ROCK or Rho-associated kinase, a serine/threonine kinase, is an effector of Rho-dependent signaling and is involved in actin-cytoskeleton assembly and cell motility and contraction. The ROCK protein consists of several domains: an N-terminal region, a kinase catalytic domain, a coiled-coil domain containing a RhoA binding site, and a pleckstrin homology domain. The C-terminal region of ROCK binds to and inhibits the kinase catalytic domains, and this inhibition is reversed by binding RhoA, a small GTPase. Here we present the structure of the N-terminal region and the kinase domain. In our structure, two N-terminal regions interact to form a dimerization domain linking two kinase domains together. This spatial arrangement presents the kinase active sites and regulatory sequences on a common face affording the possibility of both kinases simultaneously interacting with a dimeric inhibitory domain or with a dimeric substrate. The kinase domain adopts a catalytically competent conformation; however, no phosphorylation of active site residues is observed in the structure. We also determined the structures of ROCK bound to four different ATP-competitive small molecule inhibitors (Y-27632, fasudil, hydroxyfasudil, and H-1152P). Each of these compounds binds with reduced affinity to cAMP-dependent kinase (PKA), a highly homologous kinase. Subtle differences exist between the ROCK- and PKA-bound conformations of the inhibitors that suggest that interactions with a single amino acid of the active site (Ala215 in ROCK and Thr183 in PKA) determine the relative selectivity of these compounds. Hydroxyfasudil, a metabolite of fasudil, may be selective for ROCK over PKA through a reversed binding orientation.

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Topoisomerase IV and DNA gyrase are related bacterial type II topoisomerases that utilize the free energy from ATP hydrolysis to catalyze topological changes in the bacterial genome. The essential function of DNA gyrase is the introduction of negative DNA supercoils into the genome, whereas the essential function of topoisomerase IV is to decatenate daughter chromosomes following replication. Here, we report the crystal structures of a 43-kDa N-terminal fragment of Escherichia coli topoisomerase IV ParE subunit complexed with adenylyl-imidodiphosphate at 2.0-Å resolution and a 24-kDa N-terminal fragment of the ParE subunit complexed with novobiocin at 2.1-Å resolution. The solved ParE structures are strikingly similar to the known gyrase B (GyrB) subunit structures. We also identified single-position equivalent amino acid residues in ParE (M74) and in GyrB (I78) that, when exchanged, increased the potency of novobiocin against topoisomerase IV by nearly 20-fold (to 12 nM). The corresponding exchange in gyrase (I78 M) yielded a 20-fold decrease in the potency of novobiocin (to 1.0 M). These data offer an explanation for the observation that novobiocin is significantly less potent against topoisomerase IV than against DNA gyrase. Additionally, the enzyme kinetic parameters were affected. In gyrase, the ATP K m increased Ϸ5-fold and the V max decreased Ϸ30%. In contrast, the topoisomerase IV ATP K m decreased by a factor of 6, and the V max increased Ϸ2-fold from the wild-type values. These data demonstrate that the ParE M74 and GyrB I78 side chains impart opposite effects on the enzyme's substrate affinity and catalytic efficiency.Type II topoisomerases catalyze the interconversion of DNA topoisomers by transporting one DNA segment through another. Bacterial genomes encode two type II topoisomerases, DNA gyrase and topoisomerase IV (TopoIV), that function in DNA replication. DNA gyrase is unique in coupling the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. In the absence of the ATP substrate, DNA gyrase can relax negatively supercoiled plasmid DNA. These activities result from the enzyme's ability to wrap (Ϸ150 bp) DNA (23, 31) around itself upon binding the DNA substrate. This DNA wrapping preferentially presents the T-segment (transported DNA segment) to the gyrase-DNA complex so that the introduction of negative supercoils is the primary outcome. In contrast, TopoIV and other eukaryotic type II topoisomerases only bind a Ϸ30-bp region of DNA (20,35). TopoIV utilizes the energy of ATP hydrolysis to decatenate newly replicated chromosomal DNA but also has the ability to relax positive and negative DNA supercoils in an ATP-dependent manner (8,43).In prokaryotes, these type II topoisomerases are composed of two subunits. In Escherichia coli, the gyrase subunits are named A and B and the corresponding TopoIV subunits are named C and E. For each enzyme, these subunits combine into a heterotetrameric (gyrase, A 2 B 2 ; and TopoIV, C 2 E 2 ) complex to form the functional enzymes. I...

