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.

Cardiovascular effects of a novel potent and highly selective azaindole‐based inhibitor of Rho‐kinase

“…However, azaindole 1 was at least more than 250-fold selective for ROCK. This observation is supported by two recent studies (Breitenlechner et al, 2003;Jacobs et al, 2006), which describe the unique structural features of the active site of ROCKs. Accordingly, docking of azaindole 1 in a model of the catalytic domain of ROCK-1 (Jacobs et al, 2006) shows that the inhibitor interacts with residues at the ligandbinding site, which generates a uniquely shaped inhibitor binding pocket that confers the observed selectivity.…”

Activating ROCK1 somatic mutations in human cancer

“…As expected, the probe bound to the ROCK1 N-terminal residues 27-75 that mediate kinase dimerization (Figure 3b, Supplementary Figure 3a) (Jacobs et al, 2006). In addition, the kinase domain strongly bound four C-terminal regions (Figures 3a and b; regions 2d, 2f, 2h and 2i), indicating that potentially there are multiple sites engaged in kinase regulation.…”

“…ROCK1 is an unusual AGC kinase family member as its kinase domain assumes an active conformation without activation loop phosphorylation (Jacobs et al, 2006). Instead, ROCK1 activity is restrained by its inhibitory C terminus; relief from autoinhibition results from Rho-GTP binding or by caspase cleavage at a C-terminal site (Ishizaki et al, 1997;Coleman et al, 2001).…”

Roco kinase structures give insights into the mechanism of Parkinson disease-related leucine-rich-repeat kinase 2 mutations

“…4B). The first H1152 binding site, is in the nucleotide-binding pocket, as expected from its inhibitory mechanism, and is similar to the mode in which H1152 binds to ROCK1 (36). The binding site is formed by 17 residues, and the buried surface area is 280.6 Å 2 (Fig.…”