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Thrombin orchestrates cellular events after injury to the vascular system and extravasation of blood into surrounding tissues. The pathophysiological response to thrombin is mediated by proteaseactivated receptor-1 (PAR-1), a seven-transmembrane G proteincoupled receptor expressed in the nervous system that is identical to the thrombin receptor in platelets, fibroblasts, and endothelial cells. Once activated by thrombin, PAR-1 induces rapid and dramatic changes in cell morphology, notably the retraction of growth cones, axons, and dendrites in neurons and processes in astrocytes. The signal is conveyed by a series of localized ATP-dependent reactions directed to the actin cytoskeleton. How cells meet the dynamic and localized energy demands during signal transmission is unknown. Using the yeast two-hybrid system, we identified an interaction between PAR-1 cytoplasmic tail and the brain isoform of creatine kinase, a key ATP-generating enzyme that regulates ATP within subcellular compartments. The interaction was confirmed in vitro and in vivo. Reducing creatine kinase levels or its ATP-generating potential inhibited PAR-1-mediated cellular shape changes as well as a PAR-1 signaling pathway involving the activation of RhoA, a small G protein that relays signals to the cytoskeleton. Thrombin-stimulated intracellular calcium release was not affected. Our results suggest that creatine kinase is bound to PAR-1 where it may be poised to provide bursts of site-specific high-energy phosphate necessary for efficient receptor signal transduction during cytoskeletal reorganization. P rotease activated receptor-1 (PAR-1) mediates the cellular responses to thrombin during blood coagulation, cell proliferation, vascular permeability changes, tumor metastasis, and nervous system injury (1-3). PAR-1 is a seven-transmembrane G protein-coupled receptor with a novel activation mechanism. Proteolysis at a thrombin cleavage site in the extracellular amino terminus exposes a new amino terminus containing the peptide ligand SFLLRN, which binds intramolecularly to initiate intracellular signals (4). Although originally detected in platelets, endothelial cells, and fibroblasts, PAR-1 is also expressed in the nervous system in a developmentally regulated manner and by specific subpopulations of neurons and astrocytes that are especially vulnerable to neurodegeneration and ischemic injury (1,5,6).In most cells expressing PAR-1, activation of the receptor transmits signals to the actin cytoskeleton that profoundly alter cell shape. Platelets, for example, convert from a spherical to disk shape and extend filopodia (7), endothelial cells contract (8), neurons retract axons, and astrocytes resorb processes and flatten their cell bodies (9-11). These signals also regulate changes in actin-related cell motility observed in neurons (10), fibroblasts (12), and tumor cells (3). The morphological response is mediated by a key signaling pathway that uses serine͞ threonine kinases, G␣12͞13, RhoA, and myosin light chain kinase; actomyosin contraction ...

Termination of DNA replication at a sequence-specific replication terminus is potentiated by the binding of the replication terminator protein (RTP) to the terminus sequence, causing polar arrest of the replicative helicase (contrahelicase activity). Two alternative models have been proposed to explain the mechanism of replication fork arrest. In the first model, the RTP-terminus DNA interaction simply imposes a polar barrier to helicase movement without involving any specific interaction between the helicase and the terminator proteins. The second model proposes that there is a specific interaction between the two proteins, and that the DNA-protein interaction both restricts the fork arrest to the replication terminus and determines the polarity of the process. The evidence presented in this paper strongly supports the second model.

The replication terminator protein (RTP) protein is mostly a-helical, and long C-terminal a-helices from each monomer interact to form an antiparallel coiled-coil dimerization domain. The dimer also contains a pair of two-fold related 3-strand (3-sheets with associated ,-ribbons. The structure of one monomer of RTP is shown in Fig. 1. Based on pertinent biochemical data, we have proposed that one RTP dimer binds two turns of DNA. In the model, the pair of a3-helices make base-specific contacts in successive major grooves, the (3-ribbons and the connecting loops interact with the peripheral minor grooves, and the flexible N-terminal arms wrap around the central minor groove (18). The crystal structure did not provide any clue as to the location of the dimer-dimer interaction surface, but computer graphics modeling exercises using two adjacent ATP-DNA model complexes suggest that it is close to the ,3-ribbon.In this paper, we address the question of the location and structural nature of the dimer-dimer interaction surface by performing site-directed mutagenesis, purifying the mutant forms of the protein, and analyzing the biochemical properties of the mutant proteins. Initial results conclusively showed that tyrosine-88 plays a critical role in promoting dimer-dimer interaction. Substitution of the tyrosine by a phenylalanine completely abolishes dimer-dimer interaction, and fails to block E. coli DnaB helicase and replication fork progression. This result suggested that the dimer-dimer interaction involves strand (33, and additional

