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

Marc Jacobs,Koto Hayakawa,Lora Swenson,Steven Bellon,Mark Fleming,Paul Taslimi,John Doran

doi:10.1074/jbc.m508847200

Marc Jacobs, Koto Hayakawa + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m508847200

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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.

Highlights

Because desired therapeutic outcomes would likely result from a reduction of ROCK activity, much research has focused on designing small molecule inhibitors of ROCK
Two Kinase Domains Interact via a Dimerization Domain—The protein used in this study consists of the N-terminal region comprising approximately one-third of the full-length sequence of ROCK
Each monomer consists of an N-terminal helical domain, a bilobed kinase domain, and a kinase tail

Summary

Introduction

Because desired therapeutic outcomes would likely result from a reduction of ROCK activity, much research has focused on designing small molecule inhibitors of ROCK. There are two isoforms of ROCK, known as ROCK I and II, or Rhokinase ␤ and ␣, respectively [5] These two kinases regulate the activity of muscle myosin regulatory light chain (RLC) proteins by direct phosphorylation, [6, 7] and by phosphorylation and inhibition of the myosin binding subunit of myosin phosphatase. The C-terminal region of ROCK (coiled-coil and pleckstrin homology domains) has been shown to partially inhibit the kinase catalytic activity by binding directly to the kinase domain [15]. ROCK (6 – 415) proved to be highly homogenous in solution and yielded diffraction quality crystals suitable for x-ray structure determination This construct lacks the C-terminal inhibitory domains and is similar to the caspase-3-activated form of ROCK. An analogous form of truncated CRIK has been observed in vivo [14]

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Discussion

Conclusion