Abstract
The small GTPase Rho and one of its targets, Rho-associated kinase (ROCK), participate in a variety of actin-based cellular processes including smooth muscle contraction, cell migration, and stress fiber formation. The ROCK protein consists of an N-terminal kinase domain, a central coiled-coil domain containing a Rho binding site, and a C-terminal pleckstrin homology domain. Here we present the crystal structure of a large section of the central coiled-coil domain of human ROCK I (amino acids 535–700). The structure forms a parallel α-helical coiled-coil dimer that is structurally similar to tropomyosin, an actin filament binding protein. There is an unusual discontinuity in the coiled-coil; three charged residues (E613, R617 and D620) are positioned at what is normally the hydrophobic core of coiled-coil packing. We speculate that this conserved irregularity could function as a hinge that allows ROCK to adopt its autoinhibited conformation.
Highlights
Rho is a small GTPase that upon conversion from its inactive form (GDP N Rho) to active form (GTP N Rho) binds to specific effectors to exert its biological functions
The structure of Rho-associated kinase (ROCK)-CC was solved by single-wavelength anomalous dispersion (SAD) phasing at the peak wavelength using selenomethionine (SeMet) substituted protein
We describe the crystal structure of a tryptic fragment of the central coiled-coil domain of ROCK I
Summary
Rho is a small GTPase that upon conversion from its inactive form (GDP N Rho) to active form (GTP N Rho) binds to specific effectors to exert its biological functions. ROCK is able to regulate the phosphorylation of myosin light chain (MLC) by direct phosphorylation [7] and by inactivation of myosin phosphatase through the phosphorylation of its myosin-binding subunit (MBS) [8]. In non-muscle cells, the Rho/ROCK pathway is involved in stress fiber and focal adhesion formation [2,10,11], neurite retraction [12], and cell migration [13]. Increased MLC phosphorylation in non-muscle cells enhances the actin binding and actin-induced ATPase activity of myosin II. The enhanced binding of myosin to actin promotes the bundling of Factin into stress fibers and the subsequent formation of focal adhesions [14].
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