Thrombin is a serine protease that evokes various cellular responses involved in injury and repair of the nervous system through the activation of protease-activated receptor-1 (PAR-1). Signals that modulate cell morphology precede most PAR-1 effects, but the initial signal transduction molecules are not known. Using the yeast two-hybrid system, we identified Hsp90, a chaperone with known signaling properties, as a binding partner of PAR-1. The interaction was confirmed by glutathione Stransferase pull-down, overlay, and co-immunoprecipitation assays. Morphological assays in mouse astrocytes were carried out to evaluate the importance of Hsp90 during cytoskeletal signaling. Reducing Hsp90 levels by antisense treatment or disruption of the Hsp90⅐PAR-1 complex by the Hsp90-specific drug geldanamycin attenuated thrombinmediated astrocyte shape changes. Furthermore, overexpression of the PAR-1 cytoplasmic tail abrogated thrombininduced cytoskeletal changes in neuronal cells. Treatment with geldanamycin specifically inhibited activation of RhoA without affecting thrombin-mediated intracellular calcium release, revealing the regulation of a distinct signaling pathway by Hsp90. Taken together, these studies demonstrate that Hsp90 may be essential for PAR-1-mediated signaling to the cytoskeleton.Thrombin is a serine protease involved in a number of pathophysiological processes that include blood clotting, inflammation, repair processes, and tumor metastasis (1-4). In brain, thrombin regulates the viability of neurons and astrocytes by increasing survival under conditions of hypoglycemia and oxidative stress and inducing apoptosis under other conditions (5-8). Thrombin is also chemotactic for macrophages and mitogenic for smooth muscle cells, fibroblasts, and astrocytes and induces secretion of growth factors and cytokines from fibroblasts and smooth muscle cells (9). Most of the thrombinmediated effects are preceded by morphological changes in cells that follow activation of a seven-transmembrane G proteincoupled receptor called protease-activated receptor-1 (PAR-1)

The replication terminator protein (RTP) of Bacillus subtilis impedes replication fork movement in a polar mode upon binding as two interacting dimers to each of the replication termini. The mode of interaction of RTP with the terminus DNA is of considerable mechanistic significance because the DNA‐protein complex not only localizes the helicase‐blocking activity to the terminus, but also generates functional asymmetry from structurally symmetric protein dimers. The functional asymmetry is manifested in the polar impedance of replication fork movement. Although the crystal structure of the apoprotein has been solved, hitherto there was no direct evidence as to which parts of RTP were in contact with the replication terminus. Here we have used a variety of approaches, including saturation mutagenesis, genetic selection for DNA‐binding mutants, photo cross‐linking, biochemical and functional characterizations of the mutant proteins, and X‐ray crystallography, to identify the regions of RTP that are either in direct contact with or are located within 11 angstroms of the replication terminus. The data show that the unstructured N‐terminal arm, the alpha3 helix and the beta2 strand are involved in DNA binding. The mapping of amino acids of RTP in contact with DNA, confirms a ‘winged helix’ DNA‐binding motif.

The replication terminator protein (RTP) of Bacillus subtilis is a homodimer that binds to each replication terminus and impedes replication fork movement in only one orientation with respect to the replication origin. The threedimensional structure of the RTP-DNA complex needs to be determined to understand how Replication of the chromosome of Bacillis subtilis is initiated at an unique origin, and under normal conditions the forks progress bidirectionally until converging at six sequencespecific replication termini that are located approximately 1800 from the origin (1-3). The terminus IR1 (Terl) appears to be the most frequently used site for replication fork arrest in vivo (4). Each terminus arrests replication forks in vivo and in vitro in only one orientation with respect to the replication origin (4-6). The replication terminator protein (RTP) specifically binds as two interacting dimers to each terminus (7-9). RTP is functional in vivo (10) and in vitro (5, 6, 11) in the surrogate Gram-negative Escherichia coli system and arrests the replicative helicases DnaB and PriA of E. coli in a polar mode (5, 6, 9, 11).RTP is a homodimeric protein with subunit molecular mass of 14.5 kDa (12, 13). The crystal structure of RTP has been determined at 2.6-A resolution and the structure reveals a disordered N-terminal arm, four a-helices, and three antiparallel (3-strands. The (2-and (3-strands are connected by an extended loop and the two a4-helices of the two monomers form an antiparallel coiled-coil dimerization domain. The overall structure is a winged helix with the (32-and (33-strands and the connecting loop of the two monomers forming the two wings and the al-, a2-, and a3-helices forming the prototypical helices of the winged helix ( Fig. 1 and ref.14). Two interacting dimers of RTP, bound to the overlapping core and the auxiliary sites, are necessary to impede replication forks (9). We have shown that the (33-strands and the tip of the extended loop that connects P2 with (33 are both necessary for dimerdimer interaction (9). Mutational analyses and biochemical studies have shown that the N-terminal arm, the (32-strand, and the a3-helix of RTP are involved in DNA binding (15).Although the structure of the RTP dimer is symmetrical (14), the protein impedes fork movement in an asymmetric mode (5-7, 9). It is reasonable to suspect that the interaction of the protein with DNA might provide the structural basis of the functional polarity. To understand the mechanistic details of replication fork arrest, it will be necessary to determine the structure of the RTP-DNA complex. The minimum functional unit of the replication terminus of B. subtilis consists of four turns of DNA and two interacting dimers of RTP, a structure that is perhaps too large and too flexible to lend itself to cocrystallization. We have therefore resorted to an alternative approach to derive a model of the three-dimensional structure of the DNA-protein complex. The approach consisted of the conversion of RTP into a site-directed chemical...

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