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J. Biol. Chem., Vol. 281, Issue 1, 260-268, January 6, 2006

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

From the Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139

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.

Received for publication, August 10, 2005 , and in revised form, October 7, 2005.

^* This work was supported in part by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division of the United States Department of Energy under Contract DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (codes 2ETR, 2ERZ, 2ESM, 2ETK, 2ETO) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¹ To whom correspondence should be addressed: 130 Waverly St., Cambridge, MA 02139. Tel.: 617-444-6451; Fax: 617-444-6566; E-mail: marc_jacobs{at}vrtx.com.

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