The x-ray crystal structure of recombinant human renin has been determined. Molecular dynamics techniques that included crystallographic data as a restraint were used to improve an initial model based on porcine pepsinogen. The present agreement factor for data from 8.0 to 2.5 angstroms (A) is 0.236. Some of the surface loops are poorly determined, and these disordered regions border a 30 A wide solvent channel. Comparison of renin with other aspartyl proteinases shows that, although the structural cores and active sites are highly conserved, surface residues, some of which are critical for specificity, vary greatly (up to 10A). Knowledge of the actual structure, as opposed to the use of models based on related enzymes, should facilitate the design of renin inhibitors.

MAPK-activated protein kinase 2 (MAPKAPK2), one of several kinases directly phosphorylated and activated by p38 MAPK, plays a central role in the inflammatory response. The activated MAPKAPK2 phosphorylates its nuclear targets CREB/ATF1, serum response factor, and E2A protein E47 and its cytoplasmic targets HSP25/27, LSP-1, 5-lipoxygenase, glycogen synthase, and tyrosine hydroxylase. The crystal structure of unphosphorylated MAPKAPK2, determined at 2.8 Å resolution, includes the kinase domain and the C-terminal regulatory domain. Although the protein is inactive, the kinase domain adopts an active conformation with aspartate 366 mimicking the missing phosphorylated threonine 222 in the activation loop. The C-terminal regulatory domain forms a helix-turn-helix plus a long strand. Phosphorylation of threonine 334, which is located between the kinase domain and the C-terminal regulatory domain, may serve as a switch for MAPKAPK2 nuclear import and export. Phosphorylated MAPKAPK2 masks the nuclear localization signal at its C terminus by binding to p38. It unmasks the nuclear export signal, which is part of the second C-terminal helix packed along the surface of kinase domain C-lobe, and thereby carries p38 to the cytoplasm.

ATP-citrate lyase (ACLY, EC 2.3.3.8) 3 catalyzes the reaction, citrate ϩ CoA ϩ ATP 3 acetyl-CoA ϩ oxaloacetate ϩ ADP ϩ P i , in the presence of magnesium ions (1). ACLY is the cytoplasmic enzyme linking energy metabolism from carbohydrates to the production of fatty acids. Acetyl-CoA produced in the mitochondria cannot be exported to the cytoplasm. Instead, acetylCoA in mitochondria is transformed to citrate by citrate synthase, and citrate is exported to the cytoplasm where ACLY regenerates acetyl-CoA. This acetyl-CoA is an important precursor for fatty acid synthesis. In addition, ACLY has been shown to signal the metabolic state of cells, likely by providing acetyl-CoA for histone acetyltransferases in the cells' nuclei (2). The physiological importance of the enzyme is supported by knock-out experiments in which mice embryos lacking ATPcitrate lyase could not be obtained (3).Our understanding of the reaction mechanism of ACLY has been based on studies of this enzyme and of two enzymes with sequence similarity to ACLY. The first is succinyl-CoA synthetase (SCS), which catalyzes the formation of succinyl-CoA from succinate and CoA using ATP (4). The second is citrate synthase, the enzyme that generates citrate from acetyl-CoA and oxaloacetate in mitochondria. The reaction catalyzed by ACLY is thought to occur in four steps (Reactions 1-4):where E represents ACLY. Like SCS, ACLY is phosphorylated by ATP on an active site histidine residue to give E-P in step 1 (Reaction 1) (5, 6). The phosphoryl group is transferred to citrate in step 2 (Reaction 2). Citryl-phosphate is thought to remain bound to the enzyme, symbolized by E⅐citryl-P. Phosphate is released in the attack by CoA to form the citryl-CoA thioester bond in step 3 (Reaction 3) (7). In the last step (Reaction 4), citryl-CoA is cleaved to give acetyl-CoA and oxaloacetate, the reverse reaction to that catalyzed by citrate synthase.

