Structural and functional insights into the p160 Rho-associated coiled-coil-containing protein kinase ROCK

Abstract

Abstract Rho-associated protein kinases (ROCK1/2) are critical regulators of fundamental cellular processes and represent promising therapeutic targets for various pathologies, particularly cardiovascular disease. While the terminal globular kinase domain and the PH-C1 tandem (PHn-C1-PHc) are well characterized, the sequence-structure-function relationship of the central amphipathic α-helical segment remains poorly understood. Utilizing an integrative biophysical approach—including multi-angle light scattering (MALS), analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), negative-stain electron microscopy (EM), X-ray crystallography, and 3D molecular modeling—we elucidate the structural organization of the central coiled-coil region of ROCK1. Notably, the 2.2 Å resolution crystal structure of the homology region 1 (HR1) of ROCK1 reveals a canonical parallel coiled-coil dimer. This structural arrangement diverges significantly from the antiparallel coiled-coil HR1 module found in other Rho effector kinases. Combined with the structural reconstitution of full-length ROCK1, our data demonstrates that the central segment of ROCK1 forms a highly elongated coiled-coil dimer. Furthermore, EM analysis of the isolated full coiled-coil domain uncovers a dynamic oligomerization state, transitioning between a dimer and a tetramer. Together, these biophysical insights establish a foundation that opens avenues for future investigation into ROCK1 regulation, activation, and downstream signaling mechanisms.