title: The structure of dimeric ROCK I reveals the mechanism for ligand selectivity.
abstract: 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.
authors.affiliation: Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139, USA. marc_jacobs@vrtx.com
authors.forename: Marc
authors.initials: M
authors.lastname: Jacobs
authors.forename: Koto
authors.initials: K
authors.lastname: Hayakawa
authors.forename: Lora
authors.initials: L
authors.lastname: Swenson
authors.forename: Steven
authors.initials: S
authors.lastname: Bellon
authors.forename: Mark
authors.initials: M
authors.lastname: Fleming
authors.forename: Paul
authors.initials: P
authors.lastname: Taslimi
authors.forename: John
authors.initials: J
authors.lastname: Doran
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000737:chemistry; D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000737:chemistry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008969:Molecular Sequence Data; D047428:Protein Kinase Inhibitors / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000737:chemistry; D013329:Structure-Activity Relationship; D054460:rho-Associated Kinases
chemical_list: C459092:2-methyl-1-((4-methyl-5-isoquinolinyl)sulfonyl)homopiperazine; D000577:Amides; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C431085:hydroxyfasudil; C108830:Y 27632; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; C049347:fasudil