V8 protease, an extracellular protease of Staphylococcus aureus, is related to the pancreatic serine proteases. The enzyme cleaves peptide bonds exclusively on the carbonyl side of aspartate and glutamate residues. Unlike the pancreatic serine proteases, V8 protease possesses no disulfide bridges. This is a major evolutionary difference, as all pancreatic proteases have at least two disulfide bridges. The structure of V8 protease shows structural similarity with several other serine proteases, specifically the epidermolytic toxins A and B from S. aureus and trypsin, in which the conformation of the active site is almost identical. V8 protease is also unique in that the positively charged N-terminus is involved in determining the substrate-specificity of the enzyme.

Two isoforms of succinyl-CoA synthetase exist in mammals, one specific for ATP and the other for GTP. The GTP-specific form of pig succinyl-CoA synthetase has been crystallized in the presence of GTP and the structure determined to 2.1 Å resolution. GTP is bound in the ATP-grasp domain, where interactions of the guanine base with a glutamine residue (Gln-20␤) and with backbone atoms provide the specificity. The ␥-phosphate interacts with the side chain of an arginine residue (Arg-54␤) and with backbone amide nitrogen atoms, leading to tight interactions between the ␥-phosphate and the protein. This contrasts with the structures of ATP bound to other members of the family of ATP-grasp proteins where the ␥-phosphate is exposed, free to react with the other substrate. To test if GDP would interact with GTP-specific succinyl-CoA synthetase in the same way that ADP interacts with other members of the family of ATP-grasp proteins, the structure of GDP bound to GTP-specific succinyl-CoA synthetase was also determined. A comparison of the conformations of GTP and GDP shows that the bases adopt the same position but that changes in conformation of the ribose moieties and the ␣-and ␤-phosphates allow the ␥-phosphate to interact with the arginine residue and amide nitrogen atoms in GTP, while the ␤-phosphate interacts with these residues in GDP. The complex of GTP with succinyl-CoA synthetase shows that the enzyme is able to protect GTP from hydrolysis when the active-site histidine residue is not in position to be phosphorylated.The enzyme succinyl-CoA synthetase (SCS) 4 uses ATP or GTP to catalyze the formation of succinyl-CoA from succinate and coenzyme A. In animals, two different isoforms exist, one specific for ATP and the other specific for GTP (1). The two isoforms include the same ␣-subunit, but different ␤-subunits (2). The amino-terminal domain of the ␤-subunit has an ATP-grasp fold (3-5), and Mg 2ϩ -ADP was shown to bind in this domain of the Escherichia coli SCS using labeling experiments and site-directed mutagenesis (6) and by soaking the nucleotide into crystals and determining the structure of the resulting complex (7). The ATP-grasp fold has been found in a number of other enzymes (8) (24), and RNA ligase 2 (25). The determinations of the structures of several of these enzymes in complex with nucleotides, nucleotide analogues, and their other substrates has led to a good understanding of the interactions that are important in their binding and catalysis.The biological roles of the ATP-and GTP-specific SCS have not been fully delineated. Originally it was thought that the primary role for SCS was in the citric acid cycle, where it was responsible for the breakdown of succinyl-CoA to succinate and coenzyme A accompanied by the phosphorylation of nucleotide diphosphate to nucleotide triphosphate (26). This step provides the only substrate-level phosphorylation of the citric acid cycle. It was thought that some species had SCS that could use either ADP or GDP, e.g. E. coli (27), while others had SCS that coul...