Ligand-induced conformational rearrangements regulate the switch between membrane-proximal and distal functions of Rho kinase 2

István Hajdú,András Szilágyi,András Wacha,Éva Gráczer,Péter Gál,Márk Somogyi,Péter Závodszky,Dániel Györffy,Barbara M Végh

doi:10.1038/s42003-020-01450-x

Abstract

Rho-associated protein kinase 2 (ROCK2) is a membrane-anchored, long, flexible, multidomain, multifunctional protein. Its functions can be divided into two categories: membrane-proximal and membrane-distal. A recent study concluded that membrane-distal functions require the fully extended conformation, and this conclusion was supported by electron microscopy. The present solution small-angle X-ray scattering (SAXS) study revealed that ROCK2 population is a dynamic mixture of folded and partially extended conformers. Binding of RhoA to the coiled-coil domain shifts the equilibrium towards the partially extended state. Enzyme activity measurements suggest that the binding of natural protein substrates to the kinase domain breaks up the interaction between the N-terminal kinase and C-terminal regulatory domains, but smaller substrate analogues do not. The present study reveals the dynamic behaviour of this long, dimeric molecule in solution, and our structural model provides a mechanistic explanation for a set of membrane-proximal functions while allowing for the existence of an extended conformation in the case of membrane-distal functions.

Highlights

Rho-associated protein kinase 2 (ROCK2) is a membrane-anchored, long, flexible, multidomain, multifunctional protein
ROCK2 kinase was initially identified as a Rho-associated protein, the direct binding between recombinant RhoA and ROCK2 was recently questioned[29], despite the fact that even a crystal structure of a complex between RhoA and a ROCK1 fragment had been published[25]
We could not detect the ternary complex between ROCK2/RhoA/BODIPY-GTP directly, indirect evidence was obtained: BODIPY-GTP bound much more strongly to RhoA in the presence of ROCK2 than in its absence, suggesting the presence of a ternary complex similar to that measured for ROCK130, this ternary complex is weak, and the dynamic equilibrium between ROCK2 and RhoA/BODIPY-GTP is probably shifted towards the dissociated form. (Figs. 1 and S1)

Summary

Introduction

Rho-associated protein kinase 2 (ROCK2) is a membrane-anchored, long, flexible, multidomain, multifunctional protein. Rho-associated coiled-coil containing kinases (ROCK1 and ROCK2) are known to have multiple repertoires of activities in several cellular functions[1] Their downstream targets are involved in cell shape and motility, cell survival and apoptosis, vesicle dynamics, cell growth and regeneration, and cytoskeleton regulation. The two ROCK proteins share 64% identity in their amino acid sequence with the highest (92%) identity in their kinase domains[13] The functions of these kinases were initially considered highly overlapping, but knockout studies showed that disruption of either gene is sufficient to generate stillborn embryos[14,15], indicating that at least in early development, the two enzymes cannot substitute for each other, it was shown that they can compensate for each other in adult organisms[16]. The dimerization of the protein is suggested to play a role in its biological function towards dimeric substrates[13]

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