Jacobs Marc, Hayakawa Koto, Swenson Lora, Bellon Steven, Fleming Mark, Taslimi Paul, Doran John
Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139, USA.
J Biol Chem. 2006 Jan 6;281(1):260-8. doi: 10.1074/jbc.M508847200. Epub 2005 Oct 24.
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.
ROCK，即Rho相关激酶，是一种丝氨酸/苏氨酸激酶，是Rho依赖性信号传导的效应器，参与肌动蛋白细胞骨架组装以及细胞运动和收缩。ROCK蛋白由几个结构域组成：一个N端区域、一个激酶催化结构域、一个包含RhoA结合位点的卷曲螺旋结构域和一个普列克底物蛋白同源结构域。ROCK的C端区域与激酶催化结构域结合并抑制它，而这种抑制作用会因结合小GTP酶RhoA而逆转。在此，我们展示了N端区域和激酶结构域的结构。在我们的结构中，两个N端区域相互作用形成一个二聚化结构域，将两个激酶结构域连接在一起。这种空间排列使得激酶活性位点和调控序列位于同一面上，从而使两个激酶有可能同时与一个二聚体抑制结构域或二聚体底物相互作用。激酶结构域呈现出具有催化活性的构象；然而，在该结构中未观察到活性位点残基的磷酸化。我们还确定了ROCK与四种不同的ATP竞争性小分子抑制剂（Y-27632、法舒地尔、羟基法舒地尔和H-1152P）结合的结构。这些化合物中的每一种与cAMP依赖性激酶（PKA）（一种高度同源的激酶）结合的亲和力都降低。抑制剂与ROCK和PKA结合的构象之间存在细微差异，这表明与活性位点的单个氨基酸（ROCK中的Ala215和PKA中的Thr183）的相互作用决定了这些化合物的相对选择性。羟基法舒地尔是法舒地尔的一种代谢产物，可能通过相反的结合方向对ROCK的选择性高于PKA。
