Figure 1: ROCK2 forms a semi-rigid extended coiled-coil dimer.
From: A molecular ruler regulates cytoskeletal remodelling by the Rho kinases
(a) ROCK2 domain composition. N-terminal kinase and C-terminal membrane-binding PH and C1 domains are separated by 730 amino acids of predicted coiled-coil. (b) Static light-scattering analysis of recombinant ROCK2. ROCK2 has a molecular weight of 327 kDa and a polydispersity index of 1.000. (c) FCS of EGFP-labelled ROCK2. ROCK2 was isolated from mammalian cells using FSEC and its diffusion coefficient determined by FCS. The value of 25 μm2 s−1 is consistent with an extended, semi-rigid particle. (d) Rotary shadowing electron microscopy of ROCK2. Dimeric ROCK2 particles consist of globular N- and C-terminal domains separated by a long coiled-coil. (e) Zoomed image of a representative particle. The particles show a clear asymmetry between their ends: while one end appears more compact, the other end is characterized by two regions of electron density. The length of the coiled-coil is 106.7±3.8 nm (n=10). (f) Scale model of ROCK2. The N-terminal kinase domains dimerize via their capped helix bundle domains (PDB: 2F2U). A parallel coiled-coil of 107 nm joins the kinase domains to the monomeric C-terminal membrane-binding regulatory domains (PDBs: 2ROV (C1), 2ROW (split PH)), giving a maximum particle length of 120 nm. We note that the scale model gives the impression of a longer particle than the rotary-shadowed images of ROCK2, but that this is a consequence of the shadowing of the globular domains, which gives the impression of domains that are larger than they actually are.
