. Fattyacid metabolites reportedly activate PPARc through conformational changes of the X loop. Here, we report that serotonin metabolites act as endogenous agonists for PPARc to regulate macrophage function and adipogenesis by directly binding to helix H12. A cyclooxygenase inhibitor, indomethacin, is a mimetic agonist of these metabolites. Crystallographic analyses revealed that an indole acetate functions as a common moiety for the recognition by the sub-pocket near helix H12. Intriguingly, a serotonin metabolite and a fatty-acid metabolite each bind to distinct sub-pockets, and the PPARc antagonist, T0070907, blocked the fatty-acid agonism, but not that of the serotonin metabolites. Mutational analyses on receptor-mediated transcription and coactivator binding revealed that each metabolite individually uses coregulator and/or heterodimer interfaces in a ligand-type-specific manner. Furthermore, the inhibition of the serotonin metabolism reduced the expression of the endogenous PPARc-target gene. Collectively, these results suggest a novel agonism, in which PPARc functions as a multiple sensor in response to distinct metabolites.

The atomic view of the active site coupling termed channelling is a major subject in molecular biology. We have determined two distinct crystal structures of the bacterial multienzyme complex that catalyzes the last three sequential reactions in the fatty acid b-oxidation cycle. The a 2 b 2 heterotetrameric structure shows the uneven ring architecture, where all the catalytic centers of 2-enoyl-CoA hydratase (ECH), L-3-hydroxyacyl-CoA dehydrogenase (HACD) and 3-ketoacyl-CoA thiolase (KACT) face a large inner solvent region. The substrate, anchored through the 3 0 -phosphate ADP moiety, allows the fatty acid tail to pivot from the ECH to HACD active sites, and finally to the KACT active site. Coupling with striking domain rearrangements, the incorporation of the tail into the KACT cavity and the relocation of 3 0 -phosphate ADP bring the reactive C2-C3 bond to the correct position for cleavage. The ahelical linker specific for the multienzyme contributes to the pivoting center formation and the substrate transfer through its deformation. This channelling mechanism could be applied to other b-oxidation multienzymes, as revealed from the homology model of the human mitochondrial trifunctional enzyme complex.