Koto Hayakawa

The Structure of Dimeric ROCK I Reveals the Mechanism for Ligand Selectivity

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.

Structure of Mitogen-activated Protein Kinase-activated Protein (MAPKAP) Kinase 2 Suggests a Bifunctional Switch That Couples Kinase Activation with Nuclear Export

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.

Crystal Structures of Escherichia coli Topoisomerase IV ParE Subunit (24 and 43 Kilodaltons): a Single Residue Dictates Differences in Novobiocin Potency against Topoisomerase IV and DNA Gyrase

ABSTRACT 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 μM). 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 Km increased ≈5-fold and the Vmax decreased ≈30%. In contrast, the topoisomerase IV ATP Km decreased by a factor of 6, and the Vmax increased ≈2-fold from the wild-type values. These data demonstrate that the ParE M74 and GyrB I78 side chains impart opposite effects on the enzymes substrate affinity and catalytic efficiency.

Identification of the citrate-binding site of human ATP-citrate lyase using X-ray crystallography.

ATP-citrate lyase (ACLY) catalyzes the conversion of citrate and CoA into acetyl-CoA and oxaloacetate, coupled with the hydrolysis of ATP. In humans, ACLY is the cytoplasmic enzyme linking energy metabolism from carbohydrates to the production of fatty acids. In situ proteolysis of full-length human ACLY gave crystals of a truncated form, revealing the conformations of residues 2–425, 487–750, and 767–820 of the 1101-amino acid protein. Residues 2–425 form three domains homologous to the β-subunit of succinyl-CoA synthetase (SCS), while residues 487–820 form two domains homologous to the α-subunit of SCS. The crystals were grown in the presence of tartrate or the substrate, citrate, and the structure revealed the citrate-binding site. A loop formed by residues 343–348 interacts via specific hydrogen bonds with the hydroxyl and carboxyl groups on the prochiral center of citrate. Arg-379 forms a salt bridge with the pro-R carboxylate of citrate. The pro-S carboxylate is free to react, providing insight into the stereospecificity of ACLY. Because this is the first structure of any member of the acyl-CoA synthetase (NDP-forming) superfamily in complex with its organic acid substrate, locating the citrate-binding site is significant for understanding the catalytic mechanism of each member, including the prototype SCS. Comparison of the CoA-binding site of SCSs with the similar structure in ACLY showed that ACLY possesses a different CoA-binding site. Comparisons of the nucleotide-binding site of SCSs with the similar structure in ACLY indicates that this is the ATP-binding site of ACLY.

Interactions of GTP with the ATP-grasp domain of GTP-specific succinyl-CoA synthetase.

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-20β) and with backbone atoms provide the specificity. The γ-phosphate interacts with the side chain of an arginine residue (Arg-54β) and with backbone amide nitrogen atoms, leading to tight interactions between the γ-phosphate and the protein. This contrasts with the structures of ATP bound to other members of the family of ATP-grasp proteins where the γ-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 α- and β-phosphates allow the γ-phosphate to interact with the arginine residue and amide nitrogen atoms in GTP, while the β-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.

Catalytic role of the conformational change in succinyl-CoA:3-oxoacid CoA transferase on binding CoA.

Catalysis by succinyl-CoA:3-oxoacid CoA transferase proceeds through a thioester intermediate in which CoA is covalently linked to the enzyme. To determine the conformation of the thioester intermediate, crystals of the pig enzyme were grown in the presence of the substrate acetoacetyl-CoA. X-ray diffraction data show the enzyme in both the free form and covalently bound to CoA via Glu305. In the complex, the protein adopts a conformation in which residues 267-275, 280-287, 357-373, and 398-477 have shifted toward Glu305, closing the enzyme around the thioester. Enzymes provide catalysis by stabilizing the transition state relative to complexes with substrates or products. In this case, the conformational change allows the enzyme to interact with parts of CoA distant from the reactive thiol while the thiol is covalently linked to the enzyme. The enzyme forms stabilizing interactions with both the nucleotide and pantoic acid portions of CoA, while the interactions with the amide groups of the pantetheine portion are poor. The results shed light on how the enzyme uses the binding energy for groups remote from the active center of CoA to destabilize atoms closer to the active center, leading to acceleration of the reaction by the enzyme.

Crystallization of canine cardiac calsequestrin

Calsequestrin is the major Ca2+ binding protein in the lumen of the sarcoplasmic reticulum membranes. Two X-ray quality crystal forms of canine cardiac calsequestrin were obtained by the hanging drop method using KCl as a precipitant. One form is monoclinic (space group P2(1), a = 73.4 A, b = 104.4 A, c = 60.2 A, beta = 120.4 degrees) with two molecules in the asymmetric unit and a solvent content of approximately 40%. The second form is trigonal (P3(1)21 or P3(2)21, a = b = 99.3 A, c = 89.8 A) with a single molecule in the asymmetric unit and 55% solvent content. Cross rotation function calculations show that despite the different space groups the packing of the molecules in both crystals is likely to be similar suggesting the existence of a stable dimer. The monoclinic crystals diffract beyond 3 A using a laboratory rotating anode source, while under the same conditions the trigonal crystals diffract only to approximately 4.5 A. This is the first report of successful preparation of X-ray quality crystals of a high capacity Ca2+ binding protein.