Crystal structure of a coiled-coil domain from human ROCK I - PubMed (original) (raw)
Crystal structure of a coiled-coil domain from human ROCK I
Daqi Tu et al. PLoS One. 2011.
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.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Schematic view of the ROCK I domain structure.
In the inactive form, the Rho-binding domain (RBD) and the pleckstrin homology (PH) domain bind to the amino-terminal kinase domain. This autoinhibitory interaction is released either by GTP-Rho binding to RBD, or by caspase-3 cleavage of the PH domain. PH domain contains an internal cysteine-rich domain (CRD). The entire central domain (422–1102) is predicted to be coiled-coil, as indicated. ROCK-CC (in blue) is the solved crystal structure region in this report. The position of residue 617 is indicated with an arrow.
Figure 2. ROCK-CC forms a dimer in solution.
Purified ROCK (535–704) was analyzed on a Superdex 200 gel filtration column coupled to a multi-angle light scattering detector. The protein elution profile measured by refractive index is shown as a thin red trace; the horizontal thick red line is measured molar mass (∼40.5 KDa). Theoretical molar mass of a dimer is 40.4 KDa.
Figure 3. Overall structure of ROCK-CC.
(A) Ribbon diagram of the ROCK-CC dimer is shown with chain A in green and chain B in cyan. The parallel coiled-coil homo-dimer is asymmetric. (B) Crystal packing of dimer A/B with dimer C/D. The orientation is that of Fig. 3A turned 90° towards the viewer along the superhelical axis. Chain C is colored in yellow, chain D is in magenta.
Figure 4. Inter-helical interactions in ROCK-CC.
(A) In sphere representation, knobs from chain A (green) pack into holes from chain B (cyan). For clarity, only side chains are shown and knobs from chain B packing into holes from chain A are not displayed. (B) Helical wheel diagram of residues Asn-589 to Glu-665. Interhelical polar interactions are indicated by a line connecting paired residues. E613 and D620 at the d positions are colored red. R617 at the a position is colored blue. (C) Inter-Helical distance (HD) for two structures. Due to crystal packing, only residues 589 to 666 assume coiled-coil conformation for dimer A/B; for dimer C/D, residues 578 to 666 are in a coiled-coil conformation. Note that both dimers have a peak in their inter-helical distance at the E613/R617/D620 junction. (D) Average B-factors for mainchain atoms at each residue for all four chains. Since the structure was refined using TLS, the B-factor plotted represents the isotropic equivalent of the total B-factor (B_tls + B_individual).
Figure 5. Stereogram of electron density map at the coiled-coil interface in the region of the E613/R617/D620 bulge.
Chain C is shown in yellow and chain D in grey. The σA weighted 2FO – FC map is contoured at 1.5 σ and computed anisotropically to 2.3 Å. Due to the anisotropy, the map was sharpened by applying a B factor scaling of -10 Å2 (see methods).
Figure 6. Superposition of ROCK-CC and tropomyosin (PDB code 2d3e).
(A) DALI alignment matrix was based on only aligning ROCK-CC chain A to chain C in the tropomyosin structure. The tropomyosin structure is a fusion with the GCN4 leucine zipper; the tropomyosin portion of the structure (residues 176–282) is indicated below the superposition. Positions of R617 of ROCK chain A and E218 of tropomyosin chain D are indicated by arrows. (B) Detailed views of the respective unusual junctions in ROCK (left) and tropomyosin (right).
Figure 7. Sequence alignment of the ROCK coiled-coil region.
Residues 535–700 is the region used for this crystallographic study. Identical residues are indicated by the red background, similar residues by red characters. The experimentally identified _a_-g heptad repeats are displayed above the sequence for residues 589 to 665. Residues E613, R617 and D620 are marked by arrows.
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